THE UNITED REPUBLIC OF

TANZANIA

MINISTRY OF HEALTH

National Medical Laboratory

Standard Operating
Procedures for Dispensaries

Version 01:2024

JANUARY, 2024
NATIONAL MEDICAL STANDARD OPERATING PROCEDURES FOR
DISPENSARY LEVEL LABORATORY

PUBLISHED IN 2024

©
MINISTRY OF HEALTH,
MAGUFULI GOVERNMENT CITY, AFYA ROAD/STREET, MTUMBA,
PO BOX 743,
40478 DODOMA, TANZANIA.
LANDLINE: +255 (0)26 232 3267
EMAIL: [email protected]
WEBSITE: www.moh.go.tz

ISBN: 978-9912-9833-7-3

Any section(s) or clause(s) within this document, can be used by the Government of Tanzania
Departments, implementers and stakeholders, provided that, the Ministry of Health is
acknowledged
Disclaimer

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 DISCLAIMER
This document is provided on the basis that it is for the use of MoH and PO-RALG
only. MoH will not be bound to discuss, explain or reply to queries raised by any
agency/organisation other than the intended recipients of this document. MoH
disclaims all liability to any third party who may place reliance on this document and
therefore does not assume responsibility for any loss or damage suffered by any such
third party in reliance thereon.
The images and related drawings used in this document are intended solely as a
guiding support and should be considered as purely indicative, not restrictive of the
expected item characteristics nor an indication of the client’s choice and/or preference.
The views expressed in this manual are those of the individual contributors. Comments
on the usefulness of this manual, and suggestions for improvements in future editions
will be welcome, and should be addressed to the contact therein.
This communication may contain information that is proprietary, confidential, or exempt
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dissemination, distribution, use or copying of this communication is strictly prohibited.
Anyone who receives this document in a message in error should notify the sender
immediately by telephone or by return e-mail and delete it from their computer.

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Table of Contents

ABBREVIATIONS AND ACRONYMS ................................................................................................. I

TERMS AND DEFINITIONS .............................................................................................................V

FOREWORD ................................................................................................................................VI

ACKNOWLEDGEMENTS ................................................................................................................. I

EXECUTIVE SUMMARY ................................................................................................................. II

SCOPE......................................................................................................................................... III

PRIMARY BENEFICIARIES ............................................................................................................. III

SECONDARY BENEFICIARIES ........................................................................................................ III

CHAPTER ONE: SAMPLE COLLECTION ......................................................................................... 1

1.0 GENERAL CONSIDERATIONS................................................................................................. 1

2.0 COLLECTION OF BLOOD SAMPLES......................................................................................... 2
3.0 COLLECTION OF URINE SAMPLES .......................................................................................... 5
4.0 COLLECTION OF BODY FLUIDS .............................................................................................. 8
5.0 COLLECTION OF GENITAL SAMPLES .................................................................................... 10
6.0 WOUND SAMPLES ............................................................................................................. 12
7.0 COLLECTION OF THROAT AND NASAL SWABS ..................................................................... 13
7 COLLECTION OF OROPHRYANGEAL SWABS............................................................................ 15
8 SPUTUM SAMPLES ............................................................................................................... 15

CHAPTER TWO: PARASITOLOGY .............................................................................................. 17

2.1. PROCEDURE FOR MALARIA RAPID TEST (MRDT)................................................................. 17

2.2. PROCEDURE FOR MALARIA MICROSCOPY.......................................................................... 19
2.3. PROCEDURE FOR URINE MICROSCOPY .............................................................................. 23
2.4. PROCEDURE FOR STOOL ROUTINE EXAMINATION.............................................................. 26

CHAPTER THREE: BLOOD TRANSFUSION .................................................................................. 31

3.0 PROCEDURE FOR ABO AND RHESUS BLOOD GROUPING ...................................................... 31

3.1 PROCEDURE FOR ESTIMATION OF HEMOGLOBIN BY USING COPPER SULPHATE SOLUTION ... 36
3.2 PROCEDURE FOR COMPATIBILITY TESTING ......................................................................... 39

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CHAPTER FOUR: HAEMATOLOGY............................................................................................. 42

4.1 PROCEDURE FOR URIT-12 HEMOGLOBIN METER ................................................................. 42

4.2 PROCEDURE FOR DETERMINATION OF HAEMOGLOBIN LEVEL USING HEMOCHROMAX PLUS 44
4.3 PROCEDURE FOR DETREMANATION OF HEMOGLOBIN LEVEL USING HEMOCUE 201+ MACHINE
46
4.4 PROCEDURE FOR CD 4 COUNT TEST BY USING BD FACS PRESTO ........................................... 48
4.5 PROCEDUIRE FOR FULL BLOOD COUNT BY USING URIT BH – 40P HAEMATOLOGY ANALYSER. 51
4.6 PROCEDURE FOR FULL BLOOD COUNT USING OF ABX PENTRA 80 HAEMATOLOGY ANALYSER.
53
4.7 PROCEDURE FOR FULL BLLOD COUNT USING SINNOWA HB - 7021 ....................................... 56
4.8 PROCEDURE FOR FULL BLOOD COUNT USING BHA 3000 HAEMATOLOGY ANALYSER ........... 59
4.9 PROCEDURE FOR PERFORMING FULL BLOOD COUNT BY USING MS4 HAEMATOLOGY
ANALYSER.................................................................................................................................. 62

CHAPTER FIVE: CLINICAL CHEMISTRY AND IMMUNOLOGY ....................................................... 65

5.1 PROCEDURE FOR BLOOD GLUCOSE BY USING ACCUCHECK GLUOCOMETER .......................... 65

5.2 PROCEDURE FOR TESTING BLOOD GLUCOSE BY GLUCO PLUS ............................................... 68
5.3 PROCEDURE FOR URIT 50 (URINE CHEMISTRY ANALYZER) ................................................... 71
5.4 PROCEDURE FOR (URIT-560) URINE ANALYZER ................................................................... 74
5.5 ROCEDURE FOR PERFORMING URINE BIOCHEMISTRY BY USING CYBOW READER 300 .......... 76
5.6 PROCEDURE FOR DETERMINATION OF ALT BY USING DIRUI-DR 7000 CHEMISTRY ANALYZER 79
5.7 PROCEDURE FOR DETERMINATION OF AST BY USING DIRUI-DR7000 CHEMISTRY ANALYZER 81
5.8 PROCEDURE FOR SA-30 SEMI AUTOMATED CHEMISTRY ANALYZER ..................................... 84
5.9 PROCEDURE FOR OPERATING CLINDIAG FA 200 CHEMISTRY TEST ........................................ 86
5.10 PROCEDURE FOR DETERMINATION OF IMMUNOASSAYS BY USING GETEIN 1100
IMMUNOANALYSER ................................................................................................................... 89
5.11 PROCEDURE FOR OPERATING FIA 8000 ANALYSER ............................................................ 91
5.12 PROCEDURE FOR OPERATING ALERE AFFINION AS100ANALYSER ....................................... 94

CHAPTER SIX: SEROLOGY......................................................................................................... 97

6.1 PROCEDURE FOR SYPHILIS ANTIBODIES RAPID TEST ............................................................ 97

6.2 PROCEDURE FOR (HIV) TESTING BY USING BIOLINETM HIV 1/2 3.0 TEST ............................ 100
6.3 PROCEDURE FOR PERFORMING (HIV) BY USING UNIGOLD TEST ........................................ 104
6.4 PROCEDURE FOR URINE PREGNANCY TEST ....................................................................... 106
6.5 PROCEDURE FOR HEPATITIS C ANTIBODY RAPID TEST ....................................................... 109
6.6 PROCEDURE FOR CRYPTOCOCCAL ANTIGEN RAPID TEST ................................................... 113
6.7 PROCEDURE FOR PERFORMING (HBSAG) RAPID TEST......................................................... 115
6.8 PROCEDURE FOR SARS-COV-2 ANTIGEN RAPID DIAGNOSTIC TEST ..................................... 118

CHAPTER SEVEN: BACTERIOLOGY .......................................................................................... 121

7.1 POTASSIUM HYDROXIDE (KOH) WET MOUNT PREPARATION ............................................ 121

7.2 PROCEDURE FOR ZIEHL NIELSEN (ZN) STAIN ...................................................................... 124
7.3 PROCEDURE FOR AURAMINE O PHENOL STAINING ........................................................... 127
7.4 PROCEDURE FOR GRAMS STAINING ................................................................................. 131

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7.5 DIAGNOSIS OF MTB/RIF TESTING USING A TRUENAT MACHINE ......................................... 133

ANNEX 1: LIST OF PARTICIPANTS WHO DEVELOPED THE SOPS................................................ 140

ANNEX 2: BIOLOGICAL REFERENCE INTERVALS FOR FULL BLOOD COUNT ................................ 143

ANNEX 3: BIOLOGICAL REFERENCE INTERVALS FOR COAGULATION PROFILE ........................... 144

ANNEX 4: BIOLOGICAL REFERENCE INTERVALS FOR URINE BIOCHEMISTRY ............................. 145

ANNEX 5: BIOLOGICAL REFERENCE INTERVALS FOR CLINICAL CHEMISTRY AND IMMUNOASSAYS

146

ANNEX 6: CRITICAL/PANIC VALUES THAT CALLS FOR IMMEDIATE ACTIONS ............................ 150

NOTE PAD ................................................................................................................................ 151

NOTE PAD ................................................................................................................................ 152

NOTE PAD ................................................................................................................................ 153

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ABBREVIATIONS AND ACRONYMS
For the purposes of this NMLSOP document, these abbreviations and acronyms will
apply:

Abbreviations Acronyms
AIDS Acquired Immunodeficiency Syndrome
AJLM African Journal for Laboratory Medicine
AMMP-1 Adult Morbidity and Mortality Project Phase 1
AMR Antimicrobial Resistance
ANC Antenatal Care
BMC Bugando Medical Centre
BRM Bio risk Management
BSC Biological Safety Cabinet
BSc Bachelor of Science
BSL Biosafety Level
BUQ Bottom-Up Quantification
CCM Chama Cha Mapinduzi (Ruling Party in the United Republic of
Tanzania)
CD4 Cluster of Differentiation 4
CDC Centres for Disease Control and Prevention
CDs Cluster of Differentiations
CEPD Continuing Education and Professional Development
CHF Community Health Financing
CHMT Council Health Management Team
CHSB Council Health Service Board
CHWs Community Health Workers
CLS Community Laboratory Services
CLSI Clinical and Laboratory Standards Institute
CPD Continuing Professional Development
CPL Central Pathology Laboratory
CTRL Central Tuberculosis Reference Laboratories
DDHCTSU Director, Diagnostic and Health Care Technical Services Unit
DED District Executive Director
DHs District Hospitals
DLTs District Laboratory Technologists
DMO District Medical Officer
DO Data Officer
DP Development Partner
DQA Data Quality Assurance

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DTS Dried Tube Sample
eLIS electronic Laboratory Information System
eLMIS Electronic Logistics Management Information System
EOC Emergency Operations Centre
EQA External Quality Assessment
FBO Faith Based Organisations
FYDP Five Years’ Development Plan
GCLA Government Chemistry Laboratory Agency
GHSA Global Health Security Agenda
GOT Government of Tanzania
HCRF Health Commodity Revolving Fund
HCTS Health Care Technical Services
HIV Human Immunodeficiency Virus
HLI Health Links Initiative
HLPC Health Laboratory Practitioners’ Council
HMIS Health Management Information Systems
HMTs Health Management Teams
HSSP IV Health Sector Strategic Plan IV
HSSP V Health Sector Strategic Plan V
IATA International Air Transport Association
ICAP International Centre for AIDS Care and Treatment Programs
IDSR Integrated Disease Surveillance and Response
IHR International Health Regulations
ISBN International Standard Book Number
ISO International Organization for Standardization
IT Information Technology
KCMC Kilimanjaro Christian Medical Centre
KIU Kampala International University
KPI Key Performance Indicator
LEMM Laboratory Equipment Management Module
LIO Laboratory Information Officer
LIS Laboratory Information System
LMIS Logistic Management Information System
LO Logistic Officer
LQA Laboratory Quality Assurance
MDG Millennium Development Goals
MeLSAT Medical Laboratory Scientists Association of Tanzania
MMAM Mpango wa Maendeleo ya Afya ya Msingi
MNCH Mother and Neonatal Child Health
MoH Ministry of Health
MOU Memorandum of Understanding

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MS Marketing Surveillance
MSc Master of Science
MSD Medical Stores Department
MSD Medical Stores Department
MTBDR Mycobacterium Tuberculosis Drug Resistance
MTEF Medium Term Expenditure Framework
MUHAS Muhimbili University of Health and Allied Sciences
NACP National AIDS Control Programme
NACTE National Council for Technical Education
NBTS National Blood Transfusion Services
NCDs N0n-Communicable Diseases
NGO Non-Governmental Organization
NHLS National Health Laboratory Services
NHLSP National Health Laboratory Strategic Plan
NIMR National Institute for Medical Research
NMCP National Malaria Control Programme
NMLSSP III National Medical Laboratory Services Strategic Plan III
NPHL National Public Health Laboratory
NPHLA National Public Health Laboratory Agency
NRL National Reference Laboratory
NSCLQS National Sub-Committee on Laboratory Quality System
NSGHLS National Standard Guidelines for Health Laboratory Services
NSGRP National Strategy for Growth and Reduction of Poverty
NSLQMS National Sub-committee for Laboratory Quality Management
Systems
NSOPML National Standard Operating Procedures for Medical
Laboratories
NTDs Neglected Tropical Diseases
NTLP National Tuberculosis and Leprosy Programme
PEPFAR President’s Emergency Plan for AIDS Relief
PHDR Poverty and Human Development Report
PHIA Population-based HIV Impact Assessment
PHLB Private Health Laboratory Board
PHLS Public Health Laboratory Services
PLHIV People Living with HIV and AIDS
PO-RALG President’s Office-Regional Administration and Local
Government
POC Point of Care
PPM Planned Preventive Maintenance
PPP Public Private Partnership
PT Proficiency Testing

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QA Quality Assurance
QC Quality Control
QMS Quality Management System
QO Quality Officer
RAS Regional Administrative Secretary
RBF Results-based Financing
RFM Result Framework Matrix
RHMT Regional Health Management Team
RLQA Regional Laboratory Quality Assurance
RLTs Regional Laboratory Technologists
RMO Regional Medical Officer
RRH Region Referral Hospital
RTQII Rapid Testing Quality Improvement Initiative
SADC Southern African Development Community
SADCAS Southern African Development Community Accreditation
System
SCM Supply Chain Management
SD Standard Deviation
SDG Sustainable Development Goals
SLIPTA Stepwise Laboratory Improvement Process Towards
Accreditation
SLMTA Strengthening Laboratory Management Toward Accreditation
SO Safety Officer
SWOC Strength Weakness Opportunities and Challenges
TA Technical Assistance
TB Tuberculosis
TCU Tanzania Commission for Universities
THPS Tanzania Health Promotion Support
TIKA Tiba kwa Kadi
TMDA Tanzania Medicine and Medical Devices Authority
TOR Terms of Reference
TOT Trainer Of Trainee
TSPAS Tanzania Service Provision Assessment Survey
URT United Republic of Tanzania
WHO World Health Organization
ZACDS Zonal Advisory Committee on Diagnostic Services

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TERMS AND DEFINITIONS
For the purposes of this NMLSOP document, these terms and definitions will apply:

Terms Applicable Definitions

Demand Refers to consumers' desire to acquire the services, and
the willingness to pay for it.
Access Refers to the ability of people to reach places and services
and the ability of places to be reached by people and
goods
Quality Refers to the degree to which a set of inherent
characteristics of products or service fulfils requirements
Resilience Refers to the process of adapting well in the face of
adversity, trauma, tragedy, threats, or significant sources
of stress - such as relationship problems, serious health
problems, or workplace and financial stressors
Accountability Refers to responsibility of an individual to complete
assigned tasks and to perform the duties required by their
job
Learning Refers to the process of acquiring new understanding,
knowledge, behaviours, skills, values, attitudes and
preferences
Operational plan Refers to a comprehensive and actionable plan that
defines how team’s functions and activities contribute to
an organizations overall business goal

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FOREWORD
This is the first National Medical Laboratory Standard Operating Procedure
(NMLSOP).

The document outlines the Standard Operating Procedures that the laboratory tests
are to be performed in various facilities during the period of one year’s towards
strengthening the provision of quality medical laboratory services. The plan was
developed through consultative process involving various stakeholders within the
Ministry of Health and other Ministry of the government of Tanzania.

This plan describes the background of medical laboratory sciences, as well as the
context in which it was established in the country. It also features the current roles,
functions and structure of the organization. Furthermore, contents of the plan include;
stakeholders’ analysis, strengths, weaknesses, opportunities, and challenges that face
medical laboratory services. In identifying these elements, SWOC and Political,
Economic, Social, Technological, Environmental and Legal (PESTEL) analysis were
used.

The NMLSOP have been developed in line with the other medical laboratory’s technical
procedures. This NMLSOP is presented in seven (7) chapters set in a path to
overcome the weaknesses and challenges as well as proving a tool to take advantage
of organization’s strength in exploring the existing opportunities. The SOPs include
purpose, scope, responsibilities, principle and many steps. This NMLSOP is expected
to be reviewed annually from January 2024 to January 2025.

Successful implementation of this NMLSOP depends on the availability of technical

and financial resources to coordinate its execution. Hence, all stakeholders should be
aware of their roles and responsibilities at the national, regional, and council levels
outlined in this strategy. It is expected that, MoH through the Director, Diagnostic and
Health Care Technical Services Unit (DDS) will provide the required leadership and
guidance.

Dr. John A. K. Jingu

PERMANENT SECRETARY

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ACKNOWLEDGEMENTS
The National Medical Laboratory Standard Operating Procedure (NMLSOP) is a
product of dedicated efforts and contributions of various stakeholders. The MoH
acknowledges the contribution of PO-RALG, Development and Implementing Partners,
Non-Government Organisations (NGOs), Institutions or facilities (National Hospitals,
Zonal Hospitals, Specialised Hospitals, Regional Referral Hospitals, District Hospitals,
Health Centers, Dispensaries), Regulatory Agencies (Health Laboratory Practitioner’s
Council and Private Health Laboratories Board), Programmes (NASHCoP, NTLP,
NMCP, NBTS) and Individuals towards improving the quality of health care service
delivery through improved medical laboratory services.

In particular, the MoH would like to thank the Global Fund (GF) and Centres for
Disease Control and Prevention (CDC) for their financial and technical support through
the consultancy in development of this strategic plan. Special appreciations are
extended to technical experts and individuals for their active participation and
constructive inputs and comments provided in reviewing this guideline (ANNEX 01).

Our appreciations go to the Director of diagnostic and Health Care Technical Services
Unit (DDS) Dr Alex S. Magesa for steering up the whole process; Acting Head of
Laboratory Services (Ag. HLS) Mr Reuben S. Mkala for field coordination; and to all
laboratory technical team or individuals who played a pivotal role in developing this
NMLSOP by participating in the consultation workshops, meetings, providing relevant
information and offering their expert opinion when consulted. Last but not the least;
appreciations go to David Ocheng, who facilitated the whole process of developing this
document.

Prof. Tumaini J. Nagu

CHIEF MEDICAL OFFICER

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EXECUTIVE SUMMARY
In developing this National Medical Laboratory Standard Operating Procedure
(NMLSOP) for the period 2024-2025 consideration has been made on a number of
National and International guiding and operational guidelines. National Standard for
Medical Laboratories (2017); Health Care Technology Policy Guideline (2004);
Operational Plan for the National Laboratory System to Support HIV and AIDS Care
and Treatment (2005); National Laboratory Quality Assurance Framework to Support
Health Care Interventions (2007); Standard Guidelines for the Facilities and
Operations of Forensic Bureau Laboratory (2008); International Health Regulations
(IHR) of 2005; Global Health Security Agenda (GHSA) 2024 Framework; and the
Ruling Party Election Manifesto 2020.

The main objective of this SOPs is to enable the laboratories at all levels to effectively
and efficiently carry out their core functions of laboratory tests as stipulated by their
mandate, strategically coordinate and allocate the available resources by prioritising
functions with effective impact in line with the National Health Policy (2007) and the
Sustainable Development Goals (SDGs).

There are four chapters in this document. Chapter one provides; a brief introduction
and background information, justification and objectives for the NMLSOP.

This National Medical Laboratory Standard Operating Procedure has four strategic
directions:

1. Enhanced conducive Political, Social and Economic environment for Medical

Laboratory services (Resilience);
2. Improved and strengthened Medical Laboratory Diagnostic services (Access);
3. Heightened effective collaboration and partnerships for coordinated action
(Demand);
4. Strengthened Quality Management systems, Surveillance and Monitoring and
Evaluation (Quality).

For each objective above, strategies, targets, activities and performance indicators
were derived. The NMLSOP Matrix specifically describes the sequence of objectives,
strategies, targets, activities and performance indicators are considered the guiding
framework for the development and implementation of annual operational plans.

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SCOPE
The scope of this National Medical Laboratory Standard Operating Procedure
(NMLSOP) is to provide a step by step instructions for performing laboratory tests.

PRIMARY BENEFICIARIES
The primary beneficiaries are Laboratory Practitioners’ who perform examination of
biological samples in the laboratories and non-laboratory testers performed specialised
laboratory tests at point-of-care testing sites.

SECONDARY BENEFICIARIES
The users of this document will include but not be limited to:

Policy makers, medical care managers and administrators, medical devices regulatory
authorities, medical laboratories, intervention programmes, Regional Health
Management Teams and Council Health Management Teams, zonal medical
equipment workshops, biomedical engineers, healthcare technical services and end-
users engaged in strengthening the quality of medical diagnostic services in the
country.

Additionally, other beneficiaries include: Development and Implementing Partners who

provide technical assistance to support the Government of Tanzania (GOT) to
implement the medical laboratory agenda, public and private medical institutions and
laboratories, and higher learning medical laboratory training institutions, prospective
accrediting agencies, medical professional bodies and clinical laboratory medicine.

The objective of NMLSOP is to ensure that Laboratory personnel implement National

Medical Laboratory Standard Operating Procedure for continuity of quality medical
diagnostic laboratory services as needed by the clients.

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CHAPTER ONE: SAMPLE COLLECTION
1.0 GENERAL CONSIDERATIONS
The collection of samples for laboratory tests from patients consists of following steps:
i. Documentation/Registration of the patient
ii. Collection of sample
iii. Dispatch of sample to respective department
1.1.1 Handling of Biological Samples
Laboratory staffs are often confronted with the problem of handling of biological
samples from patients. The following must be observed for personal protection;
i. All biological samples must be considered hazardous and infected.
ii. Wearing of personal protective equipment (PPEs),
iii. Exercising due care to prevent spillage/splashes while transferring blood to
containers from syringe,
iv. The sample containers be labelled with adequate information.
1.1.2 Samples for Culture
i. As far as possible samples for culture should be obtained before
administration of antimicrobial agents.
ii. If it is not possible, then the laboratory should be informed about the
therapeutic agent(s) so that this fact is considered before issuing laboratory
report.
iii. Material should be collected from the appropriate site where the likelihood
and possibility of isolation of suspected organisms is high.
iv. Sometimes patient’s active participation is necessary for sample collection
(sputum or urine), so he/she should be instructed properly and accordingly.
v. Sufficient quantity of samples is to be collected to permit complete
examination.
vi. Samples are to be placed in sterile containers.
vii. Some samples are directly collected in culture media. Contact laboratory if
such collection is required.
viii. Proper labelling of samples should always be done with patient’s name, test
type, date and site of collection etc.
ix. The relevant clinical information is to be recorded on the request form.
x. Any condition, circumstances or situation that will require special procedures
should also be noted on the request from.
xi. Samples should be collected during working hours except in emergency, so
that the services of qualified microbiologist will be available to directly
supervise processing of the sample.
xii. The most appropriate samples for isolation of viral, chlamydial or rickettsial
agents depend on the nature of the illness.

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 xiii. The material should be collected as early as possible in the acute phase of
the disease, because these agents tend to disappear relatively rapidly after
the onset of the symptoms.
xiv. Vesicle fluid is preferably collected in a syringe or capillary pipette and
immediately diluted in an equal volume of skimmed milk or tissue culture
medium.
xv. All samples for viral culture should be frozen and stored at -70°C until culture
is initiated.
1.1.3 Dispatch of Samples from Reception to the Laboratory Sections
i. Match the containers and respective request forms, number them and enter
in the dispatch register/computer.
ii. Verify while handing over/taking away to respective department of the
laboratory.
iii. Notify the concerned department about urgent and special tests.
1.1.4 Sample Transportation
i. Exterior of the container should not be soiled/contaminated with the
samples.
ii. Sufficient absorbent materials must be used to pack the sample, so that it
absorbs the spilled liquid in case of leakage/breakage during transit to
reference/referral laboratory.
iii. Sample containers must be leak proof and unbreakable. Plastic containers
are preferred.
iv. Samples must be promptly delivered to the laboratory for valid results.
v. If applicable, appropriate transport media should be used.
vi. Samples are to be refrigerated or incubated at 37°C, as the case may be, if
there is a delay in transport of samples to laboratory.
vii. An appropriately filled request form should always accompany all samples
to guide the pathologist/ health laboratory practitioner in selection of suitable
media or appropriate technique.
2.0 COLLECTION OF BLOOD SAMPLES
Consider the following recommended order of veins during blood drawing;
• Median cubital vein (first choice),
• Cephalic vein (second choice),
• Basilic vein (third choice).
1.1.5 Blood sample for serology
i. Serological tests are required in most of the bacterial, viral and parasitic
diseases.
ii. A clotted blood sample is preferred.
iii. A vacuum collection system is both convenient as well as reliable.

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 iv. Wherever applicable, paired samples are to be collected during acute and
convalescent phases of illness in certain viral and other infections to
document a diagnostic rise in antibody titre.
v. Protect blood samples from extremes of heat and cold during transport.
vi. Whole blood is to be stored at 2-8°C.
vii. Serum can be frozen at -20°C or lower temperature for long term storage.
1.1.6 Blood sample for culture
i. Make sure you have the appropriate media for blood culture, as the media
may vary depending upon the type of pathogen suspected.
ii. Wash the hands with soap and water and wear sterile gloves.
iii. Withdraw the blood following the procedure described on the procedure for
collection of venous blood. Change needle before injecting the blood into
the culture bottle.
iv. Thoroughly clean the rubber bung of the culture bottle with iodine solution
and inject an amount of blood equal to 10% of the volume of medium (for
30 ml medium 3 ml blood and for 50 ml medium, 5 ml blood is needed).
v. After the needle has been removed, the site should be cleaned with 70%
alcohol/spirit swab again.
vi. Don’t store the containers and caps separately.
vii. Blood obtained for culture of suspected anaerobes should not be exposed
to air in any way.
1.1.7 Venous blood
i. Welcome and greet the patient, introduce yourself.
ii. Make the patient sit comfortably on the phlebotomy chair.
iii. Identify the patient by asking his/her particulars and compare them with the
request form.
iv. Check the request form for the requested investigations and Inform the
patient about the samples to be collected.
v. Where possible, ask the patient to remove any tight - fitting sleeved clothing,
or to roll up loose sleeves.
vi. Lable the containers before obtaining sample.
vii. Select syringe of appropriate size so that the quantity of blood required can
be obtained in single prick. If multiple or high volume of samples is required,
use a butterfly needle or a canula.
viii. Select appropriate vein (preferably antecubital) from forearm. Cleanse the
skin over the venepuncture site in a circle approximately 5 cm in diameter
with 70% alcohol/spirit swab and allow to air dry, do not blow.
ix. If the sample is to be collected for blood culture then skin is to be thoroughly
sterilised, following the procedure as follows:

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 a. Starting in the centre of a circle
b. apply 2% iodine (or povidone-iodine)
c. in everwidening circles until the entire
d. chosen area has been saturated with iodine.
e. Allow the iodine to dry on the skin for at
least 1 min.
f. Completely remove the iodine with 70% alcohol/spirit swab
following the pattern of application.
x. Apply a tourniquet tight enough to obstruct venous flow only and relocate the
vein to be punctured but don’t touch the proposed site of needle entry or
the needle itself. Ask the patient to clench the fist to make the veins
prominent. If the vein is not visible, palpate it with fingers. In case the veins
of forearm are not visible/palpable, other sites such as dorsum of the
hand may be selected.
xi. With bevel up, insert the correct needle size on the veins at an angle between
15 - 30o then draw the blood into the appropriate collection tubes, (make sure
the patient arm in a downward position to prevent reflux).
xii. Mix immediately after drawing each tube that contain an additive by gently
inverting the tube 8 - 10 times. Do not mix vigorously in order to avoid
haemolysis.
xiii. Release the torniquet from the patient, Withdraw the needle and apply
pressure to the puncture site using dry cotton balls/gauze pad. Do not
withdraw the piston too forcefully as it can collapse the vein and it may cause
frothing/ haemolysis of the sample.
xiv. Apply pressure with thumb on antiseptic swab at puncture site for 2 - 4 min
till the blood ooze stops.
xv. If syringe was used, safely remove the needle from the syringe before
distribution.
xvi. The blood from syringe is distributed to appropriate, labelled containers.
NOTE:
In case of multiple blood sample collection, consider the following recommended order
of draw;
• First tube blood culture,
• Second tube: non-additive tube (e.g. red stopper),
• Third tube: coagulation tube (e.g. blue stopper),
• Last tube: additive tube (e.g. lavender or green tube).
1.1.8 Capillary Blood Collection
i. Wear sterile gloves
ii. Assemble All Collection Tools
iii. Clean the ring fingertip with 70% isopropyl alcohol swab or 70% spirit starting
the middle and leave outward to prevent contaminating the area,

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 iv. Leave the site to air-dry,
v. Hold the finger firmly place the new sterile lancet device at the site on the
finger,
vi. Wipe first drop of blood with a clean dry gauze or cotton wool,
vii. Collect the sample with the second drop of blood using appropriate sample
collection devices such as blood capillary tube,
viii. Apply pressure with a clean dry gauze pad or cotton wool until bleeding stop,
ix. Transfer the collected blood sample into appropriate sample container or
testing devices.
1.1.9 Procedure for Performing Neonate Capillary Blood Collection
i. Use the most medial or lateral portions of the planter surface of the heel Limit
the depth of the puncture wound by using an automated lancet.
ii. Only consider using the whole plantar surface of the foot (using automated
lancets of 2.2mm in length or less) for neonates over 33 weeks’ gestation if
they are having multiple/frequent heel pricks
iii. Position the neonate: ensure the foot is lower than the body.
iv. Choose a puncture site – do not use a previous puncture site.
v. Clean the heel site (i.e. gauze and water) if the foot appears unclean (e.g.
faecal material).
vi. Encircle the foot with the palm of the hand and the index finger.
vii. Make a quick puncture with the automated lancet device
viii. Wipe off the first drop of blood with a gauze swab
ix. Collect the sample with the second drop of blood using any of the collection
devices such as slides or rapid test
x. Apply pressure with a clean dry gauze pad until bleeding stop
3.0 COLLECTION OF URINE SAMPLES
Urine samples are collected for routine and culture examinations to diagnose urinary
tract infections (UTIs), both lower UTIs (cystitis-infection of the bladder) and upper
UTIs (pyelonephritis-infection of the kidney). Unlike most other cultures, colony counts
are done on urine samples to determine the number of organisms present in the
sample. Generally, >100,000 organisms/ml of a single isolate indicate an individual
has a UTI. However, mixed UTIs do occur and some individuals with UTIs will have
counts lower than 100,000 organisms/ml.
Most organisms that cause UTIs are normal enteric flora, including E. coli, Proteus,
Klebsiella, Enterobacter, and Enterococcus species. In young, otherwise healthy,
sexually active females, Staphylococcus saprophyticus can be found to be the cause
of UTI.
UTIs caused by Proteus species can be complicated by the formation of urinary calculi
or stones. The large amount of urease that Proteus produces can alkalinize the urine.
If there are minerals such as phosphates or carbonates in the urine, the alkaline pH
can cause them to precipitate out and form stones.

5
TYPES OF URINE SAMPLE
A. First morning urine sample
It provides concentrated urine as the bladder incubated it the whole night. It is best for
nitrite, protein, good for microscopic examination and culture and sensitivity. The casts
may have deteriorated and bacteria may affect true glucose reading.
B. Random (routine) urine sample
It is the most common type and most convenient sample. It is good for observing
physical characteristics, chemical analysis and identification of casts, crystals and
cells.
C. Second-voided urine sample
The first morning sample is discarded and second sample is collected. Formed
elements remain intact.
D. Mid stream (clean catch) urine sample
The portion of urine that does not contain the first and last portions of the sample.
E. Post-prandial
It is collected after meal (usually after 2 hours). It is good for glucose and protein
estimation. Urine sugar testing now has limited diagnostic or prognostic value.
F. Timed sample
It is a combination of all voiding over a length of time. Two-hour sample is good for
urobilinogen and 24-hour sample is good for quantitative urinary components
estimation. Timed urine samples are collected in dynamic function tests.
G. Foley catheter
Disinfect a portion of the catheter with alcohol, puncturing the tubing directly with a
sterile syringe and needle and aspirate the urine. Place urine in a sterile container, it
should never be collected from drainage bag.
H. Suprapubic urine
Urine sample collected by suprapubic aspiration and cystoscopy.
1.1.10 Procedures for Collection of Urine Sample
Urine sample is often collected by patient him/herself. Therefore, the patient needs to
be properly instructed to have correct sample collection. An uncontaminated mid-
stream urine (MSU) sample is the best and following methods are to be used for its
collection:
A. Females
i. Wash the genital area thoroughly with - Clean water (may be omitted for
urine Routine Examination).

6
 ii. With two fingers of one hand, hold the outer folds of vagina (labia) apart.
With the other hand, rinse the area from the front to the back with running
tap water.
iii. Start urination so that the stream of urine should flow without touching the
skin. After a few moments, place a sterile container under the stream of
urine.
iv. Secure and tighten the cap on the container.
B. Males
i. Wash the genital, area thoroughly with Clean water (may be omitted for
urine Routine Examination).
ii. Start urination and after a few moments, place a sterile container under the
stream of urine. Collect the required amount of urine and remove the
container from urine stream. Secure and tighten the cap.
C. Infants, uncooperative and debilitated patients
i. Plastic bags may be attached after careful and thorough washing of genital
area.
ii. The bags should be watched so that they can be removed immediately after
patient has passed the urine.
iii. If the patient has not voided urine within 30 min the collecting bag is
removed.
iv. Patient needs to be re-scrubbed and a new collection device is to be
attached.
D. Urine collection for Mycobacterium tuberculosis
i. Three consecutive early morning samples (>90 ml each) collected in sterile
container are superior to 24h collection.
ii. Boric acid (1.6%) is used as preservative in case of 24h urine collection in
exceptional situations e.g., when patient cannot report daily for sampling.
iii. Suprapubic aspiration in ward by a doctor is preferred in catheterised
patients.
1.2 FAECAL SAMPLES COLLECTION PROCEDURE
Faecal samples are collected for routine and culture examinations to find the causative
agent of infectious diarrhoea. Rectal swabs are often helpful in identifying the cause
of acute bacterial diarrhoea when stool sample cannot be collected readily.
It is important to remember that there are many causes of diarrhoea other than
infectious agents such as metabolic disorders, certain drugs, and food intolerances
(allergies). In a routine stool culture, the following organisms are often isolated:
Salmonella species, Shigella species and Vibrio cholera. Viruses and parasites are
also common causes of diarrheal disease.
1.2.1 Procedure for Collection of faecal samples
i. Faeces should be passed directly into a clean, waxed cardboard container
that is fitted with a tight cover.

7
 ii. Avoid contact with residual soap/detergent, disinfectant or urine in the
bedpan.
iii. Faeces obtained are transferred to another clean, wide mouthed and screw
capped container. The sample should include any pus, blood, mucus or
formed elements that may have passed with stool.
iv. Sample (~1 ml) is added to 10 ml sterile alkaline peptone water in suspected
cholera cases.
v. If viral infection is suspected, faeces are extracted with sterile buffered
saline. Faeces (~1 ml) are mixed with 9 ml sterile buffered saline, allowed
to sediment for 30 min (or centrifuged). The supernatant is transferred to a
sterile container, frozen and kept below -40°C until processed.
4.0 COLLECTION OF BODY FLUIDS
The primary body fluid that are collected for routine and culture examinations includes;
cerebrospinal fluid (CSF), joint fluid, pleural fluid, and peritoneal (ascites) fluid.
1.2.2 Cerebrospinal fluid (CSF)
• CSF routine and culture examinations are performed to diagnose meningitis
due to Viruses, fungi, and bacteria. Acute bacterial meningitis (ABM) is a
medical emergency.
• The age and immune status of the patient influence the type of bacterial
pathogen most likely to cause ABM:
• Neonates 0 – 2 months; E. coli, Streptococcus agalactiae - Group B
streptococci, Listeria monocytogenes
• 2 months - 2 years; Haemophilus influenza and Neisseria meningitidis
• Older than 2 years; Neisseria meningitides – most common in children and
young adults, Streptococcus pneumoniae – most common in older adults
Procedures for Collection of CSF samples
CSF is normally collected from sub-arachnoid space of spinal cord at lumber level by
puncture with a long needle. A physician in the ward under strict aseptic conditions
performs the procedure.
i. Sample shall be collected in 2-4 ml quantities in 3-4 sterile screw capped
bottles that are serially numbered and must be sent to the laboratory
immediately.
ii. In case CSF is to be cultured for M. tuberculosis then at least 5 ml sample
is needed. CSF shall be tested as soon as it arrives in the laboratory.
iii. CSF in the first bottle is sometimes contaminated with blood and should be
kept aside.
iv. Fluid from second bottle is used for routine tests while fluid from third bottle
is used for bacterial culture etc.
v. If tuberculous meningitis is suspected, 4th bottle is kept in refrigerator
undisturbed to see whether a pellicle or coagulum forms.

8
 NB: CSF must never be refrigerated (if for bacterial culture as it kills H.
Influenzae) and should be kept at 37°C.
Body Effusions (Exudates and Transudates)
An effusion is fluid which collects in a body cavity or joint. Fluid which collects due to
an inflammatory process is referred to as an exudate (needs investigations) and that
which forms due to a non-inflammatory condition is referred to as a transudate (needs
no microbiological investigations). Effusions include; pleural, pericardial, synovial,
peritoneal, and hydrocele fluids.
1.2.3 Pleural and Pericardial Fluids
Main purpose of testing is to ascertain their transudative or exudative nature and to
find a causative organism if an infective process is indicated. See sputum sample for
list of organisms that can be isolated from pleural samples.
1.2.4 Peritoneal Fluid - Ascites
The common indications for paracentesis are ascites of unknown origin, suspected
intestinal perforation, haemorrhage or infarct, infections like tuberculosis,
complications of cirrhosis (spontaneous bacterial peritonitis) and suspected intra-
abdominal malignant disorders.
1.2.5 Joint fluid (synovial fluid)
Joint fluid cultures are performed to diagnose septic arthritis (most cases of arthritis
are NOT infectious; they are due to strain on a joint or immunological diseases).
The three most common causes of septic arthritis are: Staphylococcus aureus,
Neisseria gonorrhoea, and Coagulase-negative staphylococci in patients with joint
replacement prosthetics
1.2.6 Hydrocele Fluid
Usually from the sac surrounding the testes. Occasionally Wuchereria bancrofti
microfilariae and rarely Brugia species can be found in hydrocele fluid.
Collection of aspiration fluids (effusions)
• Collection of synovial, pleural, pericardial, peritoneal, or hydrocele fluid is
carried out by a medical officer or competent nurse.
• Label each container with the date and the patient’s identifiers
• After aspiration, aseptically dispense the fluid into 3 tubes as follows:
o 5 to 10 ml is in a sterile tube for microbiological examination.
o 5 ml in anticoagulant (heparin, trisodium citrate or EDTA) for estimation
of cell count and protein concentration.
o 2-3 ml in a plain tube and allowed to clot (normal fluid does not clot).
• If the sample cannot be examined immediately, fluid should be frozen and
stored at -70°C until examined.

9
5.0 COLLECTION OF GENITAL SAMPLES
• Indicated for the diagnosis of bacterial sexually transmitted diseases, primarily
gonorrhea (GC) or non-gonococcal cervicitis or urethritis (NGU). The most
common cause of NGU is Chlamydia trachomatis.
• immunological and molecular tests for the diagnosis of chlamydial infections
includes (PCR, DNA probes, etc.) .
• Vaginal secretions are also sent to the laboratory for the diagnosis of vaginitis.
• The diagnosis of vaginitis can be made with a wet mount – yeast, trichomonas,
bacterial vaginosis (BV), culture – yeast, or Gram stain – yeast and BV.
• Urethritis, cervicitis: Neisseria gonorrhoeae, Chlamydia trachomatis, Other
agents of NGU
• Vaginitis: Candida albicans, Trichomonas vaginalis and BV
1.2.7 Urethral swabs
Possible pathogens; Neisseria gonorrhoea, Chlamydia trachomatis, and Trichomonas
vaginalis.
Collection of urethral discharge from male patients
i. Cleanse around the urethral opening using a swab moistened with sterile
physiological saline.
ii. Gently massage the urethra from above downwards.
iii. Using a swab, collect a sample of discharge.
iv. Make a smear of the discharge on a microscope slide by gently rolling the
swab on the slide. This will avoid damaging pus cells which contain the
bacteria.
Note: Very few pus cells may be present if the patient has recently
passed urine. Allow 2–4 hours after urination before collecting a
samples.
v. When culture is indicated (see previous test),
Collect a sample of pus on a sterile cotton-wool swab.
If possible, before inserting the swab in a container of Amies transport
medium, inoculate a plate of culture medium.
vi. Label the samples and deliver them to the laboratory as soon as possible.
vii. Isolation of N. gonorrhoeae from urine
Note:
A rectal swab is also required from homosexual patients. A selective medium is
required to isolate N. gonorrhoea from a rectal sample.
In acute urethritis, it is often possible to detect N. gonorrhoea in pus cells passed
in urine, especially the first voided urine of the day (centrifuged to sediment the pus
cells).
Cervical swabs and possible pathogens
From non-puerperal women:
10
Neisseria gonorrhoea, Chlamydia trachomatis (serovars D-K), Streptococcus
pyogenes, herpes simplex virus.
From women with puerperal sepsis or septic abortion:
Streptococcus pyogenes, other beta haemolytic streptococci, Staphylococcus aureus,
Enterococcus species, anaerobic cocci, Clostridium perfringens, Bacteroides,
Proteus, Escherichia coli and other coliforms, Listeria monocytogenes.
1.2.8 Collection of cervical samples from female patients
i. A samples collected from the endocervical canal is recommended for the
isolation of N. gonorrhoeae by culture. Use a sterile vaginal speculum to
examine the cervix and collect the samples.
ii. Moisten the speculum with sterile warm water, and insert it into the vagina.
iii. Cleanse the cervix using a swab moistened with sterile physiological saline.
iv. Pass a sterile cotton-wool swab 20–30 mm into the endocervical canal and
gently rotate the swab against the endocervical wall to obtain a samples.
v. When gonorrhoea is suspected, before inserting the swab in Amies
transport medium, if possible inoculate a plate of culture medium. Label the
sampless and deliver to the laboratory as soon as possible. Inoculated
culture plates must be incubated within 30 minutes.
1.2.9 Vaginal Swabs
Vaginal discharge may be due to infection of the vagina or infection of the cervix or
uterus.
Pathogens causing vaginal infections include Trichomonas vaginalis, Candida
species, and Gardnerella vaginalis with anaerobes.
1.2.10 Collection of vaginal discharge to detect T. vaginalis, C. albicans and G.
vaginalis
Two preparations are required:
A. Wet preparation to detect motile T. vaginalis
i. Use a sterile swab to collect a samples from the vagina.
ii. Transfer a sample of the exudate to a microscope slide.
iii. Add a drop of physiological saline and mix.
iv. Cover with a cover glass.
v. Label and deliver to the laboratory for immediate examination
B. Dry smear for Gram staining to detect Candida and examine for clue cells
Although yeast cells can be seen in an unstained wet preparation, the Gram positive
cells and pseudohyphae of C. albicans are more easily seen in a Gram stained smear.
i. Use a sterile swab to collect a samples from the vagina.
ii. Transfer a sample of the exudate to a microscope slide and spread it to
make a thin smear.
iii. Allow the smear to air-dry, protected from insects and dust.
iv. Label and deliver to the laboratory with the wet preparation.
11
1.2.11 Collection of samples to detect T. pallidum
To detect motile T. pallidum spirochetes, a samples must be collected before antibiotic
treatment.
i. Wearing protective rubber gloves, cleanse around the ulcer (chancre) using a
swab moistened with physiological saline. Remove any scab which may be
present.
ii. Gently squeeze the lesion to obtain serous fluid. Collect a drop on a clean cover
glass and invert it on a microscope slide.
iii. Immediately deliver the preparation to the laboratory for examination by dark-
field microscopy
6.0 WOUND SAMPLES
Indicated for primarily to diagnose skin and soft tissue infections (SSTIs). SSTIs may
be caused by a variety of organisms; different organisms depending on how the wound
or injury occurred. Fungi, parasites, and viruses are also important causes of certain
types of SSTIs.
Community-acquired: Staphylococcus aureus, Streptococcus pyogenes, Clostridium
perfringens and other anaerobic bacteria).
Hospital-acquired: Staphylococcus aureus, Enteric Gram-negative rods – E. coli,
Pseudomonas aeruginosa, Acinetobacter species, and other non-fermenting Gram-
negative rods, Streptococcus pyogenes, Clostridium species and other anaerobic
bacteria
Collection of Wound sampless - General considerations
Samples should be collected by a medical officer or an experienced nurse.
Pus from an abscess is best collected at the time the abscess is incised and
drained, or after it has ruptured naturally.
When collecting pus from abscesses, wounds, or other sites, avoid contaminating
the sample with commensal organisms from the skin.
As far as possible, a samples from a wound should be collected before an
antiseptic dressing is applied.
When pus is not being discharged, use a sterile cotton-wool swab to collect a
sample from the infected site.
Immediately after collection, immerse the swab in Amies transport container.
Label the samples and as soon as possible deliver it with a completed request form
to the laboratory.
When myeloma is suspected: Obtain a samples from a draining sinus tract using a
sterile hypodermic needle to lift up the crusty surface over the sinus opening. This
method of samples collection has the advantages that the pus obtained is usually free
from secondary organisms and the draining granules can usually be seen clearly and
removed for microscopic examination. Transfer the pus to a sterile container.
When tuberculosis is suspected: Aspirate a sample of the pus and transfer it to a
sterile container.
12
When the tissue is deeply ulcerated and necrotic (full of dead cells): Aspirate a
sample of infected material from the side wall of the ulcer using a sterile needle and
syringe. Transfer to a sterile container.
Fluid from pustules, buboes, and blisters: Aspirate a samples using a sterile needle
and syringe. Transfer to a sterile container.
Serous fluid from skin ulcers, papilloma, or papules, that may contain
Treponema: Collect a drop of the exudate directly on a clean cover glass and invert it
on a clean slide. Immediately deliver the samples to the laboratory for examination by
dark-field microscopy.
Caution: Samples from patients with suspected plague or anthrax are highly
infectious. Label such samples HIGH RISK and handle them with care.
In a health centre for dispatch to a microbiology laboratory
Collect the samples using a sterile cotton-wool swab.
Insert it in a container of Amies transport medium, breaking off the swab stick to allow
the bottle top to be replaced tightly.
In a hospital with a microbiology laboratory
i. Using a sterile technique, aspirate or collect from a drainage tube up to 5
ml of pus.
ii. Transfer to a leak-proof sterile container. When the material is aspirated
fluid from a pustule, transfer the fluid to a sterile, leak-proof container.
Stopper, and seal in a leak-proof plastic or metal container.
Note: It is not possible to transport exudate from a suspected treponemal ulcer
because the Treponema remain motile for only a short time.
Make a smear of the material on a clean slide (for Gram staining) and allow to air-
dry in a safe place.
Heat-fix the smear.
Caution: Do not make a smear for transporting when the samples is from a
patient with suspected anthrax.
Send the samples with a completed request form to reach the microbiology
laboratory within 6 hours.
7.0 COLLECTION OF THROAT AND NASAL SWABS
Throat cultures are performed to diagnose streptococcal pharyngitis (infection with
Streptococcus pyogenes (Group A streptococci). The most common causes of
pharyngitis (sore throat) are viruses, which cause over 75- 80% of all cases. Of the
bacteria that cause pharyngitis, Group A streptococci is the major cause and therefore,
with few exceptions the only bacteria that is reported from a throat culture is
Streptococcus pyogenes.
Exceptions include:
Corynebacterium diphtheria which in areas of the world where vaccination is
prevalent, is a rare cause of pharyngitis
Neisseria gonorrhoea can cause pharyngitis, however many pharyngeal infections
with N. gonorrhoea are mild or asymptomatic.

13
If the physician suspects either of these two organisms, he/she must let the lab know
because the isolation of either requires special culture techniques.
Throat Swabs
Throat swab cultures are to be taken under direct vision with good light.
Areas of exudation, membrane formation, any inflammation or if not seen then tonsillar
crypts are the sites of choice.
Nasal swabs
Nasopharyngeal swabs are better taken by treating physician/surgeon himself.
For recovery of viral agents, washings are collected after gargles with nutrient broth
by the patient.
Nasal Sample for Mycobacterium leprae
The nasal sample for M. leprae can be taken as follows:
6.1 Nasal swab
i. Make the patient sit with his head bent backwards but facing the light.
ii. Insert and repeatedly rotate the swab into one of the nasal cavities, against
upper part of the nasal septum.
iii. Make 2-3 evenly spread smears.
iv. Air dry the slides, wrap in a paper and send to the laboratory.
6.2 Nasal washings and nasal blow
i. Make the patient sit.
ii. Place a few drops of sterile saline in the nose.
iii. After 3 min, ask the patient to blow hard his nose on a small sheet of plastic
or cellophane. (This plastic or cellophane can be given to the patient to take
it home and ask him to blow hard onto the sheet, the following morning,
soon after waking and before washing.
iv. The patient can bring it directly to the laboratory).
v. Transfer some of the mucus pieces from the washing to a slide with a clean
wooden stick and make thin smear.
vi. Air dry slide and send it to the testing area
6.3 Collection of Nasopharyngeal Swabs
Assemble equipment for Nasopharyngeal swab collection and PPEs for prevention of
infections.
i. Lable the VTM with required information and fill the register and all necessary
forms with all necessary information.
ii. Remove the swab from the package. Do not touch the soft end with your hand
or anything else.
iii. Insert the nasopharyngeal swab into the nasopharnx region.
iv. Leave in place for a few seconds.
v. Slowly remove swab while slightly rotating it.
vi. Break the applicator’s stick end and put tip of swab into ATM containing vial
VTM
14
7 COLLECTION OF OROPHRYANGEAL SWABS
Assemble equipment for orophryangeal throat swab collection and prevention of
infections.
i. Label the VTM with required information and fill the register and all necessary
forms with all necessary and correct information.
ii. Remove the swab from the package. Do not touch the soft end with your hand
or anything else.
iii. Have the patient open his/her mouth wide.
iv. Insert swab in the area of tonsils.
v. Use tongue depressor if the patient is not able to resist gagging and closing the
mouth while the swab touches the back of the throat near the tonsils.
vi. Rotate swab to obtain adequate sample
vii. Break the applicator’s stick end and put tip of swab into vial containing VTM
8 SPUTUM SAMPLES
Sputum cultures are performed to diagnose infections such as pneumonia and
pulmonary tuberculosis that is caused by Mycobacterium tuberculosis.
Bacteria associated with Community-Acquired Pneumonia (CAP):
• Streptococcus pneumoniae – most common bacterial cause of CAP
• Haemophilus influenzae
• Moraxella catarrhalis
• Staphylococcus aureus - particularly following a viral infection such
as influenza
• Klebsiella pneumoniae – particularly in individuals with chronic
conditions such as alcoholism
• Mycoplasma pneumonia* – particularly in young individuals in closed
quarters
• Bacteria associated with Hospital-Acquired Pneumonia:
• Streptococcus pneumoniae
• Enteric Gram-negative rods such as E. coli, Klebsiella, Enterobacter,
Citrobacter, and Serratia
• Staphylococcus aureus
• Pseudomonas aeruginosa
• Acinetobacter species
• Haemophilus influenzae
Sputum Collection Procedure
1. Label the sample container on the body of the container, not on the lid and
fill out the sputum examination request form.
2. Instruct the patient and demonstrate how she or he can produce and collect
good sputum;
a. Access to a well ventilated place (outside the laboratory working area),
b. Mouth wash (rinsing with water),
15
 c. Breathe in deeply 2-3 times, and breathe out hard each time.
d. Cough deeply from the chest and collect the sputum into the container.
e. Opening and closing the sputum container so as there are no leaks or
smearing on the exterior of the container.
f. Hand wash steps
3. Emphasize the need for the patient to supply the most useful sample, the
normally thick, yellowish (sometimes blood-streaked), purulent material
brought up from the lungs after a deep, productive cough.
4. Emphasize that saliva produced by spitting is not sputum. However, if the
only sample the patient can produce is salivary, do submit it to the laboratory
as it can still yield useful information.
5. Encourage the patient to bring the collected sample back to the unit as
quickly as possible.
6. For M.tuberculosis culture, a series of three fresh, early morning samples
(5-10 ml) are collected and kept in the refrigerator. If amount is less, the
patient is advised to collect 24 h sputum or until 50 ml is obtained.
7. M.tuberculosis can be recovered from the gastric contents in infants,
debilitated patients and those who are unable to cooperate in the collection
of sputum. This can be obtained by gastric aspiration performed as an
indoor procedure.
8. Gastric washings are better collected early in the morning, in fasting state.
These are neutralised soon after collection by N/10 NaOH.

16
CHAPTER TWO: PARASITOLOGY
2.1. PROCEDURE FOR MALARIA RAPID TEST (MRDT)
5.1. Purpose
This procedure provides instructions for In vitro qualitative screening test for detection
of malaria parasites (P. falciparum, P. vivax, P. ovale and P. malariae) in whole blood
5.2. Scope
This procedure applies to all Health Laboratory Practitioners who works in parasitology
section on performing MRDT test.
5.3. Responsibility
Head of section is responsible to ensure implementation and competence assessment
for all staff that will perform this test
5.4. Principle
Test is based on principle of immune-chromatography in which nitrocellulose
membrane is pre-coated with two monoclonal antibodies as two separates lines. One
monoclonal antibody (test line PAN), is PAN specific to lactose Dehydrogenase
(phLDH) of the plasmodium species (plasmodium falciparum, P. vivax, P. ovale and
P. malariae). And the other line (test line pf) consist of monoclonal antibody specific to
histidine rich protein 2 (HRP2) of plasmodium falciparum. When the test sample along
with assay diluent flows through intercellular membrane, monoclonal antibody
conjugated with colloidal gold which are PAN specific to plDH and falciparum specific
for HRP2 binds to plasmodium antigen released from lysed blood sample. These
antigen –conjugate complex moves through the nitrocellulose membrane and binds to
corresponding immobilised antibody at test lines, which leads to the formation of colour
band/bands indicating reactive results. The control band will appear irrespective of
reactive or non-reactive sample.
5.5. Sample Requirements
Whole blood from EDTA/Finger prick
5.6. Equipment
Timer
5.7. Materials
Gloves, Test kit, Lab Coat, and
5.8. Storage and Stability
MRDT sample should be tested within 1hour after collection if not possible stored at
2-80c for 7 days.
5.9. Safety

17
 i. All personal protective equipment (PPE) must be worn when performing this
procedure.
ii. All Samples must be regarded as potentially infectious.
iii. Refer to National infection prevention and control Guidelines (IPC) f
5.10. Calibration
Not Applicable
5.11. Quality Control
Process known positive and negative blood sample daily before performing patient
samples.
5.12. Procedure Steps
Follow the actions described below for specimen collection step-by-step.
i. Allow all kit components and specimen to room temperature prior to testing
ii. Remove the test device from the foil pouch; place it on a flat, dry surface.
iii. Label the test device with the patient identification number/name
iv. Transfer 5µl of whole blood collected in an inverted cup/special capillary
provided into sample well by touching sample pad. (see manufacture
instruction)
v. Add 4 drops of assay diluents into the squire assay diluents well. (See
manufacture instruction)
vi. Read the result within 15 min. (Do not interpret after 30 min) and read result.
(See manufacture instruction).
5.13. Biological Reference Intervals
Not Applicable
5.14. Interpretation and Reporting of Results
Negative
The presence of one colour band (“C” Control line) within the result window indicates
a negative result.
Positive
“C” and “P. falciparum the presence of two coloured bands (“Pf” test line and “C”
control line) within the result window no matter which band appears first, indicate P.F
positive result.
Positive
“C”, “P.f” and “Pan” the presence of three colored band (“Pf”, Pan “Test line and “C”
Control line) within the result window no matter which band appears first, indicate P.F
positive or mixed infection of P.F and P.V or P.M or P.O
Invalid result
If no coloured band appear, at control line “c” within stipulated time then the result is
invalid.
5.15. Limitations of the procedure and sources of error

18
Test kit cannot detect malaria antigen if parasites are less than 100. The test is limited
to detect HRP2, an antigen to Malaria Plasmodium species that may persist after
treatment or passed P. falciparum infection.
5.16. Performance Characteristics
Refer to package insert
5.17. Supporting Documents
Sample collection manual
5.18. References
Manufacturer inserts
2.2. PROCEDURE FOR MALARIA MICROSCOPY
2.2.1 Purpose
This procedure provides instructions for the examination of malaria parasite to
diagnose and monitor treatment outcome of malarial infection.
2.2.2 Scope
This procedure is used during examination of malaria parasites at the Laboratory
using Microscopy.
2.2.3 Responsibility
Qualified and trained health Laboratory Practitioners are responsible for performing
this test procedure
The Head of Parasitology unit is responsible for ensuring the effective implementation
and maintenance of this procedure.
2.2.4 Principle
Giemsa stain
Giemsa stain is a Romanowsky stain contains methylene blue which is basic stain and
eosin is acidic stain, the malaria parasites has DNA in the nucleus which is basic in
nature and RNA in the cytoplasm is acidic in nature, during staining the reactions takes
place whereby acidic part of a parasite will pick-up basic part of the stain and the basic
part of the parasite will pick-up the acidic part of the stain, that’s why the nucleus of
the parasite will show red color and the cytoplasm will stain bluish. Malaria parasites
are identified by microscopic examination of thick or thin blood films stained with
Giemsa. Thick blood films are used for detecting parasites, thin blood films are used
for more detailed morphological examination and for determining parasite species.
Thick blood films consist of several layers of blood cells, so that a large volume of
blood is examined. The thick film staining technique ruptures red cells, leaving white
cells and parasites intact. The thin film staining technique preserves the morphology
of blood cells and malaria parasites.
2.2.5 Sample Requirements
Whole blood collected in EDTA tube is required
19
Blood slides
2.2.6 Equipment
Hot plate, Tally counters or differential counter, Microscope, Timer, Weighing scale,
and PH Meter
Maintenance
To increase the life-span of microscopes, preventive maintenance, including cleaning
the objectives and replacing parts as necessary. should be part of routine internal QC
and must be properly done and documented. Microscopes should be covered when
not in use to avoid exposure to dust, and proper precautions must be taken in humid
areas to avoid fungal growth on the lenses and in the microscope. Also Hot plate, Tally
counter, Timer, weighing scale and PH meter should be maintained as per
manufacture instructions.
2.2.7 Materials
• High-quality immersion oil should be used according to the manufacturer’s
recommendations.
• High-quality microscope slides, free of surface abrasions, preferably have a
frosted end for labeling and purchased from a reputable supplier should be
used.
• 10% alcohol-based Giemsa stain,
• markers, lancets, syringes, needles, Vacutainer-type needles, alcohol
swabs, lens-cleaning solution, lens-cleaning tissues, buffer tablets, pH
calibration solutions, cotton-wool, gloves, safety glasses (including the over-
spectacle type), filter paper and glycerol. gloves, sharps, boxes, gowns and
detergents, and slide boxes.
2.2.8 Storage and Stability
Unstained blood slides should be stored at 2-8 c for 3 months
Stained blood slides should be stored at room temperature for 3 months on slide
boxes.
2.2.9 Safety
Adhere to safety precaution as stated in safety manual/IPC guideline
All personal protective equipment(PPE) should be worn when performing procedure
All samples should be regarded as potential infectious
2.2.10 Calibration
Calibration of PH meter and weighing scale should be done once per year.
Maintenance of microscopes should be done as planned.
2.2.11 Quality Control
QC slides should be used to check the quality and performance of the Giemsa stain,
Microscope and Laboratory personnel. Malaria-positive and negative blood should be
used to prepare QC thick and thin films. Before examining patient slides, the QC slides
should be checked first, If the QC slides are satisfactory, the patient slides can be

20
examined. Slides must be selected regularly for cross-checking, either by sending
them to a crosschecking center or during routine supportive supervisory visits.
2.2.12 Procedure Steps
Preparation of thick and thin blood film
i. Prepare both thick and thin film on the same microscope glass slide.
ii. Label the slides with the unique patient ID including the date of examination.
iii. Put Slide card for making thin and thick blood films, showing size of blood
drops and area of slide to cover for a thin film and thick film
iv. Put the microscope glass slide on the slide card for thick and thin film
v. Pipette 2µl of blood and pour it on the smallest circle on the slide card
vi. Place the spreader in front of the 2µl drop of blood at a 30 o- 45o angle. Use
a clean microscope slide with a smooth edge as a spreader.
vii. Pull back the spreader and hold until the blood evenly spreads along the
width
viii. Push the slide forward in a smooth continuous motion
ix. Avoid hesitation or a jerky motion when spreading the blood
x. Pipette 6µl of blood and pour on the large circle on the slide card.
xi. Using another slide, spread the drop of blood within the confined area to
make a thick film.
xii. Put the slide films on the slide rack and allow it to dry.
xiii. Fix the thin smear for one second by either spraying or dipping into absolute
methanol.
xiv. Air dry the slides on a slide rack with the fixed thin film facing down
Preparation of Giemsa Stock Solution (500ml) from Giemsa powder
i. Measure and dissolve 3.8g of Giemsa powder in 250ml of methanol
ii. Measure 250ml of glycerol and add to the solution above
iii. Ripen the stock solution by placing in direct sunlight for about 1 week or
place in water bath of temperature 56oC and shake at interval.
iv. Filter the stock solution before storing in a cool dry place with label and date
of preparation
Preparation of 10% Giemsa stain from Giemsa Stock solution
i. Add 1 part of stock solution to the 9 parts of 7.2 buffer solution
ii. Prepare 30ml of 10% working giemsa solution as follows: 3mls of Giemsa
+25ml of buffered water PH of 7.2
iii. Mix and transfer to a clean caped leakproof bottle
iv. Lable and keep in a dry place
Staining of blood films
i. Arrange the slides on a staining rack with the sample side facing up
ii. With an aid of a disposable pipette, Flood the films with 10% Giemsa
solution and leave to stain for 15 minutes.
21
 iii. Decant the Giemsa and wash in buffered water at pH 7.2
iv. Clean the back of each slide with cotton wool or gauze
v. Air dry the slides
Examination of the blood films
i. Place a drop of immersion on the thick film
ii. Place the slide on the microscope stage
iii. Swing the X10 objective into position and bring the film into focus using the
course adjustment.
iv. Use the X10 and X40 objectives to check quality of the slide(s) before reading
and reporting;
v. Swing the x 100 oil immersion objective into position and focus using the fine
adjustment
vi. Choose the correct smear reading pattern either Horizontal, start from up right
to left or Vertical, start from upright down.
vii. Systematically, Examine the thick film.
viii. If you do not see any parasites, continue examining the whole film.
ix. If parasites are seen, start counting number of white blood cells and parasites
simultaneously up to a WBC of 200.
x. If the parasitemia level is less than 10/200WBC, continue to count up 500 WBC
xi. Report the number of parasites count per 200 or 500 white blood cells in the
Blood parasites worksheet.
xii. Retain the read slides for 3 Months
2.2.13 Biological Reference Intervals
Not Applicable
2.2.14 Reporting and Interpretation of Results
Malaria parasites
Typical Malaria parasites have the following features on the Giemsa stained films;
Purple red chromatin dot, Blue cytoplasm, Brown-black/yellowish green pigment and
Distinct morphology
Interpretation
In stained blood films, trophozoites appear as red stained chromatin dots with blue
staining cytoplasm. If doubtful on parasites seen, search for definite ones. Do not
make a diagnosis on the basis of structures that resemble rings or chromatin dots
alone. Structures that may be confused with malaria parasites are platelets, portions
or other red cell inclusion bodies
Reporting
If no parasites are seen report as “No parasites seen”
If malaria parasites are seen, report species identified and count e.g. “Plasmodium
species seen 50/200WBC”
Critical values
22
The following are the Panic/Critical values for Malaria microscope test results. In case
of the patient result falling inside the indicated values, call the Doctor or relevant ward
and record the details of the conversation on the Panic Result Book
Test Critical value
Malaria for Under 5 years >100 P.falciparum
Asexual/200wbc’s
Malaria for > 5 years& adult >1000P.falciparumAsexual/2
00wbc’s
None Tropical people ANY POSITIVE
2.2.15 Limitation of the Procedure and Sources of Errors
Poor storage of reagents or using the reagents after expiry date may cause false
results.
Difficulty in distinguishing young ring-stage parasites
2.2.16 Perfomance characteristics
Refer to the method verification report of this procedure.
2.2.17 Supporting Documents
Not Applicable
2.2.18 References
• Practical Laboratory Manual-Jane Carter and Orgenes Lema
• Standard Operating Procedure Essential Laboratory Tests {AMREF-2008 }
EXT 120
• Basic Malaria Microscopy Part I. Learner’s Guide, Second edition, WHO.
2.3. PROCEDURE FOR URINE MICROSCOPY
2.3.1 Purpose
The purpose of this procedure is to provide step by step instructions for performing
macroscopic and microscopic examination of urine sediment samples
2.3.2 Scope
This procedure is used during examination of urine samples in Parasitology section in
medical laboratory.
2.3.3 Responsibility
The Registered medical laboratory personnel is responsible for effective
implementation and maintenance of this procedure.
2.3.4 Principle
Macroscopic Examination
The urine is visualized with naked eyes to determine its appearance (turbidity and
colour).
Microscopic Examination

23
The urine sediment is analyzed by a microscope to observe the presence of white
blood cells, red blood cells, parasites and other abnormalities in urine sample.
Identification of cells (WBC (pus cells), RBGs and Epithelial), casts, crystals,
amorphous phosphates, bacteria and parasites in urine is based on their different
cellular and intra-cellular morphology under light microscopy.
2.3.5 Sample Requirements
20 mL, minimum 1 mL Fresh, cleanly voided urine collected in a clean container
Early morning voided mid-stream urine. Other samples include;
Random urine (collected at any time of the day)
Terminal urine sample collected at any time of the day for demonstrating ova of
Schistosoma haematobium
First voided urine sample in the morning is used to demonstrate Trichomonas
vaginalisin males
In infants and babies, a random urine sample collected as early as possible is used
for all types of urine investigation.
2.3.6 Equipment
Microscope, Centrifuge machine, Refrigerator, Timer, and Forceps
2.3.7 Materials
Gloves, Cover slips, Glass slides, Test tubes, Centrifuge tubes, Gauze, Grease pencil,
Lens paper, Waste containers, Urine container, Laboratory coat, Marker pen
2.3.8 Storage and stability
Process urine sample within 1hour of collection, if not possible refrigerate at 2-80C
immediately and test within 12 hours.
2.3.9 Safety
i. Adhere to safety precautions as stated in the Safety manual
ii. All personal protective equipment (PPE) must be worn when performing this
procedure.
iii. All samples must be regarded as potentially infections.
iv. Refer to National infection prevention and control Guidelines for health
waste management and safety practice.
2.3.10 Calibration
Urine analyzers, , timer and centrifuge should be calibrated as per schedule
Maintenance of all equipments should be done as planed
2.3.11 Quality Control
Quality control should be performed daily before processing patient samples by
analysing known positive and negative samples.
2.3.12 Procedure
Macroscopic examination
24
 Observe the appearance of the urine for color and clarity/turbidity
Microscopic examination
i. Label the centrifuge tubes with the laboratory unit number using a grease
pencil and arrange into a rack.
ii. If the urine sample is to be tested both for urinalysis and culture, mix well by
inverting three times and aliquot the sample into a centrifuge tube and label
exactly as the original sample
iii. Transfer the centrifuge tubes containing urine sample into the centrifuge.
iv. Make sure the centrifuge tubes are balanced well.
v. Centrifuge the urines at 3000rpm for 5 minutes.
vi. Remove the centrifuge tubes from the centrifuge
vii. Pour off the supernatant into the sink with running water and leave the urine
deposit
viii. Transfer the rack containing arranged centrifuge test tubes to the working area
ix. Arrange the marked slides according to the number of urine sample
x. Re-suspend the urine deposit by tapping the bottom of the centrifuge tube.
xi. Transfer a drop of the deposit onto a clean glass slide.
xii. Put on a cover slip
xiii. Place the slide on the microscope stage and reduce the iris diaphragm
xiv. Examine the slide systematically using x 10 objective and count the cells,
casts, crystals, amorphous phosphates and parasites (these structures are
more likely to be seen around the edges of the cover slip.
xv. Swing the x40 objective into position
xvi. Open the iris diaphragm very slightly to allow just enough light to provide a
contrast of cells, casts, crystal and amorphous phosphates against the bright
back ground (these structures are more likely to be seen around the edges of
the cover slip).
xvii. structures cannot be seen if a very bright light is used
xviii. Put the used glass slide and cover slip in a container with disinfectant.
2.3.13 Biological Reference interval
Macroscopy
Color Straw - Dark yellow
Appearance Clear - Hazy
Microscopy
Red blood cells: 0–2/hpf.
WBC (PUS CELL)/Pus cells: 0–5/hpf
Casts 0-4/hpf
Bacteria Negative
2.3.14 Interpretation and Reporting of Results
Macroscopic
Report the Turbidity and color of the urine e.g. Clear yellowish, Blood stained etc.
25
 Microscopic
Report a range of the actual highest number of WBC (pus cells), RBC and Parasites
per high power field as counted under x40 objective (e.g. 0 – 2 RBC/HPF, 3 – 5
WBC/HPF, 10 – 12 S. haematobium ova/HPF) etc.
For crystals, Epithelial cells, yeasts or casts, report as +, ++ or +++. Where +
indicates 1 to 9 observed findings per field; ++ indicates 10 to 100 observed findings
per field; +++ indicates above 100 observed findings per field.
If no cells or parasites are seen report as “No parasites or cells seen.”
Results interpretation
Schistosomes Haematobium ova in urine indicates Schistosomiasis disease
Presences of more than 3 Red blood cells for males, more than 8 red blood cells
for females and any RBC or WBC (pus cell) for children and notification of more
than 8 WBC (Pus Cell) in either male or female is pathological significant.
White blood cells may indicate infection or inflammation.
Red blood cells may indicate kidney disease, a blood disorder, or bladder cancer.
Bacteria can indicate infection.
Skin cells can indicate infection or kidney disease.
Crystals may be a sign of kidney stones.
Casts, or tube-shaped proteins, may be a sign of a kidney disorder.
Parasites can indicate parasitic disease in various parts of the body.
2.3.15 Limitations of the Procedure and Sources of Errors
Inadequate centrifugation of sample
Poor collection of urine sample
Prolonged storage of urine sample at wrong temperature
Expired reagents
Technical competency level
Presence of artifacts such as plant cells
2.3.16 Performance Characteristics
Not Applicable
2.3.17 Supporting document
Sample Collection Manual
2.3.18 References
• Monica Cheesbrough: District Laboratory Practice in Tropical Countries, Vol
1,Tropical Health Technology, 1998.
• Practical Laboratory Manual-Jane Carter and Orgenes Lema
2.4. PROCEDURE FOR STOOL ROUTINE EXAMINATION
2.4.1 Purpose
This procedure provides instructions for examination of stool macroscopic and
microscopic to detect abnomalities/Parasites. The most common parasites include the

26
roundworms Ascaris lumbricoides and Necator americanus (commonly called
hookworm); the tapeworms D. latum, Taenia saginata, and, rarely, T. solium; the
amoeba E. histolytica; and the flagellate G. lamblia.
2.4.2 Scope
This procedure is used during examination of stool in Parasitology Section.
2.4.3 Responsibility
The Registered medical laboratory personnel are responsible for implementing this
test procedure.
The Head of Parasitology section is responsible for ensuring the effective
implementation and maintenance of this procedure
2.4.4 Principle
Normal saline retains the morphology of the organism in its natural shape and color,
free helminthes eggs from debris.
Iodine - stains the internal structure of cyst to brown/yellow color so to allow the study
of cyst morphology. It also kills the organisms to allow internal structure easily seen.
2.4.5 Sample Requirements
Stool collected in Clean plastic screw capped container which has spoon like. Include
macroscopic worms or worm segments as well as bloody and mucoid portions of the
sample.
About 150mg stool should be collected.
2.4.6 Equipment
Microscope, Tally counters or differential counter, Centrifuge and Fume hood
Maintenance
Maintenance of microscopes should be done as planned
2.4.7 Materials
Normal saline, 1% Iodine, 10% formalin, Wooden applicator stick, Grease pencil,
Gloves, Microscopic slides, Cover slip, Marker and Stool container
2.4.8 Storage and stability
Stool sample should be at the laboratory within two hours after collection. If a liquid or
soft stool sample can’t be examined within 30 minutes of passage, place it in a
preservative; if a formed stool sample can’t be examined immediately, refrigerate it or
place it in preservative.
2.4.9 Safety
• All sampless must be considered as potentially infectious and must be
handled and examined with care.
• All personal protective equipments (PPE) should be worn when performing
procedure
• Adhere to safety precautions as stated in the Safety manual
27
 • Refer to National infection prevention and control Guidelines for health
waste management and safety practice.
2.4.10 Calibration
Calibration of centrifuge should be done as per schedule
2.4.11 Quality Control
Quality control should be performed daily before processing patient samples by using
known positive and negative samples
2.4.12 Procedure
Macroscopic Observations
Observe the stool sample for the following; Color of the samples, Consistency,
Presence of blood, mucus, and, or, pus. Whether the samples contain worms.
Wet Preparation by Saline and 1% Iodine
i. Using marker label a microscope slide with the laboratory number
ii. Place a drop of fresh physiological saline at one end of a slide and a
drop of 1% Iodine on the other end.
iii. Using applicator stick, pick up a size of match head stool (~2 mg) and
mix with a drop of saline and a similar amount with Iodine to make a
smooth thin preparation.
iv. If stool is formed, the portion should include the inside and outside parts
of the sample
v. For mucoid or watery stool, mix the entire contents before picking a
portion to mix with saline.
vi. Cover the preparations with a cover slip.
vii. Start with 10x objective to systematically, examine the entire saline
preparation.
viii. Use the 40x objective to assist in the detection and identification of
parasitic elements (eggs, ova, cyst etc). Always examine several
microscope fields with this objective before reporting ‘No parasites
seen.’.
ix. Use the iodine preparation to assist in the identification of cysts.
x. Report the findings on Stool analysis worksheet.
xi. Discard the used microscope slide on the container with disinfectant.
2.4.13 Biological Reference Intervals
Not Applicable
2.4.14 Interpretation and Reporting of Results
Interpretation
i. Adult helminths or portions of helminths may be recovered and seen
with a naked eye. Examples include E. vermucularis adult worms,
Ascaris lumbricoides adult worms, and tapeworm proglottids.

28
 ii. Occasionally, other helminths may be recovered (hookworm,
Strongyloides stercoralis), but identification requires the use of the
microscope.
iii. The appearance of stool will yield diferrent interpretation such as;
blood and mucus in faeces: might be suggestive of amoebic dysentery,
intestinal schistosomiasis, invasive balantidiasis (rare infection), and
severe T. trichiura infections. Other non-parasitic conditions in which
blood and mucus may be found include bacillary dysentery,
Campylobacter enteritis, ulcerative colitis, intestinal tumour, and
haemorrhoids.
iv. Presence of pus: This can be found when there is inflammation of the
intestinal tract. Many pus cells can be found in faecal sampless from
patients with bacillary dysentery. They can also be found in amoebic
dysentery but are less numerous.
v. Pale coloured and frothy (containing fat) samples: might be
suggestive of giardiasis and other infections as- sociated with
intestinal malabsorption.
vi. Pale coloured faeces: (lacking stercobilinogen) might be suggestive
of an obstructive jaundice.
vii. Mucoid and blood diarrhea might be suggestive to presence of E.
histolytica
Reporting
Macroscopic Findings - Report the following:
i. Colour of the samples.
ii. Consistency, i.e. whether formed, semiformed, unformed, watery.
iii. Presence of blood, mucus, and, or, pus. If blood is present note
whether this is mixed in the faeces. If only on the surface this
indicates rectal or anal bleeding.
iv. Presence of worms, e.g. A. lumbricoides (large roundworm), E.
vermicularis (threadworm) or tapeworm segments, e.g. T. solium, T.
saginata.
Microscopic Findings – Report the Following
i. Report presence of any ova, trophozoites or cysts seen, specifying the
species, e.g. “Entamoeba histolytica trophozoites seen”.
ii. If no parasites are seen report as “ No ova or cysts seen”.
2.4.15 Limitation of the Procedure and Sources of Error
Delay in examination of stool sample may cause missing of some parasites in wet
prepation which are dectected still alive. Examples of such organisms are
Strongloides stercolaris, Giardia lamblia E. Histolytica trophozoites, etc.
Presence of urine kills trophozoites (false-negative results).
Excessive heat or cold.
2.4.16 Performance Characteristics
29
Refer to the method verification of this procedure.
2.4.17 Supporting Documents
Not Applicable
2.4.18 References
• Practical Laboratory Manual-Jane Carter and Orgenes Lema
• Monica Cheesbrough: District Laboratory Practice in Tropical Countries,Vol 1,
• Tropical Health Technology, 1998.
• Brunner & Suddarth’s Handbook of Laboratory and Diagnostic Tests, 2010.

30
CHAPTER THREE: BLOOD TRANSFUSION
3.0 PROCEDURE FOR ABO AND RHESUS BLOOD GROUPING
3.0.1 Purpose
This procedure provides instructions for performing ABO and Rhesus (D) blood
grouping using tube method.
3.0.2 Scope
This procedure is used in Blood Transfusion unit when performing ABO & Rhesus(D)
blood group typing for donors and patients.
3.0.3 Responsibility
The head of Blood Transfusion and competent medical laboratory personnel are
responsible for ensuring this procedure is effectively implemented and maintained.
3.0.4 Principle
This method is based on immunophenotyping principle. The known antibodies
A&B(antisera) react with unknown antigens on the red cells surface to form
agglutination or haemolysis; this is known as forward or cells grouping. Also known
antigens (A and B) react with unknown antibodies in the patient or donor serum/plasma
to form agglutination or haemolysis; this is known as backward or serum blood
grouping.
The D antigen on the red cells surface reacts which known D antibodies(anti-D) to form
agglutination which determines the Rhesus group of an individual, either as Rh (D)
Positive(agglutination) or Rh (D) Negative (no agglutination).
3.0.5 Sample Requirements
2-3mls of EDTA sample, 2-3mls of clotted sample from plain tube
Centrifuge the sample at 3500rpm (RCF) for 5 minutes.
3.0.6 Equipment
Centrifuge, Microscope, Refrigerator, Timers, 37˚C water bath and Thermometer
3.0.7 Materials
Reagent Consumables
Anti –A Gloves
Anti-B Laboratory coat
Anti-D (saline, IgM) Test tubes,
Incomplete anti-D (IgG) Test tube rack,
Anti-Human Globulin Serum (AGS) slides
0.85% Physiological Saline Marker pens,
Low ionic strength solution (LISS) Beakers,
Pasteur pipettes,
31
3.0.8 Storage and Stability
• Store serum or plasma at 20C -80C for 7 days.
• Whole blood is stored at 20C - 80C for 3 days
• Anti-sera should be kept at 20C – 80C or as per manufacturer instructions
3.0.9 Safety
i. Adhere to safety precautions as stated in the Safety manual/ IPC
guideline
ii. All personnel protective equipment (PPE) must be worn when performing
this procedure.
iii. All samples must be regarded as potentially infections.
3.0.10 Calibration
Calibration of auxiliary equipment should be done as per calibration schedule
3.0.11 Quality Controls
Perform Quality Controls once in a week, when new antisera received and when new
control cells are prepared.
Controlling of anti-sera
• Commercial anti-sera are quality controlled by reacting them with known cell
Suspension of A, B, O, Rh (D) Positive and Rh (D) Negative.
• Arrange 6 tubes labelled A, B, AB, O and D positive and D negative.
• Put one drop of 2-5% cell suspension of A, B, AB, O Rh (D) Positive and O
Rh (D)
Negative to the corresponding tubes above
• Add one drop Anti-A, Anti-B, Anti-AB and Anti-D into corresponding tubes.
Controlling of O sensitized cells
• Label two tubes as O sensitized cells and O un sensitised cells
• Put one drop of corresponding of 3-5% cell suspension in the two tubes
above.
• In the two tubes, add one drop of AHG each.
Controlling of physiological saline
• Label two tubes as saline and distilled water
• Put one drop of saline and one drop of distilled water to the respective tubes
above
• Add one drop of O Rh (D) Positive cells to the two tubes
Common step
• Centrifuge all tubes above at light speed (1000rpm for 1 minute).
• Agglutination or haemolysis indicates positive reactions.
• Expected reactions are shown in table below
• Record results on ABO & Rh blood.

32
 Interpretation of IQC results
Cell suspensions
Commer A B AB Rh D pos Rh D O - Un -
cial cells cells cells cells Neg sensitized sensitised
Antisera Cells cells cells

Anti A + - + -
Anti B - + + -
Anti AB + + + -
Anti D +
LISS +++
AHG + -
Saline + to ++
Distilled Haemolysis
water
Key:
+ Agglutination
- No agglutination

Note: Strength of agglutination is graded from 1+ (separate agglutination) to 3+ (one solid

agglutination)

3.0.12 Procedure Steps

Prepare of 2- 5% cell suspension
• Place 2-3 drops of donor red blood cells into a tube
• Fill the tube (3/4) with 0.85% normal saline
• Centrifuge the tube at 3400rpm for 2 to 3 minutes. Decant supernatant fluid.
(Repeat 3 times)
• Transfer a drop of packed red cells from the above tubes and add 19 drops of
saline to make 2% to 5% donor red cell suspension
Blood grouping procedure
Forward/Cell Grouping
i. For each patient/donor label 3 test tubes as Anti-A tube (A), Anti-B tube (B), and
anti-D tube (D).
ii. Arrange the labelled test tubes in the test tube rack
iii. Add one volume of anti A into tube A
iv. Add one volume of anti-B into tube B
v. Add one volume of anti-D into tube D
vi. To each of the above tubes add one drop of 2 - 5% cell suspension and mix well.
vii. Centrifuge the three tubes at 1000rpm for one minute
viii. Examine the contents of the tubes for the evidence of agglutination.

33
 ix. Read, interpret, and record the test results.
Backward/Serum Grouping
i. For each patient/donor, label 2 test tubes as A cells and B cells.
ii. Add two drops of serum to each tube.
iii. Add one drop of A reagent cells into tube labelled A cells,
iv. Add one drop of B reagent cells into tube labelled B cells
v. Mix the contents of the tubes gently
vi. Centrifuge the tubes at 1000 rpm for one minute.
vii. Examine the serum for evidence of haemolysis, gently suspend the cell bottoms
and examine them for agglutination macroscopically and microscopically.
viii. Read, interpret and record test results.
Procedure for Weak Rh (D)
i. If the above anti-D reaction is negative, confirm weak Rh (D) as follows:
ii. Add two drops of LISS
iii. Incubate at 37˚C for 15 minutes in water bath. In absence of low ionic strength
solution (LISS) incubate at room temperature for 30 minutes
iv. Spin for 1000rpm for 1 minute
v. Observe for agglutination macroscopically and microscopically or for
haemolysis
vi. If agglutination or haemolysis is observed at this stage, report result as Rh (D)
Positive and the procedure ends here.
vii. If there is still no agglutination, proceed as follows:
viii. Wash contents of the tube 3 times with physiological saline
ix. Discard supernatant after third wash
x. Add one drop Anti-Human Globulin Serum (AGS)
xi. Spin for 1000rpm for one minute
xii. Observe for agglutination (macroscopically and microscopically) or haemolysis
xiii. If agglutination is observed at this stage, report results as Rh (Du) Positive
xiv. If reaction is still negative, add one drop of O sensitised cells to the tube
xv. Spin for 1000rpm for 1minute
xvi. Observe for agglutination (macroscopically and microscopically) or haemolysis
xvii. Presence of agglutination or haemolysis indicates a valid negative result.
xviii. Absence of agglutination or haemolysis means the test is invalid; therefore, it
has to be repeated.

Note: For purposes of transfusion, patients with Rh(Du) Positive should be given rhesus
negative blood.

34
3.0.13 Biological Reference Intervals
Not Applicable
3.0.14 Interpretation and Reporting of Results
Interpretation of results
Patient/Donor Cell Grouping Patient/ Donor Serum
Grouping
Anti-A Anti-B Ant-AB Anti-D Blood group A-cells B-cells Blood
&Rhesus group
factor
+ - + + A Rh(D)Pos - + A
- + + + B Rh(D)Pos + - B
+ + + + AB Rh(D)Pos - - AB
- - - + O Rh(D)Pos + + O
KEY: - + Means Agglutination
- Means No Agglutination
Reporting of results
Results report should include the ABO Type and Rhesus D reaction results, e.g. Blood
group A Rh (D) Positive, or Blood Group A Rh(D) Negative
3.0.15 Limitations of the Procedure and Source of Error
• Avoid haemolysed samples as this may lead to false negative results.
• Patients who have had recent multiple transfusions may develop allo-antibodies
that can interfere with antigen – antibody reactions
3.0.16 Performance Characteristics
Refer to the method verification report
3.0.17 Supporting Documents
Sample collection manual, safety manual, and quality manual.
3.0.18 References
i. Technical manual of the American Association of Blood Banks
ii. Mollison P.L., Blood Transfusion in Clinical Medicine 8th Ed. Oxford. Blackwell
Scientific, Practical haematology by Decie
iii. Guidance manual on “ABO and Rh blood grouping” (Institute of Biologicals-
India)
iv. Anti-sera insert kit (Anti-A, Anti- B, Anti-AB, Anti- D Monoclonal blood grouping
antibodies for tube and slide test) T Tulip diagnostics (P) LTD.

35
3.1 PROCEDURE FOR ESTIMATION OF HEMOGLOBIN BY USING
COPPER SULPHATE SOLUTION
3.1.1 Purpose
To provide guidance on the procedure of Haemoglobin estimation level to blood donors
using the Copper Sulphate solution technique
3.1.2 Scope
This procedure provides guidance on haemoglobin estimation of blood donors using
copper sulphate (CUS04) method in NBTS Blood collection teams and its satelites.
3.1.3 Responsibility
Trained qualified and competent certified registered medical personnel and other
authorized medical personnel.
3.1.4 Principle
A blood droplet is allowed to fall into copper sulphate solution of a specific gravity 1.053
and the movement of droplet is observed, If the specific gravity is higher than solution,
the drop will sink within 15 sec or else it will remain suspended for some time.
3.1.5 Sample Requirements
Capillary Blood
3.1.6 Equipment
Not applicable
3.1.7 Materials
Reagents: 70% Ethyl alcohol, Copper Sulphate solution with specific gravity of 1.053
Consumables: Sterile swabs, Picker, Sharp container, Capillary tubes, Gloves,
Universal bottle, Waste containers and Timer
3.1.8 Preparation of Copper II Sulphate solution
i. Weigh 170 gms of hydrous Copper Sulphate Powder/Crystals
ii. Put into volumetric flask
iii. Dissolve 170gms of hydrous Copper Sulphate Powder/Crystals with 1 litre
of distilled water.
iv. Mix well until all Crystals dissolves
v. Label the solution as STOCK SOLUTION with Preparation date, Batch
number and expiry date
vi. NB: Calculate expiry date six months from date of preparation
vii. Store and keep stock solution at room temperature in a tightly capped brown
glass bottle.
3.1.9 Prepare Working Solution
To prepare 1 Litre of working solution;
36
 - Dispense 480mls of distilled water using measuring cylinder into a volumetric
flask.
- Add 520mls of prepared stock solution using measuring cylinder
- Mix thoroughly
3.1.10 Storage and Stability
Copper Sulphate working solution stored at room temperature in brown bottle in
three months
3.1.11 Safety
i. Decontaminate working surfaces twice daily, in the morning and afternoon
and when needed, all generated records are kept.
ii. Adhere to safety precautions as stated in the Safety manual
iii. All personal protective equipment (PPE) must be worn when performing this
procedure.
iv. All samples must be regarded as potentially infections.
v. Refer to National infection prevention and control Guidelines for health
waste management and safety practice.
vi. All spills should be wiped thoroughly using 0.5% sodium hypochlorite
solution.
3.1.12 Calibration
Not applicable
3.1.13 Quality Control
Reagent : Test specific gravity of copper sulphate solution using Hydrometer
Accept the copper sulphate solution if the specific gravity is 1.053
Reject copper sulphate if specific gravity is not equal to 1.053
Record findings of the copper sulphate inspection and testing report form.
Then select sample with slightly bellow and slightly high with a cut off value of High
(12.6 -13.0) g/dl and Low (12.0 – 12.4) g/dl.
Accept the copper sulphate solution, if drop of blood with haemoglobin level less than
12.5 g/dl floats and blood with haemoglobin level higher than 12.5g/dl sinks.
Perform corrective action if results are outside an acceptable limits
3.1.14 Procedure Steps
Follow the actions described below step-by-step:
i. Explain the Procedure to the Blood Donor
ii. Welcome and greet the donor, offer a chair to seat
iii. Explain the procedure and reassure the donor.
iv. Select and clean Site
v. Select donors' middle finger or the finger medial to the middle finger
vi. Clean the upper top of right-side area with cotton wool swab soaked in 70%
ethyl alcohol in a spiral movement starting at the intended site out ward

37
 vii. Leave it until dried do not blow the site
viii. Remove the lancets cover
ix. Using the index finger and thumb, squeeze and hold the donor's·fingertip at the
upper joint tightly and prick
x. Wipe off the first drop of blood once with a dry swab
xi. Draw blood into the Capillary Tube
xii. Gently press the pricked fingertip to draw blood Note; Do not Squeeze the
pricked finger because it may introduce tissue fluid, dilute blood and give false
low Hemoglobin
xiii. Hold Capillary tube at approximately 60 degrees and let the blood flow into the
capillary tube up to not less than 3/4 capacity
xiv. Avoid air entering the capillary tube by ensuring smooth flow and undisrupted
flow of blood into the capillary tube
xv. Close the upper tip of capillary tube by placing your finger tip
xvi. Give clean dry swab to donor and instruct them to press finger with thumb till
bleeding stops.
xvii. Release a drop of Blood to Copper Sulphate Solution
xviii. Hold capillary tube at least 1 cm above the surface of copper sulphate solution
xix. Release your finger tip to allow one drop of blood freely into labeled container
of fresh daily prepared and quality control copper sulphate solution.
3.1.15 Biological Reference Intervals
Above or equal 12.5 g/dl
3.1.16 Interpretation and Reporting of Results
Accept if HB is greater/equal than 12.5g/dl
Reject if HB is less than 12.5 g/dl
3.1.17 Limitations of The Procedure and Sources of Error
Not applicable
3.1.18 Performance Characteristics
Not applicable
3.1.19 Supporting Documents
Sample collection manual, Quality manual, Safety manual
3.1.20 References
AAB technical manual 12th edition, AfSBTC 4th Edition.

38
3.2 PROCEDURE FOR COMPATIBILITY TESTING
3.2.1 . Purpose
This procedure provides instructions for performing compatibility testing, which is used
to select blood and blood components that will not cause harm to the recipient (patient)
3.2.2 Scope
This procedure is used in Blood Transfusion section when performing compatibility
testing prior to issue of a unit of blood to recipient.
3.2.3 Responsibility
The head of Blood Transfusion and competent medical laboratory personnel are
responsible for ensuring this procedure is effectively implemented and maintained.
3.2.4 Principle
This method is based on immunophenotyping principle. The known red cell antigens
from donor are mixed with unknown antibodies from the recipient (patient) to detect if
there is any incompatibility caused by ABO, Rhesus and/or other blood groups
antibodies.
3.2.5 Sample Requirements
➢ Patient/ recipient serum from a clotted blood sample in plain tube
➢ Donor cells from a segment tubing of the blood unit
3.2.6 Equipment
Centrifuge, Water bath, timer, refrigerator
3.2.7 Materials
Reagent Consumables
Incomplete anti-D (IgG) Test tubes,
Anti-Human Globulin Test tube rack,
Serum (AGS) grease pencil,
Sensitized cell Beakers,
Physiological Saline,
Pasteur pipettes

3.2.8 Storage and Stability

Venous blood must be used within 3 hours at room temperature then after should be
refrigerated at 2-8˚C. Blood Donor units should be stored at 1-6˚C
3.2.9 Safety
i. Adhere to safety precautions as stated in the Safety manual
ii. All personal protective equipment (PPE) must be worn when performing this
procedure.

39
 iii. All samples must be regarded as potentially infections.
iv. Refer to National infection prevention and control Guidelines for health
waste management and safety practice.
3.2.10 Calibration
Calibration should be done as per schedule.
3.2.11 Quality control
Quality Controls for Antihuman globulin should be done on a daily basis before
performing patient samples..Internal quality control should be prepared from patient
samples or known organisation.
3.2.12 Procedure Steps
Immediate Spin Saline Technique
i. Select a unit of blood from the blood bank storage with the same group as the
recipient. If there is no unit with the same blood group as the recipient, blood
group O packed red cells can be used as universal donor. Also recipients with
AB blood group are considered as universal recipients of packed red cells.
ii. Label a tube for each donor red cell suspension being tested with the patient’s
serum.
iii. Add two drops of patient’s serum or plasma to each tube.
iv. Add one drop of donorcells suspension into appropriate test tubes
v. Rinse the Pasteur pipette 5 times during transferring of cells and serum to avoid
contamination
vi. Mix the contents of the tube(s) and centrifuge at 1000rpm for 60 seconds.
vii. Gently re-suspend the cell buttons and observe for haemolysis or agglutination.
viii. Read, interpret, and record test results.
ix. If compatible, proceed with Indirect Antiglobulin Technique
Interpretation and reporting:
Agglutination or hemolysis means a positive (incompatible) test results
A smooth suspension of red cells after resuspension of the red cells button means
negative results and indicates a compatible immediate spin cross match.

Note: In emergency cases where the blood unit is required immediately, perform Immediate Spin
Saline Technique. Issue the blood unit if it is compatible then proceed with Indirect Anti-globulin
test. If there is any incompatibility, immediately call the ward to stop the transfusion and re-call
the blood unit.

3.2.13 Biological References Intervals

Not applicable
3.2.14 Reporting and Interpretation of results
Interpretation of results

40
Results should be reported in such a way that will indicate the recipient’s blood group
and the donor’s blood unit number to which the donor’s bloods is compatible or not
compatible.
Reporting of Results
Report as compatible when there is no agglutination or incompatible when there is no
agglutination.
3.2.15 Limitation of the Procedure and Sources of Errors
Hemolysis samples may lead to false negative results.
Patients who have had recent multiple transfusions may develop allo-antibodies that
can interfere with antigen – antibody reactions
3.2.16 Performance Characteristics
Refer to the method verification report.
3.2.17 Supporting Documents
Quality manual, sample collection manual and safety manual
3.2.18 References
Pam S. Helekar, D.P. Blackall et.al. American Association of Blood Bank 15 Edition,
1985. (method 3.1) and (method 3.2.1)

41
CHAPTER FOUR: HAEMATOLOGY
4.1 PROCEDURE FOR URIT-12 HEMOGLOBIN METER
4.1.1 Purpose
This procedure is used to describe step by step on how to operate the URIT-12
Haemoglobin Meter using human whole blood sample in the laboratory.
4.1.2 Scope
This procedure is applied in testing hemoglobin parameter using human whole blood
sample in the haematology department/section in the laboratory
4.1.3 Responsibility
A trained and competent laboratory scientist, laboratory technologists and assistant
laboratory technologists are responsible for performing this procedure.
The head of section or assigned personnel will be responsible for ensuring that this
procedure is effectively implemented.
4.1.4 Principle
The URIT-12 Hemoglobin Meter utilizes optical reflectance for determination of the
total hemoglobin. A drop of whole blood is applied to the test spot on the strip, blood
immediately disperses within the membrane contacting the reagent, then reaction
product could absorb spectrum in the range of 500nm – 600nm. The meter’s optical
detector automatically measures the change in membrane reflectance. The intensity
of reflectance is inversely proportional to the hemoglobin concentration. The meter
calculates and displays the total hemoglobin concentration in gram/decilitre (g/dL) in
12 seconds based on mathematical conversion.
4.1.5 Sample Requirements
Fresh capillary or EDTA-anticoagulated venous whole blood
4.1.6 Equipment
URIT-12 Hemoglobin Meter
4.1.7 Materials
The materials required in this procedure are Clean gloves, Laboratory coats,
Micropipettes, Cuvetes,Sharp container, Pricker and Alcohol swab
4.1.8 Storage and Stability
Anticoagulated blood is stable up to 72 hours at 2-8 ⸰c
4.1.9 Safety
• Personnel Protective Equipment must be worn at all times
• All samples must be treated as potentially infectious.
• Adhere to safety precautions as stated in the Safety manual/IPC guidline
42
4.1.10 Calibration
Not Applicable
4.1.11 Quality Control
Use Hemoglobin HQ-A Control Solution or known higher and low concentration made
in-houseto run on weekly basis to determine the accuracy of the patient results.
4.1.12 Procedural Steps
i. Massage the patient’s middle or ring finger from knuckle up to the tip to stimulate
blood flow
ii. Insert the test strip into the strip holder with the notched end in first and the hole
facing up. The notched end on the top of strip should no longer be visible when
test strip is inserted correctly and fully
iii. Perform finger prick. Avoid “Milking” Apply light pressure to obtain one drop of
blood.
iv. Take 13-15µl of whole blood with capillary tube or transfer pipette
v. Rapidly drip the blood into the sample spot on the strip when the meter shows
blood symbol and ensure the test strip is covered by blood sample completely.
vi. During the test do not disturb or move the meter or strip, even press any key of
meter
vii. The test results will be displayed in less than 30 seconds
viii. Record the test results displayed on the machine.
ix. Remove the test strip and immediately dispose off into highly infectious waste
container.
4.1.13 Biological Reference Intervals
Infant 14.0 - 22.0g/dl, Children 11.1 - 14.1g/dl, Adult male 13.0 -17.0g/dl, Adult female
12.0-15.0g/dl
4.1.14 Interpretation and Reporting Of The Results
Interpretation of results
Interpretation of the results is based on the biological reference intervals.
Reporting of results
The obtained results will be reported in g/dl.
Critical results: HB ≤ (5.07) g/dl is considered as critical and communicate with
clinician
4.1.15 Limitation of the Procedure and Sources of Errors
Only whole blood or EDTA anticoagulated blood should be used.
4.1.16 Performance Characteristics
Refer into method verification report
4.1.17 References
URIT – 12 Hemoglobin meter Operation Manual

43
4.2 PROCEDURE FOR DETERMINATION OF HAEMOGLOBIN LEVEL
USING HEMOCHROMAX PLUS
4.2.1 Purpose
The procedure provides instructions to laboratory staff on operation of Hemochroma
Plus Machine.
4.2.2 Scope
This procedure is applicable when performing quantification of Haemoglobin (Hb)
concentration in Hospital Laboratory.
4.2.3 Responsibility
Qualified, trained and competent Medical Laboratory Scientist, Technologist and
Technician are responsible for doing this test procedure. Section heads are
responsible for ensuring the effective implementation of this procedure.
4.2.4 Principle
The hemochroma PLUS analyser utilizes a dual wavelength LED light sources by
which the haemoglobin absorbance is detected and converted into an electrical signal.
The signal is direct proportional to the amount of haemoglobin present in the blood
sample.
4.2.5 Sample Requirements
Whole blood, capillary or venous ant coagulated collected blood into EDTA tube.
4.2.6 Equipment
Hemochroma PLUS machine
4.2.7 Materials
Hemochromax Plus Micro calibrator cuvette, Calibrator ID chip cuvettes, , Gauze,
70% methylated spirit and Blood lancet or prickers
4.2.8 Storage and Stability
Anticoagulated blood is stable up to 3 days at 2-8 ⸰c
4.2.9 Safety.
• Adhere to safety precautions as stated in the Safety manual/IPC guideline
• All personnel must be worn protective equipment (PPE) when performing
this procedure.
• All samples must be regarded as potentially infections.
4.2.10 Calibration
It should be done when the machine is not working properly or when provides a doubt
result.
4.2.11 Quality Control

44
Use commercial IQC materials or known higher and low concentration made in-house
to run on daily basis before performing the patient sample
4.2.12 Procedure
i. Establish good relationship with the patient
ii. Make sure the patient is sitting comfortably
iii. Lightly massage to stimulate circulation. Only use the middle or ring finger. The
patient should not wear the ring on that finger
iv. Pres lightly and draw finger-prick blood into a micro cuvette by bringing the
cuvette in contact with the blood drop on the fingertip and puncture the side to
a depth of cuvette.
v. Remove any excessive blood from the outside of the cuvette.
vi. Insert the cuvette containing blood sample to the Hb machine
vii. Wait until displaying of the test results and record the findings into the system
and register book
viii. Pull the cuvette holder out to its loading position and discard the used
microcuvette in sharp box
4.2.13 Biological Reference Interval
Infant 14.0 - 22.0g/dl, Children 11.1 - 14.1g/dl, Adult male 13.0 -17.0g/dl, Adult female
12.0-15.0g/dl
4.2.14 Interpretation and Reporting of Results
Interpretation of results
Interpretation of the results is based on the biological reference intervals.
Reporting of results
The obtained results will be reported in g/dl.
Critical results: HB ≤ (5.07) g/dl is considered as critical and communicate with
clinician
4.2.15 Limitation of the Procedure and Sources of Errors
i. Only whole blood should be used
ii. Air bubbles in the optical eye caused by inadequate filling of the cuvettes may
lead into false results
4.2.16 Performance Characteristics
Refer to method verification report
4.2.17 Supporting Documents
Sample Collection Manual, Safety Manual,
4.2.18 References
• Monica Cheesbrough. District laboratory practice in tropical countries, Part 2.
2000.
• Hemochromax PLUS package insert

45
 • Hemochromax PLUS user manual
4.3 PROCEDURE FOR DETREMANATION OF HEMOGLOBIN LEVEL
USING HEMOCUE 201+ MACHINE
4.3.1 Purpose
This procedure provides instructions for the performance of Haemoglobin Estimation
using Hemocue 201+ machine.
4.3.2 Scope
This procedure applies to all competent laboratory staffs during determination of
haemoglobin level by using Hemocue 201+ machine.
4.3.3 Responsibility
Qualified, trained and competent health laboratory practitioners in the laboratory are
responsible for implementation of this procedure.
4.3.4 Principle
The reaction in the microcuvette is a modified azidemethemoglobin reaction. The
erythrocytes are haemolysis to release the haemoglobin. The haemoglobin is
converted to methoglobin and the combined with azide to form azide methoglobin. The
measurement takes place in the analyser in which the transmittance is measured the
absorbance and haemoglobin level is calculated. The absorbance is directly
proportional the haemoglobin concentration
4.3.5 Sample Requirements
Capillary whole blood sample or Anti-Coagulated Whole blood (collected in EDTA
anticoagulant)
4.3.6 Equipment
Hemocue 201+ machine
4.3.7 Materials
Hemocue Hb 201+ microcuvette, Lancet/Pricker for capillary sample, Pipette or any
other transfer device for venous sample or control materials
4.3.8 Storage and Stability
Ant-Coagulated whole blood is stable up to 4 hours at room temperature and up to 24
hours at 4°C– 8°C.
4.3.9 Safety
• Personnel Protective Equipment must be worn at all times
• All samples must be treated as potentially infectious.
4.3.10 Calibration
Not Applicable

46
4.3.11 Quality Control
The Hemocue Hb 201+ has an electronic self-test daily IQC or the use of known higher
and low concentration made in-house can also be applied.
4.3.12 Procedural Steps
i. Establish good relationship with the patient
ii. Make sure the patient is sitting comfortably
iii. Lightly massage to stimulate circulation. Only use the middle or ring finger. The
patient should not wear the ring on that finger
iv. Pres lightly on the fingertip and puncture the side to a depth of cuvette.
v. Remove any excessive blood from the outside of the cuvette.
vi. Insert the cuvette containing blood sample to the Hb machine
vii. Wait until displaying of the test results and record the findings into the system
and register book
viii. Pull the cuvette holder out to its loading position and discard the used
microcuvette in sharp box

Note: The microcuvette should be filled within 3 minutes after it has been taken out of its
package.

4.3.13 Biological reference Intervals

Infant 17.0-22.0g/dl, Children 11.0-13.0g/dl, Adult male 13.0 -17.0g/dl, Adult female
12.0-15.0g/dl
4.3.14 Interpretation and Reporting of Results
Interpretation of results
Interpretation of the results is based on the biological reference intervals.
Reporting of results
The obtained results will be reported in g/dl.
Critical results: HB ≤ (5.07) g/dl is considered as critical and communicate with
clinician
4.3.15 Limitation of the Procedure and Sources of Error
• Only whole blood should be used
• Air bubbles in the optical eye caused by inadequate filling of the cuvettes
• Delayement in transfer of filled cuvettes with blood into HB machine
4.3.16 Performance Characteristics
Refers into method verification report
4.3.17 Supporting Documents
Waste Management Procedure /IPC guideline
Quality Control Result Procedure
4.3.18 References:

47
Monica Cheesbrough. District laboratory practice in tropical countries, Part 2. 2000.
Hemocue Hb 201+ cuvette kits insert and use manual.
4.4 PROCEDURE FOR CD 4 COUNT TEST BY USING BD FACS PRESTO
4.4.1 Purpose
The purpose of this procedure is to provide detailed information on how to analyse and
detect CD4 T Cell enumeration on blood sample by using BD FACS presto.
4.4.2 Scope
This procedure applicable in haematology to analyse and detect CD4 T Cell by using
BD FACS presto.
4.4.3 Responsibility
Qualified, trained and competent Medical Laboratory Technician and Technologist are
responsible for doing this test procedure. Section heads are responsible for ensuring
the effective implementation and competency assessment for this procedure.
4.4.4 Principle
The BD FACS Presto™ cartridge the CD4/%CD4/Hb cartridge contains dried
fluorochrome-conjugated antibody reagents. When blood reacts with the reagents, the
antibodies in the reagent bind to the surface antigens on the BD FACS Presto
Cartridge: lymphocytes and monocytes. After the incubation period, the cells are
analysed on the BD FACS Presto Near-Patient CD4 Counter (the instrument). The
software identifies the cell populations of interest and calculates CD4 absolute counts,
CD4 percentages of lymphocytes, and haemoglobin concentration. The system
measures total haemoglobin by spectrophotometric method, using absorbance at an
isobestic point for oxyhemoglobin and deoxy-hemoglobin, with Correction for scatter.
4.4.5 Sample Requirements
Blood sample on K2EDTA vacuum tubes. All K2EDTA samples must be received and
set up within 24 hours from collection time.
4.4.6 Equipment
BD FACS Presto
4.4.7 Materials
Reagent Consumables
BD FACS presto Blue and yellow tips
cartridge BD FACS presto print out paper
BD disposable 100 µl Pipette
4.4.8 Storage and Stability
• Do not refrigerate whole blood SAMPLE before sample preparation.
• Do not use previously fixed and stored samples.
4.4.9 Safety
48
 • Blood samples may contain infectious agents that are hazardous to your
health. Observe Standard Universal precautions.
• Ensure the instrument and environment you working are kept clean and free
from infectious substance such as human blood to avoid contamination.
• Spills should be immediately disinfected with 0.5% Sodium Hypochlorite
Solution.
4.4.10 Calibration
Not Applicable.
4.4.11 Quality Control
BD Facs presto has internal electronic self-test for IQC or the use of known higher and
low CD4 counts made in-house can also be applied
4.4.12 Procedural Steps
i. Open the cartridge package and label the patient ID on to the cartridge.
ii. Face the inlet port up.
iii. Invert the tube 10 times to mix the contents well.
iv. Use the pipette to obtain the sample.
v. Gently squeeze the bulb on the pipette to form a drop of blood on the tip
of the pipette.
vi. Carefully dispense the sample into the inlet port. Hold the cartridge by its
ridges only.
vii. Make sure the blood reaches the top of the inlet port. If necessary, gently
squeeze the bulb on the pipette to dispense more blood.
viii. Make sure the cartridge is level, with the barcode side up, at all times.
Make sure that blood appears in the part of the channel not covered by
the channel protector, next to the containment zone.
ix. Discard the pipette into a biohazard us waste container.
x. Close the cartridge cap securely and Set the on-board timer.
xi. Place the cartridge, barcode side up, on the work station
xii. Press the Run Test tab.
xiii. Press Patient ID.
xiv. Enter the patient’s ID and press Accept.
xv. The Confirmed Patient ID screen opens.
xvi. Press Accept and Insert the cartridge:
xvii. Select your Operator ID and press Accept. Then cartridge door on the
instrument opens will open.
xviii. Note: If possible, complete the following two steps within 30 seconds.
xix. Remove the channel protector from the cartridge.
xx. Hold the cap with the channel facing upwards.
xxi. Insert the prepared cartridge into the cartridge door. The cartridge door
closes.
xxii. Press Accept to eject the cartridge within 30 seconds.
49
4.4.13 Biological Reference Interval
Analyte Gender Reference interval SI UNIT
Absolute CD 4 count Male 462 – 1306 cells/uL
Female 440 – 1602 cells/uL
%CD4 of lymphocytes Male 29 – 54 %
Female 32 – 55 %
HAEMAGLOBIN Male 13.5 – 18.0 g/dL
Female 12.0 – 16.0 g/dL
4.4.14 Interpretation and Reporting of Results
Interpretation of results
• The results are displayed on the screen and print automatically.
Reporting of Results
• Report the obtained results as displayed on the screen.
Critical values
• If the CD4 count is low (below 200cells/l for adult and below 450
cells/ l for children) the result will be regarded as critical and
communicate with clinician
4.4.15 Limitations of the procedure and sources of error
• Use the cartridge only with the BD FACS Presto instrument.
• The use of expired cartilage may result into false results
• Improper filling of the test device may not give the proper results
4.4.16 Performance Characteristics
Refer to Method verification report
4.4.17 Supporting Documents
Quality Manual, Sample Collection Manual, Safety Manual.
4.4.18 References
Becton Dickinson BD FACS presto Operating Manual

50
4.5 PROCEDUIRE FOR FULL BLOOD COUNT BY USING URIT BH – 40P
HAEMATOLOGY ANALYSER.
4.5.1 Purpose
This procedure provides instruction on how to operate urit BH-40P haematology
analyser in determining full blood count
4.5.2 Scope
This procedure applies in the Haematology section when performing FBC (Full blood
count) analysis using URIT BH - 40P Haematology Analyser.
4.5.3 Responsibility
Qualified, trained and competent health laboratory practitioners are responsible for
implementing this procedure. The Head Haematology is responsible for ensuring the
effective implementation and maintenance of this procedure.
4.5.4 Principle
The URIT BH – 40P Automated Haematology Analyser is a multi-parameter, it can
display 21 parameters and 3 histograms. Analyser adopts electrical impedance
method for WBC, RBC and PLT test and colorimetric method for HGB test. The
electrical impedance method is based on non-conductivity of blood cells. When the
blood cells in diluents pass through the ruby aperture, resistance will change, based
on that we can get the counting and volume of blood cells. The colorimetric methods
to measure and calculate HGB. Add lyse into the diluents sample, and then RBC will
be dissolve and release haemoglobin. Then the haemoglobin combines with lyse to
form cyanohemoglobin. Measure the transmission light intensity of this compound in a
sample cup through the monochromatic light of 540 nm wavelength and then compare
it with the result in blank state to get the haemoglobin concentration (blank state refers
to the state that only has diluents in sample cup).
4.5.5 Sample Requirements
Whole blood sample in EDTA-K2.2H2O tube.
4.5.6 Equipment
• URIT BH-40 Automated Haematology Analyser
• Perform start up, maintenance, troubleshooting and shut down the URIT BH-40
Automated Haematology analyser as per manufacturer’s instrument
instructions
4.5.7 Materials
Diluent, Lyse, Probe detergent, Set of Controls kit, Marker pen, Examination Gloves
Capillary tube, Plane test tube, Thermal paper, and Gloves
4.5.8 Storage and Stability
• Keep the Set of Controls at 2oC – 8oC
• Never use reagent and control beyond its expiration date
51
 • Blood sample be kept at temperature between 2oC- 8oC for 7 days.
4.5.9 Safety
• Adhere to safety precautions as stated in the Safety manual/IPC guideline
• All personal protective equipment (PPE) must be worn when performing
this procedure.
• All samples must be regarded as potentially infections.
4.5.10 Calibration
• URIT calibrates the analyser in factory before shipment.
• Use the user URIT BH-40P to recalibrate the analyser when there is shifts or
trends in some parameters
4.5.11 Quality Control
Run all quality controls; QC1 (Low), QC 2 (Normal) and QC 3 (High) before
examination of patient samples
4.5.12 Procedural Steps
Running Patient Samples
i. Pre- diluent peripheral blood mode
• Present the empty sample tube under aspiration probe.
• At main menu screen, click “Drain”; the diluents will be dispensed into the
tube.
• Remove the tube, add 20µl of the blood sample to the tube, and gently shake
the tube to make them well mixed.
• After that present the well mixed sample under the aspiration probe; make
sure the probe touches bottom slightly.
• Press RUN key on the front panel and remove the sample after hearing beep
sound. The result will be available after analysis is performed.
ii. Whole Blood Mode
• Gently shake the tube to well mix the blood sample, then present the sample
tube beneath the probe, make sure the probe touches tube bottom slightly.
• Press RUN key and remove the sample after hearing beep sound. The
results will be available after analysis is performed.
iii. Ant coagulated Peripheral Blood Mode
• Gently shake the tube to well mix the blood sample, then present the
sample tube beneath the probe, make sure the probe touches tube
bottom slightly.
• Press RUN key and remove the sample after hearing beep sound. The
results will be available after analysis is performed.
4.5.13 Biological Reference Interval
See annex 1.

52
4.5.14 Interpretation and Reporting of Results
Interpretation of results
• Normal: if the results are within acceptable range.
• Abnormal (pathological): if the results are out of acceptable range
Reporting of results
Results are automatically printed from the URIT BH-40P and then review
by section head.
Critical value
WBC > 20 x109/L, HGB <5g/dL, PLT < 50 or 1000 x109/L
4.5.15 Limitation of the Procedure and Sources of Error
• The test will be affected by hemolysed blood and coagulated blood.
• Samples with extreme lipemic, chylomicrons or extremely high bilirubin
concentrations might produce falsely elevated haemoglobin values.
4.5.16 Performance Characteristics
Refer to the method verification report of this procedure.
4.5.17 Supporting Document
Sample collection manual
4.5.18 References
URIT BH- 40P Operation manual
4.6 PROCEDURE FOR FULL BLOOD COUNT USING OF ABX PENTRA 80
HAEMATOLOGY ANALYSER.
4.6.1 Purpose
This procedure provides instructions for operation and maintenance of ABX Pentra 80
analyzer for Full blood picture
4.6.2 Scope
This procedure applies to all Full Blood Count tests done on ABX Pentra 80 analyser
in the haematology section
4.6.3 Responsibility
Qualified, trained and competent health laboratory practitioners are responsible for
implementing this test procedure.
The Head Haematology is responsible for ensuring the effective implementation and
maintenance of this procedure.
4.6.4 Principle

53
The ABX Pentra 80 is an automated haematologyanalyser used for counting and
differentiating the cellular components in whole blood using electrical impedance,
cytochemical staining, light scatter and spectrophotometer.
The principle behind cell counting is based on disruption of electric current as particles
pass through an orifice. An electric current applies on both sides of this orifice. Cells
do not conduct electric current, therefore their passage through the orifice leads to a
change of the electric current established between both electrodes. These electric
current differences are registered and increment a counter at every cell passage.
A chemical agent is used to separate erythrocyte and leukocyte populations, because
of size overlapping and quantities discrepancies. This chemical agent contained in
Lysis (ACTI-DIFF) pops the cytoplasmic membrane of the red cells. Erythrocyte
population disappears leaving the leukocytes.
A haemoglobin preservative is added in lysing agent to measure haemoglobin scaled
down in a 540nm photometric tank at the end of the counting. The haemoglobin
measurement is made from the first dilution. The lysing agent has a powerful
haemoglobin reducer (potassium cyanide) and then the haemoglobin measurement
follows Drabkin method with a 540nm reading. The integration of luminous intensity
transmitted is evaluated according to the BEER-LAMBERT formula. An enzymatic
liquid (ABX CLEANER) ensures the system cleanliness between every analysis and
prevents carryover between samples.
4.6.5 Sample Requirements
2 to 4mls of whole blood collected in K3 EDTA tube
4.6.6 Equipment
ABX Pentra 80 Analyser and Perform start up, maintenance, troubleshooting and shut
down the ABX Pentra 80 Haematology analyser as per manufacturer’s instrument
instruction.
4.6.7 Materials
ABX Pentra 80 analyser reagent pack, Laboratory coat, Biohazard waste container.
0.5% Sodium hypochlorite solution, Distilled water, Protective gloves, Methanol, A4
paper
4.6.8 Storage and Stability
Control kit is stable until expiry date and should be kept at 2 oC – 8oC. Blood sample
be kept at temperature between 2oC- 8oC for 7 days.
4.6.9 Safety
• Adhere to safety precautions as stated in the Safety manual/IPC guideline
• All personal protective equipment (PPE) must be worn when performing
this procedure.
• All samples must be regarded as potentially infections.
4.6.10 Calibration

54
Calibrate the ABX Pentra 80 analyser under the following conditions:
i. Change of software
ii. Major component replacement
4.6.11 Quality Control
Run control QC materials (Low, Normal and High) before patient samples
4.6.12 Procedural Steps
i. Click the STAT MODE key.
ii. Then write the patient ID, Age, Gender.
iii. Click the VALIDATE key.
iv. Mix well the patient sample.
v. Place the sample on the tube holder of analyser.
vi. Press the door of analyser inside to run the sample.
vii. The patient results will be printed automatically.
4.6.13 Biological Reference Interval
See annex 1.
4.6.14 Interpretation And Reporting Of Results
• Normal: if the results are within acceptable range.
• Abnormal (pathological): if the results are out of acceptable range
• Results are automatically printed from the ABX Pentra 80 and then review by
section head.
• Communicate the Critical value with clinicians
Test Critical Value
Haemoglobin Less than 5 g/dl (50 g/L)
White Blood Count Less than 2.0 or greater than 18.0 cells/mm3
Platelet Count Less than 50,000/ mm3 or greater than
800,000/mm 3

4.6.15 Limitation of the Procedure and Sources of Error

i. Sickled red blood cells may not be accurately recognised and may give
erroneous results
ii. Samples with cold agglutinins may falsely decrease the red cell count. The
indices will indicate that the haemoglobin and haematocrit values do not agree.
Thin film is recommended. These samples should be incubated for 30 minutes
at 37 oC and reanalysed.
iii. Platelet clumps and neonatal samples may interfere with Drabkins method of
haemoglobin determination.
iv. Samples with extreme lipaemia, chylomicrons or extremely high bilirubin
concentrations might produce falsely elevated haemoglobin values.

55
 v. Samples with nucleated red blood cells may falsely elevate white cell count.
Additionally, presence of nucleated red cells may interfere with white cell
differential count. Samples from patients with elevated chylomicrons ad those
receiving total parenteral nutrition (TPN) including a high lipid concentration may
falsely elevate the platelet count.
vi. Aggregated platelets may falsely elevate the white blood cell count and
percentage of lymphocytes.
vii. The presence of immature white blood cells, including blasts, may affect the
accuracy of the differential. The instrument will give an << I >> or ‘M’ error code
if the blasts are suspected. A thin film is recommended.
viii. Clinical studies have shown that the Full Blood Count is not affected by
presence of malaria parasites, Howell-Jolly bodies, cryogoblins and red cell
fragments.
4.6.16 Performance Characteristics
Refer to the method verification report of this procedure.
4.6.17 Supporting Document
Sample collection manual
4.6.18 References
ABX PENTRA 80 operator’s manual
4.7 PROCEDURE FOR FULL BLLOD COUNT USING SINNOWA HB - 7021
4.7.1 Purpose
This procedure provides instructions for operation and maintenance of SINNOWA HB
– 7021 Hematology Analyser.
4.7.2 Scope
This procedure applies to all Full Blood Count tests done on SINNOWA HB – 7021
analyser in the haematology section
4.7.3 Responsibility
Qualified, trained and competent health laboratory practitioners are responsible for
implementing this test procedure.
The Haematology section head is responsible for ensuring the effective
implementation and maintenance of this procedure.
4.7.4 Principle
The instrument adopts the method of impedance to measure and count cells. The
conductivity liquid (mainly diluents) provides constant current source to the electrode
thus they circuit can form a steady impedance circulation. When cells pass through the
aperture, the conductive liquid is replaced by cells. Change of circuit resistance
produces electrical pulse. The amplitude varies when the cells of different size pass

56
through the aperture. Consequently the number and volume of cells pass through the
aperture can be calculated based on the amplitude.
4.7.5 Sample Requirements
2 to 4mls of whole blood collected in K3 EDTA tube
4.7.6 Equipment
• SINNOWA HB – 7021 Haematology Analyzer, Refrigerator
• Perform start up, maintenance, troubleshooting and shut down the SINNOWA
HB – 7021 Haematology analyser as per manufacturer’s instrument instruction
4.7.7 Materials
Reagents consumables
SINNOWA Diluent Reagent, Laboratory coat, Biohazard waste container.
SINNOWA Lyse Reagent, 0.5% Sodium hypochlorite solution, Distilled
SINNOWA Detergent, SINNOWA water, Protective gloves, Methanol, Printing
Probe Detergent, paper
4.7.8 Storage and Stability
• Control kit is stable until expiry date and should be kept at 2 oC – 8oC
• Blood sample be kept at temperature between 2oC- 8oC for 7 days.
• Store reagents as per manufacturer instructions
4.7.9 Safety
• Adhere to safety precautions as stated in the Safety manual/IPC guideline
• All personal protective equipment (PPE) must be worn when performing this
procedure.
• All samples must be regarded as potentially infections.
4.7.10 Calibration
The SINNOWA HB – 7021Hematology Analyser System requires commercial
calibrator material or assayed whole blood for calibration.
Calibrate the SINNOWA HB – 7021Hematology Analyser under the following
conditions:
i. Change of software
ii. Major component replacement
4.7.11 Quality Control
• Run the three levels of control LOW, NORMAL and HIGH” for “SINNOWA HB –
7021 Hematology Analyzer to ensure quality of results
• Perform Quality Control:-
i. Before analyzing the samples
ii. After replacement of the reagents
iii. After maintenance component replacement, or a field service action

57
 iv. If there is any doubt in accuracy of the test results
v. After a reagent lot number change
vi. After a software change
vii. Following calibration
viii. According to your laboratory’s quality control program
ix. According to manufacturer requirements
4.7.12 Procedural Steps
i. Turn on the power on rear panel and indicator shows red light
ii. The instrument initializes test program
iii. Diluents, lyses and detergent will be sucked and tubing system cleaned
iv. If initialization is finished the display shows all parameters WBC,RBC and PLT
v. Shift to select ID and then press OK
vi. Select the test selection from the drop down menu
vii. Gently mix the sample
viii. Open the sample tube and place it under the Open Mode Probe
ix. Raise the tube until the end of the probe is deeply immersed in the sample.
Press the Touch Plate to activate aspiration
x. Remove the tube when the beep sounds and replace the cap.
xi. When the cycle is finished, the results post to the Data log and are displayed in
the Run View
xii. Print the results
4.7.13 Biological Reference Interval
See annex 1.
4.7.14 Interpretation and Reporting of Results
• Normal: if the results are within acceptable range.
• Abnormal (pathological): if the results are out of acceptable range
• Results are automatically printed from the SINNOWA HB – 7021 and then
review by section head.
• Communicate the Critical value with clinicians
Test Critical Value
Haemoglobin Less than 5 g/dl (50 g/L)
White Blood Count Less than 2.0 or greater than 18.0 cells/mm3
Platelet Count Less than 50,000/ mm3 or greater than
800,000/mm 3

4.7.15 Limitation of the Procedure and Sources of Error

i. Keep reagent away from direct sunlight and protect them from evaporation.
ii. Use reagent container cap attached to each inlet tube, the cap will minimize
evaporation and contamination.
iii. Never use reagent, control and calibrators beyond their expiration date.

58
4.7.16 Performance Characteristics
Refer to the method verification report of this procedure.
4.7.17 Supporting Document
Sample collection manual
4.7.18 References
• SINNOWA HB – 7021 Hematology Analyser user manual.
4.8 PROCEDURE FOR FULL BLOOD COUNT USING BHA 3000
HAEMATOLOGY ANALYSER
4.8.1 Purpose
This procedure provides instructions for operation and maintenance of BHA 3000
Haematology Analyser.
4.8.2 Scope
This procedure applies in haematology section for performing Full Blood Count tests
done on BHA 3000 analyser
4.8.3 Responsibility
Qualified, trained and competent health laboratory practitioners are responsible for
implementing this test procedure. The Haematology section head is responsible for
ensuring the effective implementation and maintenance of this procedure.
4.8.4 Principle
BHA -3000 Automatic Hematology Analyser provides a 3 part differential blood count
uses an electrical impedance to count red blood cells, platelets and volume
distributions, and uses calorimetry to measure the haemoglobin and relevant
parameters will be enumerated.
This system uses electrical impedance method to count red blood cells, platelets and
white blood cells. When the absorbed quantitative sample is diluted by a quantitative
conductive solution, it is sent to the detection unit of the instrument. The detection unit
has a detection aperture, with a pair of positive and negative electrodes on both sides
of the aperture, which is connected with a constant current power supply. Due to the
bad conductor characteristic of these cells, when the cells in the diluted sample pass
through the detection aperture under a constant negative pressure, the DC resistance
between the electrodes will change, thus forming a pulse change at both ends of the
electrode in portions to the size of the cell volume. When cells continuously pass
through the aperture, a series of electrical pulses are generated at both ends of the
electrodes. The number of pulse is equal to the number of cells passing through the
aperture. And the pulse amplitude is proportion to the cell volume. Amplify the collected
electrical pulses, and calculates the number of electric pulse in the red blood/platelets
channel and WBC channel. The pulses are then classified according to different
channel voltage threshold, hence determines the cell volume distribution

59
4.8.5 Sample Requirements
2 to 4mls of whole blood collected in K3 EDTA tube
4.8.6 Equipment
BHA 3000 Haematology Analyzer, Refrigerator
4.8.7 Materials
DIL-3 Diluent and HR-1 Lyse, Controls (Normal level (N), Low- level (L) and High- level
(H) Laboratory coat, Biohazard waste container. 0.5% Sodium hypochlorite
solution, Distilled water, Protective gloves, Printing paper
4.8.8 Storage and Stability
• Control kit is stable until expiry date and should be kept at 2 oC – 8oC
• Blood sample be kept at temperature between 2oC- 8oC for 7 days.
• Store reagents as per manufacturer instructions
4.8.9 Safety
i. Adhere to safety precautions as stated in the Safety manual/IPC guideline
ii. All personal protective equipment (PPE) must be worn when performing
this procedure.
iii. All samples must be regarded as potentially infections.
4.8.10 Calibration
Calibrate the BHA 3000 Haematology Analyzer Analyser under the following
conditions:
i. Change of software
ii. Major component replacement
4.8.11 Quality Control
Run the three levels of control LOW, NORMAL and HIGH” for BHA 3000 Haematology
Analyzer to ensure quality of results
Perform Quality Control:-
i. Before analyzing the samples
ii. After replacement of the reagents
iii. After maintenance component replacement, or a field service action
iv. If there is any doubt in accuracy of the test results
v. After a reagent lot number change
vi. After a software change
vii. Following calibration
viii. According to your laboratory’s quality control program
ix. According to manufacturer requirements
4.8.12 Procedural Steps
i. Turn on the power on rear panel and indicator shows red light
ii. The instrument initializes test program
60
 iii. Diluents, lyses and detergent will be sucked and tubing system cleaned
iv. If initialization is finished the display shows all parameters WBC,RBC and PLT
v. Shift to select ID and then press OK
vi. Select the test selection from the drop down menu
vii. Gently mix the sample
viii. Open the sample tube and place it under the Open Mode Probe
ix. Raise the tube until the end of the probe is deeply immersed in the sample.
Press the press Plate to activate aspiration
x. Remove the tube when the beep sounds and replace the cap.
xi. When the cycle is finished, the results post to the Data log and are displayed in
the Run View
xii. Print the results
4.8.13 Biological Reference Interval
See annex 1.
4.8.14 Interpretation and Reporting of Results
• Normal: if the results are within acceptable range.
• Abnormal (pathological): if the results are out of acceptable range
• Communicate the following Critical value with clinicians
Test Critical Value
Haemoglobin Less than 5 g/dl (50 g/L)
White Blood Count Less than 2.0 or greater than 18.0 cells/mm3
Platelet Count Less than 50,000/ mm3 or greater than
800,000/mm 3

4.8.15 Limitation of the Procedure and Sources of Error

iv. Keep reagent away from direct sunlight and protect them from evaporation.
v. Use reagent container cap attached to each inlet tube, the cap will minimize
evaporation and contamination.
vi. Never use reagent, control and calibrators beyond their expiration date.
4.8.16 Performance Characteristics
Refer to the method verification report of this procedure.
4.8.17 Supporting Document
Sample collection manual
4.8.18 References
BHA 3000 Haematology Analyser user manual.
District Level

61
4.9 PROCEDURE FOR PERFORMING FULL BLOOD COUNT BY USING
MS4 HAEMATOLOGY ANALYSER
4.9.1 Purpose
This procedure provides instructions for operation and maintenance of MS4 analyser
for Full blood count
4.9.2 Scope
This procedure applies to all Full blood count tests done on MS4 analyser in the
Laboratory hematology section
4.9.3 Responsibility
Qualified and trained Health Laboratory Practitioners are responsible for implementing
this test procedure. Section head is responsible for ensuring the effective
implementation and maintenance of this procedure.
4.9.4 Principle
The MS4-S is an automated haematology analyser used for counting and
differentiating the cellular components in whole blood using electrical impedance,
cytochemical staining, light scatter and spectrophotometer.
The principle behind cell counting is based on disruption of electric current as particles
pass through an orifice. An electric current applies on both sides of this orifice. Cells
do not conduct electric current, therefore their passage through the orifice leads to a
change of the electric current established between both electrodes. This electric
current difference is registered and increment a counter at every cell passage.
A chemical agent is used to separate erythrocyte and leukocyte populations, because
of size overlapping and quantities discrepancies. This chemical agent contained in
Lysis (ACTI-DIFF) pops the cytoplasmic membrane of the red cells. Erythrocyte
population disappears leaving the leukocytes. A Haemoglobin preservative is added in
lysing agent to measure Haemoglobin scaled down in a 540nm photometric tank at the
end of the counting. The Haemoglobin measurement is made from the first dilution.
The lysing agent has a powerful Haemoglobin reducer (potassium cyanide) and then
the Haemoglobin measurement follows Drabkin method with a 540nm reading. The
integration of luminous intensity transmitted is evaluated according to the BEER-
LAMBERT formula.
An enzymatic liquid (TRANSFLUX) ensures the system cleanliness between every
analysis and prevents carryover between samples.
4.9.5 Sample Requirements
2 to 4mls of whole blood collected in K3 EDTA purple top color tube
4.9.6 Equipment
MS4-S Hematology Analyser, and roller mixer
4.9.7 Materials

62
MS4-S analyser reagent pack, Lab coat, Biohazard waste container, 5-6% Sodium
hypochlorite, Distilled water, Protective gloves and Methanol
4.9.8 Storage and Stability
Blood is stable for about 4 hours at room temperature or 24hours at 2- 6oC
4.9.9 Safety
i. Treat all samples as potentially infectious.
ii. Keep hands away from the sample carrier when the analysis begins
iii. Some components inside the MS4s have sharp edges or angular corners,
therefore operate with caution to avoid cuts to the hands
iv. When performing maintenance procedure, take similar precautions as you
would take when handling patient samples
4.9.10 Calibration
All auxiliary equipment should be calibrated annually
4.9.11 Quality Control
Use commercially quality control or in house prepared quality control samples
Running of quality control
i. Take the QC material from the refrigerator and let them stay at room
temperature for 15 minutes.
ii. After 15 minutes’ mix well the controls one by one by inverting the tube gently
at least (x7) seven times without creating bubbles.
iii. Press ANALYSIS key (Tube like symbol) then DOWN arrow (↓).
iv. Select Quality CT (to run controls) then use RIGHT arrow ( ) to select
control levels.
v. Select LOW if you start with low control up to High control using UP and
DOWN arrows on key board or on machine. (↑) or (↓)
vi. Place the control tube on the tube holder
vii. Press ENTER key on keyboard or ( ) on the machine to validate
viii. Repeat the procedure to the Normal and High controls
ix. The QC results will be printed automatically
x. Plot the results of the QC on the Levy-Jennings chart and verify that the QC
has passed before running patients’ samples.
4.9.12 Procedural Steps
Running of Patient Samples
i. Press Analysis key in the machine
ii. Press RIGHT arrow ( )
iii. Select the gender female or male by using UP and DOWN arrows (↑) or (↓)
iv. Press the ENTER key (using key board) or ( ) on the machine to validate
v. Write the name of the patient (Full Name)
vi. Mix well the sample

63
 vii. Place the sample on the tube holder of the machine.
viii. Press ENTER on keyboard or ( ) on the machine to start running the
sample.
ix. The patient results will be printed automatically.
4.9.13 Biological Reference Intervals
See annex 1.
Critical value/results should be recorded and immediately communicated to the
requesting clinician.
4.9.14 Interpretation and Reporting of Results
Results are reported and communicated through the appropriate locally established
procedure
4.9.15 Limitation of the Procedure and Sources of Errors
i. Exposing reagents to direct sunlight may cause them to deteriorate
ii. Sickled red blood cells may not be accurately recognised
iii. Samples with cold agglutinins may falsely decrease the red cell count.
iv. Platelet clumps and neonatal samples.
v. Samples with extreme lipaemia, chylomicrons or extremely high bilirubin
concentrations might produce falsely elevated Haemoglobin values.
vi. Samples with nucleated red blood cells may falsely elevate white cell count.
vii. Samples from patients with elevated chylomicrons and those receiving total
parenteral nutrition (TPN) including a high lipid concentration may falsely
elevate the platelet count.
viii. Circulating micro megakaryocytes may be counted as white cells
ix. Aggregated platelets may falsely elevate the white blood cell count and
percentage of lymphocytes.
4.9.16 Performance Characteristics

Method verification should be carried out and its report will be referred to fulfil this
requirement.
4.9.17 Supporting Documents

Laboratory quality policy manual, Laboratory safety policy manual and Laboratory
sample collection manual
4.9.18 References

MS4S operator’s manual, 2013-07

64
CHAPTER FIVE: CLINICAL CHEMISTRY AND
IMMUNOLOGY
5.1 PROCEDURE FOR BLOOD GLUCOSE BY USING ACCUCHECK
GLUOCOMETER
5.1.1 Purpose
This procedure provides instructions for use of blood glucose test strips for
determination of Blood Glucose level. Blood glucose is measured mainly in the
diagnosis and maNot applicablegement of diabetes mellitus.
5.1.2 Scope
This procedure will be used for Blood Glucose testing in the laboratory and at point of
care testing sites (POCT).
5.1.3 Responsibility
Qualified and competent registered and licensed Health laboratory practititioners and
Trained health care providers respectively are responsible for doing this test
procedure.
The head of clinical chemistry section is responsible for ensuring the effective
implementation of this procedure.
5.1.4 Principle
The test strips contain a capillary that sucks up a reproducible amount of blood.
Glucose in the blood reacts with an enzyme electrode containing Glucose oxidase (or
Glucose dehydrogeNot applicablese).The enzyme is reoxidized with an excess of a
mediator ferricyanide ion, a ferrocene derivative or osmium bipyridyl complex. The
mediator in turn is reoxidised by reaction at the electrode, which generates an electrical
current. The total charge passing through the electrode is proportioNot applicablel to
the amount of glucose in the blood that has reacted with enzyme.
5.1.5 Sample requirements
i. Capillary or 1ml Fluoride-oxalate venous anticoagulated blood (fasting, post-
prandial, or random samples). Do not collect blood from an arm receiving an
I.V. infusion. Fasting samples: This refers to blood collected after a period of
no food intake. For adults the fasting time is usually 10 to 16 hours. For
children the fasting time is 6 hours unless a longer time is indicated, e.g.
when investigating hypoglycaemia. The drinking of plain water is permitted.
ii. Post-prandial samples: This describes blood collected after a meal has been
taken. The sample is usually taken as a 2 hour post - prandial samples.
iii. Random samples: This refers to a blood sample collected at any time,
regardless of food intake.

65
5.1.6 Equipment
Glucometer
Maintenance
Conduct maintenance as required by manufacturer instructions
5.1.7 Materials
Glucose test strips, Lancets, Cotton wool or gauze or alcohol swab, Sharp box or
Container, waste bin, Disposable gloves, Laboratory coat
5.1.8 Storage and stability
All related materials should be stored as the per manufactures instructions.
Sample should be processed within 1hour after collection
5.1.9 Safety
i. All samples must be considered as potentially infectious and must be handled
and examined with care.
ii. All person applicable protective equipment (PPE) should be worn when
performing procedure
iii. Adhere to safety precautions as stated in the Safety manual
Refer to National infection prevention and control Guidelines for health waste
management and safety practice.
5.1.10 Calibration
Equipment should be calibrated as per schedule.
5.1.11 Quality control
Process internal quality control before examing the patient samples on daily base
5.1.12 Procedure Steps
i. Compare the code number on the chip with the corresponding code number on
the label of the test strip container where applicable.
ii. The three-digit number on the code chip (e.g.689) must match the three-digit
number on the label. (Leave the meter turned off).
iii. Gently slide the code chip into the slot on the side of the meter. (You must feel
the code lock into place)
iv. To turn on the Glucose meter, press the S button and hold it down for more than
3 seconds until the depicted display appears.
v. Wear gloves clean the patient’s finger using the alcohol swab and allows it to
dry.
vi. Take one strip from the container. Close cap tightly and make sure the yellow
color in the round window on the back of the test strip matches the yellow color
above 0 mg/dL on the container. If it looks green do not use it.

66
 vii. Insert the test strip, with the orange pad facing up, until it will go no further into
the meter. Do not bend the test strip. (The arrow heads are almost no longer
visible when the test strip is inserted correctly.)
viii. Make sure the code on the meter matches the code on the test strip container.
ix. When you see the flashing blood drop, hold the lancet device against the side
of patient fingertip and press the release button.
x. Gently squeeze patient fingertip to get a drop of blood.
xi. Once the the blood drop appears on the screen, you have 2 minutes to apply
the drop of blood.
xii. Touch the blood drop to the center of the square orange pad. Do not bend the
test strip.
xiii. An hourglass symbol appears on the screen, and then the test result appears.
xiv. To remove the lancet, take off the lancet device cap and point the lancet end
away.
xv. Slide out the rejector to discharge the lancet into an appropriate container for
sharp objects.
xvi. Applying Blood with Test Strip outside of the Meter.
xvii. Take the test strip out of the meter
xviii. Touch the center of the square orange pad to the drop of blood. Do not bend
the test strip.
xix. Within 20 seconds, insert the test strip, with the orange pad to the drop of blood.
5.1.13 Biological Reference Intervals
Fasting Blood glucose (mmol/L) Random Blood Glucose
Blood/Plasma: 3.9 – 5.6 mmol/L (70 - 100 mg/dl) ≤6.9 (125 mg/dl)
5.1.14 Interpretation and reporting of results
i. Results are displayed in either mg/dl or mmol/liter depending on which unit of
measurement is selected. Report the value in the agreed SI unit.
ii. If the result is lower than 10mg/dL (0.6mm/L) “Lo” is displayed instead of a
result.
iii. “Lo” may indicate that your blood is very low.
iv. If the result is higher than 600mg/dL (33.3mmol/L), “Hi” is displayed instead of
a result.
v. Fasting blood glucose between 5.6 – 6.9 mmol/L (100 – 125 mg/dl) indicates
high risk to diabtes.
vi. Two separate test results of 7.0 mmol/L (126 mg/dl) or higher indicate diagnosis
of diabetes.
Critical value
Fasting blood glucose <2mmol/L
>20mmol/L
5.1.15 Limitation of the Procedure and Sources of Error

67
 i. Falsely elevated glucose results may be obtained when a person’s blood
contains bilirubin (unconjugated) >340 µmol/l (>20 mg/dl), triglycerides >57
mmol/l
ii. Abnormal uric acid levels may also interfere with test results. Caution is needd
in the interpretation of neoNot applicablete blood glucose values <2.8 mmol/l
(<50 mg/dl).
iii. Abnormal haematocrit values may affect test results.
iv. Haematocrit levels below 0.20 may cause falsely low glucose values when the
glucose concentration is less than or equal to 11.1 mmol/L. Values above 0.55
may cause falsely low glucose values when the glucose is above 11.1 mmol/l.
5.1.16 Perfomance Characteristics
Refer to the verification report
5.1.17 Supporting Document
Not applicable
5.1.18 References
ACCU-CHEK Active user’s manual.
5.2 PROCEDURE FOR TESTING BLOOD GLUCOSE BY GLUCO PLUS
5.2.1 Purpose
This Standard Operating Procedure (SOP) is aimed to describe step by step on how
to operate Gluco plus device
5.2.2 Scope
This procedure for Gluco-plus device will be used for blood glucose chemistry testing
in health facility in Tanzania
5.2.3 Responsibility
Trained, qualified and competent laboratory registered practitioners are responsible for
performing this procedure.
The head of section for chemistry is responsible for ensuring the effective
implementation and competency assessment for this procedure .
5.2.4 Principle
Not applicable
5.2.5 Sample Requirements
Whole blood
5.2.6 Equipment
Perform the procedure for start-up, maintenance, troubleshooting and shut down the
Gluco-plus as per manufacturer’s instrument instructions
5.2.7 Materials

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Test strips and Lancing device
5.2.8 Storage and Stability
• Gluco-plus strips should be stored at room temperature
• All reagents should be protected from direct sunlight, extreme heat, and freezing
during shipment and storage.
• Temperatures below 32° F (0°C) may cause reagent layering that changes the
tonicity and conductivity of the reagents.
• Sample stability after collection of venous whole blood:
✓ Run Samples within one hour of collection.
5.2.9 Safety
• Decontaminate working surfaces twice daily, in the morning and afternoon
• All personal protective equipment (PPE) must be worn when performing this
procedure.
• All samples must be regarded as potentially infections.
• Refer to Manufacturer instruction, National infection prevention and control
• Guidelines for health waste management and safety practice.
5.2.10 Calibration
Not Applicable
5.2.11 Quality Control
Control solution
Test prepared GLUCOPLUS control Solution;
i. Once per week
ii. When opening new strips kit
iii. When you suspect the meter or test strips are not working properly.
iv. If you drop/damage the meter.
v. Record results on QC Form.
5.2.12 Procedural Steps
Step Action
Changing strip code
1 Check the code on the test strip vial before inserting the test strip.
2 Insert the test strip to turn ON the meter and match the code on the meter
with the code on the strips vial.
3 If the code is already matched press OK to go to APPLY SAMPLE screen.
4 If the code in Meter does not match the code on the test strip vial, Press S button
until you hear a beep sound. Press S or M to match the code number on the
test strips vial.
Setting time
1 Switch on the analyser
2 Press S button until you hear a beep sound. Press off button to save the strip
code.
69
 3 Month number will blink use S and M button to select the required month
4 Press the off button to save month and date number will blink and use S and M
button to select the required date.
5 The time hours will blink use S and M to set time .
Performing a Quality Control test
1 Perform weekly and as per meter (machine) protocol.
2 Control used is GLUCOPLUS Selected Control solution.
3 Prepare and Apply Control solution.
4 Touch the sample tip of the test strip to the control drop. Verify check window
filled.
5 Meter display will count down from 5 to 1 and then display result.
6 Compare the control solution results with correct Control range printed on test
strips vial. (Example; 6.2 – 8.2 mmol/L). If the result are not within control
range repeat the control solution test.
Testing Blood
1 Use lancing device to puncture site, normally fingertip.
2 Insert a test strip into the strip port to turn on meter.
3 Touch the blood sample to the sample tip at the end of the test strip.
4 The meter display will count down from 5 to 1 and then display result.
5 If the “HI” or “LO” message appears on the meter, the result is above 33.3 or
below 1.1. Repeat test to verify.
Error Codes
E-1 Problem with Meter – Do not use the meter
E-2 Meter or strip Problem – Repeat the test with New strip.
E-3 Meter was not ready – Repeat the test with a new strip. Apply SAMPLE or
CONTROL appears on the display.
E-4 Strip Problem – Repeat test with a new strip.
E-5 Strip problem or Sample too small – Repeat the test with a new strip and
new sample.
HI.E Temperature too high—repeat test in a cooler area.
LO.E Temperature too low—repeat test in a warmer area.
E-6 Battery LOW — replace battery soon.
5.2.13 Biological Reference Interval
Fasting Blood glucose (mmol/L) Random Blood Glucose
Blood/Plasma: 3.9 – 5.6 mmol/L (70 - 100 mg/dl) ≤6.9 (125 mg/dl)
5.2.14 Interpretation and Reporting of Results
vii. Results are displayed in either mg/dl or mmol/liter depending on which unit of
measurement is selected. Report the value in the agreed SI unit.
viii. If the result is lower than 10mg/dL (0.6mm/L) “Lo” is displayed instead of a
result.
ix. “Lo” may indicate that your blood is very low.
x. If the result is higher than 600mg/dL (33.3mmol/L), “Hi” is displayed instead of
a result.
xi. Fasting blood glucose between 5.6 – 6.9 mmol/L (100 – 125 mg/dl) indicates
high risk to diabtes.
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xii. Two separate test results of 7.0 mmol/L (126 mg/dl) or higher indicate diagnosis
of diabetes.
Critical value
Fasting blood glucose <2mmol/L
>20mmol/L
5.2.15 Limitation of the Procedure and Sources of Errors
If the “HI” or “LO” message appears on the meter, the result is above 33.3 or below
5.2.16 Perfomance Characteristics
Refer the method verification reports from for this procedure and equipment
manufacturer user manual
5.2.17 Supporting document
Sample Collection Manual, Safety Manual, Quality Manual
5.2.18 References
1. User manual for Gluco-plus
2. Manufacturers package insert
5.3 PROCEDURE FOR URIT 50 (URINE CHEMISTRY ANALYZER)
5.3.1 Purpose
This procedure is provides description for performing urine biochemistry by using URIT
50 semi-automated Urine analyser.
5.3.2 Scope
This procedure is applied for testing Urine sample at the health facilities in Tanzania.
5.3.3 Responsibility
A trained and competent health laboratory practitioners are responsible for performing
this procedure. The head of section of biochemistry and parasitology is responsible of
ensuring the implementation of this procedure.
5.3.4 Principle
The analyser measures change of the reflectance of reagent strips pads, A detector
integrated in the system is composed of light source and a light receiver, the light from
which goes through in the spherical integrator and reflect to the reagent pads on strip.
The absorbance (reflectance) varies according to the color of reagent pads, the darker
of the reagent pads higher the absorbance is and less light is reflected. Conversely the
lighter the reagent pad is the lower the absorbance is and more light is reflected degree
of color developed is direct proportion to the concentration of analyte in urine” .
5.3.5 Sample Requirements
4mls of uncentrifuged mild stream urine sample is used.
5.3.6 Equipment

71
Urit-50
5.3.7 Materials
Disposable gloves, Laboratory coats, Urine container, Waste container, Marker pen,
Urine strips from urit G10, G11 or G14, Gauze
5.3.8 Storage and Stability
i. Store urine sample at room temperature for 30 minutes to 2 hours or 24 hours
in refrigerator.
ii. Reagent strips and calibrator should be stored to free and clean area at 37°C
iii. Control materials should be stored as per the manufacturers instructions

72
5.3.9 Safety
Samples and control materials at this section should be treated as infectious material
and should be handled careful.
5.3.10 10.0. Calibration
Not applicable
5.3.11 Quality Control
i. Put on PPE
ii. Install the strip holder into machine
iii. Switch on the machine and wait the machine for initialization
iv. Put the dry or calibrator strip on the strip holder till D sound
v. Wait the machine to scan and print result
vi. Record the QC result.

Note: Return calibrator strip into its container and discard other used materials according to
standard operating procedures

5.3.12 Testing Procedures

i. Deep the reagent strips of G series into urine sample and put it on the dry gauze
to remove excess urine on the back of the strip.
ii. Put the sample strip on the strip holder till D sound
iii. Read the patient result on the machine
iv. Record result to the register
5.3.13 Biological Reference Intervals
See annex 3.
5.3.14 Interpretation And Reporting Of Results
Refer to the insert which present on the reagents strips of G series G10, G11, G14.
Report result according to insert present on the reagent strip bottle
5.3.15 Limitation of the Procedure and Sources of Errors
The test relies on correct collection of sample by the patient, and if this is not done
properly the results may not be accurate
5.3.16 Performance Characteristics
Refer to method verification
5.3.17 Supporting Documents
URIT 50 user amnual
5.3.18 References
URIT 50 urine user manual.

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5.4 PROCEDURE FOR (URIT-560) URINE ANALYZER
5.4.1 Purpose
This procedure provides instructions for determining urine biochemical test using the
URIT-560 analyzer
5.4.2 Scope
This procedure is used in Clinical chemistry section of the user when performing urine
using the URIT-560 analyzer
5.4.3 Responsibility
The section head of Clinical Chemistry is responsible for ensuring this procedure is
effectively implemented and maintained
5.4.4 Principle
The analyzer measures changes in reflectance of the reagent strips pads. A detector
integrated in the system is composed of a light source and a light receiver, the light
from which goes through spherical integrator and reflect at the reagent pads on the
strips. The absorbance varies according to the color of the reagent pads. The darker
the reagent pads is the higher the absorbance is and less light is reflected. Conversely,
the lighter the reagent pad is the lower the absorbance is, and more light is reflected;
ie. The degree of color development is proportion to the concentration of analyte in
urine.
The reflected light goes in to the optical-electronic detector system, which transforms
the optical into electrical. The strength of the electricity correlates which reflectance.
Then the electrical cables will be processed by CPU after being transformed by I/V
converter, and the test results can be printed out by printer.
5.4.5 Sample Requirements
Dry, wide-necked, leak proof container 10-20 ml of urine specimen.
5.4.6 Equipment
URIT-560, Printer
5.4.7 Materials
Urine strips (URIT G Series), Printer thermal paper, Urine container, Gloves
5.4.8 Storage and Stability
Sample is stable for 2hrs at room temperature or 24hrs at 2-80C
5.4.9 Safety
i. Personnel Protective Equipment must be worn at all times
ii. Samples must be treated as potentially infectious.
5.4.10 Calibration
Perform calibration as per manufacturer instructions

74
5.4.11 Quality Control
Currently no control
5.4.12 Procedural Steps.
i. Give the patient a sterile, dry, wide-necked, leak proof container and request10-
20 ml of urine specimen.
ii. Explain to the patient the need to collect the urine with as little contamination as
possible i.e. a clean-catch specimen.
iii. On the instrument, press (image test) to enter into the image test interface.
iv. Insert or Pour urine on the test strip, put test strip into test strip holder until the
sound of alarm raised.
v. Then wait the printer out results.
5.4.13 Biological Reference Intervals.
Parameter Abbreviation Biological Reference Intervals
Urobilinogen URO Normal
Glucose GLU Negative
Bilirubin BIL Negative
Ketones KET Negative
Specific gravity S.G 1.003-1.029
Occult blood BLD Negative
Ph Ph 4.5 - 7.8
Protein PRO Negative
Nitrite NIT Negative
Leukocytes LEU Negative
5.4.14 Interpretation And Reporting Of Results
Results are automatically printed from the machine. Attach results printout with report
form
5.4.15 Limitation of the Procedure and Sources of Errors.
• Urine must be processed within 2hr to avoid growth of bacteria which consuming
glucose and developing ammonia in urine, loose of ketone bodies, Increase of
PH
• Urine if not processed on time store in refrigerator 2oC – 8oC for 24
5.4.16 Performance Characteristics
Refer into method verification report
5.4.17 Supporting Documents
Equipment maintenance form, sample collection manual, quality manual
5.4.18 References
• URIT-560 operator’s manual

75
 • Standard guard line for Health Laboratory 2007 Edition.
5.5 ROCEDURE FOR PERFORMING URINE BIOCHEMISTRY BY USING
CYBOW READER 300
5.5.1 Purpose
This Standard Operating Procedure (SOP) is aimed to describe step by step on how
to operate the CYBOW™ READER 300 semi-automated Urine analyser using Urine
sample at the health facilities in Tanzania
5.5.2 Scope
This procedure applies to all staff who works in parasitology section on performing
urinalysis test.
5.5.3 Responsibility
A trained, qualified and competent laboratory registered practitioner are responsible
for performing this procedure. The head of sections is responsible for ensuring the
implementation of this procedure.
5.5.4 Principle
The CYBOW reader 300 are reflectance photometer. The strip is illuminated by white
light, and the reflected light from the strip is detected by the sensor. The RGB signal is
digitized, and this digitized image is interpreted by the processor. The intelligent image
analyser SW locates the strip and the pads, and based on this colour data the
parameter values are determined. The results including the date and the time of
measurement, sequence number and the ID are stored printed out by the internal
printer.
5.5.5 Sample Requirements
10-20mls of mild stream urine sample collected in sterile wide mouth container is
required for performing this test. Do not centrifuge urine sample before bio chemical
test.
5.5.6 Equipment
Perfom the CYBOWTM READER 300 procedure for start-up, maintenance,
troubleshooting and shut down the urine analyser as per manufacturer’s instrument
instruction.
5.5.7 Materials/REAGENTS
Gloves, Marker pen, Laboratory coat, Waste container, Gauze, Urine container and
CYBOW strip.
5.5.8 Storage and Stability
Sample, reagents, calibrators and control materials should be stored as per the
manufacturer instructions.
5.5.9 Safety

76
 • Adhere to safety precautions as stated in the Safety manual
• All personal protective equipment (PPE) must be worn when performing this
procedure.
• All samples must be regarded as potentially infections.
• Front cover of machine should be covered during operation to avoid sample
contamination.
• Used only power cord specified for CYBOWTM READER 300
• Avoid excessive dust, wet/damp condition and provide proper ventilation.
• Do not wipe the body clean with benzene, thinner, gasoline. This may discolour
5.5.10 Calibration
The calibration of instrument should be done prior to first time use and then the 2 nd
calibration process is recommended in every 4 weeks with calibration strip provided
in the package.
5.5.11 Quality Control
Run internal Quality Control samples daily before examing patient samples to ensure
quality of examination results. Other conditions that drive controls include:
✓ After a reagent lot number change
✓ After maintenance, component replacement, or a field service action
✓ After a software change
✓ Following calibration.
✓ According to regulatory requirements
5.5.12 Procedural Steps
•
Check if the urine was received within 1 hour of collection and in a sterile
universal container
• Mix the urine by swirling the container and dip a CYBOW strip into urine,
making sure that the entire measuring region of the strip is immersed then
running strip on instrument.
Mode of running strip on CYBOWTM READER 300
1. General mode
• Select the general mode by pressing direction key(◄) and press
Enter button to return to the standby mode.
• After the 1st strip dipped in urine and placed on a plate, press start
key(►).
• Put 1st-10th (max.) strip on the plate one by one after dipping each in
selected urine.
• Once last reagent strip on batch is placed, press Enter button
• After incubation time of the 1st strip, it will start to loading the results
of the strip on by one.
2. One by one mode

77
 •
Select one by one mode by pressing Direction key (◄) and press
Enter button.
• After the 1st strip is placed on the loading plate press start key (►).
• Press direction key whenever each of the next strip is placed on the
plate one by one.
• Once last strip is place on strip loading plate, press Enter button.
• After incubation time of the 1ststrip, it start to read and print result of
the strip one by one.
3. Quick mode
• Select the quick mode by press direction key(◄) and press Enter
button
• Put the strips (incubation is done) once strip loading plate
continuously.
• Once the last strip is placed on the loading plate, press Enter button.
• Test result is shown on the LCD and automatically saved in the
memory
5.5.13 Biological Reference Intervals
See annex 3.
5.5.14 Interpretation And Reporting Of Results
Chemical urinalysis
Report the reading when the immersed strip is compared to the colours on the strips
container
Macroscopic examination
Report whether the urine is Clear, Slightly cloudy, Cloudy or turbid. Report the colour
of the urine which will range from Light yellow, Yellow, Amber, Red to Brown
Microscopic examination of urine sediment
• White Blood Cells/Pus Cells/ leucocytes.
Report the average number of cells per High Power Field, example 2-5
WBCs/HPF
• Red Blood Cells
Report the average number of cells per High Power Field, example 2-5
RBCs/HPF
• Casts
Identify the type of cast and report as number per High Power Field
• Crystals
Identify the type of crystals and report their presence
• Epithelial cells
Identify the type whether squamous, transitional or renal epithelial cells, quantify
them and report per high power field. Otherwise report the presence of epithelial
cells if can’t be identified.

78
 • Trichomonas Vaginalis
Report as “seen” or “not seen”
• Yeast
Report as “seen” or “not seen”
• Spermatozoa
Report for males and not for females
5.5.15 Limitation of the Procedure and Sources of Errors
Urine samples should be tested within one hour of collection
If any delay happen put the urine sample in the refrigerator to avoid bacterial growth.
Do not use urine dipsticks beyond expiry date
5.5.16 Performance Characteristics
Refer to the method verification report of this procedure
5.5.17 Supporting Documents
Safety manual and Sample collection manual
5.5.18 References
Manufacturer’s Package Insert in multistrips kit
Cheesbrough, Monica Health Laboratory Manual for Tropical Countries
Graff’s Text Book for Urinalysis and body fluids, Second edition, Lillian A. Mundt and
Kristy Shanahan
5.6 PROCEDURE FOR DETERMINATION OF ALT BY USING DIRUI-DR 7000
CHEMISTRY ANALYZER
5.6.1 Purpose
This procedure provides instructions for determining Alanine Aminotransferase (ALT)
using the DIRUI-DR7000 Analyzer
5.6.2 Scope
This procedure is used in Clinical chemistry section for analysing ALT using the
DIRUI-DR7000 clinical chemistry Analyzer.
5.6.3 Responsible personnel
Qualified, trained and competent Health Laboratory Assistant technologists,
Technologists, and Scientists are responsible for performing this procedure..
The section head of Clinical Chemistry is responsible for ensuring this procedure is
effectively implemented and maintained.
5.6.4 Principle
Kinetic method for the determination of ALT activity according to the recommendations
of the Expert Panel of the International Federation of Clinical Chemistry (IFCC).
Without pyridoxalphosphate activation. ALT is measured by the reagent rate analysis
by the coupled reaction with lactate dehydrogenase (LDH) to reduce NADH (measured
79
at a wavelength of 340nm) to NAD+. The rate of decrease in absorbance at 340 nm
due to NADH depletion is proportional to the ALT activity in the sample.
5.6.5 Sample requirement
30µl serum or plasma, sample free from hemolysis.
5.6.6 Equipment
DIRUI DR7000 Semi Automated Chemistry Analyzer, Centrifuge machine
5.6.7 Materials
Reagent Kits, Calibrator/Stsndard, Control Kits (LEVEL I and II), Disposable gloves
Laboratory coat, Sample Cups, Reaction Wells, Transfer Pipettes
5.6.8 Storage and stability
Refer to the facility laboratory sample collection manual
5.6.9 Safety
i. Adhere to safety precautions as stated in the facility laboratory Safety
manual/IPC guidline
ii. All personal protective equipment (PPE) must be worn when performing this
procedure.
iii. All samples must be regarded as potentially infections.
5.6.10 Calibration
i. Perfom equipment calibration when; -
ii. There is a change in the reagent lot number.
iii. If the QC result falls outside the acceptable ranges.
iv. The machine blinks on the QC-calibration indicating that the calibration is
expired.
v. There is a change in the system software
vi. System maintenance/ component replacement procedure is performed
5.6.11 Quality control
QC shluld be performed before patient samples, after calibration of reagent
5.6.12 Procedure Steps
Running the Calibrator, controls or samples;
i. Label the tubes for blank, calibrator, Controls or samples
ii. Prepare the working reagents as indicated by the industrial manufacturer
iii. Pipette the working reagents R1 480µl into the labelled tubes
iv. Pipette 30µl of distilled water into the tube labelled blank.
v. Incubate for 300s
vi. Add 120µl of R2 reagent
vii. Incubate for 60s
viii. Read the result

80
5.6.13 Biological reference intervals
See annex 4.
5.6.14 Interpretation and Reporting of Results
Interpretation of results
Perfomed results will be displayed on the machine screen
Result reporting
Once all of the results are accepted or validated, a final report will automatically be
printed out.
5.6.15 Limitation of the Procedure and Sources of Error
Hemolyed, lipemic samples, icterus and anticoagulants such as citrate, oxalate and
fluoride (for other tests except Glucose) and drugs such as hydroxocobalamin and
Cephalosporin antibiotics.
5.6.16 Performance Characteristics
Refer to the verification report
5.6.17 Supporting document
Sample collection manual, quality manual
5.6.18 References
DIRUI-DR7000 Chemistry analyser user manual
5.7 PROCEDURE FOR DETERMINATION OF AST BY USING DIRUI-DR7000
CHEMISTRY ANALYZER
5.7.1 Purpose
This procedure provides instructions for determining ASAT using the DIRUI-DR7000
Analyzer
5.7.2 Scope
This procedure is used in Clinical chemistry section of the user when performing blood
analysis using the DIRUI-DR7000 clinical chemistry Analyzer
5.7.3 Responsibility
The section head of Clinical Chemistry is responsible for ensuring this procedure is
effectively implemented and maintained.
5.7.4 Principle
Kinetic method for the determination of Aspartat-Aminotransferase (AST) activity
according to the recommendations of the Expert Panel of the International Federation
of Clinical Chemistry (IFCC). Without pyridoxalphosphate activation. AST is measured
by the reagent rate analysis by the coupled reaction with Malate dehydrogenase (MDH)
to reduce NADH (measured at a wavelength of 340nm) to NAD+. The rate of decrease
in absorbance at 340 nm due to NADH depletion is proportional to the AST activity in
the sample.
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5.7.5 Sample Requirements
Serum or plasma, sample free from hemolysis and not contaminated
5.7.6 Equipment
DIRUI DR7000 Semi Automated Chemisry Analyzer•
Cleaning and Maintenance
i. Use a large amount of distilled water to rinse the tubing by click the rinse
interface.
ii. And drain the liquid from the tubing if necessary.
iii. Remove the waste liquid bottle from the back of the analyzer.
iv. Keep the instrument vertical during move and transport.
v. Try best to avoid vibration.
vi. And check and debug the instrument before use.
5.7.7 Materials
Reagent Kits, Calibrator/Standard,Control Kits (LEVEL I and II), Supplies, Disposable
gloves, Laboratory coat, Sample Cups, Reaction Wells and Transfer Pipettes
5.7.8 Storage and Stability
• Reagent Should be kept at temperature of 2-8°C and sealed in dry place without
sunshine. The shelf life is 18 months .
• Under condition of 2-8°C, the open vial stability is 30 days
5.7.9 Safety
Personnel Protective Equipment must be worn at all times and samples must be
treated as potentially infectious.
5.7.10 Calibration
It is suggested to use supplementary calibrator as instructed. When lot number is
changed or QC is invalid, calibration shall be conducted again.
Procedures for reagent, Calibration, QC and Sample preparation
5.7.11 Quality Control
It is suggested to use QC products produced by DIRUI.
5.7.12 Procedural steps
i. Label the tubes for blank, calibrator, Controls or samples
ii. Prepare the working reagents as indicated by the industrial manufacturer
iii. Pipette the working reagents R1 480µl into the labelled tubes
iv. Pipette 30µl of distilled water into the tube labelled blank.
v. Incubate for 300s
vi. Add 120µl of R2 reagent
vii. Incubate for 60s
viii. Read the results

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5.7.13 Biological Reference Intervals.
See annex 4.
5.7.14 Interpretation and Reporting of Results
Reporting of results
Results are automatically printed from the machine
Attach results printout with report form.
5.7.15 Limitations of the procedure and sources of error.
Gross hemolysis, Lipemic AND Icterus specimen
5.7.16 Performance Characteristics
Refer verification report
5.7.17 Supporting Documents
Equipment Maintenance Form.
18.0 References
DIRUI DR7000 Analyser operator’s manual
National standard guard line for Health Laboratory 2007 Edition

83
5.8 PROCEDURE FOR SA-30 SEMI AUTOMATED CHEMISTRY ANALYZER
5.8.1 Purpose
This Procedure describes step by step on how to operate the SA-30 a semi-automated
chemistry analyser to perform basic chemistry tests using human serum, plasma or
cerebral spinal fluid (CSF) sample.
5.8.2 Scope
it is applied in testing biochemistry parameters using human serum or plasma sample
in the biochemistry department/section.
5.8.3 Responsibility
All qualified, trained and competent laboratory scientist, laboratory technologists and
assistant laboratory technologists are responsible for performing this procedure.
The head of section of biochemistry is responsible of ensuring the implementation of
this procedure.
5.8.4 Principle
The principle of the instrument is based on the phenomenon of different wave band
absorbance from substance, which is in line with Lambert-Bill Law.
(The greater the concentration of the sample, the more light is absorbed, the less light
is transmitted, and the darker of the color)
5.8.5 Sample Requirements
The 2 - 4ml of whole blood collected in plain tube (red top) for serum or EDTA (purple
top) for plasma or in heparinized tube free from hemolysis. Cerebral spinal fluid (CSF)
when required.
5.8.6 Equipment
SA-30 semi- automated chemistry analyser, Centrifuge
5.8.7 Materials
Micropipettes and tips, Marker pen, Thermal paper and PPE
5.8.8 Storage and Stability
i. Store serum/plasma at room temperature 25 – 35 °C for 8 hours
ii. Tested sample stored at 2 -8 °C to 7 days.
iii. Store reagent,calibrator and controls per manufacture recommendation
5.8.9 Safety
Treat all Samples and control materials as infectious material and should be handled
careful as per IPC guidelines
5.8.10 Calibration
Quality Control procedure is the same as analytic procedure of unknown sample

84
(Use Randox Calibrator (pipette 500µl of reagent to tube and add 25µl calibrator mix
gently and incubate at 370C for 8-10 minutes for end point test method, no need of
extra incubation for kinetics method)
5.8.11 Quality Control
Quality Control procedure is the same as analytic procedure of unknown sample (Use
Randox QC ,pipette 500µl of reagent to tube and add 25µl control mix gently and
incubate at 37C for 8- 10 minutes end point test method, no need of extra incubation
for kinetics test method)
5.8.12 Procedural Steps
i. Prepare for sample and reagent.
ii. For End point test method, Add R1 + R2 + Sample in ratio and mix thoroughly
and incubate at 37℃ for 8 – 10 Min (refer to reagent user manual)
iii. For kinetics test method, Add R1 + R2 + Sample in ratio and mix thoroughly,
no need of extra incubation.
iv. For HDL, LDL add R1 + Sample in in ratio, mix them thoroughly incubate at
37℃ in 2 min then add R2 and incubated them in 5-7 Min,
v. Click “Test” on main menu to enters next page to start testing.
vi. Press PUSH button to aspirate distilled water, to calibrate AD value. The AD
value should be 45000 to 60000.
vii. Click “Continue” to test reagent blank.
viii. Select “YES” to aspirate reagent blank to test reagent blank absorbance.
ix. Press PUSH button to aspirate reagent blank to test reagent blank
absorbance.
x. Click “Continue” to test STD
xi. Select “NO”, device will use last factor and perform the sample test directly.
Select “YES”, device will aspirate standard to test STD
xii. Press PUSH button to aspirate standard, then device will test standard
absorbance and calculate factor automatically
xiii. Click “Continue” to next to test the sample directly or perform control test.
xiv. Press PUSH button to aspirate sample or control, then device will test the
sample or control and display test result automatically
5.8.13 Biological Reference Intervals
See annex 4.
5.8.14 Interpretation And Reporting Of Results
Interpretation
i. If the result of the particular parameter lies within the established
reference range, it means that the patient has normal particular
parameter.
ii. If the result of the particular parameter lies below or above the established
reference range, it means that the patient has abnormal particular

85
 parameter and requires intervention as per the clinical history and the
laboratory findings.
Reporting results
Report the results as they are displayed on the screen of the machine
5.8.15 Limitation of the Procedure and Sources of Errors
i. Avoid using the haemolysed and lipemic sample as this will cause falsely
elevated values. In this case inform the requesting physician and ask for another
specimen.
ii. Avoid exposure of the freshly dissolved substrate to strong sunlight, since the
reagent is light sensitive. The change in absorbance will increase with an
increase in temperature, since the pH of the reagent will be different at different
temperatures
iii. Serum must be separated by centrifugation as soon as possible after collection
of the patient's blood sample, preferably <2 hours, otherwise, phosphate
present in erythrocytes will be released into the serum causing falsely elevated
values
iv. Grossly bloody CSF may give spuriously elevated values. Undue delay in
analysis may give low values. The report to the requesting physician should
include the appearance of the CSF before and after centrifugation.
5.8.16 Performance Characteristics
Refer to method verification report
5.8.17 Supporting Documents
Sample collection manual, Safety manual, Quality manual
5.8.18 References
SA-30 semi automated chemistry analyser user manual
5.9 PROCEDURE FOR OPERATING CLINDIAG FA 200 CHEMISTRY TEST
5.9.1 Purpose
This procedure provides instructions for determining basic Chemistry tests using
Clindiag FA 200 analyzer.
5.9.2 Scope
This procedure is used at Clinical chemistry section for processing basic chemistry
tests using Clindiag FA 200 analyzer.
5.9.3 Responsibility
Qualified and trained Health Laboratory Personnel are responsible for doing this test
procedure.
5.9.4 Principle

86
The test principle of the biochemistry analyser is mainly based on the lambert Beer
law.
The reagents and the samples to be tested are mixed at a certain proportion. The
mixture is placed in a calorimetric dish at a certain temperature for incubation, its
absorption of light of specific wavelength is continuously measured, and finally the
concentration of the measured substance is automatically calculated according to the
value of absorbance (change).The procedure uses Lambert Beers Law which state
that the absorptive capacity of a dissolved substance is direct proportional to its
concentration to the solution.
5.9.5 Sample Requirements
Serum is most preferred sample
Refer to the facility Laboratory sample collection manual.
5.9.6 Equipment
Clindiag FA200 Chemistry analyser.
5.9.7 Materials
Reagent kits, cleaning solution, HD- high efficiency cleaning agent, Sample cups,
Calibrators and controls, Gloves, Laboratory coat, A4 paper, Micropipette, Micropipette
tips
5.9.8 Storage and Stability
Samples are stored at 2-8oC after testing for 3days.
5.9.9 Safety
i. Adhere to safety precautions as stated in the Safety manual.
ii. All personal protective equipment (PPE) must be worn when performing this
procedure.
iii. All samples must be regarded as potentially infections.
iv. Refer to National infection prevention and control Guidelines for health care.
v. Avoid any contact between hands and eyes and nose during sample collection
and testing.
vi. Do not use kit beyond the expiration date.
vii. Do not reuse the test device.
viii. All spills should be wiped thoroughly using 1% sodium hypochlorite solution.
5.9.10 Calibration
Machine should be calibrated when
• There is a change in the reagent lot number.
• If the QC result falls outside the acceptable ranges.
• There is a change in the system software
• System maintenance/ component replacement procedure is performed
5.9.11 Quality Control
87
Follow the following steps to run internal Quality Control.sample
i. Click on task.
ii. Click on add Quality control(QC.)
iii. Click on ADD
iv. Select QC batch number.
v. Select a QC item in QC list
vi. Select type of container
vii. Input the position of QC material on the sample plate
viii. Click on OK
ix. Allow the amchine to perfom test till final results
5.9.12 Procedural Steps
i. Click Task
ii. Click Add sample
iii. Click ADD
iv. Enter patient information
v. Select the test item
vi. Select the sample cup number
vii. Click OK
viii. Click Test on left side
ix. Select test sample
x. Then click start test
xi. When sample processing is complete, Select the results from the menu bar,
patient result review will display, select name of patient on the left side.
xii. All the undone tests will be shown on the results as NA with a reason on the right
side.
xiii. Re-running patient sample, repeat the patient order with no change in a sample
cup number

88
5.9.13 Biological Reference Intervals
See annex 4.
5.9.14 Interpretation And Reporting Of Results
Results interpretation
Click on <Results> then <sample Results>. The results appear on the computer
screen, from here you can select the desired results and release them.
Reporting of the results
To print the results, select the sample interface in browse result and click print the test
results. Results.
Critical results
Critical results should be immediately communicated to the clinicians requested the
examination. Refer to the annex …. For more details on critical results for clinical
chemistry assays.
5.9.15 Limitation of the Procedure and Sources of Errors
Do not proces hemolysed samples as they might lead to falsely high results of
potassium and low results for glucose.
Samples for glucose investigation should be processed within 2 hours of collection;
any delay would cause falsely low results.
Potential operator errors and clindiag FA 200 system technology limitations.
Communicate the following Panic/Critical values to the clinicians
5.9.16 Performance Characteristics
Method verification of this procedure should be done and that the report should be
referred to verify compliance to this requirement.
5.9.17 Supporting Documents
Sample collection manual
5.9.18 References
Refer to equipment instruction manual Clindiag FA 200
5.10 PROCEDURE FOR DETERMINATION OF IMMUNOASSAYS BY USING
GETEIN 1100 IMMUNOANALYSER
5.10.1 Purpose
This procedure provides description on determination of immunoassays by using
Getein 1100 Immunofluorescence Quantitative analyser.
5.10.2 Scope
This procedure is used for processing and analysis of immunol assay tests in the
biochemistry department/section at the hospital laboratory.
5.10.3 Responsibility

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A qualified,trained and competent laboratory scientist, laboratory technologists and
assistant laboratory technologists are responsible for performing this procedure. The
head of section of biochemistry is responsible of ensuring the implementation of this
procedure.
5.10.4 Principle
The detection element scans the binding area and converts the optical signal to
electrical signal. The voltage variation between the test line and background has a
linear relationship with the antigen concentration which can be used to calculate the
concentration. In conclusion the antigen concentration in whole blood, plasma, serum,
urine can be calculated quantitatively according to optical signal of the test line.
5.10.5 Sample Requirements
The 2 - 4ml of whole blood collected in plain tube (red top) for serum or EDTA (purple
top) for plasma or in heparinized tube free from hemolysis.
urine sample will be collected in urine container if required.
5.10.6 Equipment
Getein1100 Immunofluorescence Analyzer, stop watch
5.10.7 Materials
Disposable gloves, Micropipettes and its tips, Containers for waste segregation,
Marker pen, Getein test card
5.10.8 Storage and Stability
Store unproceesed samples at room temperature for 12 hours. Store performed
samples at 2 - 8°c up to 7 days.
Calibrator, controls and test kit devices should be stored as per manufactures
instruction
5.10.9 Safety
i. Samples and control materials should be treated as infectious material
ii. Worn PPE all of the time while working
5.10.10 Calibration
Calibration of the assay should be peformed as per manufacturer instruction.
5.10.11 Quality Control
Use commercial available controls or inhouse controls to run QC as per schedule
5.10.12 Procedural Steps
i. Refer to the user manual or material data sheet to specific items including
reaction time and sample volume carefully for accurate information
ii. Add patient information including ID, name, age, gender, types of sample and
test mode to be used

90
 iii. Click start after inserting the card, test item will be auto-recognized and the
result will be shown on the screen. User can also see the voltage waveform by
slide to the left side
iv. Normally, the test card will auto-quit after testing if not please click on “Quit”
icon to quit manually
5.10.13 Biological Reference Intervals
See annex 4.
5.10.14 Interpretation And Reporting Of Results
Interpretation of results
If the result of the particular parameter lie within established reference range, it means
that the patient has normal particular parameter
If the results of the particular parameter lie below or above established reference
range, it means that the patient has abnormal particular parameter and requires
intervention as per the clinical history and the laboratory finding.
Reporting of results
Report the result as they are displayed on the screen of the machine.
5.10.15 Limitation of the Procedure and Sources of Errors
Only used for in vitro analysis of human whole blood, serum, plasma, urine or stool
freezed samples can not be used for testing due to loss of enzyme or hormone
activities
5.10.16 Performance Characteristics
Reffer to verification report.
5.10.17 Supporting Documents
• Sample collection manual
• Material data sheet or reagent manual
• Safety manual
5.10.18 References
Getein 1100 user manual
5.11 PROCEDURE FOR OPERATING FIA 8000 ANALYSER
5.11.1 Purpose
This procedure provides instructions for operating FIA 8000 Quantitative Immunoassay
Analyzer for biomarkers.
5.11.2 Scope
This FIA8000 is an analyzer that used to measure biomarkers in human whole blood,
serum, plasma or urine samples.
5.11.3 Responsibility

91
Qualified and competent registered Health Laboratory practititioners are responsible
for doing this test procedure. The head of section of chemistry is responsible for
ensuring the effective implementation and competency assessment for this procedure.
5.11.4 Principle
The combination of the antigensin the sample, the gold-labelantibodyin the colloidal
gold pad or nitrocellulose membrane, and theantibody pre-coated on the test linecan
form a purplish red streak on the test line. The colour intensity of the test lineis
proportionate to the quantity of antigens detectedin the sample. The analysersystem
can obtain the photo-electric signal intensity of the complexby scanning the test line
with a photo-electric component. Then the voltage difference between the voltage of
the test line and the background is obtained. The voltagedifference has a linear
relationship with the antigen concentrationwhichcan be used to calculate the antigen
concentration. The relationship has been established and varying from the measured
parameter. In conclusion, the antigen concentration in whole blood, plasma, serum,
urine can be calculated quantitatively in one-step according to the colour intensity of
the test line.
5.11.5 Sample Requirements
Centrifuged Serum and plasma
5.11.6 Equipment
FIA 8000 Quantitative Immunoassay Analyzer
5.11.7 Materials
Test kit, Power source, Printing paper, QC Card, QC SD, Gloves
5.11.8 Storage and Stability
Fresh sample is preferred however if can not be done sample can be stored at 2-8⁰C
not more than 3days.
5.11.9 Safety
i. Decontaminate working surfaces twice daily, in the morning and afternoon
ii. Adhere to safety precautions as stated in the Safety manual
iii. All personal protective equipment (PPE) must be worn when performing this
procedure.
iv. All samples must be regarded as potentially infections.
v. Avoid any contact between hands and eyes and nose during sample collection
and testing.
vi. All spills should be wiped thoroughly using 1% sodium hypochlorite solution
vii. Decontaminate the biohazpus waste before disposal.
5.11.10 Calibration
Perfom equipment calibration when; -
i. There is a change in the reagent lot number.
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 ii. If the QC result falls outside the acceptable ranges.
iii. The machine blinks on the QC-calibration indicating that the calibration is expired.
iv. There is a change in the system software
v. System maintenance/ component replacement procedure is performed
5.11.11 Quality Control
Quality Control (QC) card should be run before processing patient sample for each day
5.11.12 Procedural Steps
i. Centrifuge collected whole blood samples to obtain serum or plasma.
ii. Mix urine samples thoroughly before testing.
iii. Allow samples to reach room temperature before testing.
iv. Turn on the analyser and select the test.
v. Touch the screen to turn on the analyzer.
vi. Select the test you want to perform from the list of available tests.
vii. Insert the test card.
viii. Open the test card slot.
ix. Carefully insert the test card into the slot, making sure that the arrows on the
test card are pointing in the same direction as the arrows on the analyzer.
x. Close the test card slot.
xi. Add the sample.
xii. Follow the on-screen instructions to add the sample to the test card. Be
careful not to overfill the sample well.
xiii. Touch the screen to start the test.
xiv. The analyzer will automatically begin to process the sample.
xv. Read the results.
xvi. The analyzer will automatically read the results and display them on the
screen.
xvii. The results may be displayed in a variety of formats, such as quantitative
results, qualitative results, or graphs.

5.11.13 Biological Reference Intervals

See annex 4.
5.11.14 Interpretation And Reporting Of Results
Interpretation of results
Interpretate results based on the Biological reference interval.
Normal results are patient results which fall within the reference range for the particular
test. Abnormal results are those that fall below or above the reference range. The test
report is labeled H: High and L: Low to show the abnormality obtained.
Reporting of results
Report the obtained and displayed results in request form/register
Critical results

93
 Analyte Less Than Greater Than
Amylase 25U/L 150U/L
Chloride 85 mmol/L 115mmol/L
CK 30U/L 200U/L
Creatinine 26umol/L 120umol/L
Glucose(fasting) 2.5mmol/L 20.0mmol/L
Potassium 2.5mmol/L 6.0mmol/L
Sodium 120mmol/L 160mmol/L
Bilirubin Total 3.4umol/L 20.5umol/L
Biliribun Total for new Newborn
Born 24hours ≥ 1374umol/L
48hours ≥ 2224umol/L
84hours ≥2904umol/L
One week to one month ≥3424umol/L
Urea (BUN) ≤1.0mmol/L ≥ 54mmol/L

5.11.15 Limitation of the Procedure and Sources of Errors

Haemolyzed sample should not be used since the color changes caused by
haemolysis may result to wrong results. Refer to package insert for interfering
substances for specific test
5.11.16 Performance Characteristics
Refer to the method verification report.
5.11.17 Supporting Documents
Sample collection manual
5.11.18 References
User manual for FIA 8000 Analyser.
5.12 PROCEDURE FOR OPERATING ALERE AFFINION AS100ANALYSER
5.12.1 Purpose
The purpose of this procedure is to provide instructions on how to operate ALERE
AFFINION AS100 analyser for HbA1c, Lipid panel and C - reactive protein.
5.12.2 Scope
This procedure is used in Clinical chemistry section for processing HbA1c,Lipid panel
and C-Reactive Protein.
5.12.3 Responsibility
A trained, qualified and competent laboratory registered practitioners are responsible
for performing this procedure. The head of section for chemistry is responsible for
ensuring the effective implementation and competency assessment for this procedure
.

94
5.12.4 Principle
A Test Cartridge with patient sample or control is placed in the cartridge chamber of
the Analyzer. By manually closing the lid, the Test Cartridge is transported into the
analysis compartment of the Analyzer. Test and lot-specific information is obtained
from the barcode label (Figure 2). When the Test Cartridge enters the Analyzer, the
integrated camera reads the barcode. The calibration data for the actual lot are read,
which then initiates the processing of the Test Cartridge. The sample and reagents are
automatically transferred between the wells. An integrated camera monitors the entire
process. Light-emitting diodes (LEDs) illuminate the reaction area, which can be either
a colored membrane or a reaction well. The camera detects the reflected or transmitted
light, which is converted to a test result and displayed on the touch screen. When the
user accepts the result, the lid covering the cartridge chamber opens automatically and
the used Test Cartridge can be removed and discarded. The Analyzer is then ready
for the next run.
5.12.5 Sample Requirements
Whole blood/Serum / plasma as specified in the reagents insert or as stated in the
sample collection manual.
5.12.6 Equipment
Alere Affinion AS100
5.12.7 Materials
Test cartridge, Calibrators, Controls, PPEs, Pipette tips 100 -200ul, Pipette tips 100,
1000ul, Sample container rack, waste bin
5.12.8 Storage and Stability
• Test cartridge should be stored in refrigerator at 2-8oC in sealed foil pouches
and only stable until expiration date. If not refrigerated they can be stored at
room temperature(15-25oC) for four weeks.
• Test cartridges should not be exposed to direct sunlight at relative humidity
below 90%.
• Whole blood samples can be stored refrigerate at 2-8o C for 3 days. Plasma
and serum samples can be refrigerated for 10 days or frozen up 1 year if the
tubes are properly sealed.
5.12.9 Safety
i. Adhere to safety precautions as stated in the Safety manual/IPC guidline
ii. All personal protective equipment (PPE) must be worn when performing this
procedure.
iii. All samples must be regarded as potentially infections.
5.12.10 Calibration
Perform calibration as per Alere Afinion™ AS100 user manual.

95
5.12.11 Quality Control
Quality control should be done as prescribed in the quality management procedure.
Use commercial or in-house made quality control materials to perform on daily basis
before testing patient samples. Commercially available quality materials should be
used to verify performance of the procedure at least after 100 patient samples have
been tested. Lot to lot verification should also be used to check performance
acceptability of reagents.
5.12.12 Procedural Steps
Analyzing a patient/control sample
1 Touch to enter the patient sample mode.
Touch to enter the control mode. A “C” in the upper left
corner indicates that the Analyzer is in the control mode. The lid opens
automatically.
If the lid is left open from the previous run and “Insert Cartridge” is
displayed, this step is omitted and you can start with step 2.
2 Insert the Test Cartridge with the barcode label facing left.
Be sure that the Test Cartridge is correctly placed in the cartridge
chamber.

3 Close the lid manually. The Analyzer will start processing the Test
Cartridge.
The processing time depends on the test in use.

4 Touch and enter the patient ID.

Touch to confirm.
Touch and enter the control ID or Alere Afinion™ Control Data.
Touch to confirm.
Entering the patient ID, control ID or Alere Afinion™ Control Data will not
interrupt the processing.
5 Record the result, then touch to accept.
If a printer is connected, touch to print the result.
The lid opens automatically.
The result will be saved in the result records.

6 Remove the used Test Cartridge from the cartridge chamber and
discard it in a suitable waste container. Insert a new Test Cartridge or
close the lid manually.
Keep the lid closed to protect the cartridge chamber when the Analyzer
is not in use.

5.12.13 Biological Refences

96
See annex 4.
5.12.14 Interpretation And Reporting Of Results
Interpretation of results
• Interpretation of results is based on the Biological reference interval;
• Normal results are patient results which fall within the reference range for the
particular test.
• Abnormal results are those that fall below or above the reference range.
Reporting of Results
Report the displayed/printed results into register/request form
5.12.15 Limitation of the Procedure and Sources of Error
i. Icteric samples that appears with yellow colour of the serum or plasma due to
bilirubin accumulation.
ii. Samples tested after 24 hours may give unreliable results Avoid using lipemic
samples
5.12.16 Performance Characteristics
Refer the method verification reports .
5.12.17 Supporting Document
i. Sample Collection Manual, Safety Manual, Quality Manual
5.12.18 References
ALERE AFFINION AS100analyzer user manual
Manufacturer package insert

CHAPTER SIX: SEROLOGY

6.1 PROCEDURE FOR SYPHILIS ANTIBODIES RAPID TEST
6.1.1 Purpose
This procedure provides instructions for Qualitative detection of antibodies of all
isotopes against Treponema pallidum
6.1.2 Scope
The procedure is used in all Laboratory areas for screening syphilis infection
6.1.3 Responsibility
Qualified and trained Medical Laboratory Technicians, Technologists and Scientist are
responsible for implementing this test procedure.
The Head serology is responsible for ensuring the effective implementation and
maintenance of this procedure.
6.1.4 Principle
97
The syphilis Ab Rapid test strip (Serum/plasma) is a lateral flow chromatographic
immunoassay based on the Principle of the double antigens–sandwich technique, In
this test syphilis recombinant antigen is immobilized in the test line region of the strip
test device, After sample is added to the sample well of the device it react with syphilis
recombinant antigen coated particles in the test. This mixture migrates
chromatographically along the length of the test strip and interacts with immobilized
syphilis antigens.
If the sample contains syphilis antibodies a coloured line will appear in the test line
region indicating positive results. If the sample does not contain syphilis antibodies a
coloured line will not appear in the region, indicating a negative result
6.1.5 Sample Requirements
Whole blood/plasma sample in purple tube (EDTA)
Serum from clotted blood sample in plain tube.
6.1.6 Equipment
Centrifuge, Timer, Micropipette
Maintenance
Maintenance of the equipment should be performed as per schedule
6.1.7 Materials
Reagents Consumables
Syphilis Ab Rapid Test Strips kit Marker pen
Known Positive control, Examination Gloves
Known Negative control

98
6.1.8 Storage and Stability
• The kit should be stored at 2-30 oC until the expiry date printed on the sealed
pouch or as instructed by the manufacturer.
• Do not freeze the kit or exposing it over 300C.
• Store Serum and plasma sample at 2-80C for up to 3 days.
• For long term storage, serum/plasma should be kept below -20oC
6.1.9 Safety
i. Adhere to safety precautions as stated in the Safety manual/IPC guideline
ii. All personnel protective equipment (PPE) must be worn when performing
this procedure.
iii. All samples must be regarded as potentially infections.
6.1.10 Calibration
Perform calibration of equipment as per calibration schedule
6.1.11 Quality Control
A known Negative and Positive in-house controls once every week and whenever a
new kit is opened.
6.1.12 Procedural Steps
i. Bring the test kit and sample to room temperate before use.
ii. Remove the test from its sealed pouch, and use it as soon as possible.
iii. Place the test strip on a clean, dry flat surface
iv. Label the test strip with the Patient ID
v. For serum or plasma specimen;
vi. Hold the dropper vertically and transfer 2 drops of serum or plasma
(approximately 60µl) onto the specimen pad of the test strip.
vii. Read test results in 15 minutes. Do not interpret results after 15 minutes.
viii. For whole blood specimen;
ix. Hold the dropper vertically and transfer 2 drops of whole blood
(approximately 50ul) onto the specimen pad of the test strip.
x. Then add 1 drop of buffer (approximately 30ul) and start the timer.
xi. Read test results in 15 minutes.
xii. Do not interpret test results after 15 minutes
6.1.13 Biological Reference Interval
Not applicable
6.1.14 Interpretation and Reporting of Results
Interpretation of results
Negative - Only one coloured band appears on the control(C) region. No
apparent band on the test (T) region

99
 Positive - In addition to a pink coloured control (C) band, a distinct pink coloured
band will also appear in the tests (T) region
Invalid – a total absence of colour in both regions or no coloured line appears on
the control (C) region is an indication of procedure error or the tests deterioration.
Repeat the test with a new kit.
Reporting of results
Report results as: Syphilis - Negative or syphilis – Positive
Critical value
Not applicable
6.1.15 Limitation of the Procedure and Sources of Error
i. The syphilis Ab rapid test strip should be stored at room temperature (15-
30°c)
ii. Humidity and temperature can adversely affect results.
iii. Do not use test if pouch is damaged or broken
iv. Do not use it beyond expiration date.
v. Do not perform the test in a room with strong air flow. i.e. an electric fan
strong air-condition
vi. Test is for single use only. Do not re use test.
6.1.16 Performance Characteristics
Refer manufacture kit insert for specificity and sensitivity.
6.1.17 Supporting Document
Sample collection manual, Safety manual
6.1.18 References
Manufacturer Kit insert for syphilis
6.2 PROCEDURE FOR (HIV) TESTING BY USING BIOLINETM HIV 1/2 3.0 TEST
6.2.1 Purpose
The purpose of this procedure is to describe the method of performing Bio line HIV-
1/HIV-2 rapid test assay.
6.2.2 Scope
This procedure is applicable to all HIV-1/HIV-2 rapid test using Bio line HIV-1/HIV-2
6.2.3 Responsibility
Qualified, registered, licenced and trained Medical personnel are responsible for
implementing this test procedure.
The Head of Serology is responsible for ensuring the effective implementation and
maintenance of this procedure.
6.2.4 Principle

100
SD Bioline HIV-1/HIV-2 is a rapid HIV qualitative immune-chromatographic assay used
to detect antibodies to HIV in human blood, serum or plasma as the sample had been
added to sample pad. As the sample migrates through the conjugate pad, it
reconstitutes and mixes with the selenium colloid-antigen conjugate. This mixture
continues to migrate through the solid phase to the immobilized recombinant antigens
and synthetic peptides at the sample window site. If antibodies to HIV-1 and/or HIV-2
are present in the sample, the antibodies bind to the antigen-selenium colloid and to
the antigen at the client window, forming a red line at the client window site. If
antibodies to HIV-1 and/or HIV-2 are absent, the antigen selenium colloid flow past the
client window and no red line is formed at the client window site.
6.2.5 Sample Requirements
2-3mls Whole blood/plasma/ serum
6.2.6 Equipment
Timer, Centrifuge, Micropipette, Refrigerator.
Maintenance
Maintenance of equipment should be performed as per schedule
6.2.7 Materials
Abbott Bioline TM HIV 1/2 3.0 Test/kit, Assay diluent, Disposable gloves, Laboratory
coat
6.2.8 Storage and Stability
The test kit should be stored at a temperature between 1 oC and 30oC or as per
manufacturer claims
Whole blood; If the blood sample is not immediately tested, it should be refrigerated
at 2-80C for 3days
Plasma or serum; If plasma or serum sample is not tested immediately, it should be
refrigerated at 2-80C for 7 days
For storage period longer than 2week, freezing below -200C is required. They should
be brought to room temperature 15-300C prior to use.
6.2.9 Safety
Adhere to safety precautions as stated in the facility Safety manual /IPC guideline
All personal protective equipment (PPE) must be worn when performing this
procedure.
All samples must be regarded as potentially infectious.
6.2.10 Calibration
Perform calibration of equipment as per calibration schedule
6.2.11 Quality Control
Run known Negative and Positive in-house controls daily before performing patient
samples or when new test kit is opened.

101
6.2.12 Procedure Steps
i. Bring reagents and samples to room temperature before use.
ii. Tear off the desired number of test strips from the 10-test card by bending
and tearing off along the perforated line.
iii. Label the strips with sample identification number or patient/client
identification number.
iv. Peel the foil cover from the reagent area of the test strips.
For serum or plasma samples;
v. Apply 10 µl of sample using a precision pipette to the sample pad (marked
by the arrow symbol).
vi. In the absence of precision pipette apply 1 drop of sample using plastic
Pasteur pipette provided by manufacture in the kit.
vii. Then apply 4 drop of buffer to the sample pad.
viii. Wait for a 10 to 20 minutes and read results.
For whole blood collected by finger prick method;
ix. Apply 20 µl of sample (collected by EDTA capillary tube) to the sample pad
(marked by the arrow symbol).
x. In the absence of precision pipette or EDTA capillary tube, apply 1 drop of
sample using plastic Pasteur pipette provided by manufacture in the kit.
xi. then apply 4 drops of buffer to the sample pad. Wait for 10 to 20 minutes
and read results.
For whole blood collected by venepuncture method;
xii. Apply 20 µl of sample using a precision pipette to the sample pad (marked
by the arrow symbol).
xiii. Then apply four (4) drops of buffer to the sample pad.
xiv. Wait for 10 minutes (up to 20 minutes) and read results.
6.2.13 Biological Reference Interval
Not applicable
6.2.14 Interpretation and Reporting of Results
Result interpretation
Negative Result
The presence of only control line(C) within the result window indicate a negative
result
Positive Result
The presence of two lines as C and T -1(1) within the window indicates positive results
for HIV-1
The presence of two lines as C and T -2 (2) within the window indicates positive results
for HIV-2
The presence of three lines as C, T-1(1) and T-2(2) within the result window indicates
a positive result for HIV-1 and/or HIV -2

102
Invalid results
No presence of control line (C) or/and pink/purple band observed in the result window
Indicate an invalid result. The direction may not have been followed correctly or the
test
may have deteriorated. It is recommended that the specimen be retested.
Reporting of results
i. Reactive test Results will be reported as POSITIVE
ii. Non-reactive test results will be reported as NEGATIVE
Critical value
Not applicable
6.2.15 Limitation of the Procedure and Sources of Error
• Avoid haemolysed sample and beware of lipemic samples
• Samples other than blood have not been validated to give accurate results.
• Intensity of the patient bar does not necessarily correlate to the titre of antibody
• A negative result with BIOLINE HIV-1/2 does not exclude the possibility of an
infection with HIV.
6.2.16 Performance Characteristics
Refer to the method verification report of this procedure.
6.2.17 Supporting Documents
Sample collection Manual, HIV rapid testing algorithm
6.2.18 References
Package insert Abbott Bioline TM HIV-1/2 3.0

103
6.3 PROCEDURE FOR PERFORMING (HIV) BY USING UNIGOLD TEST
6.3.1 Purpose
The purpose of this procedure is to describe the method of testing HIV-1and HIV-2
using Trinity Biotech Uni-Gold test assay.
6.3.2 Scope
This procedure is applicable in all sites that perform Trinity Biotech Uni-Gold HIV test
6.3.3 Responsibility
Qualified, registered, licenced and trained Medical personnel are responsible for
implementing this test procedure.
The Head serology is responsible for ensuring the effective implementation and
maintenance of this procedure.
6.3.4 Principle
Recombinant proteins representing the immune-dominant regions of the envelope
proteins of HIV-1 and HIV-2, glycoprotein gp-41, gp120 (HIV-1) and glycoprotein gp36
(HIV-2) respectively are immobilized at the test region of the nitrocellulose strip. These
proteins are also linked to colloidal gold and impregnated below the test region of the
device. A narrow band of the nitrocellulose membrane is also sensitized as a control
region. Antibodies to HIV-1 and HIV-2 react with the colloidal gold linked antigens. The
antibody protein-colloidal gold complex moves chromatographically along the
membrane to the test and control regions of the test device.
6.3.5 Sample Requirements
Whole blood/plasma sample collected in purple tube (EDTA)
Serum from clotted blood sample in plain tube
Centrifuge sample at 3000rpm for 5 minutes to obtain serum of plasma.
6.3.6 Equipment
Stop watch, Micropipette, Centrifuge, refrigerator
6.3.7 Materials
Uni-GoldTM HIV test-kit, Disposable gloves, Laboratory coat, 70% alcohol
6.3.8 Storage and stability
Uni-GoldTM HIV test device and wash solution should be stored between 2-270C or as
per manufacturer instructions
Whole blood specimen should be stores at 2-80C for up to 3 days at -200C or below
6.3.9 Safety
• Adhere to safety precautions as stated in the Facility Safety manual/ IPC
guideline
• All personal protective equipment (PPE) must be worn when performing
this procedure.
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 • All samples must be regarded as potentially infections.
6.3.10 Calibration
Perform equipment calibration as per Schedule
6.3.11 Quality Control
Run known Negative and Positive in-house controls once a week.
The test strips contain a control line, which turns colored if the run is valid.
6.3.12 Procedure Steps
i. Bring reagents and samples to room temperature at least 20 minutes before
use.
ii. Remove the test device from its protective wrapper.
iii. Label the device with sample identification number or patient/client
identification number.
iv. Peel the foil cover from the reagent area of the test strips.
v. For serum or plasma or whole blood collected by finger prick or venipuncture
samples;
vi. Using one of the disposable pipettes supplied with the kit, fill it with the
sample.
vii. Holding the pipette over the sample port, add two drops of sample
(approximately 60 µl) carefully to the sample port of the test device.
viii. Add two drops (approximately 60 µl) of wash reagent to sample port and
start the timer.
ix. Wait for a minimum of 10 minutes (up to 12 minutes) and read results
6.3.13 Biological Reference Intervals
Not Applicable
6.3.14 Interpretation and Reporting of Results
Results interpretation
Reactive test results
Two pink/red lines of the intensity in the device window, the first adjacent to
letter ‘T’ (test) and the second adjacent to ‘C’ (control).
Non – reactive test results
A pink/red line of the intensity adjacent to the letter ‘C’ (control). But no pink/red
line adjacent to ‘T’ (test) this indicates a Non-Reactive result.
Invalid results
No pink/red line appears in the device window adjacent to the letter “C” (control)
irrespective of weather or not a pink /red line appears in the device window
adjacent to “T” (test). This is an INVALID result that cannot be interpreted. An
invalid result must be repeated
Results reporting

105
 • Reactive test results: Report as HIV TEST POSITIVE
• Non-reactive test results-repeat 1st and 2nd tests following the national
HIV testing algorithm
• If the test results are still discordant report - INCONCLUSIVE then inform
the patient for retesting after 14 days
• If after 14 days, the test is still discordant report - INCONCLUSIVE
Collect fresh venous blood sample, refer the sample for ELISA testing
Critical Values
Not Applicable
6.3.15 Limitation of the Procedure and Sources of Error
i. Avoid hemolyzed sample and beware of lipemic samples when interpreting
results.
ii. The BIOLINE HIV-1/2 test is designed to detect antibodies to HIV-1 and
HIV-2 in human serum, plasma and whole blood. Other body fluids or
pooled samples may not give accurate results.
iii. Intensity of the patient bar does not necessarily correlate to the titer of
antibody in the sample.
iv. A negative result with BIOLINE HIV-1/2 does not exclude the possibility of
an infection with HIV.
6.3.16 Performance Characteristics
Refer to the method verification report
6.3.17 Supporting Documents
Sample collection manual
HIV rapid testing algorithm
6.3.18 References
Manufacture package insert (Trinity Biotech Uni-Gold HIV)
6.4 PROCEDURE FOR URINE PREGNANCY TEST
6.4.1 Purpose
This procedure provides instructions for Qualitative detection of HCG in urine
6.4.2 Scope
The procedure is used in the serology section in detection of pregNot applicablency
6.4.3 Responsibility
Qualified,trained and competent Medical Laboratory Technicians, Technologists and
scientist are responsible for implementing this test procedure.
The Head Microbiology is responsible for ensuring the effective implementation and
maintenance of this procedure.
6.4.4 Principle

106
The Human Chorionic GoNot applicabledotropin One Step PregNot applicablency Test
Strip (urine) is rapid chromatographic immunoassay for the qualitative detection of
Human Chorionic GoNot applicabledotropin in urine to aid in early detection of pregNot
applicablency. The test uses two lines to indicate results. The test line is pre coated
with a monocloNot applicablel Human Chorionic GoNot applicabledotropin antibody to
selectively detect elevated level of Human Chorionic GoNot applicabledotropin. The
control line is pre coated with goat anti-mouse IgG antibody. The test also includes a
burgundy coloured conjugate paid containing another monocloNot applicablel HCG
antibody conjugated with colloidal gold. The assay is conducted by immersing the test
strip in a urine specimen and observing the formation of coloured lines .The specimen
migrate via capillary action along the membrane to reach with coloured conjugate.
Positive specimen reacts with the specific antibody HCG coloured conjugate to form a
coloured line at the test line region of the membrane. Absence of this coloured line
suggest a negative results
6.4.5 Sample requirements
Fresh Urine collected from either morning,evining or any other time
6.4.6 Equipment
Stop watch
Refrigerator
6.4.7 Materials
Reagents Consumables
HCG test kit Disposable gloves,
Laboratory coat
6.4.8 Storage and stability
Test strips reagent are stable at 2 to 30o C up to expiration date or as per manufacturer
instruction
If sample canot be tested within 1 hour of collection, it should be stored at 2 - 8oC for
24hrs
6.4.9 Safety
i. Adhere to safety precautions as stated in the Facility Safety manual/ IPC
guideline.
ii. All personal protective equipment (PPE) must be worn when performing this
procedure.
iii. All samples must be regarded as potentially infections.
6.4.10 Calibration
Perform equipment calibration as per schedule
6.4.11 Quality control

107
Run known Negative and Positive in-house controls (known patient or EQA sample)
once a week or when the new kit is opened
6.4.12 Procedure Steps
i. Remove the test strip from the sealed pouch and use it as soon as possible.
ii. With arrow pointing towards the urine specimen immerse the test strip
vertically in the urine specimen for at least 10 to 15 seconds. Do not pass
the maximum line (MAX) on the test strip when immersing the strip.
iii. Place the test strip(s) on the non absorbent flat surface, start the timer and
wait for the colored line(s) to appear. The results should be ready in 5
minutes.
6.4.13 Biological reference intervals
Not applicable
6.4.14 Interpretation and reporting of results
Results interpretation
POSITIVE: two distinct colored lines appear. One line should be the control line region
(C) and another line should be on test line region (T).
NEGATIVE: one colored line appears in control line region (C) no apparent colored
line appears in the first line (T).
INVALID: control line fails to appear in both the control region.
Reporting of results
Report results as PregNot applicablency test Negative or PregNot applicablency test
Positive.
6.4.15 Limitations of the Procedure and Sources of Error
i. The hCG one step pregnancy test strip (urine) is preliminary qualitative test
therefore neither the quantitative nor the rate of increase of hCG can be
determined by this test.
ii. Very dilute urine specimen as indicated by low specific gravity may not
contain representative level of hCG. If pregnancy is still suspected the first
morning urine specimen should be collect 48 hours later and tested.
iii. first trimester pregnancies terminate for natural reasons, a test result that is
weakly positive should be confirmed by retesting with first morning urine
specimen collected 48 hours later.
iv. This test may produce false positive results. A number of conditions other
than pregnancy including trophoblastic disease and certain non
trophoblastic neoplasms including testicular tumours, prostate cancer,
breast cancer and lung cancer, causes elevated level of hCG. Therefore the
presence of hCG in the urine should not be used for the diagnosis pregnancy
unless this condition has been ruled out.
v. This test may produce false negative results. False negative results may
occur when the levels of hCG are below the levels of sensitivity level of the

108
 test. When pregnancy is still suspected a first morning urine specimen
should be collected 48 hours later and tested. In case pregnancy is
suspected and the test continue to produce negative result see a physician
for further diagnosis.
6.4.16 Performance Characteristics
Refer to the method verification report of this procedure
6.4.17 Supporting documents
Sample collection manual
6.4.18 References
HCG package insert
6.4.19
6.5 PROCEDURE FOR HEPATITIS C ANTIBODY RAPID TEST
6.5.1 Purpose
The purpose of this procedure is to give instructions on how to perform Hepatitis C
Virus Antibody (HCV Ab) rapid test.
6.5.2 Scope
This procedure is applicable to all site perform hepatitis C Virus Antibody rapid tests
6.5.3 Responsibility
It is the responsibility of the Head of serology Section to ensure effectively implemented
by all personnel working in the serology section.
6.5.4 Principle
The HCV Ab Rapid test Strip is a lateral flow chromatographic immunoassay based on
the Principle of the double antigen- sandwich technique. The test strip consists of 1) a
burgundy colored conjugate pad containing HCV antigens conjugated with colloidal
gold (HCV Ag conjugates) and rabbit IgG-gold conjugates, 2) a nitrocellulose
membrane strip contain a test band (T band) and a control band (C band). The T band
is pre-coated with non- conjugated HCV antigens, and the C band is pre-coated with
goat anti-rabbit IgG. When an adequate of test specimen is dispensed into the sample
well of the strip, the specimen migrates by capillary action across the strip. The
antibodies: either the IgG, the IgM, or the IgA, to HCV if present in the specimen will
bind to the HCV Ag conjugates. The immunocomplex is then captured on the
membrane by the pre coated HCV antigens, forming a burgundy colored T band,
indicating a HCV Ab positive test result. Absence of the T band suggests a negative
result. The test contains an internal control (C band) which should exhibit a burgundy
colored band of the immunocomplex of goat anti rabbit IgG / rabbit IgG-gold conjugates
regardless the presence of any antibodies to HCV. Otherwise, the test result is invalid
and the specimen must be retested with another device.
6.5.5 Sample requirement
109
2-3mls whole blood/serum/plasma. To obtain serum, Centrifuge blood collected in
plain red top tube at 3000rpm per 3 minutes. To obtain Plasma, Centrifuge blood
collected in EDTA tube at 3000rpm per 3 minutes
6.5.6 Equipment
Timer, Centrifuge and Refrigerator
6.5.7 Materials
Reagents Consumables
HCV Ab test kit (Test strips, disposable Disposable gloves,
dropper and HCV Ab buffer) Laboratory coat
Micropipette
6.5.8 Storage and stability
a. The test device in the sealed pouch can be stored at 2-400C or as instructed by
manufacturer up to the expiration date. The test device must remain in the
sealed pouch until use. DO NOT FREEZE
b. Store whole blood at 2-80c for up 3 days
c. Serum and plasma maybe stored at 2-80c for up 7 days, for long term storage,
serum and plasma specimens should be kept at-200c or below.
6.5.9 Safety
i. Adhere to safety precautions as stated in the Facility Safety manual/ IPC
guideline
ii. All personal protective equipment (PPE) must be worn when performing this
procedure.
iii. All samples must be regarded as potentially infections.
6.5.10 Calibration
Listed equipment will be calibrated as per calibration schedule.
6.5.11 Quality control
Run known Negative and Positive in-house controls once a week or when new test kit
is opened
6.5.12 Procedure Steps
Allow test strip, specimen, buffer and/or controls to equilibrate to room temperature
prior to testing.
i. Remove the test device from the foil pouch and use it as soon as possible. best
results will be obtained if the assay is performed within one hour.
ii. Place the test device on a clean and level surface.
iii. Label the test device with sample ID
iv. For venepuncture whole blood samples; Hold the dropper vertically and
transfer 2 drops of venipuncture whole blood (approximately 50ul) to the sample

110
 pad of the strip, then add 1 drop of buffer (approximately30ul) and start the
timer.
For finger stick whole blood sample; allow 2 hanging drops of fingerstick whole
blood (approximately 50ul) to fall into the center of the sample pad on the test
strip, then add 1 drop of buffer(approximately 30ul) and start the timer.
For serum or plasma sample; Hold the dropper vertically and transfer
1 drop of serum or plasma(approximately30ul) to the sample pad of the
test strip, then add 1 drop of buffer (approximately 30ul) and start the
timer.
v. Wait for the red line(s)to appear. the result should be read in 15 minutes.do not
interpret the result after 15 minutes

111
6.5.13 Biological Reference Intervals
Not Applicable
6.5.14 Interpretation and Reporting of Results
Interpretation of results
Positive; two colored lines should be observed. The line in the test region (T) is the
prone line; The line in the control region (C) is the control line, which is used to indicate
proper performance of the device.
Negative; The control line appears in the test, but the test line is not visible.
Invalid; No line appears in the control region. Under no circumstances should a
positive sample be identified until the control line forms in the viewing area if the control
line does not form, the test result is inconclusive and the assay should be repeated
Reporting of results
Reactive test result - HCV Ab rapid test positive
Non - reactive test results - HCV Ab rapid test Negative
6.5.15 Limitations Of The Procedure And Source Of Error
i. The HCV Ab Rapid Test cassette (whole blood, serum, plasma) is for in vitro
diagnostic use only. This test should be used for the detection of antibodies
to HCV in whole blood, serum or plasma sample.
ii. The HCV Ab Rapid Test cassette (whole blood, serum, plasma) will only
indicate the presence of antibodies to HCV in the sample and should not be
used as the sole criteria for the diagnosis of hepatitis C viral infection.
iii. A negative result can occur if the quantity of the antibodies to HCV present
in the specimen is below the detection limits of the assay, or the antibodies
that are detected are not present during the stage of disease in which a
sample is collected.
6.5.16 Performance Characteristics
Refer to method verification report of this procedure
6.5.17 Supporting Document
Sample collection manual
6.5.18 References
HCV Ab Package inserts
Kit manufacturer paper insert

112
6.6 PROCEDURE FOR CRYPTOCOCCAL ANTIGEN RAPID TEST
6.6.1 Purpose
The purpose of this procedure is to give instructions on how to perform cryptococcal
antigen rapid test.
6.6.2 Scope
This procedure will be used by all staff and students perform CrAg test
6.6.3 Responsibility
It is the responsibility of the Head of serology Section to ensure effectively implemented
and maintained.
6.6.4 Principle
The CrAg Lateral Flow Assay is a dipstick sandwich immune-chromatographic assay.
Specimens and specimen diluent are added into an appropriate reservoir, such as a
test tube, and lateral flow device is placed into the reservoir. The test uses specimen
wicking to capture gold- conjugated, anti-Crag monocloNot applicablel antibodies and
gold conjugated control antibodies deposited on the test membrane. If Crag is present
in the specimen, then it binds to the gold conjugated, anti-CrAg. The gold labelled
antibody antigen complex continues to pick up the membrane where it will interact with
the test line, which has immobilized ant Crag monocloNot applicablel antibodies. The
gold labelled antibody-antigen complex forms a sandwich at the test line causing a
visible line o form. With proper flow and reagent reactivity, the wicking of any specimen,
positive or negative, will cause the gold- conjugated control antibody to move to the
control line. Immobilized antibodies at the control line will bind to the gold conjugated
control antibody and form a visible control line. Positive test results create two lines
(test and control). Negative test results from only one line (control). If control line fails
to develop then the test is invalid.
6.6.5 Sample Requirement
2-3mls serum, plasma, whole blood (venous and finger prick) and cerebral spinal fluid;
NOTE 1: To obtain serum, Centrifuge blood collected in plain red top tube at 3000rpm
per 3 minutes
NOTE 2: To obtain Plasma, Centrifuge blood collected in EDTA tube at 3000rpm per
3 minutes
6.6.6 Equipment
Timer, Centrifuge and Refrigerator
6.6.7 Materials
Reagents Consumables
CrAg test kit (CrAg LF Test strip, LF Disposable gloves,
Specimen diluents, LF titration diluents, Laboratory coat
CrAg positive control) Micropipette

113
6.6.8 Storage And Stability
i. If a delay is encountered in sample processing, store at 2-8ºc for up to
72 hours is permissible.
ii. CSF, plasma, serum may be stored for longer period at -20ºc provided
they are not repeatedly thawed and refrozen
iii. Whole blood in transit should be maintained at 2-8ºc not -20ºc.
6.6.9 Safety
i. Adhere to safety precautions as stated in the Facility Safety manual/ IPC
guideline
ii. All personal protective equipment (PPE) must be worn when performing this
procedure.
iii. All samples must be regarded as potentially infections.
6.6.10 Calibration
Perform Equipment calibration as scheduled.
6.6.11 Quality Control
i. To ensure assay validity, a procedural control bar is incorporated in the
assay device.
ii. Run known positive and negative in-house controls weekly and when the
new kit of test strips is opened to verify kit.
6.6.12 Procedure Steps
i. Add one drop /40µ pipette LF Sample diluents to an appropriate labeled
reservoir. (Test tube) it is also a good practice to label the strip.
ii. Add 40µ of sample to the reservoir and mix.
iii. Submerge the white end of a CrAg LF test strip into the sample.
iv. Wait 10 minutes after inserting the strip
v. Read and record the results
6.6.13 Biological Reference Intervals
Not applicable
6.6.14 Interpretation And Reporting Of Results
Interpretation of results
Positive Results
Two red bands appear on the membrane. One band appear on the control region (C)
and another band appears on the test region (T)
Negative Results
Only one red band appears on the control region C. No apparent red band appears in
the test region T.
Invalid Results

114
No visible band at all or there is visible band only in the test region and not in the control
region, Repeat the procedure.
Reporting of results
For the positive result report as Cryptococcal antigen rapid test Positive
For the Negative result report as Cryptococcal antigen rapid test Negative
6.6.15 Limitations Of The Procedure And Source Of Error
i. The assay Performance Characteristics have not been established for matrices
other than serum, plasma, whole blood and CSF
ii. Depending on the disease and organism prevalence, testing should not be
performed as screening procedure for general population. The predictive value
of a positive or negative serologic result depends on the pre-test likelihood of
cryptococcal disease being present. Testing should only be done when clinical
evidence suggests the diagnosis of cryptococcal disease
iii. Testing hemolyzed serum samples could lead false negatives due to the high
background color on the strip
iv. This assay was not evaluated for potential interference related to specimen pre-
treatment with 2-mercatoethanol or with specimens including the following
substances: Vaginal cream, caffeine, ascorbic acid, intraconazole, amphotericin
B, acetaminophen, or acetylsalicylic acid
6.6.16 Performance Characteristics
Refer to method verification report of this procedure
6.6.17 Supporting Document
Sample collection manual
6.6.18 References
6.7 PROCEDURE FOR PERFORMING (HBsAg) RAPID TEST
6.7.1 Purpose
The purpose of this procedure is to give instructions on how to perform Hepatitis
B Virus Surface Antigen Rapid test in human whole blood, plasma or serum.
6.7.2 Scope
This procedure is applicable in all sites that perform Hepatitis B Virus surface
antigen in whole blood ,serum or plasma sample qualitatively.
6.7.3 Responsibility
It is the responsibility of the Head of serology section and all laboratory
personnel to ensure effective implementation of this procedure.
6.7.4 Principle
HBsAg is an antibody sandwich immunoassay. Colloidal gold conjugated monoclonal
antibody reactive to HBsAg is dry-immobilized onto a nitrocellulose membrane strip.
When the sample is added, it migrates by capillary diffusion through the strip
115
rehydrating the gold conjugate. If present, HBsAg will bind with the gold conjugate
antibody to form particles. These particles will continue to migrate along the strip until
the test zone (T) where they are captured by ant-HBs antibody immobilized there and
a visible red line appears. If there is no HBsAg in sample, no red line will appear in the
T zone. The gold conjugate will continue to migrate alone until is captured in the control
zone (C) by immobilized goat, ant-mouse IgG antibody aggregating a red line, to serve
as an internal process control, a control band should always be seen after test is
completed. Absence of a colored control line in the control region is an indication of an
invalid result.
6.7.5 Sample requirements
2-3mls Whole blood, centrifuged Serum or plasma samples
6.7.6 Equipment
i. Timer
ii. Refrigerator
iii. Centrifuge
6.7.7 Materials
Reagents Consumables
HBsAg test kits (HBsAg Device, Disposable gloves,
disposable specimen droppers) Laboratory coat
Assay diluent Micropipette
6.7.8 Storage And Stability
a. The test device in the sealed pouch can be stored at 2-400C or as
instructed by manufacturer up to the expiration date. The test device
must remain in the sealed pouch until use.DO NOT FREEZE.
b. Store whole blood at 2-80c for up 3 days
c. Serum and plasma maybe stored at 2-80c for up 7 days, for long term
storage, serum and plasma specimens should be kept at-200c or
below.
6.7.9 Safety
i. Adhere to safety precautions as stated in the Facility Safety manual/ IPC
guideline
ii. All personal protective equipment (PPE) must be worn when performing this
procedure.
iii. All samples must be regarded as potentially infections.
6.7.10 Calibration
Perform Equipment calibration as scheduled.
6.7.11 Quality Control

116
Run known Negative and Positive in-house controls once a week or when new test kit
is opened .
6.7.12 Procedure Steps
i. Remove the test strip from foil pouch and use as soon as possible.
Note: Check and verify the device’s integrity before and after opening the
foil pouch
ii. Label the test device with patient ID
Whole blood specimen:
iii. Hold the dropper vertically and transfer 2 drops of whole blood (approximately
50-60 ul) to the specimen area, then add one drop of buffer
Serum/ plasma;
i. Immerse the strip into the sample tube with the arrow end pointing towards the
sample.
ii. Let it stay immersed until you see liquid traveling up past the MAX word.
iii. Lay the strip (MAX side facing up) flat on a clean, dry, non-absorbent surface
iv. If cassette; Add 60µl (2 drop) of serum or plasma in to the sample window and
allow to soak in.
v. Read the results at 15-20 minutes. Ensure that the background of the test area
is white before interpreting the results
6.7.13 Biological Reference Intervals
Not Applicable
6.7.14 Interpretation And Reporting Of Results
14.1 Interpretation Of Results
NEGATIVE: if only one line (control line) appears in result line area, interpret
the result as negative. This shows that the concentration of HBsAg in the sample
is under the detection limit.
POSITIVE: if only two line (control line and test) appears in result line area,
interpret the result as positive.
INVALID: Control line fails to appear. Insufficient sample volume or incorrect
procedural technique is the most likely reasons for control line failure. Review
the procedure and repeat the test with a new test strip.
Reporting of results
Reactive test Results. Report as HBsAg rapid test positive
Non-reactive test results: Report as HBsAg rapid test negative
6.7.15 Limitations Of Procedure And Source Of Error
i. HBsAg Rapid Test Kit detects HBsAg in human serum or plasma and is
only screening test. All reactive samples should be confirmed by
supplemental assays like PCR OR ELISA.
ii. A non -reactive result does not exclude the possibility of exposure or
infection with Hepatitis B virus.
117
 iii. Patients with auto-immune liver diseases may show falsely reactive
results.
iv. This test is standardized to work best when the test procedure mentioned
in the package insert is strictly followed. Any deviation from the test
procedure may lead to erroneous results.
6.7.16 Performance Characteristics
Refer to method verification report of this procedure
6.7.17 Supporting Documents
Sample collection manual
6.7.18 References
HBsAg Package insert kit
6.8 PROCEDURE FOR SARS-COV-2 ANTIGEN RAPID DIAGNOSTIC TEST
6.8.1 Purpose
The purpose of this standard operating procedure (SOP) is to provide guidelines to be
followed for performing Rapid Antigen Detection Test for COVID-19 using the Standard
COVID-19 Ag detection assay kit
6.8.2 Scope
This procedure is to be performed at point of care or any health facility
6.8.3 Responsibility
The Head of Serology is responsible for ensuring the effective implementation and
maintenance of this procedure
Qualified, competent and registered Medical Laboratory practitioners are responsible
for implementing this test procedure.
6.8.4 Principle
It is a rapid chromatographic immunoassay for qualitative detection of specific antigens
to SARS-CoV-2. When the liquid sample is dropped on the sample pad, the antigen in
the sample forms an immunocomplex with the antibody labelled with colloidal gold. Its
complex moves along with the liquid sample, and makes a contact with the antibody
immobilized on the membrane, followed by forming an immunocomplex with the
immobilized antibody, resulting in generation of a coloured red purple line. Appearance
of red purple line on the membrane indicates the presence of antigen in the sample.
Since the liquid of the sample migrates through the membrane very fast, it makes it
possible to detect the presence or absence of antigen within 15 minutes.
6.8.5 Sample Requirements
Nasopharyngeal swab sample collected from nostril of the suspect individual.
Oropharyngeal swab sample collected from the posterior pharynx and tonsillar area of
the suspect individual.

118
6.8.6 Equipment
Stop watch, Micropipette/Supplied capillary
6.8.7 Materials
Reagent Consumables
Test devices Disposable gloves
Buffer Laboratory coat
70% alcohol
Mask
6.8.8 Storage And Stability
Store Covid 19 rapid kit devoice 2-30OC Protected from sunlight and should not be
frozen
6.8.9 Safety
i. Adhere to safety precautions as stated in the facility Safety manual
ii. All personal protective equipment (PPE) must be worn when performing
this procedure.
iii. All samples must be regarded as potentially infections.
iv. Refer to National infection prevention and control Guidelines for health
waste management and safety practice.
6.8.10 Calibration
Perform equipment calibration as per schedule
6.8.11 Quality Control
Run known Negative and Positive in-house controls once a week.
The test strips contain a control line, which turns coloured if the run is valid.
6.8.12 Procedural Steps
i. Peel off the aluminium foil seal from the sample processing tube containing the
extraction buffer.
ii. After the sample collection, plunge the swab up and down in the sample
processing tube for at least 15 seconds, taking care not to spill the contents out
of the tube.
iii. Remove the swab while pinching wall of the tube with the swab and rotating the
swab, to extract the liquid from the swab.
iv. Firmly attach the dropper lid to the top of the sample processing tube.
v. Remove the test cassette from the sealed pouch.
vi. Sample adding: Reverse the sample processing tube, holding the tube upright,
and slowly add 3-4 drops to the sample ole (S) of the test cassette then start
the timer.
vii. Timing observation: judge the result 15 minutes after sample adding; do not
observe the results after 30 minutes later.
viii. After the test, put the medical wastes into the biosafety bag.
119
6.8.13 Biological Reference Interval
Not Applicable
6.8.14 Interpretation and Reporting of Results
Results interpretation
A Positive: Two distinct coloured bands appear on the strip.
Negative: Only one distinct coloured band on the strip.
Invalid: If no control band is seen.
Reporting of results
Report negative results as SARS-COV-2 – Negative.
Report positive result as SARS-COV-2 – Positive
Critical value
Any positive results
6.8.15 Limitation of the Procedure and Sources of Error
i. The kit is not intended for testing liquid sample such as wash or aspirate
sample or swab in transport media as a result can be compromised by
over dilution.
ii. Insufficient sample volume or incorrect procedural techniques are the
most likely reason for control line failure
6.8.16 Performance Characteristics
Refer manufacturer reagent kit insert for sensitivity and specificity
6.8.17 Supporting DocumentsS
Sample collection manual
6.8.18 References
Manufacture package insert kit for SARS COV-2
ASOT Test Kit insert:
Atlas Medical,

120
CHAPTER SEVEN: BACTERIOLOGY
7.1 POTASSIUM HYDROXIDE (KOH) WET MOUNT PREPARATION
7.1.1 Purpose
This procedure provides instructions for examination of wet preparations that is mainly
used to examine samples and cultures for motile bacteria, C.S.F for capsulated yeast
cells and for fungi.
7.1.2 Scope
This procedure applies to the microbiology section and health laboratory practitioners
in the laboratory settings.
7.1.3 Responsibility
Competent Health Laboratory Practitionersare responsible for implementing this test
procedure.
The Head of Microbiology/who asned is responsible for ensuring the effective
implementation and maintenance of this procedure.
7.1.4 Principle
A KOH preparation is used for Samples such as skin scrapings, nail, infected hairs, or
for other Samples such as sputum to clear out background debris that may be confused
with fungal elements. KOH dissolves proteinaceous tissues, including keratin, and
renders them transparent. This enables fungi to be visualized more easily. The use of
KOH is not necessary for Samples such as CSF where background debris is minimal.
7.1.5 Sample Requirements
KOH preparation is used ideally in suspected cases of dermatophytosis, i.e., fungal
infection of skin, hair, or nails. Also used for specimen such as sputum, pus, and urine
sediment.
7.1.6 Equipment
Microscope
7.1.7 Materials
Potassium hydroxide 10 or 20%, Potassium hydroxide 10 g, Glycerol 10ml, Deionized
water 80ml, (optional) helps prevent the KOH mount from drying. Dispense working
solution in a dropper bottle. Microscope slides, 24 x 50 mm cover slips, sterile forceps,
mounting needle
7.1.8 Storage and Stability
Potassium hydroxide, 10 or 20% solution is stable for one year at room temperature
7.1.9 Safety
Observe standard safety precautions when handling Samples. Refer to Safety Manual

121
Discard all used materials (e.g., sticks, pipettes, disposable forceps) in a bucket
containing bleach.
7.1.10 Calibration
Not applicable
7.1.11 Quality Control
Fungal spores may contaminate the KOH solution, and may give false positive results.
Solution must be examined for signs of contamination (e.g., turbidity) before each use.
7.1.12 Procedure Steps
i. Place the material to be examined on a glass slide, add a drop of 10% KOH
ii. Place a cover slip over the preparation.
iii. Let stand for five to ten minutes.
iv. If clearing is not complete, an additional ten minutes is necessary.

Note: Keratinous (skin, nails) Samples should be left at room temperature for 20 to 30 minutes
to allow digestion and “clearing” of the keratin, after clearing, press coverslip gently to make a
thin mount.

v. Examine the slide microscopically on low power and confirm observations with
high power. Observe for hyphae, conidia, budding yeasts, spherules or sclerotic
bodies, etc. Consult photomicrographs in appropriate references when
necessary, to identify fungal structure.
7.1.13 Biological Reference Intervals
Not applicalbe
7.1.14 Interpretation and Reporting of Results
Interpretation
Dermatophytes in skin or nail are seen as branching hyphae or arthrospores and often
appear slightly greenish in color, with hyphae running across the colorless host cells.
Most hyphae will be parallel-sided and around 2 µm in width.
Yeasts are present as budding cells, pseudohyphae, or yeast mycelium.
In infections caused by dematiaceous fungi, the hyphae are often brown
(dematiaceous septate hyphae).
Artifacts, such as fibers, may be distinguished from hyphae from the lack of septa,
tapering ends, and size differences.
Reporting of Results
Report the type of fungal structure seen. Do not report quantity.
Examples: “Fungal elements seen (septate hyphae)”
“Fungal elements seen (yeast cells and pseudohyphae)”
Report negative results as: “No fungal elements seen”
Report as: “Fungal elements seen (Malassezia spp.)”

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7.1.15 Limitations of the Procedure and Sources of Errors
i. Cotton swabs should not be used in preparing these slides as the cotton strands
may resemble hyphae.
ii. The contrast between unstained fungal elements that may be present and the
background mounting fluid can be accentuated by narrowing the iris diaphragm
to reduce the amount of incident light; or use of phase-contrast microscopy that
can greatly enhance visualization of organisms.
iii. KOH preparations are presumptive and should not be substituted for culture and
further identification.
iv. KOH should not be stored in a glass bottle as it will leach minerals from the glass.
The minerals will result in a cloudy, flocculant solution. This can be avoided by
storing the KOH in plastic, polystyrene or other non-glass container.
v. Gentle heating may speed the activity of the KOH, but it may be harmful to the
specimen if overdone.
vi. KOH preparations are not permanent; the reagent will eventually destroy the
fungi. The addition of small amount of glycerol to the preparation will preserve it
for several days.
vii. KOH combined with calcofluor white is a more sensitive method, but a fluorescent
microscope with appropriate filter is required.
viii. India ink, added to the KOH, stains fungal elements and helps them stand out
against the background. Reagent is prepared as follows: Make a 10% KOH
solution in Parker Super Quink Ink, permanent blue black (50 ml of ink + 5 g of
KOH pellets). Centrifuge KOH-ink solution at 2,000 x g for ten minutes. Pour
supernatant into plastic (not glass) sterile tube. Store at room temperature.
7.1.16 Performance Characteristics
Not applicable
7.1.17 Supporting Documents
Not applicable
7.1.18 References
✓ Lynne S. Garcia, Henry D. Isenberg. Clinical Microbiology Procedures Handbook.
3rd edition.
✓ American Society for Microbiology. Washington DC, USA. 2010.
✓ Guidelines on Standard Operating Procedures for Laboratory Diagnosis of
HIV/AIDS Opportunistic Infections in Patients. WHO Regional Office for Southeast
Asia. New Delhi. June 2001
✓ Bailey and Scott’s Diagnostic Microbiology. 13th edition. Mosby, Inc. St. Louis,
Missouri, USA. 2013.

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7.2 PROCEDURE FOR ZIEHL NIELSEN (ZN) STAIN
7.2.1 Purpose
This procedure provides instructions for performing Ziehl-Nielsen stain of sputum or
other body fluid samples.
7.2.2 Scope
This procedure is to be used for detection of Acid Fast Bacilli in sputum or other body
fluid samples in the Laboratory
7.2.3 Responsibility
The section heads and technical staffs are responsible for implementing this
procedure.
7.2.4 Principle
This procedure is used to stain mycobacterium tuberculosis and mycobacterium
leprae. These bacteria are also called acid fast bacilli. They stain with carbolfuschin,
which is a red dye. They retain the dye when treated with acid, which is because of the
presence of mycolic acid in their cell wall.
7.2.5 Sample Requirements
The Sputum and body fluids such as CSF, synovial, pericardial, synovial, ascitic, blood,
pus, bone marrow, tissue biopsies or pleural fluid samples.
7.2.6 Equipment
Centrifuge (when necessary), Bunsen burner/spirit lamp, Light Microscope and
Biosafety cabinet
7.2.7 Materials
ZN stains reagents: 1% Carbol Fuchsin stain, 20% Sulphuric acid and 0.125%
Methylene blue. Acid Fast Bacilli Positive and negative Control slides for zn.
Personal protective gears, Gauze, Glass slides, Staining racks, Drying rack, Clean
Glass slides, Applicator stick, Micropipette or Pasture pipettes,sputum container,timer
7.2.8 Storage and Stability
Sputum samples should be proccesed within 3days if stored at room temperature and
if not possible store it at 2 to 8°C for 10 days.
7.2.9 Safety
Decontaminate working surfaces as recommended by IPC Guidelines.
All personal protective equipment (PPE) must be worn when performing this
procedure.
All samples must be regarded as potentially infections.
Refer to National infection prevention and control Guidelines for health waste
management and safety practice.
7.2.10 Calibration
124
Centrifuge and safety cabinety should be calibrated as per schedule
7.2.11 Quality Control
Perform IQuality control by Smearing and staining known Negative and positive
sample /EQA before examining any patient sample every day.
Perform lot to lot for every new batch of reagent received
7.2.12 Procedure Steps
i. Place the fixed slides with smear upwards on a staining rack over a sink about
1 cm apart.
ii. Flood the smear with filtered carbol fuchsin staining solution.
iii. Prepare a torch by dipping its cotton wool end in burning spirit and light it. Use
only a few drops on acid alcohol or 70% v/v ethanol or methanol.
iv. Heat the slide keeping the torch a little below the slide and moving it
continuously forth and back along the line until steam arises, repeat twice at
intervals of 3-5 minutes. Do not overheat. Allow slides to stand for at least 30
minutes.
v. Tilt the slide using forceps to drain off the staining solution.
vi. Rinse the slide well with clean water from a beaker (or running tap water).
vii. Pour the 20% Sulphuric acid or 3%Hcl over the smears, covering them
completely. Allow to act for 3 minutes.
viii. Tilt the slide with forceps to drain off the acid solution; then gently rinse the slide
again with clean water.
ix. Repeat covering by sulphuric acid or Hcl solution and rinsing once for smears
that are still red.
x. Flood smear with methylene blue solution for 1 minute.
xi. Tilt the slide with forceps draining off the Methylene blue solution.
xii. Wash with clean water.
xiii. Using forceps take the slide from rack, drain off water and stand the slide on the
edge to air dry on the drying rack.
xiv. Examine the smear microscopically first with the 40x objective to see the
distribution of material, then with the oil immersion objective to look for Acid Fast
Bacilli . Open fully the condenser iris when using the oil immersion lens. After
examining a positive smear, the oil immersion objective must be wiped clean.

Note: The stained smear should show a light blue colour from methylene blue. If the smear is
dark blue, it usually indicates that the smear is too thick.

7.2.13 Biological Reference interval

Not applicable

7.2.14 Interpretation and Reporting of Results

125
Acid Fast Bacilli appears as Red, straight or slightly curved rods, occurring singly or
in small.
If any definite red bacilli are seen, report the smear as Negative If no red bacilli are
seen in at least 100 fields and ‘Acid Fast Bacilli positive’ if give an indication of the
number of bacteria present as follows:
More than 10 AFB/HPF field Report + + + OR +3
1 – 10 AFB/HPF field Report + + OR +2
10 - 99 AFB/100 fields Report + OR +1
-9 AFB/100 fields Report the exact number/100
If no red bacilli are seen in at least 100 Report as negative
fields
7.2.15 Limitation of the Procedure and Sources of Error
i. Re use of containers or positive slides.
ii. Contaminated stain prepared with water containing environmental mycobacteria.
iii. Use of scratched slides.
iv. Acid Fast Bacilli floated off one slide and became attached to another during
staining procedure because of no distance between each slide.
v. Poor quality of staining solutions.
vi. Taking improper portion of sample for smear preparation.
vii. Improper focal distance for examination.
viii. Use of poorly prepared staining solution.
ix. Overheating during fixing.
x. Too long interval between staining and reading, especially when slides not kept
in dark or poorly stained.
7.2.16 Performance Characteristics
Not applicable
7.2.17 Supporting Document
Patients Results Register - TB 05
Internal Quality Control review form
7.2.18 References
✓ Clinical Microbiology Procedures Handbook. American Society for Microbiology.
Washington D.C., USA, 2nd edition, 2007.
✓ Monica cheesbrough (2005). District Laboratory Practice in Tropical countries.
Cambridge University Press, New York, USA, 2nd edition, 2005.
✓ WHO, (2003). Mannual of basic techniques for a health laboratory. Geneva. 2 nd
edition, 2003.
✓ Manual of Clinical Microbiology. American Society for Microbiology (ASM),
Washington D.C., USA. 9th edition, 2007.

126
7.3 PROCEDURE FOR AURAMINE O PHENOL STAINING
7.3.1 Purpose
The Auramine O staining technique applies to identification of Acid Fast Bacilli in
patient sample using fluorescence Microscopy which increase the rate of detection
compared to the light microscopy.
7.3.2 Scope
This procedure is to be used for detection of Acid Fast Bacilli in sputum or other body
fluid samples in the Laboratory by using Auramine O reagents.
7.3.3 Responsibility
Competent Health Laboratory Practitioners are responsible for implementing this test
procedure.
The Head Microbiology is responsible for ensuring the effective implementation and
maintenance of this procedure
7.3.4 Principle
The fluorochrome dye, Auramine-Rhodamine, forms a complex with mycolic acids
contained in the acid fast cell wall of organisms which resist decolorization by acid-
alcohol. The counterstain, Methylene blue 0.3%, renders tissue and its debris non
fluorescent, thus reducing the possibility of artifacts. The cells visualized under
ultraviolet light appear bright yellow or reddish orange.
7.3.5 Sample Requirements
Mucoid, purulent or blood stained samples.
7.3.6 Equipment
Biosafety cabinet, Fluorescent microscopes, Timer, Bunsen burner/ Spirit lamp
7.3.7 Materials
Auramine –O phenol 0.1%, Hydrochloric acid in Alcohol 0.5%, Methylene blue 0.3%,
Microscopic slides, Slide holding box, Gloves, Gauze/Cotton wool, Beaker, Forceps
Applicator sticks/Disposable, Pencil, Dry rack, Slide racks, wide mouth sputum
container for routine samples or Falcon tube 50ml for referral samples.
7.3.8 Storage and Stability
Sputum samples should be proccesed within 3 days if stored at room temperature and
if not possible store it at 2 to 8°C for 10 days.
7.3.9 Safety
Decontaminate working surfaces as recommended by IPC Guidelines.
All personal protective equipment (PPE) must be worn when performing this
procedure.
All samples must be regarded as potentially infections.

127
Refer to National infection prevention and control Guidelines for health waste
management and safety practice.
7.3.10 Calibration
Centrifuge and safety cabinety should be calibrated as per schedule
7.3.11 Quality Control
Internal Quality Control should be done by smearing and staining known Negative and
positive sample before examining any patient sample every day. Perform lot to lot for
every new batch of reagent received/prepared.
7.3.12 Procedure
Film Preparation
i. Label the slides properly using a unique laboratory number
ii. Place the labeled slides, the samples and the applicator stick /Pasteur pipette
in the Biological safety cabinet
iii. Match each slide with the corresponding sputum or sample container.
iv. Proceed to smearing, taking the labeled slides and opening containers one by
one, do the smearing from the center of the slide, outwards making small coil –
like movements (A thin smear ,allow to air dry).
v. Select a small portion of purulent or muco-purulent material with the applicator
stick for a direct sputum smear and then transfer it to the slide, if a stick is used,
break it in two pieces and used ragged ends for dissecting sputum and for
smearing.
vi. Spread the material carefully over the area equal to about 2×1 cm using
repeated coil like movements, without touching the margins of the slides and
should be in the middle.
vii. Make the smear as even as possible by continuing this process until no thick
parts remain. Remove excess material with the second stick and discard in the
biohazard bag.
viii. The thickness of the smear should be such that a newspaper can bared bye
read through the dried smear held about 10cm above it (Translucent).
ix. Warm the slides on the slide warmer in the biological safety cabinet to dry and
fix the smear for at least 30minutes.
x. Re-fix the smears by passing a flame under the slides before staining.
xi. If the slide warmer is not functioning, air dry the smear then fix them by passing
a flame under the smear ,the smear should face upward ,do not over heat ,or
else Acid Fast Bacilli staining will be poor.
Fluorescent Microscopy Staining Procedure
Place the slides on staining rack. IT IS A MUST to keep distance of at least 1cm
between every slide. Otherwise there is a possibility that acid fast bacilli may cross
contaminate the negative smears due to over flooding or splashes from positive smear
to a negative smear

128
 i. Cover the smear completely with filtered 0.1% auramine solution Do not heat
ii. Leave for 15 minutes
iii. Wash the slides well with distilled water or running water
iv. Pour the acid alcohol solution over the slides.
v. Allow to act for 2- 3 minutes.
vi. Gently rinse each slide with distilled or running water.
vii. Repeat decolourization if macroscopically visible stains are still visible.
viii. Flood smear with 0.5% methylene blue counter solution for 1 minute .Time is
critical because counterstaining for longer time may quench the acid fast bacilli
ix. Gently wash off counterstain with distilled or running water.
x. Stand the slides on edge to drain and air dry on the slide rack away from direct
sun light.
Examination
i. Keep stained smears in the dark (box or folder) till reading, and read as soon
as possible (within 24hours) since fluorescence fades quickly out of the box.
ii. Use 20 x objectives for scanning and 40x confirmation; scan the stained smear
systematically from one side to another side
iii. One length has to be scanned before reporting a Negative.
iv. Acid-fast bacilli appear bright yellow against the dark background material.
v. Store the slides in a slide box according to the study, following the laboratory
Number as they will be needed for EQA.

Note: Acid fast bacilli appear bright yellow against dark background, report as possible for Acid
Fast Bacilli if at least one acid-fast bacillus was seen in a well stained smear, even if you think
they might be other mycobacterium other than tubercle bacilli. Tubercle bacilli are quite variable
in shapes from very short fragments to elongated types. They may be uniformly stained or with
one or many gaps, or even granular. They occur singly or in small groups (coded), and rarely in
large clumps. The typical appearance of bacillli are usually rather long and slender, straight or
slightly curved rods.

7.3.13 Biological Reference interval

Not applicable
7.3.14 Interpretation and Reporting of Results
If fluorescent Acid Fast Bacilli are seen, report the smear as Acid Fast Bacilli positive,
and give an indication of the number of bacilli present in plus signs (+ to +++).
The results have to be reported on the working sheet and on patients result register
(MTB Register 05 if available).
If no fluorescent rods are seen, report the smear as (NO AFB SEEN).
Report Fluorescence (200magnigication, Fluorescence
one length 20x=field 200=HPF (400magnigication,
one length 40x =field 200=HPF
Negative Zero AFB/1 length Zero AFB/1 length
Scanty 1-29 AFB/1 Length 1-19 AFB/1 Length

129
1+ 30-299 AFB/1 Length 20-199 AFB/1 Length on average
2+ 10-100 AFB/ 1field on average 5-50 AFB/1 field on average
3+ >100 AFB/ Field on average >50 AFB/ Field on average

Critical values
Presence of Acid Fast Bacilli
7.3.15 Limitation of the Procedure and Sources of Error
i. Direct reagents to Sun light
ii. Poor reagent quality
iii. Using wrong reagent Concentration
iv. Using unfiltered reagent
v. Using expired reagent
vi. Poor sample quality
7.3.16 Perfomance characteristics
Not applicable
7.3.17 Supporting documents
Patients Results Register - TB 05
Internal Quality Control
7.3.18 References
✓ International union against tubercle and lung diseases. The public health service
national tuberculosis Reference laboratory and the national laboratory Network
,Paris 1998
✓ Smithwick R.W laboratory Manual for acid fast microscopy .US Department of
Health ,Education and Welfare,CDC,1979
✓ Angra P, Becx-Bleumink M,Glipin C,et al,Ziehl Neelsen staining ,strong red on
week blue, or weak red under strong blue Int J Tuberic Lung Dis 2007:11:1160-1

130
7.4 PROCEDURE FOR GRAMS STAINING
7.4.1 Purpose
This procedure provides instructions on the steps to be followed when performing
Gram staining
7.4.2 Scope
This procedure will be used by all laboratory personnel perform gram staining to
identify bacteria
7.4.3 Responsibility
It is the responsibility of the Head of Microbiology Section to ensure effectively
implemented and maintained .
7.4.4 Principle
Gram stain based on the ability of bacteria cell wall. When the bacteria are stained with
primary stain (crystal violet) and fixed by the mordant (Iodine), Gram Positive bacteria
retain the primary stain when decolorized by ethanol (alcohol) because the cell walls
of gram positive bacteria have THICK layer of protein-sugar components called
peptidoglycan and low lipid contents. Upon decolourization Gram positive bacteria
causes thick cell wall to dehydrate and shrink, which closes the pores in the cell wall
and prevent the stain from existing the cell, therefore ethanol cannot remove crystal
Violet-Iodine complex that is bound to the thick layer which peptidoglycan and appears
BLUE. While Gram Negative bacteria are decolorized by ethanol. For the gram
negative bacteria cell wall takes up the crystal violet-iodine complex but due to the thin
layer of peptidoglycan and thick outer layer with form of lipids, crystal violate- iodine
complex gets washed off, when they are exposed to decolorizer dissolves the lipids in
the cell walls which allow the crystal violet-iodine complex to lead out of the cells then
when again stained with counterstain (safranin) they pick up the safranin and appears
red in color.
7.4.5 Sample Requirement
Fresh collected Pus, urine sediment, CSF, sputum, other body fluids
7.4.6 Equipment
Microscope, Timer, Bunsen burner/hot plate
7.4.7 Materials
Reagents Consumables
• Crystal violet as primary stain • Gloves
• Lugol’s iodine as mordant • Gauze
• 10% Acid/Acetone as • Waste bins
decolourizer
• Grass slides
• Neutral red/safranin as counter
• Applicator stick
stain

131
7.4.8 Storage and Stability
i. Samples are stable at 2-80C for 7 daysdays
ii. store reagentas instructed by manufacturer
7.4.9 Safety
i. Adhere to safety precautions as stated in the Facility Safety manual/ IPC
guideline
ii. All personal protective equipment (PPE) must be worn when performing this
procedure.
iii. All samples must be regarded as potentially infections.
7.4.10 Calibration
Perform Equipment calibration as scheduled.
7.4.11 Quality Control
Quality control is done daily when receiving/preparing a new batch of reagents. A
known sample ATCC for gram positive Staphylococcus aureus and for gram negative
Escherichia coli bacteria respectively are used.
7.4.12 Procedural Steps
Smear preparation and staining
i. Slide with one end frosted should be used for making smears so that a lead
pencil can be used to label the slide clearly.
ii. Use a sterile applicator stick to add one drop of normal saline on the slide.
iii. Use a sterile applicator stick to transfer one pure colony on the slide and make
a smear
iv. Allow smear to air dry on a flat safe place.
v. Flood the fixed smear with crystal violet for 30 seconds
vi. Decant crystal violet and rinse gently slide with running tap water
vii. Flood the slide with Lugol’s iodine for 30 seconds
viii. Rinse off iodine gently with flowing tap water
ix. Decolorize by letting the reagent flow over the smear while the slide is held at
an angle or tilt slide.
x. Adjust decolourisations time to thickness of smear
xi. Remove excess decolorizer with gentle flow of tap water
xii. Flood with neutral red for 30 seconds
xiii. Remove excess neutral red with gentle flow of tap water
xiv. Drain slide and air dry in an upright position
Reading smears
Place the slide on the microscope and ensure that the smear is facing upward
7.4.13 Biological Reference Intervals
Not Applicable

132
7.4.14 Interpretation And Reporting Of Results
Interpretation of results
• If Bacteria pick the color of the counter stain (Neutral red/dilute
Carbolfuschin/safranin) THEN report as Gram Negative (Cocci, Bacilli/Rod
depending on morphological observed/identified cells)
• IF stained bacteria pick the color of the primary stain (crystal violet) THEN
report as Gram Positive (Bacilli/Rods or cocci depending on observed identified
cells)
Reporting of results
• Results should be reported as Gram Positive Rods or Cocci according to the
morphology of the bacteria or
• Gram NEGATIVE Rods or Bacilli according to the shape of bacteria
7.4.15 Limitations Of The Procedure And Source Of Error
Avoid over staining,
Avoid over decolourization
Do not use expired reagent.
7.4.16 Performance Characteristics
Refer to method verification report of this procedure
7.4.17 Supporting Document
Sample collection manual
7.4.18 References
Districtlaboratory practice in tropical countries. Part 2: AuthorMonica Cherbourg 6th
edition

7.5 DIAGNOSIS OF MTB/RIF TESTING USING A TRUENAT MACHINE

7.5.1 Purpose
The purpose of SOP is to describe the stepwise procedure for the rapid detection of
Mycobacteria tuberculosis (MTB) and detection of rifampicin resistance using Truenat
MTB/RIF test for use with the Truenat Dx system is a semi-quantitative real-time PCR
in-vitro diagnostic test for: The detection of MTB DNA in sputum samples, detection of
rifampicin resistance-associated mutations of the rpoB gene. The purpose of SOP is
to describe the stepwise procedure for the rapid detection of Mycobacteria tuberculosis
(MTB) and detection of rifampicin resistance using Truenat. MTB/RIF test for use with
the TrueNat Dx system is a semi-quantitative real-time PCR in-vitro diagnostic test for;
detection of MTB DNA in sputum samples and detection of rifampicin resistance-
associated mutations of the rpoB gene.
7.5.2 Scope

133
 This SOP describes the use of the Truenat™ MTB Plus assay, a chip-based Real-
Time Polymerase Chain Reaction (PCR) test, for the semi-quantitative, detection and
diagnosis of Mycobacterium tuberculosis complex bacteria (MTBC) in human sputum
samples
7.5.3 Responsibility
Qualified and competent health laboratory practitioners are responsible for
implementing this test procedure.
The section head is responsible for ensuring the effective implementation and
maintenance of this procedure.
7.5.4 Principle
The TrueNat assays utilize chip-based real-time micro-polymerase chain reaction
(PCR) for detection of TB and RIF-resistance from Deoxyribonucleic Acid (DNA) that
is extracted from sputum sample within an hour.
Truenat™ MTB Plus works on the principle of Real-Time Polymerase Chain Reaction
where the sputum sample is first liquefied and lysed thereafter the extracted DNA from
the sample is then amplified by the Truelab Real-Time micro-PCR analyser. The
purified DNA is then dispensed into the reaction well of the Truenat™ MTB Plus chip
and the test is started.
Description of the apparatus

Trueprep AUTO Sample Prep device Truelab™ Real Time micro PCR Analyser
7.5.5 Sample Requirements
Sputum sample type, collected as either Spot or morning sputum sample
7.5.6 Equipment
True prep AUTO Sample Prep device, Truelab Real Time micro PCR Analyser and
Refrigerator
7.5.7 Materials
Extraction of DNA Amplification of Materials required Reagents and
purified DNA but not provided Solutions
• Trueprep AUTO v2
• Truelab Duo Real
Universal Cartridge • Truenat™ Positive • Liquefaction
Based Sample Prep Time Quantitative Control Kit - Panel I buffer
Device micro PCR Analyzer• Powder • Lysis buffer
free
disposable gloves

134
• Trueprep AUTO • Truenat™ MTB Plus • Two waste disposal • Conc. bleach
MTB Sample Pre- micro PCR chip containers, with lids, and 70%
treatment Pack • Truelab™ micro containing bleach alcohol
• Trueprep AUTO v2 PCR printer solutions
•
Universal Cartridge Truepet SPA fixed • Timer
Based Sample Prep volume (6 •
µl) Two waste bags
Kit Precision • Micro tube Stand
micropipette • . Cartridge Holder
• • DNase and RNase- • Two cryovials racks
free pipette tips with
filter barrier

Note: Connect a new reagent pack to the Trueprep Auto v2 device by inserting the Plug-in
Connector into the slot provided.

7.5.8 Storage and Stability

store at room temperature for 3 days
Store at 2°C -8°C
7.5.9 Safety
• Treat all biological samples, including used cartridges, as if capable of
transmitting infectious agents. Because it is often impossible to know
which might be infectious, all biological samples should be treated with
universal precautions.
• Wear protective disposable gloves, laboratory coats and eye protection
when handling samples and reagents.
• Wash hands thoroughly after handling samples and test reagents.
Follow safety procedures for working with chemicals and handling
biological samples, (See safety manual)
• Dispose used cartridges according to infectious waste material
disposal guidelines.
7.5.10 Calibration
Calibration is perfumed as per schedule
7.5.11 Quality Control
• Use Truenat Positive Control Kit- Panel containing Positive Control and
Negative Control or use PBS as a negative control and a known positive
culture sample.
• Run positive and negative controls at least one time per month or
➢ When opening a new o test kit lot.
➢ If the temperature of the storage area falls outside of 2-30°C.
➢ New user prior to performing testing on the clinical sample.

135
 ➢ Accept patient results if the positive controls give positive results
while negative controls give negative results. Corrective action should
be taken in case of QC failure either by repeating the control and/or
informing the supervisor.
➢ Whenever a new shipment of test kits is received.
• Records QC results in the TB register.
7.5.12 Procedural Steps
A. Equipment start-up procedure
Press the “Power” button to switch on the Truenat device.
Press ‘start’ and ‘eject’ simultaneously to reset when prompted to change the
Reagent Pack and reset
B. Sample Processing procedure
Prepare Sample and Extract DNA
i. Wear gloves for sample handling.
ii. Label Sputum sample with patient details or lab ID
iii. Add 2 drops of liquefaction buffer to the sputum sample
iv. Close the cap and swirl gently to mix
v. Incubate for 10 minutes at room temperature. If sample is not pipetteable after
10 minutes, incubate for another 5 minutes with swirling at 2-minute intervals
vi. Transfer 0.5 ml of liquefied sputum sample into the lysis buffer bottle using a 1
ml transfer pipette
vii. Add 2 drops of liquefaction buffer into the lysis buffer bottle, swirl gently to mix
and incubate for 3-5 minutes
viii. Remove the cartridge from the pouch, label it and place it on the cartridge stand.
Take out the elute collection tube (ECT) and label it appropriately. Keep it aside
for later use. Keep the elute transfer pipette in the pouch for later use.
ix. Transfer the entire contents of the lysis buffer tube to the sample chamber (black
cap) of the cartridge using 3 ml transfer pipette
x. Switch “on” the Trueprep® AUTO v2 device. Press “eject” button to open and
gently pull out the cartridge holder
xi. Place the cartridge in the tray, and gently push to close the cartridge holder.
Press “start.”
xii. The device will beep at the end of the DNA extraction process (20 minutes), and
the cartridge holder will eject automatically.
xiii. Gently pull out the cartridge holder, remove the cartridge, and place it on the
cartridge stand.
xiv. Carefully pierce the elute chamber with the provided elute transfer pipette, and
transfer the entire elute into the ECT. Discard the transfer pipette and cartridge
C. Running a PCR TB Test

136
 i. Switch “on” the Truelab microPCR analyser by pressing the red button in the
back right corner for 2 seconds. LED will glow in Green. Wait for 30-50 seconds
for “boot-up screen” to appear followed by “home screen.”
ii. Select USER ID, enter password. Press “Sign in” to Log in
iii. Select test profile “MTB” or “MTB Plus”. To confirm selection tap “PROCEED”
and enter patient details (referred by, patient ID, gender, patient name & age)
iv. Select sample type (sputum).
v. Open a TRUENAT™ MTB Plus chip pouch *Pull out the orange desiccant pouch
and confirm that it is orange in colour.
vi. Gently take out the chip without touching white well portion and place it on the
chip tray by aligning it in the slot provided
vii. Press “START TEST” on the screen. Chip tray opens. “Please Load Sample”
will appear. (Don’t press “YES” until chip loading is complete.
viii. Open the master mix tube, discard the stopper and place the tube in the micro
tube stand. *Check for white cake at the bottom of the micro tube.
ix. Attach the 6ul micro tip provided in the pouch to the single push pipette.
x. Transfer 6ul of the elute from ECT into the master mix tube
xi. Allow the master mix to stand for 30 SECONDS to get a clear solution. *Do not
mix by tapping, shaking or reverse pipette. *Do not discard the pipette tip.
xii. Transfer the elute from the master mix tube to the white reaction well of the chip
(Figure 16). *Avoid spillage of the clear solution outside the white reaction well.
*Discard the pipette tip and master mix tube.
xiii. Click “YES” on the device screen to start the test. The PCR will be completed
in 35 minutes.
xiv. Tap the “Open/Close Tray” button to eject the chip tray and discard the used
chip immediately after the reaction.
xv. If MTB is detected test the same elute for RIF resistance using the Truenat MTB
RIF Dx chip as a follow-on test. The test takes about 55 minutes.
D. Running a RIF-Resistance Test
i. If MTB is detected in a sample, Run a RIF resistance test.
ii. Use a portion of the same DNA eluate to test for RIF resistance using a Truenat
MTB-RIF Dx chip.
iii. Start by returning to Step 3 in the PCR TB test process and repeat for RIF-
resistance by Selecting “MTB RIF” as the test type in the Truelab micro–PCR
Analyser.
iv. RIF-resistance testing takes an additional 60 minute.
7.5.13 Biological Reference Interval
Not applicable
7.5.14 Interpretation and Reporting of Results
Interpretation of results
At the end of the test run, the result screen will display;
137
 i. “DETECTED” for Positive result.
ii. “NOT DETECTED” for Negative results.
iii. MTB load as “HIGH”, “MEDIUM”, “LOW” or “VERY LOW” for positive.
iv. The result screen also displays the validity of the test run as “VALID” or
“INVALID”.
NOTE: Invalid samples have to be repeated with fresh samples from the sample
preparation stage.
IPC will co-amplify in most positive cases also, in some samples having a high
target load, the IPC may not amplify, however, the test run is still considered valid
Sample with MTB DETECTED should be tested for MTB RIF
Reporting of results
Click “VIEW RESULTS” on the menu bar. The View Results window appears.
Optional: Press “Print” to print the result page using Truelab® microPCR printer.
Critical value
MTB Detected RIF Resistance Detected
7.5.15 Limitation of the Procedure and Sources of Error
i. Optimal performance of this test requires appropriate sample collection,
handling, storage and transport to the test site.
ii. Though very rare, mutations within the highly conserved regions of the target
genome where the Truenat™ assay primers and/or probe bind may result in the
under-quantitation of or a failure to detect the presence of the concerned
pathogen.
iii. The instruments and assay procedures are designed to minimize the risk of
contamination by PCR amplification products. However, it is essential to follow
good laboratory practices and ensure careful adherence to the procedures
specified in this package insert for avoiding nucleic acid contamination from
previous amplifications, positive controls or samples.
iv. A sample for which the Truenat™ assay reports “Not Detected” cannot be
concluded to be negative for the concerned pathogen. As with any diagnostic
test, results from the Truenat™ assay should be interpreted in the context of
other clinical and laboratory findings.
v. The performance of the test has not been evaluated with samples processed by
methods other than those described in the package insert.
vi. Do not open the cartridge lid except when adding sample.
vii. Do not use a cartridge if it appears wet or if the lid seal appears to have been
broken.
viii. Do not use a cartridge that has a damaged reaction tube.
ix. Each single-use cartridge is used to process one test. Do not reuse spent
cartridges.
7.5.16 Performance Characteristics
Refer to the method verification report of this procedure.
138
7.5.17 Supporting Document
Sample collection manual
7.5.18 References
• Truenat MTB Plus package insert version 5.
• The Trueprep™ AUTO v2 Universal Cartridge Based Sample Prep Device user
manual.
• TBRL Bamenda Biosafety manual, Version 4.0, section 10.
• Truenat™-A Point-of-care Real Time PCR Test for Tuberculosis, video by
Molbio available at https://youtu.be/ydR2I5S2v3

139
ANNEX 1: LIST OF PARTICIPANTS WHO
DEVELOPED THE SOPS

SN NAME TITILE FACILITY

1. Adam Mwakyoma Laboratory Scientist Kilimanjaro Christian
Medical Centre
(KCMC), Moshi
2. Amedeus Mushi Laboratory Scientist HLPC, MoH, Dodoma
& Epidemiologist
3. Anjela Maufi Laboratory Scientist Kitete RRH, Tabora
4. Antusa Massawe Laboratory Scientist Dunga H/C, Tanga
5. Baraka Mathias Laboratory Scientist Maranatha Hospital,
Mbeya
6. Betrand Msemwa Microbiology & Catholic University of
Immunologist Health and Alliened
Sciences (CUHAS),
Mwanza
7. Bezard Ngumbuchi Quality Officer PHLB, Dodoma
8. Ceif Abdul Quality Officer NPHL, Dar es Salaam
9. David Ocheng Laboratory Expert/ Ilala, Dar es Salaam
Consultant
10. Desmond Kileo ICTO DHCTSU, MoH,
Dodoma
11. Dickson Charles Kakuntebe Laboratory Scientist Cardinal Rugambwa
Hospital, Dar es Salaam
12. Dominic Fwiling’afu Registrar PHLB, MoH, Dodoma
13. Dr. Alex Magesa DDS DHCTSU, MoH,
Dodoma
14. Dr. Goodluck Tesha Epidemiologist Jhpiego, Dar es Salaam
15. Dunstan Haule Laboratory Quality NBTS-HQ, Dar es
Officer Salaam
16. Edward Lushishi Laboratory Singida RRH, Singida
Technologist
17. Emmanuel Ndaki Laboratory Scientist NBTS Lake zone,
Mwanza
18. Eng. Gervas H. Swai Biomedical Engineer DHCTSU, MoH,
Dodoma
19. Eng. Suniva S. C. Haule Head Health Care DHCTSU, MoH,
and Technical Dodoma
Services
20. Ezekiel Mng’ong’o Laboratory Scientist Maranatha Hospital,
Mbeya
21. Farhiya Mohamed Laboratory Quality NBTS Eastern Zone,
Officer Dar es Salaam

140
SN NAME TITILE FACILITY
22. Ferdinand Matata Head of Diagnostic PORALG, Dodoma
Services
23. Frank Changala Laboratory Scientist NPHL, Dar es Salaam
24. Dr Haji Hussein Technical officer ICAP, Dar es Salaam
25. Frank Mushi Laboratory Scientist Manyara RRH,
Manyara
26. Frank Shemhande Laboratory Quality SBNRH-Ndanda,
Officer Mtwara
27. Godfrey Mahundi Laboratory Manager Dodoma MC Hospital
28. Helman Mhangala Deputy Laboratory NBTS Central Zone,
Manager Dodoma
29. Ibrahim Mauki Laboratory Scientist NPHL, Dar es Salaam
30. Idrissa Hassan Laboratory Service PORALG, Dodoma
Coordinator
31. Innocent A. Chinguile Laboratory Scientist Training, MoH, Dodoma
& Epidemiologist
32. Jackson Luvamunda Quality Officer Sinza HC, Dar es
Salaam
33. Jackson Zengo Deputy National DHCTSU, MoH,
Laboratory Quality Dodoma
Officer
34. Jacob Lusekelo National Laboratory DHCTSU, MoH,
Quality Officer Dodoma
35. James Mziray Laboratory Service PO-RALG, Dodoma
Coordinator
36. Joakim Chacha LS-HSEEP DHCTSU, MoH,
Dodoma
37. Joyce Samson Laboratory Quality Sekou-Toure RRH,
Officer Mwanza
38. Kadogosa Saimon Laboratory Singida RRH, Singida
Technologist
39. Lanja Kahemela Laboratory Quality MNH, Mloganzila, Dar
Officer es Salaam
40. Maige Ngeleja DSCMC MoH, Dodoma
41. Mary F. Mtui Registrar HLPC, MoH, Dodoma
42. Mayala Lushina Laboratory Quality MNH, Upanga, Dar es
Officer Salaam
43. Michael Mazoya Laboratory Scientist NBTS, Dar es Salaam
44. Mzelifa Daudi Microbiologist University of Dodoma
(UDOM), Dodoma
45. Pius R. Tarimo Laboratory Scientist Kilimanjaro Christian
& Epidemiologist Medical University
College (KCMUCo),
Moshi
46. Rajabu Muninge Laboratory Scientist Maranatha Hospital,
Mbeya

141
SN NAME TITILE FACILITY
47. Rashid Nassoro Laboratory Quality Morogoro RRH,
Officer Morogoro
48. Reuben Abednego Laboratory Scientist NPHL, Dar es Salaam
49. Reuben Lema Deputy Laboratory Morogoro RRH,
Manager Morogoro
50. Reuben S. Mkala Ag. Head of DHCTSU, MoH,
Laboratory Service Dodoma
51. Richard Kinyaha Laboratory Scientist Kibong’oto IDH,
Kilimanjaro
52. Robert Makala Regional Laboratory RAS - Manyara
Technologist (RLT)
53. Sabra Rashid Laboratory Scientist MoH, Unguja-Zanzibar
54. Said Lunemhya Laboratory Scientist Benjamin Mkapa ZRH,
Dodoma
55. Scolastica S. Manyama Laboratory Quality Mirembe Hospital,
Officer Dodoma
56. Susu Jeremiah Susu Laboratory Quality Benjamin Mkapa ZRH,
Officer Dodoma
57. Tamaly Lutufyo Laboratory Manager CCBRT, Dar es Salaam
58. Ulfat Rahim Laboratory TMJ, Dar es Salaam
Technologist
59. William S Mollel Laboratory MOLLEL LABORATORY,
Technologist Dodoma
60. Yahya Mnung’a MeLSAT-President MeLSAT, Dar es
Salaam
61. Yohana Shirima Laboratory NBTS Northern zone,
Technologist Moshi
62. Yulitha Barnabas Laboratory Manager Dodoma RRH, Dodoma
63. Zacharia Omary Laboratory Manager Sekou-Toure RRH,
Mwanza

142
ANNEX 2: BIOLOGICAL REFERENCE
INTERVALS FOR FULL BLOOD COUNT
Reference Range
Parameter Adult Infants Children
Males Females Birth 1 month 1 year SI Unit
WBC 4.00 - 10.0 4.0 - 10 10 - 26 5.0 - 9.0 6.0 - 16.0 x103 /µL
NEU 40 - 80 40 - 80 40 - 80 40 - 80 40 - 80 %
LYM 20 - 40 20 - 40 20 - 40 20 - 40 20 - 40 %
MON 2 - 10 2 - 10 2 - 10 2 - 10 2 - 10 %
EOS 1–6 1-6 1-6 1-6 1-6 %
BASO <1 - 2 <1 - 2 <1 - 2 <1 - 2 <1 - 2 %
RBC 4.5 - 5.5 3.8 - 4.8 5.0 - 7.0 3.0 - 5.4 3.9 - 5.1 x106 /µL
HGB 13.0 - 17.0 12.0 -15.0 14.0 - 22.0 11.5 - 16.5 11.1 - 14.1 g/dL
MCV 83.0 - 99.0 83.0 - 99.0 100 -120 92 - 116 72 - 84 Fl
MCH 27.0 - 32.0 27.0 - 32.0 31.0 - 37.0 30.0 - 36 25.0 - 29.0 Pg
MCHC 31.5 - 34.5 31.5 - 34.5 30.0 - 36.0 29.0 - 37.0 32.0 - 36.0 g/dl
Hct 40 – 50 36 - 46 45 - 75 33 - 53 30 - 38 %
RDW
PLT 150 - 410 150 - 410 100 - 450 200 - 500 200 - 550 x103 /µL
MPV

143
ANNEX 3: BIOLOGICAL REFERENCE
INTERVALS FOR COAGULATION PROFILE
TEST RANGE SI UNIT
Prothrombin Time 9.40 - 12.50 Sec
Activated Prothrombin Time 25.40 – 36.90 Sec
Fibrinogen 2.20 – 2.80 g/l
Factor V 0.62 -1.39 IU
Factor VII 0.50 -1.29 IU
Factor VIII 0.50 -1.50 IU
Factor IX 0.65 -1.50 IU
Free protein S (male) 74.10 - 145.10 %
Free protein S (female) 54.70 - 123.70 %
Protein S activity 63.50 - 149.00 %
Protein C may be less in neonates, infants and 70.00 – 140.00 %
increase in adolescence
Plaminogen (activity) 80.20 - 132.50 %
Plamin inhibitor 98.00 – 122.00 %
Homocysteine 4.30 - 11.10 μmol/L
D-dimmer ≤ 232 ng/ml
von will brand factor ristocetin 480 - 201.90 %
cofactor activity (blood group O)
Von Will brand Factor ricostein 60.80 - 239.80 %
factor activity (blood A+B+AB)

144
ANNEX 4: BIOLOGICAL REFERENCE
INTERVALS FOR URINE BIOCHEMISTRY
Parameter Abbreviation Biological Reference Intervals
Urobilinogen URO Normal
Glucose GLU Negative
Bilirubin BIL Negative
Ketones KET Negative
Specific gravity S.G 1.003-1.029
Occult blood BLD Negative
Ph Ph 4.5 - 7.8
Protein PRO Negative
Nitrite NIT Negative
Leukocytes LEU Negative

145
ANNEX 5: BIOLOGICAL REFERENCE
INTERVALS FOR CLINICAL CHEMISTRY AND
IMMUNOASSAYS
Test Name Normal range SI Unit
Parameter/Analyte Sub category
Alanine aminotransferase
0 - 55 U/L
(ALT)
Albumin 35 – 50 g/l
Male 15-125 U/L
Female 15-125 U/L
Male child 0 - 500 U/L
Female Child 0 - 500 U/L
Children U/L
Aged 1 day <250 U/L
Aged 2 -5 days <231 U/L
Alkaline Phosphate
Aged 6 days – 6 months <449 U/L
Aged 7months – 1 year <462 U/L
Aged 1-3years <281 U/L
Aged 4– 6years <269 U/L
Aged 7 – 12 years <300 U/L
Aged 13 – 17 years (M) <390 U/L
Aged 13 – 17 years (F) <187 U/L
Aspatate Aminotransferase 5 - 34 U/L
Bilirubin – Direct 0 – 8.6 µmol/L
Bilirubin – Total 3.4 - 20.5 General µmol/l
Total Protein 64 - 83 g/l
Male 12- 64 U/L
Female 9-36 U/L
Gamma Glutamyl Transferase
Male child 9-36 U/L
Female child 9-36 U/L
CSF protein 0-4.3 lumbar fluid g/L
CSF glucose One third of Glucose g/L
Asciti Protein 60-80 g/L
Ascitic Glucose 70 - 100 mg/dL
Adult Male 63.6-110.5 µmol/L
Adult Female 50.4 – 98.1 µmol/L
Creatinine
Male child 27 - 88 µmol/L
Female child 27-88 µmol/L

146
Test Name Normal range SI Unit
Male 3.2- 7.4 µmol/L
Female 2.5-6.7 µmol/L
Blood Urea Nitrogen (BUN)
Male child 3.2-7.4 µmol/L
Female Child 2.5- 6.7 µmol/L
Cholesterol Total < 5.2 mmol/L
HDL - Cholesterol 1.04 – 1.55 mmol/L
LDL – Cholesterol 0 - 3.34 mmolL
Triglycerides 0 - 1.69 mmol/L
Sodium ( Na ) 136 – 145 mmol/L
Potassium ( K ) 3.5 – 5.1 mmol/L
Chloride (Cl ) 98 - 107 mmol/L
Amylase Total 25- 125 U/L
Male 30 – 200 U/L
Female 29- 168 U/L
Creatine Kinase ( CK )
Male child 30 – 200 U/L
Female child 29-168 U/L
Lactate dehydrogenase (LDH) 125 - 220 IU/L
Lipase 13- 60 U/L
Male 2.1 – 2.55 mmol/L
Famale 2.1-2.55 mmol/L
Calcium
Male child 2.2 – 2.7 mmol/L
Female child 2.2 – 2.7 mmol/L
Glucose 3.3 - 6.1 mmol/L
Male 5.5 – 25.8 µmol/L
Female 4.5 - 25.8 µmol/L
Iron
Male child 5.5 – 25.8 µmol/L
Female child 4.5 – 25.8 µmol/L
% Saturation (Iron saturation) 20 - 50 %
Male 0.21 - 0.42 mmol/L
Female 0.15 - 0.35 mmol/L
Uric Acid
Male child 0.21 - 0.42 mmol/L
Female child 0.15 - 0.35 mmol/L
Phosphorus 0.74 - 1.52 mmol/L
Sodium 24hrs Urine 27 - 287 mmol/24 hours
Potassium 24hrs Urine 25 - 125 mmol/24hrs
Male 1.74 - 3.64 g/L
Female 1.8 - 3.82 g/L
Transferrin
Male child 1.86 – 3.88 g/L
Female child 1.86 - 3.88 g/L
Alpha Feto Protein 0.0 - 1.09 ng/ml
High Sensitive Troponin 13.8-17.5 Female pg/ml

147
Test Name Normal range SI Unit
28.9-39.2 Male pg/ml
Vitamin B12 187-883 pg/ml
Ferritin 10 - 250 ng/ml
Folate 3.72 - 50.4 ng/ml
PSA 0.0 - 4.0 ng/ml
TSH 0.49 - 4.67 IU/ml
T4 0.47 - 4.67 ng/L
T3 1.45 - 3.48 pg/ml
CK-MB 0.0 - 6 %
Tacrolimus 3 - 20 ng/ml
BNP 0-142 pg/ml
Cyclosporine 30.0-1500 ng/ml
CEA 0-5 ng/ml
CA-125 0-35 IU/mL
Less than 5 for non pregnant mIu/mL
B-HCG
25 for early pregnancy mIu/mL
Vitamin D 0 - 160 ng/ml
Immunoglobulin G 5.40-18-22 Male g/l
5.52-16.31 Female g/l
1-12 months <15 IU/mL
1-5 years <60 IU/mL
Immunoglobulin E 6-9 years <90 IU/mL
10-15 years <200 IU/mL
Adults <100 IU/mL
Male 63 - 645 mg/dl
Female 65 - 517 mg/dl
Immunoglobulin A
Male child 21- 291 mg/dl
Female child 21 - 281 mg/dl
Male 0.22-2.40 g/l
Female g/l
Immunoglobulin M
0.33-2.93 g/l
Either 0.22-2.93 g/l
D - Dimer 0 .0 - 198 ng/L
CRP 0.0-5.0 mg/L
Follicular phase 21-251 pg/ML
Midcycle phase 38-649 pg/ML
Lueal phase 21-312 pg/ML
Estradiol
Postmenopausal female 10-
pg/ML
28
Male 11-44 pg/ML
Prolactin Male ng/ml

148
Test Name Normal range SI Unit
3.46-19.40 ng/ml
Female5.18-26.53 ng/ml
Male 4.94-32.01 nmol/L
Testosterone
Female 0.38-1.97 nmol/L
Follicular phase 0.1-0.3 ng/ml
Luteal phase 1.2-15.9 ng/ml
Postmenopausal 0.1-0.2 ng/ml
Progesteron First trimester 2.8-147.3 ng/ml
Second trimester 22.5-95.3 ng/ml
Third trimester 27.9-242.5 ng/ml
Male 0.1-0.2 ng/ml
Male 1.14-8.75 ng/ml
Follicular phase 2.39-6.60 ng/ml
Midcycle peak-9.06-74.24 ng/ml
LH
Luteal phase 0.909.33 ng/ml
Postmenopausal ng/ml
10.39-64.57 ng/ml
Male 0.95-11.95 mIu/mL
Follicular phase 3.03-8..08 mIu/mL
Midcycle peak 2.55-16.69 mIu/mL
FSH
Luteal phase 1.38-5.47 mIu/mL
Postmenopausal mIu/mL
26.72-133.41 mIu/mL
Male 0.66 - 1.07 mmol/L
Female 0.66 - 1.07 mmol/L
Magnesium
Male child 0.70 - 0.86 mmol/L
Female 0.70 - 0.86 mmol/L
ADA 0 - 15 U/L
Glycated haemoglobin
4-6 %
(HBA1C)

149
ANNEX 6: CRITICAL/PANIC VALUES THAT
CALLS FOR IMMEDIATE ACTIONS
Analyte Less Than Greater Than
Amylase 25 U/L 150 U/L
Chloride 85 mmol/L 115 mmol/L
CK 30 U/L 200 U/L
Creatinine 26 umol/L 120 umol/L
Glucose(fasting) 2.5 mmol/L 20.0 mmol/L
Potassium 2.5 mmol/L 6.0 mmol/L
Sodium 120 mmol/L 160 mmol/L
Bilirubin Total 3.4 umol/L 20.5 umol/L
Biliribun Total for new Newborn
Born 24hours ≥ 1374 umol/L
48hours ≥ 2224 umol/L
84hours ≥2904 umol/L
One week to one month ≥3424 umol/L
Urea (BUN) ≤1.0mmol/L ≥ 54 mmol/L
HGB < 5 mg/dl > 20 g/dl
CD4 200 cells/l

150
NOTE PAD

151
NOTE PAD

152
NOTE PAD

153
 FOR FURTHER INFORMATION CONTACT:

PERMANENT SECRETARY
MINISTRY OF HEALTH,
MAGUFULI GOVERNMENT CITY, AFYA ROAD/STREET, MTUMBA,
PO BOX 743,
40478 DODOMA, TANZANIA.
LANDLINE: +255 (0)26 232 3267
EMAIL: [email protected]
WEBSITE: www.moh.go.tz