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TA B L E O F C O N T E N T S
Acknowledgements 3
Introduction 4
Amendment Procedure 6
Process Flow Chart 7
1. Title 8
2. Purpose 8
3. Introduction 8
4. Scope 9
5. Staff Competency Requirements 9

6. Safety Instructions 9
7. Pre-Examination Procedures 10

7.1 Sample Type 10
7.2 Sample Collection 10
7.3 Sample Transport & Storage 11
7.4 Rejection Critiria 11

7.5 Relevant Clinical Information 11

8. Table of Media, Reagents, Materials & Equipment 12
9. Examination Procedures 12
9.1 Quality Control 12
9.2 Microscopy 12
9.3 Culture 13

9.4 Identification 13
9.5 Susceptibility Testing 14
9.6 Sample Referral 14
10. Limitations 14
11. Post-Examination Procedures 14
11.1 Interpretation of Results 14
11.2 Reporting 14
11.3 Sample Retention, Storage & Disposal 15
12. Limitations and Pitfalls of the Procedure 16
13. References 16

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INTRODUCTION

The Caribbean Regional Standard Methods include a variety This initiative should enable the region to implement a
of standard, validated methods, produced as a single standardized and constructive method for ensuring that
standard operating procedure (SOP) for use in a variety of validated methods are available for the region, and that they
levels of microbiology laboratory service. It is intended that are updated as required.
these methods provide detailed instructions for microbiology
services for microbiological investigations, in order to Advantages of using regionally validated methods are to
provide accurate, reliable and reproducible results which improve quality, make better use of resources, reduce costs,
will have clinical utility. These methods may be adopted by enable central procurement & media preparation, facilitate
laboratories within the region, or adapted, provided that such staff training and transfers due to horizontal integration, a
adaptations use an evidence-based validation process. reduction in variability of service provision, an improved
quality of surveillance data, and the purchase of appropriate
These methods have been developed by the Caribbean equipment. A major advantage is that the availability of
Regional Microbiology Standard Methods Drafting Group regional standard methods would assist microbiology
(CSMDG) in response to a request by the Caribbean laboratories with documentation for accreditation
Regional Microbiology Council (CRMC), which was set up by
the CARIFORUM Project entitled ‘Strengthening of Medical Although the CSMDG has taken every care with the
Laboratories in the Caribbean’ to strengthen specifically the preparation and issue of these standard procedures, and they
microbiology services in the Caribbean Region. The Project have been validated regionally, nationally and internationally,
was initiated in response to findings which indicated that the CSMDG, or any other organization, cannot be responsible
there was an unacceptable level of error in laboratories within for the accuracy of any statement or representation made
the region. External quality assessment results revealed that or the consequences arising from the use of or alteration to
microbiology laboratories were not performing well and any information contained in them. These procedures are
feedback from the region via laboratory staff, lab managers intended solely as a resource for practicing microbiology
and directors was that they felt that guidance in microbiology professionals in the field, operating in the Caribbean region,
requirements was required. and specialist advice should be obtained where necessary.
If changes are made to the original publication, it must be
The background for this initiative is a worldwide move to made clear where changes have been made to the original
implement standards in all areas, which has now extended document. When referring to these SOPs in successive
to include medical laboratories. As tourism is so vital to documentation, the CSMDG should be acknowledged.
the region’s economy, the need for accurate diagnosis and
treatment is paramount. It was accepted that there is a
requirement for validated methods for accreditation purposes
and providing validated standard methods will assist in the
move towards accreditation.

The methods will be chosen for standardization by the

Caribbean Regional Microbiology Council, and this selection
will be based on a review of EQA results, most common and/
or critical tests. Part of the method standardization process
will be an ongoing review and amendment procedure. The
CSMDG consists of microbiology laboratory representatives
from most of the CARIFORUM countries, all of whom were
nominated to the task by the CRMC.

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AMENDMENT PROCEDURE

Controlled Document Reference CRM-SOP 16

Controlled Document Title Standard Operating Procedure for Urine Microscopy

Each Regional Standard Method should be reviewed annually by the Caribbean Standard Methods Drafting
Group. Any amendments should be validated and authorized by an agreed process, and referenced.

Each Regional Standard Method has an individual record of amendments. The current amendments are listed
on this page.

On issue of revised or new pages, each controlled document should be updated by the copyholder in the
laboratory.

Amendment Issue Number Insert Page Section(s) Amendment

Number / Date Discarded Issue Number involved

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PROCESS FLOW CHART

URINE MICROSCOPY

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1. Title

Urine Microscopy.

2. Purpose

To ensure that the correct, validated procedure is followed for the microscopic examination of urine to provide accurate,
reliable, reproducible results having clinical utility.

3. Introduction

The diagnosis of Urinary Tract Infection (UTI) relies on the microscopic examination of the cellular components of urine and
qualitative culture. Clinical evaluation may also be required in addition to laboratory results.

Microscopy is used to identify white blood cells (WBCs), red blood cells (RBCs), casts, squamous epithelial cells, bacteria,
yeast, Trichomonas vaginalis and other cellular components in the urine.

Microscopy need not be performed on all urines when screening for asymptomatic bacteruria is required (e.g. ante-natal clinic
screening), and may be omitted if in compliance with local protocols.

Microscopy of uncentrifuged, unstained urine may be used as a method of screening for bacteriuria without the need for culture,
but is unreliable to detect counts <107 colony forming units per litre (cfu/L) ie <104 colony forming units per millilitre (cfu/mL).

Significant pyuria is defined as the occurrence of 107 or more WBC/l (104 WBC/mL) although higher numbers of WBC are
often found in healthy asymptomatic women. A level of >108 WBC/l (>105 WBC/mL) is possibly more appropriate in indicating
infection. Pyuria is present in 96% of symptomatic patients with significant bacteriuria of >108 cfu/L (105 cfu/mL), but only in
<1% of asymptomatic, abacteriuric patients. It should be determined accurately in uncentrifuged urine.

Pyuria alone is not a reliable indicator of infection and may be present as a result of other conditions such as genital tract
infection, catheterisation, calculi (stones) or bladder neoplasms.

Pyuria without apparent bacteriuria (ie no growth on routine culture media) may also be a result of prior treatment with
antimicrobial agents, extreme frequency, infection with fastidious organisms or sexually transmitted diseases such as C.
trachomatis. Renal tuberculosis is far less common as a cause of sterile pyuria, but may still need to be considered if clinically
appropriate (eg in high risk populations).

Pyuria may be absent in symptomless bacteriuria (eg in pregnancy) and neutropenia, and apparently absent in UTI caused by
Proteus species as a result of leucocyte lysis at alkaline pH.

Significant pyuria and/or bacteriuria are suggestive of UTI and may be used as criteria for selection of samples for direct
sensitivity testing.

Haematuria may be seen in 40-60% of patients with acute cystitis but is rarely seen in association with other dysuric syndromes.
Numbers of RBCs up to 107/L are normal. Haematuria may be caused by non-infective pathological renal conditions or by renal
mycobacterial infection, with or without associated pyuria. Apparent haematuria may be the result of menstruation.

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3. Introduction continued

RBC lysis may occur in hypertonic and hypotonic urine, rendering them undetectable by microscopy.

Casts are cylindrical protein mouldings formed in the renal tubules and often give clues to renal pathology. Recognition of casts
is important in helping to establish the existence of renal disease, but is less useful in differentiating among renal disorders.

Hyaline casts: Large numbers of hyaline casts are associated with renal disease but may be found in patients with fever or
following strenuous exercise.

Cellular and densely granular casts: indicate pyelonephritis or glomerulonephritis.

RBC casts: usually indicate glomerular bleeding and are excreted in large numbers in the acute phase of post-streptococcal
nephritis or rapidly progressive nephritis.

Epithelial cell and fatty casts: these are less common and accompany acute tubular necrosis and nephrotic syndrome.

Crystals may be asymptomatic or may be associated with the formation of urinary tract calculi. Some crystals such as cystine
are rarely seen and may indicate an underlying disease. The presence of triple phosphate crystals indicates alkaline urine and
is commonly associated with the presence of Proteus sp.

Squamous epithelial cells (SECs) are a useful indicator of the degree of contamination from the perianal region

Trichomonas vaginalis in the urine of women is abnormal and is usually indicative of acute vaginitis. This organism can also
be seen in the urine of infected men.

4. Scope

This procedure provides detailed instructions for Microbiology services performed on urines, as offered by regional laboratories.
It may be adopted or adapted by any laboratory as needed, provided that such adaptations use an evidence based validation
process.

5. Staff Competency Requirements

Laboratory personnel, trained and assessed to be competent to perform this procedure.

6. Safety Instructions

Please refer to CRM-SOP 20: Safety In The Microbiology Laboratory.

Keep all books, forms and papers away from technical work surfaces.

Level 2 containment; require all personal protective equipment (PPE).

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6. Safety Instructions continued

All microbiological samples for processing should be considered a potential source of transmissible infections so universal
safety precautions should be observed.

Hands should be thoroughly washed with soap and water before and after handling all samples.

Disinfect all work surfaces with 70% alcohol or a freshly prepared 10% bleach solution prior to testing and after processing.

All samples and reagents should be properly discarded according to the current standards for disposal of hazardous waste.

Samples and culture plates should be autoclaved before finally discarding.

7. Pre-Examination Procedures

7.1 Sample Type

Mid-stream urine, clean-catch urine, suprapubic aspirate (SPA), catheter urine, bag urine.

7.2 Sample Collection

Samples should be collected before the initiation of antimicrobial therapy wherever possible.

7.2.1 Mid-stream urine.

The first urine sample passed at the beginning of the day is preferred if possible, since this is the most concentrated
and therefore ideal for microscopy and culture.

1. The first part of voided urine is discarded and without interrupting the flow, approximately 10mls.is
collected into a suitable, sterile, wide-mouth, leak-proof container and the remaining urine is discarded.

7.2.2 Clean-catch urine

Thorough peri-urethral cleaning is recommended. The whole sample is collected into a sterile container and then an
aliquot sent for examination.

7.2.3 Supra-pubic aspirate.

Urine is obtained aseptically, directly from the bladder by aspiration with a needle and a syringe. This invasive procedure
is usually performed by a specialist staff for the clarification of anomalous culture results in infants and children.

7.2.4 Catheter urine.

May be obtained either from a transient (in-out) catheterization or from an indwelling catheter. In the latter, the sample
is obtained aseptically from a sample port in the catheter tubing or by aseptic aspiration of the tubing. Samples
should NOT be taken from the collection bag as they are frequently contaminated.

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7. Pre-Examination Procedures continued

7.2.5 Bag urine

Used mainly for infants and young children. Sterile bags taped over the freshly cleaned and dried genitalia, and the
collected urine is transferred to a sterile leak-proof container.

7.3 Sample Transport & Storage

Samples should be transported and processed within 4 hours if possible. If processing is delayed for up to 48 hours,
refrigeration at 2-8°C is essential. Boric acid preservative can be used.

7.4 Rejection Criteria

Reject samples:

• That are not labeled or that are incorrectly labeled.

• That are sent to the laboratory without an accompanying requisition form.
• If information on form and sample do not correlate exactly.
• If incorrect sample received.
• Samples received in inappropriate containers older than 48 hours samples that were not properly stored
(refrigerated or preserved) prior to arrival at the laboratory received in leaking, damaged containers are
unsuitable.

7.5 Relevant Clinical Information

Antimicrobial therapy, if any.

Symptoms such as frequency, dysuria, urgency, nocturia.
Chronic urinary tract infection.
Clinical syndromes (pyelonephritis, cystitis, urethritis, epididymitis, prostatitis) with fever.
Clinical conditions which may influence interpretation of results eg pregnancy, diabetes.

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8. Table of Media, Reagents, Materials and Equipment

Equipment Materials Reagents (Media)

Either an inverted Slides and cover slips Commercial normal

microscope or light Centrifuge tubes and abnormal control
microscope capable Marker/ grease pencil to label tubes samples
of providing optimal Pasteur pipette or transfer pipette
conditions of light to
Reference book or aids to assist in
enhance and contrast
identification of crystals and casts
the small differences of
density in urine elements Paper towels or tissues
Lens cleaner
Centrifuge Lens paper
Control samples
bulb for Pasteur pipette

9. Examination Procedures

9.1 Quality Control

Please refer to CRM-SOP 18: Media Preparation and Quality Control.

Please refer to CRM-SOP 21: Quality Control of Reagents and Tests.

Please refer to CRM-SOP 19: Propagation and Maintenance of Quality Control Organsims.

Commercial control samples are available.

Check all reagents and stains before use to ensure that they are free from contamination, debris or deposits.

9.2 Microscopy

9.2.1 Direct Method

a. Place 3 loopfuls of fresh, thoroughly mixed urine onto a clean glass slide.
b. Gently cover with a cover glass, taking care to avoid air bubbles.
c. Carefully examine the preparation using the 10X and the 40X objectives with the iris condenser sufficiently
closed to give good contrast. Examine for the presence of bacteria, white blood cells, red blood cells,
crystals, casts, yeasts and epithelial cells. Record T. vaginalis if present.

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9. Examination Procedures continued

9.2.2 Inverted Microscope Method

a. Mix urine gently, to avoid foaming.

b. Pipette approximately 60µl (refer to Appendix 1) of well mixed urine into a numbered well in a flat bottomed
microtitre tray making sure the sample covers the whole bottom area.
c. Allow to settle for a minimum of 5 minutes, but preferably 10-15 minutes before reading with an inverted
microscope.
d. Examine with the 20x objective.
e. Scan several fields in each well to check for even distribution of cells and urine.

This SOP contains a table of multiplicative factors to correct for variability in microtitre tray well size based on varying
volumes of urine dispensed, diameter of well and field of vision diameter. (see Appendix 1). The number of WBCs
counted should be multiplied by the multiplicative factor to take into account all the variables. If the well size, volume
of urine dispensed, diameter of well or field of vision diameter are altered then the multiplicative factor must be re-
calculated.

Microscopy should not be performed on screening samples sent exclusively for the isolation of S. typhi and S.
paratyphi.

9.2.3 Deposit Method

a. Describe the appearance of the urine sample: color, and whether clear or cloudy.
b. Gently invert the urine to mix and aseptically transfer approximately 10 mls into a clean, labeled, plastic
conical centrifuge tube.
c. Centrifuge at 1500-2000 rpm for 5 minutes.
d. Pour off the supernatant.
e. Re-suspend the sediment in the small volume of remaining urine.
f. Place a drop of well mixed, re-suspended sediment onto a clean, glass slide, and cover with a cover slip.
(Do not discard the remaining sediment until the entire testing procedure is completed).
g. Examine using the x10x and the x40x objectives with the iris condenser sufficiently closed to give good
contrast.
h. Report the presence of the following: bacteria, white blood cells, red blood cells, casts, crystals, yeasts,
T. vaginalis and epithelial cells. Ova of Schistosoma. haematobium may be seen as a chance finding but
an MSU is not the recommended sample for its detection.

9.3 Culture

N/A.

9.4 Identification

N/A.

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9. Examination Procedures continued

9.5 Susceptibility Testing

N/A.

9.6 Sample Referral

N/A.

10. Limitations

Cloudiness may develop in some urine due to the precipitation of urates (in acidic urine)or phosphates and carbonates (in
alkaline urine). Urates may impart a pink-orange colour to the urine. These precipitates may be dissolved before microscopy
by warming the urine to 37°C, thus facilitating the differentiation of any cellular components.

The accuracy of urine microscopy usually decreases with time due to lysis of the cells and multiplication of bacteria, particularly
if the sample is left unrefrigerated or is not in a boric acid container.

11. Post Examination Procedures

11.1 Interpretation of Results

The most accurate method of determining the numbers of White Blood Cells (WBCs) and Red Blood Cells (RBCs) is
using the inverted microscope technique or the kova-slide method where a fixed volume of urine is examined and
numbers of cells counted. This may then be converted to the number of cells per ml.

The direct and deposit method only allow an estimation of the numbers of cells per field of vision in an unknown
volume of urine.

11.2 Reporting

Identify and Quantify Urine components as follows:

Direct method

White Blood Cells:

Report average number per HPF

Red Blood Cells:

Report average number per HPF

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11. Post Examination Procedures continued

Epithelial Cells:

Few 2-5 in every HPF

Moderate 5-10 in every HPF
Many > 10 in every HPF

Crystals:
Report presence of significant crystals such as cystine only.

Casts:
Identify type and report numbers per LPF.

Bacteria:
Negative, light, moderate, heavy, or none seen, scant, 1+, 2+, 3+, 4+.

Yeast Cells and T. vaginalis:

Report presence only.

Deposit method

Report as for direct method but indicate that results are from spun deposit.

Inverted microscopy method

Count or estimate the range of WBCs and RBCs and convert to number of cells per ml (by multiplying the number of
cells counted by the multiplicative factor indicated in Appendix 1).

Interpretation of the significance of the numbers each cell type should be indicated on the report and will vary with
the method used.

Interpretation of results should be made in conjunction with the culture result.

Urgent requests should be telephoned or sent electronically as soon as results become available.

A written report should be sent out within 16-24 hours of receipt of a sample.

11.3 Sample Retention, storage & Disposal

Keep samples for a minimum of 3 days, and preferably a week, in the refrigerator (2-8°C) after results are sent out.

Samples should be autoclaved before final discarding according to the current standards for disposal of hazardous
waste.

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12. Limitations and Pitfalls of the Procedure

Delay in processing may give falsely low WBC due to lysis of the cells unless boric acid containers are used.

Delay in processing and/or storage at ambient temperature for prolonged periods may result in bacterial overgrowth.

Bag Urine: Artificially elevated leucocyte counts may be seen as a result of vaginal reflux or recent circumcision with round
epithelial cells found in urines from neonates.

Catheter Urine: WBCs may be present in catheter urine as a result of the natural defence mechanism against a foreign body.

Mid-stream Urine: Improper cleaning of peri-urethral area may cause sample contamination with vaginal WBCs and by
bacterial flora.

13. References

1. Health Protection Agency (2004). Investigation of Urine. National Standard Method CRM-SOP 41 Issue 5.
2. Cheesbrough Medical Laboratory Manual for Tropical countries.

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