Type verification sheet (scope A) / validation

(scope B) of a medical biology method

Note: the laboratory will refer to the table in § 9 of the Cofrac SH GTA 04 rev 01 document for
know the parameters to be determined as part of an on-site verification (scope A) or a
validation (scope B) and will complete a form for each medical biology exam

MEDICAL BIOLOGY EXAM

Identification of the parameter (as identified in the detailed list of examinations):
Malaria diagnosis.
Simple process Complex process (subprocess name: …2)

DESCRIPTION OF THE PROCESS

Verification/validation procedures1:
5 smears/1 operator 1. Repeatability
6 smears / 5 operators Intermediate loyalty
3 smears / 12 operators 3. Inter-operator variability
NA Justness
EEQ: BP and Kalidiv (19 5. Exactitude
programmes)
Under- 6. Analytical sensitivity and specificity
Bibliographie
process 1:
Risk assessment 7. Uncertainties
Thin smear
Bibliography 8. Measurement extent
Analysis of results: 9. Comparison of methods
1166 smears
NA 10. Interferences
NA 11. Contamination
Bibliography 12. Robustness and reliability of reagents
NA 13. Reference interval
Verification/Validation Procedures:
2 drops / 5 operators 1. Repeatability
5 drops / 4 operators 2. Intermediate fidelity
1 drop / 5 operators 3. Inter-operator variability
NA 4. Justness
EEQ: BP and Kalidiv (11 5. Exactitude
Under
programmes 6. Analytical sensitivity and specificity
process 2:
Bibliography 7. Uncertainties
Drop
Risk assessment 8. Measurement extent
thick
Bibliography 9. Comparison of methods
Data analysis
10. Interferences
NA
11. Contamination
NA
12. Robustness and reliability of reagents
Bibliography
NA 13. Reference interval
For each step, the laboratory will proceed to verify/validate the expected items, and
duplicate as many copies as needed of pages 2 to 8 (performance evaluation of the method) of
present document. If another element of the process seems critical to him, he must verify/validate this
step and specify it in the reasoned conclusion. It is this verification that will allow him to
master this critical point.

Argumentation (if applicable):

1
Note: For the verification/validation of quantitative methods, the information for items 1, 2, 4, 5, 7, 8, 9, 10, 11, 12, and 13
is expected at a minimum. For the verification/validation of qualitative methods, the information for items 3, 6, 8, 9, 10, 11, 12
and 13 is expected, at a minimum.
The type of verification (bibliographic or trials) must be indicated.
The non-applicability of certain items (NA) must be justified in the body of the document.

SH FORM 43 rev 01 - April 15, 2015 Page 1 of 25

 Type verification sheet (scope A) / validation
(scope B) of a medical biology method

SUB-PROCESS 1: Diff Quick stained thin smear

Scope A Scope B (to be justified)

DESCRIPTION OF THE METHOD

Analyte / Measured Item : Hematozoa responsible for malaria
Principle of the Method: Microscopic observation of a thin blood smear
colored by a rapid panoptic coloring, RAL 555
with the RAL Stainer colorant (or by the coloring of
Diff Quick in degraded solution: research and
identification of Plasmodiums.
Calculation of parasitemia: percentage of the number
of parasitized red blood cells in relation to the total number
of observed red blood cells
Type of primary sample: Total amount
Type of container, additives: Tube with EDTA or ACD (citric acid-dextrose)
Preprocessing of the sample: After homogenization you withdrawal by
twisting, spreading of a total blood smear
in thin layer under PSM II
Units: % for the parasitemia
Interpretation criteria2: Negative if no parasite observed after observation of
40,000 red blood cells

Marquage CE (Oui/Non) : YES

C.N.Q. coding (if it exists): PAR
Equipment (instrument, analyzer, etc.): RAL Stainer Colorator Series No. 1641
Optical microscope with white light, Zeiss, objective at
immersion X100
Reagent reference: Methanol
Reference: 20846.292
Supplier: VWR International
Storage conditions: store between 15 and
25°C
RAL Stainer Kit 555
Référence : 362870, 361640, 361645, 361650
Supplier: RAL Diagnostics
Stability: storage between 15°C and 25°C (tolerance
up to 40°C) until the expiry date, 30 cycles of
coloration or 14 days on board the RAL Stainer
Diff Quick Solution 1 Medion Diagnostics
Diff Quick Solution 2 Medion Diagnostics
Référence : 726445, 726446
Supplier: Medion Diagnostics AG
Conservation conditions: storage between 15 and
30°C
Calibration material (references): NA
Type of calibration, number of levels and
NA
values:

2
Indicate the reference values if different depending on the anticoagulant. Take into account sex, age...

SH FORM 43 rev 01 - April 15, 2015 Page 2 of 25

 Typical verification form (scope A) / validation
(scope B) of a medical biology method
IMPLEMENTATION
Qualified and recognized operator(s) Technicians, biologists, and interns authorized according to
competent(s) who carried out the
on-call planning
method verification/validation:
Validation procedure/operating mode: PN_VAM_P_001: Method validation procedure
Flexible Scope Management Procedure: PN_VAM_P_002 : flexible scope management
Study period: Method used since 1990 with change of
colorant in 2016 after internal validation.
Retrospective study with the use of EEQ since
2013 and on-site study 2016-2017.
Date of 1erausage: 1990 with the implementation of the RAL Stainer in 2016

SH FORM 43 rev 01 - April 15, 2015 Page 3 of 25

 Type of verification sheet (scope A) / validation
(scope B) of a medical biology method

RISK MANAGEMENT
(the laboratory will adapt the critical points to be mastered based on the table below for each
verified/validated parameter
Means of mastery (training of
personnel, verification
Scale experimental, trial games, ...
5M Critical points of Elements to master Documents (procedure,
criticality3 instruction, recording, ...)
with the references of the QMS of
laboratory
Identity 5 Training and information of Non-Conformity Management:
Personnel PN_DYS_P_001: Procedure of
management of non-conformities and
complaints, risk management
and tracking of actions
PN_REC_P_009 : Management of a
pre-analytical non-conformity (NCA)
in the laboratory
PN_REC_M_034: Management of non-
compliance of welcome in
Parasitology
Preparation you 3 Patient Information and Sampling Instructions:
patient : collectors hospital withdrawal manual
Information Bichat
epidemiological
Type of 4 Training of collectors Sampling instructions:
containers: catalogue of analyses, sheets of
Tube EDTA, tube request of the exams of
ACD Parasitology - Mycology
Nature and volume 3 Control à
of the sample: acceptance reception/of PN_PRA_M_028 : pre-analytical
total sang, on ACD refusal of collection: malaria diagnostics
(filled à 75%) refusal of dry tube and exemption PN_REC_F_077 : registration
minimum) or EDTA potential yes others malaria requests
anticoagulants
non-compliance if deadline Empowerment of the agents of the
transmission >2h (emergency) reception
PN_TOUS_E_011 : tutoring grid
Deadline and 4 Logistics management (shuttles,reception agents in
temperature before transport speakers) : parasitology
treatment pneumatic transport PN_GRH_E_786 criteria
analytical <2h or directly by the of authorization for reception agents
after sampling staff nurse of the PN_GRH_E_233initial assessment
to meet the deadline care services the skills of the agents of
of rendering (4h); reception
transport à PN_GRH_E_234Evaluation form
temperature of skills maintenance -
ambient Reception and pre-analytical agent

3
To be specified by the laboratory, for example 1 non-critical - 5 very critical;

SH FORM 43 rev 01 - April 15, 2015 Page 4 of 25

 Type verification sheet (scope A) / validation
(scope B) of a medical biology method
RISK MANAGEMENT
(the laboratory will adapt the critical points to be mastered based on the table below for each
parameter checked/validated
Means of control (training of
personnel, verification
Scale experimental, test games, ...
5M Critical points of Eléments à maîtriser Documents (procedure,
criticality instruction, recording, ...
with the references of the QMS of
laboratory
Conditions of 2 The samples are taken PN_TOUS_P_009: diagnostic of
conservation of in charge upon receipt then malaria
samples (t°, stored between 2 and 8°C PN_ANA_M_049 : malaria : smear
) : 24h à 20- for 30 days or before blood
30°C, 72h at + 4°C transmission to CNR PN_VAB_M_090 : validation
Conditions of 2 Malaria. biological of diagnostic you
conservation and The reagents used for malaria
of use of the colorations are preserved Records metrological:
reagents (temperature, ...): between 18°C and 25°C in thePN_MAI_M_008Management of units
15°-30°C solvent cabinets. watched and connection
Temperatures of metrological of the loudspeakers
conservation of thermostats
samples and reagents reported of temperatures
are monitored using the atmosphere:
Sirius software. PN_MAI_E_016Table of statements
Stress sheet for manual reactants of temperatures
transmits by thePN_MAI_E_085Drift analysis
suppliers. from a surveillance probe
PN_MAI_F_015 degraded mode in
sensor failure cases
temperature
Connection of the enclosures:
PN_MAI_M_028 Management and
connection metrological of the
surveillance probes
Technical data sheets of reagents:
PN_ANA_F_078 : technical sheet of
RAL Stainer
PN_ANA_F_082Technical sheet
staining by Diff Quick
Water quality 3 pH measurement (pH paper) Traceability of the verifications:
Monitoring of pH of
tap water

SH FORM 43 rev 01 - April 15, 2015 Page 5 of 25

 Standard verification sheet (scope A) / validation
(scope B) of a medical biology method
RISK MANAGEMENT
(the laboratory will adapt the critical points to be mastered based on the table below for each
checked/validated parameter
Means of mastery (training of
staff, verification
Scale experimental, test games, ...
5M Critical points of Elements to master Documents (procédure,
criticality instruction, registration, ...
with the references of the QMS of
laboratory
Surveillance of the 3 Daily cleaning of records of the
drifts objectives, maintenance of microscopes and of
Microscope annual of microscopes colorist in: Kalilab
Automate of
coloration Monitoring of PN_GCQ_E_134 colorations: monitoring of CQI
Blades smear: CQI of the Smears Malaria Smear
of observation PN_GCQ_E_150: monitoring table
degreased of CQI malaria
Annual EQ: ANSM + Recording of the EEQ in Glims
Biology perspective orPN_GCQ_E_053Individual collection
Kalidiv results of the EEQ
Monitoring the maintenance of PN_GRH_E_735: criteria
skills of the standby authorization malaria.
staff participating in PN_GRH_E_489: evaluation sheet
constraints of the diagnostic of maintaining skills:
malaria by reading the diagnosis of malaria
EEQ blades
Management of stocks de
Mastery of equipment: consumables
absence of breaks PN_GFO_F_030Process sheet -
Purchasing and Inventory Management
Contamination Individual NA tests
Computer Science 4 Registration and entryPN_SIL_M_005Qualification of
manual information systems
PN_REC_F_077 : registration
Traceability of the malaria request observer
and the validating biologist on PN_ANA_F_165: print list of
the workbench sheet malaria work
PN_TOUS_F_022: Acquisition of
Transmission of malaria results in GLIMS
by phone and on server PN_VAB_M_090: validation
computer science after biological validation of the diagnosis of
biological malaria

SH FORM 43 rev 01 – April 15, 2015 Page 6 of 25

 Standard verification sheet (scope A) / validation
(scope B) of a medical biology method
RISK MANAGEMENT
(The laboratory will adapt the critical points to be mastered based on the table below for each)
verified/validated parameter
Means of control (training of
staff, verification
Scale experimental, trial games, ...
5M Critical points of Elements to master Documents (procedure,
criticality instruction, recording, ...
with the references of the QMS of
laboratory
Conservation and 2 The reagents used for the PN_GFO_E_045: tracking sheet for
conditions colorations are preserved batches of malaria reagents
of use between 18°C and 25°C in thePN_HSL_F_062Process sheet -
solvent cabinets. Hygiene, safety, and vigilance
The temperatures of
conservation ofPN_PIL_F_016Organization of the
samples and laboratory metrology reagents
are monitored using thePN_MAI_M_028Management et
Sirius software. metrological connection of the
Stress sheet of monitoring probe reagents
transmits by thePN_MAI_M_028 Management and
suppliers connection metrological of the
monitoring probes
PN_MAI_E_016Statement table
manuals of temperatures

Inventory Management3 Inventory Management PN_GFO_F_030Process sheet -

Monthly orders Purchasing and Inventory Management
Monitoring of reagents PN_HSL_P_003: Procedure
reactovigilance
PN_GFO_E_045 : tracking sheet of
lots of malaria reagents

Reconstitution of Ready-to-use reagents

reagents

Water quality 3 pH measurement (pH paper) Traceability of the checks:

PN_PREP_E_016: Monitoring of pH of
tap water
Follow-upof the lots of reagents:
PN_GFO_E_045 : tracking sheet of
Malaria reagent lots
Achievement you 3 Froth bloody conducted PN_ANA_M_049: malaria: smears
smear manually under PSM 2 blood
according to the operating procedure

Achievement of the 4 Fixation of the smear by the PN_ANA_M_049: malaria: smear

coloration methanol after drying sanguine
Coloration with RAL PN_ANA_DE_015: user manual
Staining with the reagents from the RAL Stainer kit
RAL 555 according to protocol PN_ANA_P_032: user manual of the
validated in the laboratory (or RAL Stainer
manually with PN_ANA_F_078: technical data sheet kit
quick colorants in RAL Stainer 555
degraded procedure.
Quality of the coloring PN_GCQ_M_054: CQI Smear
verified during blood observation for malaria
microscopic of the smear PN_GCQ_E_134: monitoring of CQI
Quality control of Malaria Smears
regular smears

SH FORM 43 rev 01 - April 15, 2015 Page 7 of 25

 Type of verification sheet (scope A) / validation
(scope B) of a medical biology method
RISK MANAGEMENT
(the laboratory will adapt the critical points to master based on the table below for each
parameter checked/validated
Means of control (training of
staff, verification
Scale experimental, test games, ...
5 million Critical points of Elements to master Documents (procedure,
criticality instruction, recording, ...
with the references of the QMS of
laboratory
2 Observation of 40 000 PN_ANA_M_049 : paludisme : frottis
Detection threshold red blood cells
realization and blood
reading of thick drop PN_VEI_DE_073: Update 2017
if negative or weakly some CPR 2007 on management
positive. Sensitivity: 100p/µl and prevention you malaria
importation
Causes 2 Withdrawal dilute, Training and habilitation you
of uncertainty of anomalies of the personal globules; color control;
measure reds; coloration of respect for operating procedures
bad quality PN_GRH_E_490 : habilitation
malaria diagnostic assessment
Render from Deadline for submission from PN_TOUS_P_009: diagnosis of
results résultats : dans les 2 heures paludisme
following the receipt of PN_VAB_M_090: validation
biological sampling you diagnostic of
initial diagnosis. malaria

Service of Systematic call of PN_TOUS_P_009: diagnosis of

advice results: transmission to malaria
prescribers for the PN_VAB_M_090: validation
biological hospital samples you diagnostic of
Bichat; transmission to malaria
laboratories transmittersPN_VAB_M_006Validation UF of
for the GH and the parasitology hospitals
exteriors.
Traceability of the
communication of the result
in GLIMS and on the sheet
of the workbench

SH FORM 43 rev 01 – April 15, 2015 Page 8 of 25

 Type verification sheet (scope A) / validation
(scope B) of a medical biology method
RISK MANAGEMENT
(The laboratory will adjust the critical points to be mastered based on the table below for each)
verified/validated parameter)
Means of mastery (training of
staff, verification
Scale experimental, trial games, ...
5M Critical points of Elements to master Documents (procedure,
criticality instruction, recording, ...
with the references of the QMS of
laboratory
Skill and 4 Malaria Diagnosis Skills Records
maintenance of carried out according to the staff schedule:
competence you penalty by personnel - authorization file
staff qualified: biologist, intern PN_GRH_E_487: tutoring sheet
You technician. diagnostic constraint of malaria
Staff training: training of personnel
initial training recognition of blades
maintenance of skills PN_GRH_E_735 : criteria
malaria standby authorization
PN_GRH_E_490 : habilitation
malaria diagnostic constraint
PN_GRH_E_488: evaluation sheet
initial skills: on-call duty
of malaria
maintenance of skills / EEQ :
reading by all staff
individually (4/year)
PN_GRH_E_489: evaluation sheet
of maintaining skills:
malaria diagnosis
Traceability of occupancy
workstations : PN_SOI_E_008 :
planning for parasitology on-call duty

SH FORM 43 rev 01 – April 15, 2015 Page 9 of 25

 Type of verification form (scope A) / validation
(scope B) of a medical biology method

EVALUATION OF THE METHOD'S PERFORMANCE

Specify the type and reference of sample (control sample, serum pool, ...): blood spread
Quality of the single-layer smear
Quality of the coloration: gray-violet erythrocytes, neutrophilic poly-nuclear cells with a purple nucleus
dark and well-visible granules, dark nucleus for the lymphocytes.
Recognition of Plasmodiums (red nucleus and blue cytoplasm) and identification of the species
(see operating procedure)
Parasitemia (see operating procedure)
Observation of other pathogens such as Trypanosomes, Babesia, microfilariae

REPEATABILITY
Applicable not applicable (to be justified)
Retention CV(%)
Name
Cart- CV(%) by the
Samples of values Average CV(%) Conclusion5
type supplier laboratory (cf.
(N)
source4)
30-60
15%
Smear limit
(CV variability
sanguine 5 0.95 0.14 14.7 acceptability correct
inter-operator
1708020268 biology
on EEQ)
prospective
Rationale for the conclusion: Visual reading on 100 red blood cells / field

INTERMEDIARY LOYALTY
Applicable not applicable (to be justified)

The laboratory assessed the reproducibility of parasitemia from slides made from samples.
blood samples received for a diagnosis of malaria, read by different technicians. The variability of parasitemia must
to be integrated into its clinical meaning and its consequences on the therapeutic management of the patient.
CV(%) retained
Name
Cart- CV(%) by the
Samples of values Average CV(%) Conclusion5
type supplier laboratory (cf.
(N)
source4)
Precision of
Smear
the reading of
sanguine
30-60 the
1708014656 4 3.85 0.17 4.5 15%
(limit parasitemia
1708014944 4 1.95 0.13 6.62 (CV variability
acceptability CV included
1708017621 4 0.92 0.19 20.4 inter-operator
biology between 4.5% and
1507031555 5 0.57 0.13 22.8 on EEQ
prospective 23%, without
1708012457 4 0.015 0.001 8.5
consequences
clinics.

INTER-OPERATOR VARIABILITY
Applicable ; non applicable

Evaluated operators: technicians, Individual results of EEQ (4/year); 8 to 12 operators:

interns and biologists negative results: no variability
- positive results: no variability in species identification
- parasitemia: CV from 13 to 47.5% depending on the parasitic load
Echantillon 2014-2 : parasitémie : m = 3,97% ; CV = 13% (n=11)

4
Scholarly societies, publications (SFBC, GEHT, RICOS, QUALAB, CLIA…). Specify the reference used.
5
Compliant/Non-compliant

SH FORM 43 rev 01 – April 15, 2015 Page 10 of 25

 Type of verification sheet (scope A) / validation
(scope B) of a medical biology method
Sample 2013-4: parasitemia: m = 0.04%; CV = 47.5% (n=12)
Sample 2013-4: parasitemia: m = 0.06%; CV = 33% (n=8)

The assessment of parasitemia is imperfect, particularly in low cases.

values, but the estimated values remain within the limits of their importance in clinical practice.

JUSTICE (from outsourced CIQs)

Applicable not applicable (to be justified)
Name Average
Target Bias(%) Biais(%) Biais(%)
of Values general
Samples (group group average Conclusion5
values Lab
of pairs of pairs
(all
general Limit4
(N) techniques
Sample
CIQ level 1
Sample
CIQ level 2
Argumentation of the conclusion: microscopic examination, no quantitative result

ACCURACY (based on occasional external checks: EEQ/CNQ)

Quantitative controls Quality controls

Target Biais(%)Biais(%)/ Bias

Value
Samples Target (all / group whole (%) Conclusion5
Lab
techniques of pairs technique limit4
Biology
prospective
F2013-1 Pf 0.5 Pf 0.5
F2013-2 Po 0.13 Po 0.2
F2013-3 Pm 0.023 Pm <0.1
F2013-4 Po 0.06 Po <0.1
F2014-1 Negative Negative
F2014-2 Pf 3.5 Pf 2.6
F2014-3 Negative Negative
F2014-4 Pf 0.75 Pf 0.7
F2015-1A Po 0.014 Po 0.15
F2015-2A Pf 1.03 Pf 0.5 NA To be filled out for quantitative controls Compliant
F2015-3A Pf 0.4 Pf 1.4
F2015-4A Pf 2.2 (NR) NC
F2016-1A Po 0.06 After 0.1
F2016-2A Negative Negative
F2016-3A Pf 0.08 Pf 0.1
F2016-4A After 0.3 After 0.3
Kalidiv
2017-1 Pf 0.07 Pf 0.22±-0.08
2017-2 Pv 0.4 Pv
2017-3 Pf 0.13 Pf

Argument of the conclusion: results compliant (or letter A) for the EEC on blood smears since 2015,
for the qualitative result (positive/negative) or identification of the species and the parasitemia.

ANALYTICAL SENSITIVITY and SPECIFICITY

(essential experimental study in scope B)
(experimental study possible if relevant in scope A)
Applicable not applicable (to be justified)
True positives Spécificité, sensibilité, VPN, VPP
False positives
True negatives
False negatives
Argument for the conclusion: 146 malaria diagnoses in 2016, 168 in 2017. Analytical performance
preserved from year to year despite staff turnover and the change of staining reagent.

MEASUREMENT UNCERTAINTY (levels, choice of calculation method, interpretation):

Chosen methodology: risk analysis (absence of residual interference) calculation
Requirement for
Calculated uncertainties
performances

SH FORM 43 rev 01 – April 15, 2015 Page 11 of 25

 Type of verification form (scope A) / validation
(scope B) of a medical biology method
Calculation method (see SH GTA 14): Formula used Reference
Argument for the conclusion: qualitative method; no calculation possible if negative

SH FORM 43 rev 01 - April 15, 2015 Page 12 of 25

 Type of verification form (scope A) / validation
(scope B) of a medical biology method

DETECTION LIMIT (experimental study essential in scope B)

(experimental study possible if relevant in scope A)
Applicable not applicable
Detection limit: Theoretical sensitivity: 100p/µl under the conditions of
reading of smears on 40,000 erythrocytes, considering
4.5 T/L of red blood cells.
Bibliography: WHO manual on diagnosis of
malaria
Argument for the conclusion: 100p/µl

COMPARISON OF METHODS:
Applicable; non-applicable (to be justified)
Data bibliographic (suppliers, 2017 update of the consensus conference)
publications,...) : 2007 ; WHO Malaria Diagnostic Manual
Previous method, another method used in thick drop on negative smears
laboratory, mirror or backup device, EBMD:
Name of measures: In 2017, out of 426 F/GE: 421 negative GE, and 5 GE
positives (1.18%) don't 2 withdrawals under
treatments.
In 2016, out of 740 F/GE: 728 negative GE, and 12 GE
positives (1.6%) don't 6 withdrawals under
treatments.
Comparison interval adapted to the extent of Absence or presence of Plasmodium
laboratory measurements: Identification of the species involved
Parasitemia: 0.0025%
Method of exploiting results: Comparison of qualitative results
Equation of the regression line: NA
Diagram of differences and/or relationships: NA
Argument of the conclusion: The reading of the smears is sensitive, 98% of negative results are confirmed in
thick drop. The 2017 results are comparable to those of 2016: the change of dye and personnel
does not influence our performance.

MEASUREMENT EXTENT (essential experimental study in scope B)

(experimental study possible if relevant in scope A for:
troponin, microalbumin, platelets, PSA, TSH, ...
Applicable not applicable (to be justified)
Detection limit: Literature review (sources and Experimental study (values)
values)
Limit of quantification: Bibliographic study (sources and Experimental study (values)
values
Upper limit of linearity: Bibliographic study (sources and Experimental study (values)
values)
Argument of the conclusion: microscopic reading, non-linear quantification. Detection limit associated with
our reading criteria: observation of 40,000 red blood cells, which corresponds to a detection limit of one parasitized red blood cell on
40000, or about 100 parasites/µl for an average blood count of 4.0T/L.

INTERFERENCES (essential experimental study in scope B)

(experimental study possible if relevant in scope A for: Hemolysis, turbidity, bilirubin,
medications, ... - to take into account in the variability factors - to evaluate if necessary)
Applicable not applicable (to be justified)
Hemolysis Specify supplier data or overload test (LIH module)
Turbidity Specify supplier data or overload test (LIH module)
Bilirubin, jaundice Specify supplier data or overload test (LIH module)

SH FORM 43 rev 01 – April 15, 2015 Page 13 of 25

 Type of verification sheet (scope A) / validation
(scope B) of a medical biology method
Medications Specify supplier data or overload test
… Specify supplier data or overload testing
Rationale of the conclusion: The observation of a blood smear stained with Giemsa is not sensitive to these
interference.

CONTAMINATION (essential experimental study in scope B)

(experimental study possible if relevant in scope A for sensitive parameters)
Applicable not applicable (to be justified)
Inter-sample for sensitive parameters (by Specify the supplier data or trial of
example Ag HBS, βHCG, ... : surcharge
Reactive inter if necessary (for example: LDH and Specify the supplier data or on-site testing
ALT, cholesterol and phosphate, lipase and
triglycerides, ...
Argument for the conclusion: individual manual test, no possible contamination

ROBUSTNESS and STABILITY of REAGENTS

(indispensable experimental study in scope B)
(experimental study possible if relevant in scope A for sensitive parameters)
Applicable ; not applicable (to be justified)

Sensitive parameters tested (temperature, pH, position on a The RAL 555 reagents for the RAL Stainer have a
support, …) RFID chip that allows monitoring their use.
A quality control of the coloring is conducted.
during each change of reagent kit.
Stability of reagents after opening, onboard, ... The reagents are valid for 14 days after opening.
or for 300 coloring cycles. They are kept
at room temperature before use. Their
the optimal storage temperature is between 15-
25°C but the laboratory confirms their stability
up to 40°C (RAL mail).
Reasoning of the conclusion: The quality of the reagents used is controlled.

REFERENCE INTERVALS and/or threshold values based on demographic data (study

indispensable experimental in scope B)
Applicable not applicable
Reference values Specify supplier data or on-site testing
Argument for the conclusion:

DECLARATION of FITNESS
Conclusion: compliant method used since 15/12/2017...

Authorized by: S. Houzé, reference biologist of the sector

Signature

SH FORM 43 rev 01 - April 15, 2015 Page 14 of 25

 Fiche type de vérification (portée A) / validation
(scope B) of a medical biology method

SUB-PROCESS 2: Thick drop

Range A Scope B (to be justified)

DESCRIPTION OF THE METHOD

Analyte / Measured Variable: Hematzoa responsible for malaria
Principle of the Method: Microscopic observation of a colored thick droplet
by a Giemsa solution: research and identification
of Plasmodium. Observation of 1000 leukocytes before
to produce a negative result.
Calculation of parasitemia: number of observed parasites
for 1000 leukocytes counted.
Type of primary sample: Total song
Type de récipient, additifs : Tube with EDTA or ACD (citric acid-dextrose)
Sample pre-treatment: Spreading a thick drop from whole blood
under PSM II
Units: Number of parasites per 1000 white blood cells (WBC)
Interpretation criteria6: Negative if no parasite observed after observation of
1000GB
Marquage CE (Oui/Non) : YES
C.N.Q. coding (if it exists):
Equipment (instrument, analyzer, etc.): Optical microscope with white light, Zeiss, objective at
immersion X100
Reagent reference: - Giemsa R
Reference: 320310-0125
Fournisseur : RAL Diagnostics
Storage conditions: between 15°C and 25°C
(tolerance up to 40°C) until expiration date
- Tap water
Supplier: city of Paris
Calibration material (references): NA
Type of calibration, number of levels and
NA
values:

IMPLEMENTATION
Qualified and recognized operator(s) Technicians, biologists, and interns authorized according to
competent(s) who carried out the
on-call planning
method verification/validation:
Validation procedure/work method: PN_VAM_P_001 : Method validation procedure
Flexible scope management procedure: PN_VAM_P_002 : flexible scope management
Study period: Method used since 1990
Retrospective study with use of EQs since
2013
Date of 1erause : 1990

6
Indicate the reference values if different depending on the anticoagulant. Take into account sex, age...

SH FORM 43 rev 01 – April 15, 2015 Page 15 of 25

 Typical verification form (scope A) / validation
(scope B) of a medical biology method

RISK MANAGEMENT
(the laboratory will adapt the critical points to master based on the table below for each
parameter checked/validated
Means of control (training of
staff, verification
Scale experimental, trial games, ...
5M Critical points of Elements to master Documents (procedure,
criticality7 instruction, enregistrement, …)
with the references of the QMS of
laboratory
Identity 4 Training and information on NC Management:
personnel PN_DYS_P_001: Procedure of
management of non-conformities and
claims, risk management
and the tracking of actions
PN_REC_P_009: Management of a
pre-analytical non-conformity (NCA)
in the laboratory
PN_REC_M_034 : Management of non-
compliance welcome in
Parasitology
Information 3 Patient information and collection instructions: manual
epidemiological collectors from the Bichat hospital
Result you smear and GLIMS laboratory software
priorities of requests
Type of 4 Training of collectors Instructions of withdrawal:
containers : catalog of analyses, sheets of
EDTA tube, tube request for examinations of
ACD Parasitology - mycology
Nature and volume 3 Control à
of the sample: receipt of acceptance/from PN_PRA_M_028 : pre-analytical
sang total, tube refusal of the withdrawal des diagnostics du paludisme
ACD filled to 75% refusal dry tube, and exemptionPN_REC_F_077 : recording
minimum or EDTA possible yes others malaria requests
anticoagulants no
compliance if deadline of Authorization of agents of the
transmission >2h (emergency) reception
PN_TOUS_E_011 : tutoring grid
Deadline and 4 Logistics management (shuttles,reception agents in
temperature before transport speakers: parasitology
treatment pneumatic transport PN_GRH_E_786 criteria
analytical <1h or directly by the of authorization of agents of
after sampling staff nurse of reception
to meet the deadline care services PN_GRH_E_233initial assessment
of rendering (2h); skills of the agents of
transport à reception
temperature PN_GRH_E_234: Sheet
ambient of evaluation of maintenanceof the
skills - Reception agent and
pre-analytical

SH FORM 43 rev 01 – April 15, 2015 Page 16 of 25

 Standard verification sheet (scope A) / validation
(scope B) of a medical biology method
RISK MANAGEMENT
(the laboratory will adapt the critical points to be mastered based on the table below for each
checked/validated parameter
Means of mastery (training of
staff, verification
Scale experimental, test games, ...
5 million Critical points of Elements to master Documents (procedure,
criticality instruction, recording, ...
with the references of the QMS of
laboratory
Conditions of 2 Samples are taken PN_TOUS_P_009: diagnosis of
conservation of in charge upon receipt then malaria
samples (t°, stored between 2 and 8°C PN_ANA_M_050: thick drop
24h à 20- for 30 days or earlier for malaria
30°C, 72h à + 4°C transmission to CNR PN_VAB_M_090 : validation
Conditions of 2 Malaria. biological diagnosis of
conservation and The reagents used for malaria
of use of the colors are preserved Records metrological:
reagents (temperature, ...): between 18°C and 25°C in thePN_MAI_M_008: Management of
15°-30°C solvent cabinets. monitored units and connection
The temperatures of metrology of the speakers
conservation of thermostated
samples and reagents Recorded of the temperatures
are monitored using the atmosphere:
Sirius software. PN_MAI_E_016: Tableau of
Stress sheet of manually recorded reagent temperatures
transmits by thePN_MAI_E_085Drift analysis
suppliers. from a surveillance probe
PN_MAI_F_015Reduced mode in
sensor failure cases of
temperature
Connection of the speakers:
PN_MAI_M_028 Managementand
metrological connection of
surveillance probes
Technical sheets of the reagents:
PN_PREP_DE_018technical sheet
Giemsa R RAL
Surveillance of the 3 Daily cleaning of the records of the
derivatives objectives, maintenance of maintenances
microscopes
Microscope annual of microscopes in Kalilab
Blades
of observation Coloring time: 20 Stopwatch recording
degreased minutes in Kalilab and connection
Chronometer metrological of the speakers
thermostated

EEQ annual Biology Recording of the EQEs in Glims

perspective or Kalidiv PN_GCQ_E_053Individual collection
results of the EEQ
Monitoring of the maintenance of PN_GRH_E_735: criteria
skills of standby authorization malaria.
staff participating in PN_GRH_E_489: evaluation form
constraints of the diagnostic of the maintenance of skills:
malaria through the reading of malaria diagnostics
EEQ blades

Mastery of equipment: Management of the stocks of

absence of breaks consumables : PN_GFO_F_030
Process sheet - Purchases and Management
des stocks
Contamination Individual NA tests

SH FORM 43 rev 01 – April 15, 2015 Page 17 of 25

 Standard verification sheet (scope A) / validation
(scope B) of a medical biology method
RISK MANAGEMENT
(the laboratory will adapt the critical points to master based on the table below for each
verified/validated parameter
Means of mastery (training of
staff, verification
Scale experimental, trial games, ...
5M Critical points of Elements to master Documents (procedure,
criticality instruction, recording, ...
with the references of the QMS from
laboratory
Computer science 4 Recording andentryPN_SIL_M_005Qualification of
manual information systems
PN_REC_F_077 : registration
Traceability of the malaria request observer
and from the validating biologist on PN_ANA_F_165: print list
the laboratory bench sheet malaria work
PN_TOUS_F_022: Acquisition of
Transmission of malaria results in GLIMS
by phone and on server PN_VAB_M_090: validation
computer science after biological validation of the diagnosis of
biological malaria
Conservation and 2 The reagents used for PN_GFO_E_045: tracking sheet of
conditions Colorations are preserved lots of malaria reagents.
of use between 18°C and 25°C in thePN_HSL_F_062Process sheet -
solvent cabinets. Hygiene, safety and vigilance
The temperatures ofPN_PIL_F_016Organization of the
conservation laboratory metrology
samples and reagentsPN_MAI_M_028 Managementand
are monitored using the metrological connection of the
Sirius software. surveillance probes
Stress sheet of reagentsPN_MAI_M_028Management and
transmits by the connections metrological of
suppliers surveillance probes
PN_MAI_E_016: Tableau of
manual temperature readings

Inventory Management3 Inventory Management PN_GFO_F_030Process sheet -

Monthly orders Purchasing and Inventory Management
Tracking of reagents PN_HSL_P_003Procedure
pharmacovigilance
PN_GFO_E_045: tracking sheet of
lots of malaria reagents
Reconstitution of Giemsa dilution before PN_ANA_M_050: thick drop
reagents coloration for malaria
PN_TOUS_E_037: Giemsa Monitoring
diluted
Water quality 3 pH measurement (pH paper) Traceability of the checks:
PN_PREP_E_016
Realization of the 3 Thick drop made PN_ANA_M_050: thick drop
thick drop manually under PSM 2 for malaria
according to the operating procedure

SH FORM 43 rev 01 – April 15, 2015 Page 18 of 25

 Type verification sheet (scope A) / validation
(scope B) of a medical biology method
RISK MANAGEMENT
(the laboratory will adapt the critical points to master based on the table below for each
checked/validated parameter
Means of control (training of
staff, verification
Scale experimental, test games, ...
5M Critical points of Elements to master Documents (procedure,
criticality instruction, registration, ...
with the references of the QMS of
laboratory
Realization of the 4 After drying, PN_ANA_M_050: thick drop
coloration dehemoglobinization and for malaria
staining with Giemsa R
diluted to 7% in PN_TOUS_E_037 water: Giemsa monitoring
faucet in a diluted box
Laveran pendant 20
minutes.
Quality of the coloring
verified during the observation
microscopic of the drop
thick
2 Observation of 1000 PN_ANA_M_050 : thick drop
Detection threshold leukocytes. for malaria
Sensitivity 10p/µl PN_VEI_DE_073 : Mise à jour 2017
from the RCP 2007 on care management
and prevention you malaria
importation
Causes 2 Withdrawal diluted, Training and habilitation you
of uncertainty of anomalies of personal globules; color control;
measure red; coloration of respect for operating procedures
poor quality PN_GRH_E_490 : habilitation
diagnostic assessment of malaria

Render of Delay of rendered of PN_TOUS_P_009: diagnostic of

results résultats : dans les 2 heures paludisme
following the receipt of PN_VAB_M_090: validation
biological sampling you diagnosis of
initial diagnostic. malaria

Performance of Systematic call of PN_TOUS_P_009: diagnosis of

advice results: transmission to malaria
prescribers for the PN_VAB_M_090 : validation
biological hospital samples you diagnosis of
Bichat; transmission of malaria
laboratories transmittersPN_VAB_M_006Validation UF of
for the GH and parasitology hospitals
exteriors.
Traceability of the
communication of the result
in GLIMS and on the sheet
of workbench

SH FORM 43 rev 01 – April 15, 2015 Page 19 of 25

 Standard verification sheet (scope A) / validation
(scope B) of a medical biology method
RISK MANAGEMENT
(the laboratory will adapt the critical points to master based on the table below for each
verified/validated parameter
Means of mastery (training of
staff, verification
Scale experimental, test games, ...
5M Critical points of Elements to master Documents (procedure,
criticality instruction, recording, ...
with the references of the QMS of
laboratory
Competence and 4 Malaria diagnosis Skills records
maintenance of carried out according to the staff schedule:
competence of penalty by personnel - authorization file
staff enabled: biologist, intern PN_GRH_E_487: tutoring sheet
you technician. penalty for diagnosis of malaria
Staff habilitation: staff training
initial training recognition of blades
maintaining skills PN_GRH_E_735 : criteria
malaria standby authorization
PN_GRH_E_490 : habilitation
malaria diagnostic penalty
PN_GRH_E_488: evaluation sheet
initial skills: on-call
of malaria
maintenance of skills / EEQ:
reading by all staff
individually (4/year)
PN_GRH_E_489: evaluation sheet
of skills maintenance:
malaria diagnosis
Traceability of occupancy
workstations: PN_SOI_E_008:
parasitology on-call planning

SH FORM 43 rev 01 – April 15, 2015 Page 20 of 25

 Standard verification sheet (scope A) / validation
(scope B) of a medical biology method

EVALUATION OF THE METHOD'S PERFORMANCE

Specify the type and reference of sample (control sample, serum pool, ...): thick drop
- Quality of the spreading after coloring
- Quality of the staining: light bluish background without residual erythrocytes, well-stained leukocytes
with dark core
- Recognition of plasmodiums by the dark nucleus and light blue cytoplasm for the
asexual forms, presence of pigment according to the parasitic stage
- Parasitemia reported relative to the number of leukocytes
- Possible observation of other blood parasites: Trypanosomes, Babesia, microfilariae.

REPEATABILITY
Applicable not applicable (to be justified)

The laboratory assessed the repeatability of parasitemia quantification on 2 thick smears read 5 times.
by the same operator

Retention (%)
Name
Cart- CV(%) by the
Samples of values Average CV(%) Conclusion9
type supplier laboratory (cf.
(N)
source8)
15 to 100% (for
the techniques
microscopic,
Drops the observation
thick of a cell
NA
1712015491 5 194 49 25 additional
1712003392 5 128 19 15 on a number
from 0 to 2 cells
train a CV
of 100%)
The CV ranges between 15 and 25%, indicating the often observed distribution.
inhomogeneous parasites on the thick drop. This variation has no clinical consequence but must be
known in order to integrate it during the eventual comparison of 2 results.

INTERMEDIATE LOYALTY
Applicable not applicable (to be justified)

The laboratory evaluated the reproducibility of parasitemia quantification on 5 thick drops of

different parasitemias read by 4 different operators

Name CV(%) retained by

Cart- CV CV(%)
Sample values Average the laboratory (cf. Conclusion5
type (%) supplier
(N) source4)

8
Scholarly societies, publications (SFBC, GEHT, RICOS, QUALAB, CLIA…). Specify the reference used.
9
Compliant / Non-compliant

SH FORM 43 rev 01 - April 15, 2015 Page 21 of 25

 Type of verification sheet (scope A) / validation
(scope B) of a medical biology method
15 to 100% (for
Drops the techniques
thick microscopic,
196 6 3
1711076512 4 the observation of a
172 30 17
1711076632 4 NA cell As agreed
3 1 35
1712006013 4 additional on
23 10 41
1712001988 4 a number from 0 to 2
22 2 10
1711091552 4 cells leads to a
CV of 100%)
Argument of the conclusion: The CV is between 35 and 3%, depending on parasitemia: the observation of a
additional parasite on the thick drop when the parasitemia is 3 leads to a mathematical CV of
35% without clinical significance. However, the absence of a negative result despite this low paresthesia confirms
the laboratory's ability to diagnose malaria regardless of the operator.

INTER-OPERATOR VARIABILITY
Applicable not applicable

The laboratory assessed the reproducibility of parasitemia quantification on a thick drop.

positive (1712003392) read by 5 different operators

Operator 1 120 parasites/1000 leukocytes

Operator 2 140 parasites/1000 leukocytes
Operator 3 155 parasites/1000 leukocytes
Operator 4 114 parasites/1000 leukocytes
Operator 5 110 parasites/1000 leucocytes
The average value of parasitemia is 128. 19 parasites / 1000 leukocytes
with a CV of 15% for this inter-operator variability assessment. This variability has no
clinical consequence since parasitemia is assessed through the smear for therapeutic management.

SH FORM 43 rev 01 - April 15, 2015 Page 22 of 25

 Type of verification sheet (scope A) / validation
(Scope B) of a medical biology method

JUSTICE (from the outsourced CIQs)

Applicable not applicable (to be justified)
Name Average
Target Bias (%) Biais(%) Biais(%)
of Values general
Samples (group group average Conclusion5
values Laboratory
de pairs of pairs
(all
general Limit4
(N) techniques
Sample
CIQ level 1
Sample
CIQ level 2
Argument for the conclusion: microscopic examination, no numerical result

ACCURACY (based on one-off external checks: EEQ/CNQ)

Quantitative controls Quality controls
Bias
(%)
Target Target Bias(%)
/ Biais(%)Conclusion
Samples Value Lab (group of (all all 5
group limit4
pairs) techniques) technique
of
pairs
Biology
prospective
G2015-1A Post trophozoites Post trophozoites
G2015-2A Post trophozoites Post trophozoites
G2015-3A P gametocytes P gametocytes
G2015-4A Negative (NR) Negative
G2016-1A Negative Negative
To be filled in for the checks Compliant
G2016-2A Pos Pf trophozoite. Pos trophozoites
quantitative or letter A
G2016-3A Non Pf 12/00 Cancelled
G2016-4A Negative Cancelled
Kalidiv
2017-1 Pos Pfalciparum Pos Pfalciparum
2017-2 Pos Pvivax Pos Pvivax
2017-3 Pos Pfalciparum Pos Pfalciparum

Argument for the conclusion: results of the EEQ consistent with the expected results qualitatively
(positive/negative), and for the identification of parasitic stages. The EQC programs do not evaluate the
quantification on thick drop.

ANALYTICAL SENSITIVITY and SPECIFICITY

(experimental study essential in scope B)
(experimental study possible if relevant in scope A)
Applicable not applicable (to be justified)
True positives Spécificité, sensibilité, VPN, VPP
False positives
False negatives
False negatives
Argumentation of the conclusion:
In 2016, 1353 GE of which 1233 were negative, 120 positive (including 12 negative smears and 82 where the smears were)
positives)
En 2017, 1302 GE dont 1225 négatives, 76 positives (dont 5 frottis négatifs et 41 dont les frottis étaient positifs).
The method is correlated with the smear, confirming negative diagnoses and confirming low parasitemias.
detected by the blood smear. The discrepancies between the smear and the thick drop are few and
correlated with parasitemia.

MEASUREMENT UNCERTAINTY (levels, choice of calculation method, interpretation):

Chosen methodology: risk analysis (absence of residual interference) calculation

SH FORM 43 rev 01 – April 15, 2015 Page 23 of 25

 Type of verification sheet (scope A) / validation
(scope B) of a medical biology method
Requirement of
Calculated uncertainties
performances
Calculation method (cf. SH GTA 14): Formula used Reference
Argument for the conclusion: qualitative method; no calculation possible if negative

DETECTION LIMIT (experimental study essential for scope B)

(experimental study possible if relevant in scope A)
Applicable not applicable
Detection limit: With an average leukocyte count
estimated at 8G/L, the theoretical detection limit is
of 10 p/µL after observing 1000 white blood cells.
Patients with malaria are frequently
leukopenic, with a white blood cell count
an average of 4.5G/L, sensitivity is decreased, by
around 20 p/µL.
Argument for the conclusion: result following the observation of a given number of leukocytes whose distribution
the blade is not regular. Sensitivity is a function of the patient's white blood cell count.

COMPARISON OF METHODS:
Applicable not applicable (to be justified)
Bibliographic data (suppliers, 2017 Recommendations for Management of
publications,...) : imported malaria, WHO guidelines for
diagnosis of malaria
Compared methods: previous method, other
method used in the laboratory, devices in Smear: thick drop
mirror or back-up, EBMD, ...
Number of measures: Results 2016 and 2017
Comparison interval adapted to the range of NA
laboratory measurements:
Method of exploiting results: NA
Equation of the regression line: NA
Exploitation of comparison results Comparison of results
difference diagram, concordance
categorical) :
Reasoning of the conclusion:
In 2016, 1353 GE of which 1233 were negative, 120 positive (including 12 negative smears and 82 for which the smears were)
positive)
In 2017, 1302 GE of which 1225 were negative, 76 positive (including 5 negative smears and 41 where the smears were positive).
The method is correlated with the smear, confirming negative diagnoses and confirming low parasitemias.
detected by the blood smear. The discrepancies between the smear and the thick drop are few and
correlated to parasitemia

SCOPE OF MEASUREMENT (essential experimental study in scope B)

(experimental study possible if relevant in scope A for:
troponin, microalbumin, platelets, PSA, TSH, ...
Applicable not applicable (to be justified)
Detection limit: Bibliographic study (sources and Experimental study (values)
values)
Quantification limit: Etude bibliographique (sources et Experimental study (values)
values
Upper limit of linearity: Bibliographic study (sources and Experimental study (values)
values)
Argument of the conclusion: microscopic reading, nonlinear quantification. Detection limit associated with
our reading criteria: observation of 1000 leukocytes, which is a detection limit of one parasite for one
blood volume containing 1000 leukocytes, which is about 10 parasites/µl for a leukocyte count
average of 6G/L.

SH FORM 43 rev 01 – April 15, 2015 Page 24 of 25

 Type verification sheet (scope A) / validation
(scope B) of a medical biology method

INTERFERENCES (indispensable experimental study in scope B)

(possible experimental study if relevant in scope A for: Hemolysis, turbidity, bilirubin,
medications, ... - to be taken into account in the variability factors - to be evaluated if necessary)
Applicable not applicable (to be justified)
Hemolysis Specify the supplier data or overload test (LIH module)
Turbidity Specify the supplier data or overload test (LIH module)
Bilirubin, jaundice Specify supplier data or overload test (LIH module)
Medicines Specify supplier data or load test
… Specify supplier data or overload test
Argument of the conclusion: The observation of a thick drop colored by Giemsa is not sensitive to
these interferences.

CONTAMINATION (essential experimental study in scope B)

(experimental study possible if relevant in scope A for sensitive parameters)
Applicable not applicable (to be justified)
Inter-sample for sensitive parameters (by Specify the supplier or trial data of
example Ag HBS, βHCG, ... : surcharge
Interactive feedback if necessary (for example: LDH and Specify supplier data or on-site testing
ALAT, cholesterol and phosphate, lipase and
triglycerides, ... :
Argument for the conclusion: individual manual tests, no possible contamination.

ROBUSTNESS and STABILITY of REAGENTS

(essential experimental study in scope B)
(experimental study possible if relevant in scope A for sensitive parameters)
Applicable not applicable (to be justified)

Sensitive parameters tested (temperature, pH, position on a The monitoring of the pH of the tap water in the city of Paris
support, ... shows that it is stable and within the limits of
compliance for the dilution of the reagent.
Stability of the reagents after opening, embedded, ... Stability of undiluted Giemsa between 15 and 25°C, but
supports temperatures between 0 and 40°C (mail
RAL)
Renewal of diluted Giemsa every 4 hours
Argument for the conclusion: the reagents are stored and used under compliant conditions

REFERENCE intervals and/or threshold values based on demographic data (study

essential experimental in scope B)
Applicable not applicable
Reference values Specify the supplier data or on-site test
Argumentation of the conclusion:

DECLARATION OF FITNESS
Conclusion: compliant method used from 15/12/2017.
Authorized by: Sandrine Houzé, reference biologist of the sector
Signature

SH FORM 43 rev 01 - April 15, 2015 Page 25 of 25