SMILE
Johns Hopkins University
Baltimore, MD USA
Parallel Testing and Reagent Lot Validation - Guidelines
Guideline Number
Pro40-06
Effective Date
03/21/08
Page
1 of 1
Parallel Testing and Reagent Lot Validation - Guidelines
Supersedes
1.0
Review by
Review date
7-Feb-12
Parallel Testing and Reagent lot Validation
Subject
Heidi Hanes
SMILE Comments: This document is provided as an example only. It must be revised to accurately reflect your lab’s
specific processes and/or specific protocol requirements. Users are directed to countercheck facts when considering
their use in other applications. If you have any questions contact SMILE.
Background Information:
Audit Shell:
Questions pertaining to parallel testing and reagent lot validation can be found in
section– Test and Control Articles item
CAP Accreditation Checklist
Questions pertaining to parallel testing and regent lot validation can be found in the
Chemistry and Toxicology checklist – Reagents -CHM.12900, p. 25.
Hematology checklist – Reagents – HEM.24575, p. 44
Background Information
Clinical laboratory reagents and control materials are exposed to many variables due to
conditions during transportation and storage environments in different laboratory
settings. The validation of new reagents kits with old reagent kits is performed to ensure
that, in spite of varying environmental conditions, there are no clinically significant
differences in the results obtained when different lot numbers of reagents are used.
Control materials are parallel tested to ensure that the mean of the values obtained are
within the ranges specified by each manufacturer. The data gained during parallel
testing should then be utilized to establish QC ranges for each individual laboratory.
The procedure outlines the parallel testing and reagent lot validation testing required for
different sections of the laboratory including chemistry, hematology, coagulation, flow
cytometry and HIV viral load testing.
Resources
1. College of American Pathologists (CAP) 2006. Commission on Laboratory
Accreditation, Laboratory Accreditation Program; Laboratory General Checklist
Revised 7/27/2007.
Pro40-06 Parallel Testing
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Parallel Testing and Reagent Lot Validation
Author(s), Name &
Title
Document
Number
Jo Shim MBA, MT(ASCP)
International QA/QC Coordinator
(SMILE) Program
Pro40-06
Effective
Date
4/2/2008
SMILE Comments: This document is provided as an example only. It must be revised to accurately
reflect your lab’s specific processes and/or specific protocol requirements. Users are directed to
countercheck facts when considering their use in other applications. If you have any questions contact
SMILE.
Name, Title
Signature
Date
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Date
Approved
By
SOP
Annual
Review
Revision
History
Version # [0.0]
Revision Date
[dd/mm/yy]
Description (notes)
2.0
17/02/10
Updated CD4/CD8 Criteria & added appendix K
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I acknowledge that I have read, understand and agree to follow this SOP.
Electronic Signature
Pro40-06 Parallel Testing
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Purpose:
The purpose of this procedure is to provide a procedural template for, SMILE monitored,
international sites to use when developing a program for parallel testing. The procedure is
intended to be used by sites as a guide while developing their own parallel testing procedures.
Principle:
Clinical laboratory reagents and control materials are exposed to many variables due to
conditions during transportation and storage environments in different laboratory settings. The
validation of new reagents kits with old reagent kits is performed to ensure that, in spite of
varying environmental conditions, there are no clinically significant differences in the results
obtained when different lot numbers of reagents are used.
Control materials are parallel tested to ensure that the mean of the values obtained are within
the ranges specified by each manufacturer. The data gained during parallel testing should then
be utilized to establish QC ranges for each individual laboratory.
Responsible Personnel:
Responsible personnel may vary according to location but should include the following positions
or their equivalents –

Laboratory Manager or Director

QC/QC Coordinator

Department/Section Heads or Chief Technologist

Staff Technologist/Technicians
Precautions:
Standard precautions should be followed when conducting parallel testing and reagent lot
validation (refer to appropriate safety SOP). In addition, personnel performing the testing should
use Personal Protective Equipment (PPE) that is appropriate for the task and follow all safety
rules established for their institution.
Procedure:
The requirements for parallel testing of controls can vary according to the test being performed.
Follow the guidelines for different testing systems as outlined below.
CBC Controls:
1. The new lot of controls should ideally be run in parallel with the old lot of controls 2-3
times for 5-8 days before the old lot # expires
2. The mean for the new control and standard deviation for the new lot of the controls will
be approved by the Laboratory Supervisor or QC/QA coordinator before the new control
is used. Labs may have other ways of establishing QC ranges see Appendix A:
Establishing Hematology QC Ranges.
3. The laboratory Director or QC/QA coordinator should review and sign off on the QC
parallel testing data before the new control is put into operation.
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Chemistry Controls:
1. The new control lot number should be run in parallel with the old lot number before it
expires. The new control should be run a minimum of 20 times over 3-5 days or over a
longer time period if possible.
2. The mean for the new control and standard deviation for the new lot of the controls
should be approved by the Laboratory Supervisor or QC/QA coordinator before the new
control is put into use. Labs may have other ways for establishing QC ranges see
Appendix B: Establishing Chemistry QC Ranges.
3. The laboratory Director or QC/QA coordinator should review and sign off on the QC
parallel testing data before the new control is put into operation.
Coagulation Controls:
1. The procedure for the parallel testing of coagulation controls is very similar to the
procedure for chemistry. The new control lot number should be run in parallel with the
old lot number before it expires. The new control should be run a minimum of 20 times
over 3-5 days but a longer period of time is recommended. .
2. The mean for the new control and standard deviation for the new lot of the controls
should be approved by the Laboratory Supervisor or QC/QA coordinator before the new
control is put into use.
Reagent lot validation:
All new lots of reagents should be validated by running them in parallel with the old lot numbers
as indicated and the results obtained should be within the acceptability range defined.
HIV EIA and other EIA Assays:
1. A minimum of 3 patient samples (negative, low positive and high positive if available) or
an entire strip from a previous run are tested in parallel with QC on both the old and the
new lot numbers.
2. The QC and patient results should be reproducible between the two lots (reproducibility
includes both the OD readings and the interpretations).
3. The Laboratory Director, QA/QC Coordinator or designated technologist is responsible
for defining acceptability limits for parallel testing. (Example: OD variance of 1SD or less
and agreement on the interpretation).
4. Develop a form for the documentation of parallel testing that includes appropriate space
for entering the following data:


Lot numbers (old and new lot numbers) and expirations dates
Results obtained from the old and new lots.
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SMILE
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Baltimore, MD USA



Criteria for acceptance and space to indicate if the results obtained on the new
lots were acceptable.
QC values on both runs (include QC lot numbers).
Space for the person completing the parallel testing to sign and date the form
and a place for a reviewer to sign and enter the date.
HIV RNA PCR Quantitative Assay:
1. A minimum of 3 patient samples (not detected, low positive and high positive, if
available) or an entire strip from a previous run are tested in parallel with the QC on both
the old and the new kit or reagent lot number.
2. The QC and patient results should be reproducible between the two lots.
3. The Laboratory Director, QA/QC Coordinator or designated technologist is responsible
for defining acceptability limits for parallel testing (typical criteria for the acceptability of
Quantitative PCR assays would be that any variation should not be greater than threefold or they should be within 2 fold or 0.3 Log - HIV Prevention Trial Network (HPTN)
criteria)
4. Develop a form for the documentation of reagent lot validation that includes appropriate
space for entering the following data:





Lot numbers (old and new lot numbers) and expirations dates
Results obtained form the old and new lots.
Criteria for acceptance and space to indicate if the results obtained on the new
lots were acceptable.
QC values on both runs (include QC lot numbers).
Space for the person completing the parallel testing to sign and date the form
and place for a reviewer to sign and enter the date.
GC, Chlamydia, HIV PCR Qualitative Assays:
1. A minimum of 3 patient samples should be run in parallel on both the old and the new
lots.
2. The QC and patient results should be reproducible between the two lots.
3. The Laboratory Director, QA/QC Coordinator or designated technologist reviews the
results and confirms that there is agreement between the results for the two kits (i.e.
negative results are negative on the new kit and positive results are positive).
4. Develop a form for the documentation of reagent lot validation that includes appropriate
space for entering the following data:




Lot numbers (old and new lot numbers) and expirations dates
Results obtained from the old and new lots.
Criteria for acceptance and space to indicate if the results obtained on the new
lots were acceptable.
QC values on both runs (include QC lot numbers).
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SMILE
Johns Hopkins University
Baltimore, MD USA

Space for the person completing the parallel testing to sign and date the form
and a place for a reviewer to sign and enter the date.
CD4/CD8 Assay:
1. A minimum of 2 patient samples (The IQA recommends using one normal and one
abnormal sample) should be run in parallel when antibody lot numbers, reagent lot
numbers or reagent kits, such as truecount , lot numbers are changed.
2. The QC and patient results should be reproducible between the two lots.
3. The Laboratory Director, QA/QC Coordinator or designated technologist defines the
acceptability limits for parallel testing (The IQA recommends a difference of <10% for
both percent and absolute counts).
4. Develop a form for the documentation of the reagent lot validation that includes
appropriate space for entering the following data:





Lot numbers (old and new lot numbers) and expirations dates.
Results obtained from the old and new lots.
Criteria for acceptance and space to indicate if the results obtained on the new
lots were acceptable.
QC values on both runs
Space for the person completing the parallel testing to sign and date the form
and a place for a reviewer to sign and enter the date
Chemistry Assays:
1. New lot numbers of chemistry reagents are run in parallel with the old lot to check the
performance of the new reagent.
2. A minimum of 3 patient samples should be run on the old and new lot number.
3. The QC and patient results should be reproducible between the two lots.
4. The Laboratory Director and QA/QC Coordinator or designated technologists are
responsible for defining the acceptability limits for reagent parallel testing. (Suggested
acceptability limits are within +/- 1SD or within+/-10%).
5. Develop a form for the documentation of chemistry reagent lot validation that includes
the following data:


Lot numbers (old and new lot numbers) and expirations dates.
Results obtained from the old and new lots.
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SMILE
Johns Hopkins University
Baltimore, MD USA



Criteria for acceptance and space to indicate if the results obtained on the
new lots were acceptable.
QC values on both runs (include QC lot numbers).
Space for the person completing the parallel testing to sign and date the form
and a place for a reviewer to sign and enter the date
CBC Analyzer Reagents
1. Comparison of materials of known value prior to and following changing or priming of
new lots or shipments is required.
2. Comparison studies must be performed before or at the same time that new reagent lots
are placed in service
3. Material of known value may include a patient samples or control material.
4. Background checks must be performed on inert materials such as diluent to ensure that
new lots do not interfere with patient results.
5. Develop forms for the documentation of reagent lot validation and diluent background
checks that include:





Lot numbers (old and new lot numbers) and expirations dates.
Results obtained from the old and new lots.
Criteria for acceptance and space to indicate if the results obtained on the
new lots were acceptable.
Space for the person completing the comparison testing to sign and date the
form and a place for a reviewer to sign and enter the date
A place for background counts to be recorded.
Coagulation Reagents:
PT Reagent
1. Parallel testing of a new lot of PT reagent should be completed well in advance of the
expiration date of the old lot. Parallel testing of new lots of PT reagents also includes
verifying the reference range, geometric mean and programming the correct ISI
(international Sensitivity Index) into the coagulation analyzer.
2. To verify the reference range and geometric mean it is necessary to collect specimens
from 20 “normal” patients and to run a PT with the new lot of thromboplastin reagent.
90% of the samples must fall within the current range in order to verify the range and
geometric mean. If they do not, a new reference range study must be conducted to
determine them. Microsoft Excel or other appropriate clinical reference range software
must be used to calculate the new range and geometric mean.
3. Perform comparison studies between the old and new lot number to verify the
consistency of patient results and controls. The R value for the correlation study should
be >0.97.
4. Validate the PT reference range with 20 specimens. If the reference range does not
validate perform a new reference range study using at least 60 specimens.
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SMILE
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Baltimore, MD USA
5. Finally, perform a manual check of the INR and compare with the instrument generated
INR result.
PTT Reagents:
1. Parallel testing of PTT reagents should be conducted well in advance of the expiration of
the old reagent.
2. Perform comparison studies between the old and new lot number using patient samples
and controls. The R value for the correlation study should be R=>0.97.
3. To verify the PTT reference range it is necessary to collect specimens from 20 “normal”
patients and to run a PTT with the new lot of reagent. 90% of the samples must fall
within the current range in order to verify the range. If they do not, a new reference
range study must be conducted to determine them. Microsoft Excel or other appropriate
clinical reference range software must be used to calculate the new range.
4. Please note that if you monitor patients on heparin therapy you should perform a new
heparin curve with each change of reagent lot.
5. Develop a form for the documentation of PT/PTT reagent lot validation that includes the
following data:





Lot numbers (old and new lot numbers) and expirations dates.
Results obtained from the old and new lots.
Criteria for acceptance and space to indicate if the results obtained on the
new lots were acceptable.
QC values on both runs
Space for the person completing the reagent validation testing to sign and
date the form and a place for a reviewer to sign and enter the date
Semi quantitative Tests UA strips:
1. A minimum of 3 patient samples are run in parallel on both the old and the new lots (The
three samples should demonstrate varying results across the range for different strip
analytes).
2. The QC and patient results should be reproducible between the two lots.
3. Acceptance ranges should be established by the Laboratory Director, QC/QA coordinator or
designated technologist. (Generally negative results should remain negative, ,positive
results should give the same results or be one level up or down from the original result)
4. Develop a form for the documentation of the reagent lot validation that includes appropriate
space for entering the following data:


Lot numbers (old and new lot numbers) and expirations dates.
Results obtained from the old and new lots.
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Version#: 2.0
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SMILE
Johns Hopkins University
Baltimore, MD USA



Criteria for acceptance and space to indicate if the results obtained on the
new lots were acceptable.
QC values on both runs
Space for the person completing the parallel testing to sign and date the form
and a place for a reviewer to sign and enter the date
Qualitative Testing (Rapid HIV test kits, urine/serum qualitative hCG etc.)
1. A minimum of 3 patient samples are run in parallel on both the old and the new lots.
2. The QC and patient results should be reproducible between the two lots.
3. The Laboratory Director, QA/QC Coordinator or designated technologist reviews the
results and confirms that there is agreement between the results for the two lots (i.e.
negative results are negative on the new lot and positive results are positive).
4. Develop a form for the documentation of reagent lot validation that includes appropriate
space for entering the following data:
 Lot numbers (old and new lot numbers) and expirations dates
 Results obtained form the old and new lots.
 Criteria for acceptance and space to indicate if the results obtained on the
new lots were acceptable.
 QC values on both runs
 Space for the person completing the parallel testing to sign and date the
form and a place for a reviewer to sign and enter the date
References
Westat Checklist General required elements – Parallel Testing
Westgard website - www. Westgard.com
Mercy Medical Center –Baltimore, Maryland: Yearly Coagulation Lot changes
Grove N., Rotzoll, K. CLIA Corner: Prothrombin Time and INR Testing. University of Iowa
Hygienic Laboratory
Appendices
Appendix A: Establishing Hematology QC Ranges
Appendix B: Establishing Chemistry QC Ranges
Examples of Forms for Parallel testing Documentation
Appendix C: PCR Reagent Lot Validation Form
Appendix D: Qualitative Reagent Lot Validation Form
Appendix E: EIA Test Reagent Lot Validation Form
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Appendix F: Urine hCG Reagent Lot Validation Form
Appendix G: PT Reagent Lot Validation Form
Appendix H: PTT Reagent Lot Validation Form
Appendix I: Hematology Reagent Lot Validation Form
Appendix J: Chemistry Reagent Lot Validation Form
Appendix K: Flow Cytometry Reagent Lot Validation Form
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