SCYM ASCP COMPREHENSIVE QUESTIONS AND
ANSWERS SURE A+
✔✔Limited of Blank/Limit of Detection - ✔✔For flow cytometric methods, the major
challenge in sensitivity validation is finding or creating validation samples for Limited of
Blank/Limit of Detection, (LOB/LOD) and Lower limit of quantitation, LLOQ. The
LOB/LOD is similar to the "buffer blank" used in spectrophotometric methods.
Approaches to creating surrogate matrix samples that lack the population of interest
include: partially staining a sample by omitting antibodies in such a way that the
population of interest will not be detected; depleting the population of interest with
immunomagnetic beads; or using patient or healthy donor samples that lack the
population of interest. Similar strategies would be used to create samples with low
levels of the population of interest to assess LLOQ: admixing partially stained samples
into fully stained samples; partially depleting the population of interest; and, for leukemia
and lymphoma, admixing disease‐state samples into normal donor samples.
✔✔Stability - ✔✔Specimen stability assessment is critical in all cases where samples
will not be assayed within a few hours of collection. Certain markers or cellular subsets
may be lost or altered during the storage/shipment of the sample (typically whole blood
or bone marrow). Antigen expression, cellular composition, and viability can change
over time in the anti‐coagulant tube. The time points for the stability evaluation should
be based on when the samples are expected to arrive at the testing laboratory, and
should include at least one time point beyond the expected transit time. The generally
accepted change for a particular marker from the baseline specimen value and the
stored specimen value is 20% difference or a change within the acceptable assay
precision (10% to 30% CV).
✔✔Carryover - ✔✔It is critical to evaluate instrument carryover from one sample tube to
the other when evaluating high‐sensitivity assays reporting rare events. Carryover can
be assessed during the initial validation by placing a tube with buffer between sample
tubes. Data from the blank tubes would be evaluated in the same gating template as the
samples.
,✔✔Reference Ranges - ✔✔Reference ranges (age, gender, disease specific ranges,
healthy ranges) are essential in the interpretation of clinical chemistry laboratory results
but are not always required for flow cytometric methods. Procedure for establishing
reference ranges can be found in the Clinical and Laboratory Standards Institute (CLSI)
guideline EP28‐A3c
✔✔Documentation - ✔✔Validation should follow a three‐step approach:
1. Say It (the Validation Plan or Protocol)
2. Do It (the Experimental Phase)
3. Prove It (the Validation Report)
Documentation is critical because in a regulated environment, "if you don't document
what you did, it didn't happen."
✔✔Method Validation Plan - ✔✔The Method Validation Plan or Validation Protocol
provides detailed documentation of the validation experimental design. The Validation
Plan should include:
1. A full description of the method including gating strategy and list of the assay read‐
outs to be validated.
The source (disease, healthy) and type of validation samples, including anticoagulants
when using whole blood or bone marrow samples.
2. Quality control material, if applicable. The control material should mimic the actual
sample as closely as possible.
3. Critical reagents (manufacturer, catalog number):
For monoclonal antibodies, the fluorochrome and clone designation must be specified.
✔✔Method Validation Report - ✔✔Method validation report describes the results of the
experimental phase. Validation Report should mirror the Validation Plan. Any deviations
from the Validation Plan which occurred during the experimental phase should be
explained clearly. The validation report should include:
1. A reagent table with the lot number and expiration dates of all reagents.
2. The statistical results clearly summarized with table summaries and/or figures:
a- If any of the parameters did not meet the acceptance criteria, it should be discussed
within that section of the document.
b- if any outliers were identified, then exclude them from the statistical analysis—
justification must be provided along with the statistical tool used to identify outliers. Note
that the outliers must be included in the report.
3. Individual results should be presented in an appendix in table format.
Changes in the gating strategy or other deviations from the validation plan must be
clearly described in the validation report. The documentation for deviations typically
includes an impact assessment but must follow the laboratory's institution's quality
processes.
4. Copies of the gated data (from the data analysis software) for every sample should
be available in a validation binder and/or electronic format. In addition, the location of
the list mode files should be either included in the Validation Report or described in an
SOP.
, ✔✔Standard operating procedure (SOP) - ✔✔Information included in the SOP:
The reagent tables including storage and handling instructions.
Complete details for sample accessioning, processing, and reporting.
Detailed instrument setup and compensation procedure.
Detailed instrument acquisition procedure (e.g., how many events to collect).
Detailed gating instructions.
Additional specific requirements for a method SOP depend on the regulatory
environment in which the testing is conducted.
✔✔Limited Assay Validation - ✔✔The Limited Assay Validation represents the minimal
recommended parameters for research environments and non‐regulated laboratories. In
this example, it is assumed that the samples will not be shipped to a testing facility and
assumes one operator and one instrument; thus, specimen stability as well as inter‐
operator and inter‐instrument variation are not included in this validation plan. Note that
although sample stability is not included in this validation plan, it is assumed that
stability was demonstrated during assay optimization and that samples are analyzed
within that window.
✔✔Initial Assay Validation - ✔✔Initial Assay Validation is performed for a new assay in
a laboratory. Using the fit‐for‐purpose validation approach, the validation parameters
should be selected based on the intended use of the assay. The example shown below
represents the minimal validation protocol for low and moderate risk assays in regulated
laboratories.
✔✔CD34+ Absolute Counts - ✔✔Direct measurement of CD34+ blood stem cell
absolute counts by flow cytometry. ... Whole blood was stained with a phycoerythrin-
conjugated anti-CD34 monoclonal antibody, and, after the lysis of red blood cells,
CD34+ cells were counted in a fraction of the lymphocyte and monocyte gate.
✔✔What is Flow Cytometry - ✔✔Flow cytometry is a technology that facilitates
multiparametric analysis at the single‐cell level and is widely used in research settings,
diagnostic laboratories, and drug development. It is also used in a variety of other
settings, such as marine biology and food science. In order to ensure that the data
generated are reliable, every laboratory should perform some level of analytical method
validation. This protocol provides a roadmap for validation which can be applied in any
type of laboratory setting and will answer the questions, "what is method validation?"
and "why should I care about it?"
✔✔Solid Organ Transplant (HLA Crossmatch) - ✔✔This test is used primarily for solid
organ transplant candidates to assess the suitability of a potential donor. Positive cross
matches detected by flow cytometry suggest the presence of preformed anti-donor HLA
antibodies and carry a risk of accelerated rejection and damage to the graft. These
sensitive cross matches are interpreted in terms of the patient's sensitization history and
the quality of the donor organ.
ANSWERS SURE A+
✔✔Limited of Blank/Limit of Detection - ✔✔For flow cytometric methods, the major
challenge in sensitivity validation is finding or creating validation samples for Limited of
Blank/Limit of Detection, (LOB/LOD) and Lower limit of quantitation, LLOQ. The
LOB/LOD is similar to the "buffer blank" used in spectrophotometric methods.
Approaches to creating surrogate matrix samples that lack the population of interest
include: partially staining a sample by omitting antibodies in such a way that the
population of interest will not be detected; depleting the population of interest with
immunomagnetic beads; or using patient or healthy donor samples that lack the
population of interest. Similar strategies would be used to create samples with low
levels of the population of interest to assess LLOQ: admixing partially stained samples
into fully stained samples; partially depleting the population of interest; and, for leukemia
and lymphoma, admixing disease‐state samples into normal donor samples.
✔✔Stability - ✔✔Specimen stability assessment is critical in all cases where samples
will not be assayed within a few hours of collection. Certain markers or cellular subsets
may be lost or altered during the storage/shipment of the sample (typically whole blood
or bone marrow). Antigen expression, cellular composition, and viability can change
over time in the anti‐coagulant tube. The time points for the stability evaluation should
be based on when the samples are expected to arrive at the testing laboratory, and
should include at least one time point beyond the expected transit time. The generally
accepted change for a particular marker from the baseline specimen value and the
stored specimen value is 20% difference or a change within the acceptable assay
precision (10% to 30% CV).
✔✔Carryover - ✔✔It is critical to evaluate instrument carryover from one sample tube to
the other when evaluating high‐sensitivity assays reporting rare events. Carryover can
be assessed during the initial validation by placing a tube with buffer between sample
tubes. Data from the blank tubes would be evaluated in the same gating template as the
samples.
,✔✔Reference Ranges - ✔✔Reference ranges (age, gender, disease specific ranges,
healthy ranges) are essential in the interpretation of clinical chemistry laboratory results
but are not always required for flow cytometric methods. Procedure for establishing
reference ranges can be found in the Clinical and Laboratory Standards Institute (CLSI)
guideline EP28‐A3c
✔✔Documentation - ✔✔Validation should follow a three‐step approach:
1. Say It (the Validation Plan or Protocol)
2. Do It (the Experimental Phase)
3. Prove It (the Validation Report)
Documentation is critical because in a regulated environment, "if you don't document
what you did, it didn't happen."
✔✔Method Validation Plan - ✔✔The Method Validation Plan or Validation Protocol
provides detailed documentation of the validation experimental design. The Validation
Plan should include:
1. A full description of the method including gating strategy and list of the assay read‐
outs to be validated.
The source (disease, healthy) and type of validation samples, including anticoagulants
when using whole blood or bone marrow samples.
2. Quality control material, if applicable. The control material should mimic the actual
sample as closely as possible.
3. Critical reagents (manufacturer, catalog number):
For monoclonal antibodies, the fluorochrome and clone designation must be specified.
✔✔Method Validation Report - ✔✔Method validation report describes the results of the
experimental phase. Validation Report should mirror the Validation Plan. Any deviations
from the Validation Plan which occurred during the experimental phase should be
explained clearly. The validation report should include:
1. A reagent table with the lot number and expiration dates of all reagents.
2. The statistical results clearly summarized with table summaries and/or figures:
a- If any of the parameters did not meet the acceptance criteria, it should be discussed
within that section of the document.
b- if any outliers were identified, then exclude them from the statistical analysis—
justification must be provided along with the statistical tool used to identify outliers. Note
that the outliers must be included in the report.
3. Individual results should be presented in an appendix in table format.
Changes in the gating strategy or other deviations from the validation plan must be
clearly described in the validation report. The documentation for deviations typically
includes an impact assessment but must follow the laboratory's institution's quality
processes.
4. Copies of the gated data (from the data analysis software) for every sample should
be available in a validation binder and/or electronic format. In addition, the location of
the list mode files should be either included in the Validation Report or described in an
SOP.
, ✔✔Standard operating procedure (SOP) - ✔✔Information included in the SOP:
The reagent tables including storage and handling instructions.
Complete details for sample accessioning, processing, and reporting.
Detailed instrument setup and compensation procedure.
Detailed instrument acquisition procedure (e.g., how many events to collect).
Detailed gating instructions.
Additional specific requirements for a method SOP depend on the regulatory
environment in which the testing is conducted.
✔✔Limited Assay Validation - ✔✔The Limited Assay Validation represents the minimal
recommended parameters for research environments and non‐regulated laboratories. In
this example, it is assumed that the samples will not be shipped to a testing facility and
assumes one operator and one instrument; thus, specimen stability as well as inter‐
operator and inter‐instrument variation are not included in this validation plan. Note that
although sample stability is not included in this validation plan, it is assumed that
stability was demonstrated during assay optimization and that samples are analyzed
within that window.
✔✔Initial Assay Validation - ✔✔Initial Assay Validation is performed for a new assay in
a laboratory. Using the fit‐for‐purpose validation approach, the validation parameters
should be selected based on the intended use of the assay. The example shown below
represents the minimal validation protocol for low and moderate risk assays in regulated
laboratories.
✔✔CD34+ Absolute Counts - ✔✔Direct measurement of CD34+ blood stem cell
absolute counts by flow cytometry. ... Whole blood was stained with a phycoerythrin-
conjugated anti-CD34 monoclonal antibody, and, after the lysis of red blood cells,
CD34+ cells were counted in a fraction of the lymphocyte and monocyte gate.
✔✔What is Flow Cytometry - ✔✔Flow cytometry is a technology that facilitates
multiparametric analysis at the single‐cell level and is widely used in research settings,
diagnostic laboratories, and drug development. It is also used in a variety of other
settings, such as marine biology and food science. In order to ensure that the data
generated are reliable, every laboratory should perform some level of analytical method
validation. This protocol provides a roadmap for validation which can be applied in any
type of laboratory setting and will answer the questions, "what is method validation?"
and "why should I care about it?"
✔✔Solid Organ Transplant (HLA Crossmatch) - ✔✔This test is used primarily for solid
organ transplant candidates to assess the suitability of a potential donor. Positive cross
matches detected by flow cytometry suggest the presence of preformed anti-donor HLA
antibodies and carry a risk of accelerated rejection and damage to the graft. These
sensitive cross matches are interpreted in terms of the patient's sensitization history and
the quality of the donor organ.
