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Cellular Immunology Proficiency Testing Program

Welcome

Links to CLEP

Frequently Asked Questions

On this page we have posted information on some topics pertaining to questions that are frequently posed to laboratory personnel. If you do not find the information you are looking for here, please contact us.

Method approval/validations

Instrument Quality Control

Assay Quality Control

Proficiency Testing

All assays

Please note:When submitting material for test approval or method validations, please refer to the Submission Guidelines for Test Approval. This is a PDF file that can be downloaded from the CLEP website. For a copy of the submission guidelines in an alternate format, please contact CLEP.

In-house developed assays or modified commercial assays require well-written instructions that thoroughly describe the procedure.

Please note: The NYS Cellular Immunology PT Program requires notification of the validation including a cover letter and the data summary. Validation documentation must be available for the next NYS on-site survey. The laboratory may start using the new procedure provided the validation results are within the above stated requirements and notification documentation has been forwarded to the NYS Cellular Immunology PT Program. All subsequent PT events must be completed using the newly validated procedure.

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New assays

All assays (with the exception of CD34 testing and Malignant Leukocyte immunophenotyping):

  • Laboratory-derived normal ranges from at least 25 "healthy" individuals similar to the expected patient population in age, sex, and ethnicity
  • Assay validations with at least 25 specimens must include a minimun of 5 abnormal specimens. The abnormal specimens must be analyzed in parallel with a NYS-approved laboratory for the pertinent category.

CD34 immunophenotyping:

  • Assay validations with at least five specimens per specimen type (peripheral blood, mobilized peripheral blood, leukapheresis products, cord blood, bone marrow) as the laboratory expects to test. The specimens must be analyzed in parallel with a NYS-approved laboratory.
  • Normal ranges are not required if analyses are conducted for transplantation/graft purposes.

Malignant leukocyte Immunophenotyping:

  • Assay validations with at least five normal specimens and five abnormal specimens
  • The determined reference (normal) range should be similar to published values, and the expected reactions obtained with normal samples using the Malignant Leukocyte Immunophenotyping reagents. For example, a normal blood sample should have kappa and lambda positive (kappa>lambda) cells in the CD19 or CD20 subset or CD3+/CD4+cells should not express CD10, CD19, or CD33.

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Modifications to existing assays and instrument additions or changes

All assays (with the exception of CD34 testing and Malignant Leukocyte immunophenotyping):

  • Perform a split sample analysis utilizing the old and the revised procedure with a minimum of 25 samples for all categories
  • Results from the two assays should produce comparable patient outcomes. If not, the laboratory must calculate new normal ranges for the new procedure and indicate on patient reports that a new procedure has been implemented (include date), which could result in a non-biological change in the patient's longitudinal status.

CD34 immunophenotyping:

  • Perform a split sample analysis utilizing five specimens per specimen type (peripheral blood, mobilized peripheral blood, leukapheresis products, cord blood, bone marrow) as the laboratory expects to test.

Malignant Leukocyte immunophenotyping:

  • Perform a split sample analysis with five abnormal and five normal specimens

Results from the two assays should produce comparable patient outcomes. If not, the laboratory must calculate new normal ranges for the new procedure and indicate on patient reports that a new procedure has been implemented (include date), which could result in a non-biological change in the patient's longitudinal status.

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Daily instrument QC:

  • Verify optical alignment for non-fixed optical rail flow cytometers
  • Calibrate and standardize according to the manufacturer's recommendation (sofware and bead product)
  • Stain and analyze a normal blood or validated commercial control for Lymphoid/T-Lymphoid analysis

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Monthly instrument QC:

  • Check fluorescence/PMT linearity
  • Stain and analyze a normal blood or validated commercial control for Malignant Leukocyte analysis

As the manufacturer recommends:

  • Clean system fluidics
  • Perform preventive maintenance
  • Verify opitical alignment on fixed optical rail flow cytometers

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Moving an instrument

If an instrument is moved:

  • Verify optical alignment
  • Calibrate and standardize according to the manufacturer's recommendations for software and bead product
  • Check fluorescence/PMT linearity
  • Stain and analyze a normal blood or validated commercial control

These guidelines should also be followed when evaluating instrument performance following an instrument repair.

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QC requirements for all assays

  • Normal control results must be within the laboratory-derived normal ranges
  • Commercial control results must be within the expected lot range(s)

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Functional assay QC

For both Lymphoid and Non-lymphoid functional assays:

  • A control must be run on each assay plate or within each run
  • Specimen age must not be greater than 24 h
  • Viability must be greater than 80% at cell culture initiation

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Lymphoid immunophenotyping QC

General QC

  • A normal blood or validated commercial control must be run daily
  • Specimens containing EDTA have a maximum acceptable specimen age of 30 h
  • Specimens containing Heparin or ACD have a maximum acceptable specimen age of 48 h

2 Color Analysis (SS/FS gating with CD45/CD14):

  • The percentage of cells within the lymphocyte collection gate that are lymphocytes are minimally ≥85%/optimally ≥90% (Purity)
  • The percentage of lymphocytes in the sample that are collected in the lymphocyte gate are minimally ≥90%/optimally ≥95% (Recovery)

2, 3, and 4 Color Analysis

  • Delta CD3: The difference between the highest and lowest CD3 values within a patient's analysis panel; tube to tube variability should not exceed 3%.
  • T-sum: The sum of CD3+CD4+ and CD3+CD8+ cells should equal the total % of CD3, optimally ±5% with a maximal variability of 10%; exceptions include patients with high T delta/gamma cells.
  • Lymphosum: The sum of %CD3+ (T cells), %CD19+ (B cells), and %CD3-(CD56+±CD16+) (NK cells). If the data has not been corrected, this should equal the purity of lymphocytes in the gate (2-color analysis), optimally ±5%, with maximum variation of 10%. If the data has been corrected by purity (2-color), or if the laboratory is using Side Scatter/CD45 gating (3- or 4-color analysis), the lymphosum should optimally equal 95-105% (maximally 90-110%).

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Non-Lymphoid immunophenotyping QC

General QC

  • Specimens have a maximum acceptable age of 48 h

CD34 immunophenotyping

  • The viability of CD34+ cells should always be determined
  • Positive controls are commercially available and they should be run in each assay run to ensure that the reagents and equipment are performing properly, as well as assessing the operator's ability to properly gate the cells.

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Malignant leukocyte immunophenotyping QC

  • A normal blood or validated commercial control must be run monthly
  • Maximum acceptable specimen age of 48 h
  • Specimen must have a viability of 50% or greater
  • If the specimen is irreplaceable,results can only be reported if an abnormal population is definitively determined by the combination of the flow cytometric results with other clinical and technical features of the case taken into consideration.
  • Values for the same marker(s) (clone, manufacturer, fluorochrome) used in multiple panel assay tubes must produce similar results for the same gated cell population.
  • Specimen analysis should include examination of a normal cell population (i.e., lymphocytes) if the specimen composition permits such analysis. Marker presence or absence with qualitative intensity descriptions on both populations supports the aberrant results when the results for the normal cells are as expected.
  • Surface immunoglobulin analysis must include a pan B cell marker (i.e., CD19 or CD20) to assist in accurate assessment by eliminating non-specific staining of other cell types.
  • Positive staining reactivity for surface immunoglobulin heavy chains (i.e., IgM and IgD) support the presence of immunoglobulin light chains. Light chain determination is important in establishing clonality, however, their analysis can be problematic. Blood cells must be washed free of plasma immunoglobulins before performing B cell analysis. Inconsistencies between surface immunoglobulin light chain and heavy chain assays is indicative of analytical difficulties and should be reported as such.

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Available proficiency testing

Lymphoid Functional Assays:

  • Currently, testing is offered for mitogen (PHA)-induced lymphocyte proliferation only. Two specimens are provided.

Non-Lymphoid Functional Assays:

  • No proficiency testing is currently offered for this category.

Lymphoid Immunophenotyping

  • Five specimens for the immunophenotyping of non-malignant lymphocytes and/or T-cell subsets are provided.

Non-Lymphoid Immunophenotyping:

  • One specimen for the immunophenotyping of viable CD34+stem cells is provided.

Malignant Leukocyte Immunophenotyping:

  • Three specimens for the immunophenotyping of malignant leukocytes (leukemia/lymphoma) are provided.

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Proficiency testing grading policy

Lymphoid Functional Assays (PHA-induced lymphocyte proliferation only)

  • Proficiency test is graded on a pass/fail basis; the grade is based on the inclusion of all required parameters in the patient report and the interpretation of specimen function. Occasionally, a conditional pass is given in which case the laboratory is required to submit a letter addressing the issues of concern.

Lymphoid/T-Lymphoid Immunophenotyping

  • The accurate evaluation of each specimen represents 20% of the total score. A score of 80% or greater is required to pass.
  • For Lymphoid Immunophenotyping: Acceptable values (percentages and absolute counts) for all lymphocyte subsets are required (including CD3+/CD4+, CD3+/CD8+, CD3+, CD19+, and CD3-CD56+± CD16+).
  • For T-lymphoid Immunophenotyping: Acceptable values for CD4 and CD8 T-lymphocytes (percentage and/or absolutes, dependent upon instrumentation) are required (including CD3+CD4+ and CD3+CD8+)

Please note: At this time, while we are requesting absolute values be reported for the Lymphoid/T-lymphoid Immunophenotyping PT, laboratory performance on the absolute values is not currently graded. However, comments on absolute performances and the range of reported values will be provided to participating laboratories.

CD34 Immunophenotyping

  • Accurate evaluation of the number of viableCD34+ stem cells per microliter is required to pass.

Malignant Leukocyte Immunophenotyping

  • Accurate and appropriate immunophenotyping of the leukemia/lymphoma (lineage, stage) is required.
  • Scoring of the test is pass/fail. A temporary grade of conditional pass is used when additional information is required. Depending on the response given, a grade of either pass or fail will be assigned.

PT failures

In addition to poor performance in the requirements listed for a specific category, the following factors may result in a PT failure:

  • Failure to submit required signatures (Laboratory Director or Assistant Director, as well as the CQ holder).
  • Results received with a date postmark after the required postmark date
  • Absence of Physician's Report

Laboratories that receive a failure must submit problem evaluation data and corrective action documentation. The NYS Cellular Immunology Lab PT Program is available to offer assistance in the investigation of the basis for the unsatisfactory performance. Laboratories that receive failures on 2 out of 3 consecutive PT events may be required to cease testing. The lab must successfully pass 2 PT events before testing can resume.

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Proficiency Test participation

Laboratories must participate in proficiency test events when offered; failure to submit results for a proficiency test event will result in a score of zero for non-participation. However, a laboratory may be excused from participation in a PT event if all of the following conditions are met:

  • Patient testing has been suspended over the timeframe of the PT event
  • The laboratory has performed successfully on the two previous events
  • The proficiency testing program was notified of the temporary suspension of testing prior to the PT shipment

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Recording PT results

In addition to completeing the attestation form and PT result form(s), the results also must be provided as the typical Physician's Report. The Physician's report must contain the following information:

  • Specimen viability prior to analysis (required for lymphoid function, non-lymphoid function, CD34 immunophenotyping, and malignant leukocyte immunophenotyping)
  • Collection time and date
  • Time and date of specimen receipt
  • Time of date of specimen processing
  • Specimen type
  • Special notes
  • Test results, including normal ranges
  • Interpretation of results

For more detailed information, please refer to Regulation 10 of the NYCRR, part 58.1.11. The absence of a Physician's report may lead to a failure if left uncorrected.


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Shipment information

Frequency:

  • Shipments are sent 3 times a year; usually in February, June, and October. Specific dates can be obtained from the CLEP webpage.
  • PT shipments are shipped using UPS overnight service. If you have not received your package by noon of the day following shipment, or if the specimens that arrive are unsuitable for analysis, you must notify us by phone. Failure to do so may result in a grade of zero.

Shipment notification:

  • Notification of an upcoming PT shipment is sent to the e-mail address on file with CLEP. This notification is usually sent approximately a month prior to the shipment date.

Please note:If your PT specimens arrive in an unacceptable manner, indicate how a routine patient specimen would be handled if it were to arrive in the same condition. If there is a problem with the analysis of your PT specimen(s), indicate how the results obtained would be handled with regard to your typical patient's report. All PT specimens are comprised of fresh, whole blood collected the day of shipment. Because of the nature of the specimens, replacement specimens cannot be provided.

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