Cellular Immunology Proficiency Testing Program

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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

Questions Regarding Lymphoid Immunophenotyping of Subsets

Proficiency Testing

All assays

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.

New assays

All assays (with the exception of CD34 testing, PNH assays, 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.

PNH Assays

An assay validation with five normal and five abnormal specimens must be performed.

Malignant leukocyte Immunophenotyping:

For the category of Cellular Immunology-Malignant Leukocyte Immunophenotyping, please submit a copy of your standard operating procedure manual for review and approval along with results for at least five normal and five abnormal specimens. The five abnormal specimens must be split with a laboratory that has already been approved and holds a New York State permit. Results need to be submitted with copies of patient reports and instrument printouts.

Quantification of double-positive and double-negative T-cells

Why should double positive (DP) CD4/CD8 and double negative (DN) CD4/CD8 T cells be quantified?

With regard to the DP T cells, it is not mathematically appropriate to add the same subset to two different subsets. However, this is what most clinical labs have been doing, in that the number of DP T cells are being included in the number of CD4⁺ T cells and in the number of CD8⁺ T cells. In the majority of healthy individuals, the number of DP or DN T cells is less than 2 % of the total T cell population; however each of these subsets may be as high as 10 % of the total T cell population. Furthermore, these populations can be predictive of health problems. The number of CD4⁺ T cells may be used clinically to make an assessment of the therapies necessary for HIV-infected individuals. It will not be known if better therapeutic decisions could be made if labs continue to provide a number that is the sum of single positive (SP) CD4⁺ CD8^- T cells and DP CD4⁺CD8⁺ cells when these subsets have functional differences beyond the expression of CD8. Although some reports list the number of DP (CD4⁺CD8⁺) T cells, they do not subtract this number from the number of CD4⁺ or CD8⁺ T cells. To access references about DP T cells please click here.

The number of DN T cells also is known to have clinical significance, e.g., in patients with autoimmune lymphoproliferative syndrome (ALPS), the number of DN T cells is significantly increased, and this subset is known to promote autoimmunity. However, most labs do not separately list this subset. In order to obtain the number of DN T cells, the health care provider must know to subtract the numbers of SP CD4 T cells, SP CD8 T cells, and DP T cells from the number of total (CD3⁺) T cells. To access references about DN T cells please click here.

NKT cells

Do T cells expressing CD56 and/or CD16 have different activities from T cells not expressing these markers, and if so, why are they not being reported?

The first answer is YES. CD3⁺ cells are T cells and those cells that are CD3⁺CD56⁺ or CD3⁺CD56⁺CD16⁺ are NK-T cells. NK-T cells do have different activities and different developmental patterns from T cells. Some labs separately report this subset, which as with T cells could be further subdivided into subsets with CD4 and/or CD8. Since many labs are already performing 4- or 6-color immunophenotyping, there is no reason why the NK-T cell subset should not be included in the final report. To access references about NK-T cells please click here.

Clinical usefulness of reporting additional subsets

Why should lymphoid subsets be further divided when there is little clinical data to substantiate the need for such division?

If we reflect on the history of flow cytometry usage for clinical diagnostics, we can note that only about three decades ago many oncologists did not see the need to type leukemias/lymphomas much beyond CD19 (B cells), CD3 (T cells), and possibly a marker for monocyte and granulocyte lineages. As the data was gathered on additional markers, it became clear that further knowledge about the specific stage in a lineage could be useful for therapeutic decisions. Thus, the same is likely for reporting the number of B, T, and monocyte/dendritic cell subsets with regard to immune-associated diseases. However, we clearly do not want to incur additional financial expenses on health care before we have a more firm basis for adding additional subsets, but labs already performing 4- or 6-color immunophenotyping could begin to report additional subsets without any increased cost.

Modifications to existing assays and instrument additions or changes

All assays (with the exception of CD34 testing, PNH assays, 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.

PNH Assays:

An assay validation with five normal and five abnormal specimens must be performed.

Malignant Leukocyte immunophenotyping:

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

Results from the two assays must produce comparable patient outcomes before the new assay can be used.

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

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

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.

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)

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

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%).

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.

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 exceeds the 48h maximum age and/or the minimum 50% viability, but is irreplaceable, then the 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.

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.

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 CQ holder, for the pertaining category).
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.

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

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.

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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