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Cellular Immunology Proficiency Testing Program

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. Complete program information is available in the New York State Department of Health Clinical Laboratory Reference System Guide to Program Requirements and Services.

Method approval/validations

Please refer to the Cellular Immunology Checklist on the Clinical Laboratory Evaluation Program Test Approval webpage. This form is a fillable PDF file and lists the necessary documentation that must be submitted for assay approval in each Cellular Immunology category.


Instrument Quality Control


Assay Quality Control


Proficiency Testing


Questions Regarding Lymphoid Immunophenotyping of Subsets


Daily flow cytometer quality control requirements:

  • 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 flow cytometer quality control requirements:

  • 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. Please refer to the Cellular Immunology Checklist on the Clinical Laboratory Evaluation Program Test Approval webpage for instrument validation requirements.

Quality Control 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)

Leukocyte Function Assay Quality Control

  • A control must be run on each assay plate or within each run
  • Specimen age must not exceed 24 hours unless departmental approval has been given
  • Viability must be greater than 80% at cell culture initiation

Non-Malignant Immunophenotyping Quality Control

General Quality Control Requirements

  • 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
  • For Stem Cell testing, the viability of CD34+ cells should always be determined.

Specimen age guidelines must be adhered to unless departmental approval has been given.

3, 4, and 6 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% if the testing panel has multiple tubes.
  • 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 laboratory is using side scatter/CD45 gating (3- or 4-color analysis), the lymphosum should optimally equal 95-105% (maximally 90-110%).

Malignant Leukocyte immunophenotyping Quality Control

  • A normal blood or validated commercial control must be run monthly
  • Maximum acceptable specimen age is 48 h
  • Specimens 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

*Please note that all Cellular Immunology proficiency testing is now offered twice a year*

Leukocyte Function Assays:

Assays covered under this subcategory are those which assess the cellular function of the immune system. Lymphocyte function is tested using in vitro assays, including mitogen- and antigen-stimulated proliferation, alloantigen-stimulated proliferation, cytolytic assays, and cytokine or immunoglobulin production. Monocytic and myeloid function is examined using in vitro assays such as respiratory burst, phagocytosis, and cytokine production. Please note: The analysis of cytokines in plasma, serum, or cerebrospinal fluid is covered under the Cytokines category.

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


Non-Malignant Immunophenotyping

The category covers testing performed for the identification and enumeration of non-malignant leukocytes, usually for the purpose of assessing the immunological status of an individual. Examples of assays covered under this category include:

  • Lymphoid and T-Lymphoid Immunophenotyping (e.g., quantifying CD4+ T-lymphocytes
  • Stem Cell analysis (quantifying viable Lin-/CD34+ stem cells)
  • PNH (paroxysmal nocturnal hemoglobinuria) analysis (the assessment of GPI-linked antigen deficiencies)
  • LAD (Leukocyte Adhesion Deficiency) analysis (assessing CD15s, CD11a, b, c and CD18 expression)
  • TLR (Toll-like receptor) expression
  • Evaluation of intracellular signaling molecules (e.g., MyD88, STAT-1)

Proficiency testing is offered for Lymphoid and T-Lymphoid Immunophenotyping (5 specimens per event) as well as for Stem Cell analysis (1 specimen per event).


Malignant Leukocyte Immunophenotyping:

This category covers testing for the identification and characterization of leukemias or lymphomas in blood, bone marrow, and tissue specimens based on cell phenotype (including cell surface and cytoplasmic antigens) with or without ploidy analysis

Three specimens for the immunophenotyping of malignant leukocytes are provided per shipment.


Proficiency testing grading policy

Leukocyte Functional Assays (PHA-induced lymphocyte proliferation only)

  • Proficiency testing available for PHA-induced lymphocyte proliferation only. The results are graded as either satisfactory or unsatisfactory; this grade is based on the inclusion of all required parameters in the patient report and the interpretation of specimen function. The results for this proficiency test are submitted by laboratories via the Electronic Proficiency Testing Reporting System (EPTRS)

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+)

Stem Cell Analysis

  • Accurate evaluation of the number of viable CD34+ stem cells per microliter is required to pass. The results for this proficiency test are submitted by laboratories via the Electronic Proficiency Testing Reporting System (EPTRS)

Malignant Leukocyte Immunophenotyping

  • Accurate and appropriate immunophenotyping of the leukemia/lymphoma (lineage, stage) is required.
  • Scoring of the test is satisfactory or unsatisfactory.

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). For PT results that are submitted via EPTRS the laboratory must retain the attestation form for review during the next on-site inspection.
  • Results received with a date postmark after the required postmark date
  • Absence of a Physician's Report or other required documentation

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 two 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 completing 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:

  • Beginning in 2012 PT shipments will be sent twice a year; shipment dates are posted yearly on the CLEP webpage.
  • PT shipments are shipped via 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, please call 518-408-2107; failure to do so may result in a grade of zero.

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.

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.