Non-Malignant Leukocyte Immunophenotyping covers testing performed for the identification and enumeration of non-malignant leukocytes 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)
• T cell subset (Th1, Th2, Th17, Treg) quantification with analysis of intracellular cytokine or transcription factor expression
• 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) and cytokines. 

Please note: The ex vivo analysis of cytokines in plasma, serum, or cerebrospinal fluid is covered under the Cytokines category.

General Quality Control Requirements for Non-Malignant Leukocyte Immunophenotyping

• A normal blood and/or validated commercial control(s) must be run daily. 
  • Cellular Immunology Standard CINM-S11
• Specimens must be processed within the manufacturer’s recommended timeframe. If not defined by manufacturer, then specimens containing EDTA have a maximum acceptable specimen age of 30 hr and specimens containing Heparin or ACD have a maximum acceptable specimen age of 48 hr.  Laboratories may establish and validate maximum specimen ages, but testing conducted beyond the previously defined limits, requires viability testing to determine quality of results.
  •  Cellular Immunology Standard CINM-S2 and CINM-S3
• For Stem Cell testing, the viability of CD34⁺ cells should always be determined. 
  • Cellular Immunology Standard CINM-S24

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

Lymphoid Immunophenotyping Analysis Quality Control Requirements

• 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. 
  • Cellular Immunology Standard CINM-S17
• T-sum: For four or more color analysis, the summation of the single positive T cells (CD3⁺CD4⁺CD8^- and CD3⁺CD4^-CD8⁺ cells), the double positive T cells (CD3⁺CD4⁺CD8⁺) and the double negative T cells (CD3⁺CD4^-CD8^-) shall not exceed a difference of 3 for the mean CD3 percentage.  For three color analysis, the summed % 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. 
  • Cellular Immunology Standard CINM-S19
• Lymphosum: The Lymphosum is the sum of % CD3⁺ (T cells), % CD19⁺ (B cells), and % CD3^-(CD56⁺± CD16⁺) (NK cells). If the laboratory is using side scatter/CD45 gating, the lymphosum must be within 90-105%. 
  • Cellular Immunology Standard CINM-S18

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.