YNHH Laboratory Manual

When a sample is sent for flow cytometry, the sample is always first reviewed morphologically by a clinical pathologist (Laboratory Medicine physician). This is essential in determining the proper group of immunophenotypic markers and flow cytometry assays to analyze as well as for determining which cell subpopulations (lymphocytes, myeloid cells, erythroid cells, "blast" cells, etc) should be studied. The pathologist also reviews past flow cytometry results on the same patient (if available in the YNHH laboratory) and, when relevant, past surgical or clinical pathology reports on the patient (again, if available at YNHH). Finally, the clinical history is reviewed - in some cases, in order to render the most comprehensive and accurate diagnosis, the ordering clinician will be called prior to performing the flow assay to ascertain key elements of the history. Thus each flow cytometry specimen is highly "individualized" with respect to its analysis

Because of these considerations, when flow cytometry is ordered, it is most often best to order a "Flow Cytometry Consultation". Under these circumstances, the pathologist will combine all the available information, including morphologic review of the specimen and review of old records and clinical history, and determine the most effective but parsimonious analysis to be carried out in order to render the proper diagnosis. For example, in the case of repeat flow cytometry analysis looking for minimal residual disease in a patient with leukemia, it would be wasteful to carry out a full 'leukemia panel' analysis; instead, a more limited but customized panel targeting the patient's unique leukemic immunophenotypic and/or DNA ploidy "fingerprint" is most appropriate. Specific designated marker panels should only be ordered if the referring physician has already carried out the morphologic, clinical and old flow cytometry review and thereby feels comfortable with determining an analysis panel on that basis

Blood, bone marrow aspirate, lymph node, tissue, cerebrospinal fluid, pleural fluid, peritoneal fluid, pericardial fluid, bronchoalveolar lavage, and apheresis products are the usual specimen types analyzed. CSF specimens generally need to have a minimum white cell count of 10-20 cells per microliter and a minimum volume of 3 ml (at that white count) for adequate analysis but the laboratory should be contacted about specific patient samples

Immunophenotypic studies are carried out as either a "3-color" or "4-color" correlated data analysis. Note that for ease of reporting, only dual antigenic combinations are reported on the final form but the final diagnosis is rendered on the basis of the more 'sophisticated' analysis. Similarly, although numerical results from a single 'gate', are reported, analysis of a minimum of two gates is actually carried out and, again, used for determining the final flow cytometric diagnosis

A summary of many of the 'standard' flow cytometry panels used is given below. Again, it is worth emphasizing that 'customized' panels are more appropriate for many patients and it is generally a better idea to leave the selection of the panel up to the consulting pathologist

[Flow Cytometry Panels]

Acute Leukemia. This is most appropriate for samples with a large population of blasts. Markers include: CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD11b, CD13, CD14, CD19, CD20, CD33, CD34, CD41a, CD45, CD56, CD64, CD22(c), CD117, myeloperoxidase, TdT, glycophorin, kappa, lambda, HLADr. If the disease is pre-B cell, then the following are added: CD58, CD38, intracellular IgM. If the disease is mature B, then the following are added: IgM, IgD, IgG, FMC7, CD23, CD11c, CD43. If the disease is T cell, then the following are added: cytoplasmic CD3, CD1. If the disease is acute lymphoblastic leukemia, then a DNA ploidy study is also added. Note that for followup evaluation of acute leukemia for possible minimal residual disease, customized flow panels to detect an individual patient's unique leukemic "fingerprint" (sometimes including ploidy analysis) are usually most appropriate

Lymphoma/Lymphocytosis This is most appropriate when lymphoproliferative disease is suspected and the sample includes a significant lymphocyte population. Markers include: CD3, CD4, CD5, CD8, CD10, CD14, CD16/56, CD19, CD20, CD23, CD45, kappa, lambda. If monoclonal B cells are identified, then the following are added: CD11c, CD38, CD43, CD103, FMC7, IgM, IgD, IgG. If there is a CD4 lymphocytosis or a disease such as CTCL or HTLV-1 NHL is suspected from the clinical history, then the following are added: CD2, CD7, CD45RA, CD45R0. If a CD8 lymphocytosis or NK lymphocytosis is identified or the clinical history suggests LGL or other relevant disease, then the following are added CD16, CD57, CD56, TcR delta, HLADr, CD25. If there is a suspicion of anaplastic large cell lymphoma, then granzyme, NPM-ALK, EMA, CD25, CD30, CD38 are added. Note that for follow-up evaluation of someone with known lymphoma, generally the basic panel or a customized panel for the patient's particular disease is most appropriate

Myeloma/Lymphoma Appropriate if myeloma and/or lymphoma are suspected (often on the basis of an M-component in serum). Markers include: CD3, CD4, CD5, CD8, CD10, CD14, CD16/56, CD19, CD20, CD23, CD38, CD45, CD56, surface kappa and lambda for mature B cells, cytoplasmic kappa and lambda for plasma cells. If monoclonal B cells are identified then evaluation proceeds as indicated under Lymphoma/Lymphocytosis. If a protocol that subdivides patients on the basis of DNA ploidy results is to be used for someone with myeloma or in those cases in which stem cell transplant therapy is contemplated, a DNA ploidy study may also be appropriate. It should be noted that flow cytometry is NOT a good quantitative technique in the differentiation between plasma cell dyscrasias (eg, MGUS vs myeloma) for a variety of reasons; estimates of the percent plasma cell involvement should be made by morphology from the bone marrow aspirate and/or biopsy and not by flow cytometry techniques

CTCL Appropriate when a clinical diagnosis of CTCL has been made and initial organ involvement is being determined. Markers include: CD2, CD3, CD4, CD5, CD7, CD8, CD14, CD16/56, CD19, CD25, CD45, CD45RA, CD45R0, kappa, lambda. Followup studies on a diagnosed patient should usually involve either a customized panel or the above markers without the kappa and lambda studies

Immunodeficiency This is most appropriate for detailed evaluation of a non-HIV+ immunodeficient patient. This is NOT appropriate for simple T&B cell subsets used to follow patients with HIV disease. When T&B cell subsets are ordered, only the numbers (percent and absolute count) are reported (CD3+, CD3+ CD4+, CD3+ CD8+, CD4/CD8 ratio (calculated), CD16/56+ CD3-, and CD19+). There is no routine morphologic review by the pathologist nor is an interpretive report issued. In the case of T&B subsets, the case will be reviewed by the pathologist if any unusual phenotype cells are identified by the standard immunophenotyping. For the extensive immunodeficiency evaluation panel, the markers included are: CD1, CD3, CD4, CD5, CD8, CD10, CD11b, CD16, CD18, CD19, CD23, CD43, CD45, CD45RA, CD45R0, CD62L, TcR delta, HLA ABC, kappa, lambda. A full interpretation accompanies such an extensive Immunodeficiency evaluation. For followup of these patients, the most appropriate is usually standard T&B cell subsets plus evaluation of any specific abnormalities identified in the initial evaluation

PNH Markers include CD14, CD45, CD55, CD59. Flow cytometry is considered to be the most sensitive way to diagnose PNH. See also the section on pancytopenia below

BAL Analysis. If BAL analysis is undertaken to look for lymphoma or to assess for hypersensitivity and/or granulomatous disease, then the markers include: CD3, CD4, CD8, CD16/56, CD19, CD45, kappa, lambda. If lymphoma is not in the differential diagnosis, then the kappa and lambda analysis is eliminated

Post Stem Cell Transplant. In blood, the usual most appropriate panel includes T cell subsets and an evaluation for monoclonal B cells (especially in the case of HLA-mismatched allo transplants or T-cell depleted allo transplants in which the risk for development of post-transplant lymphoproliferative disease is high) and rarely a 'customized' one for the patient's underlying disease, e.g. circulating blasts for leukemia. In marrow, the most appropriate panel is generally a 'customized' one for the patient's underlying disease

Pancytopenia/Myelodysplasia/Possible Lymphoproliferative Disease. For some bone marrow samples, the clinical history and the morphologic review leaves open a relatively wide differential diagnosis. In these cases, the most appropriate panel is often one that examines for the following markers: CD3, CD4, CD7, CD8, CD10, CD11b, CD16, CD19, CD13, CD33, CD34, CD45, CD57, CD64, CD71, CD117, glycophorin, kappa, lambda. This constitutes a reasonable screening procedure for myelodysplasia, lymphoma, and large granular lymphocytosis. Additional evaluation may be undertaken depending on those results. For a clear case of myelodysplasia on morphologic review where characterization of the blasts is critical, then an evaluation similar to "acute leukemia" is undertaken (albeit carried out in a slightly different technical fashion in order to best define the relatively less frequent blast population). In suspected aplastic anemia, the first panel noted above is often most appropriate in order to rule out lymphoma, LGL disease and myelodysplasia - in that case, PNH evaluation may also be appropriate to add

Mastocytosis Usually the most appropriate evaluation for suspected mastocytosis is to look for lymphoproliferative disease as outlined above and then to look for normal phenotype and abnormal phenotype mast cells by additional evaluation of: CD2, CD25, CD34, CD33, CD64, CD117

Eosinophilia Patients with eosinophilia may have the disorder on the basis of abnormal clones of T cells producing appropriate cytokines. Usually, the most appropriate panel is: CD3, CD4, CD5, CD7, CD8, CD14, CD16/56, CD19, CD25, CD45, CD64, TcR delta. If other lymphoproliferative disease and/or myeloproliferative disease is in the differential based on clinical history and morphologic review, then it may be appropriate to further modify this panel

Neuroblastoma Flow cytometry has been used as a means of looking for minimal disease in neuroblastoma. The most appropriate panel is usually: CD3, CD14, CD16/56, CD19, CD33, CD34, CD38, CD45, CD56, CD81, EMA

Specific Antigen Expression for Assessment of the Potential Utility of a Therapeutic Agent. In some cases, a diagnosis may already be known and tissue involvement by disease may be known, but there is a need to assess the presence or absence of a specific antigen on the diseased tissue in order to gauge the potential for therapeutic effectiveness of a specific agent, often a monoclonal antibody. For example, it may be necessary to assess the CD20 status of a tumor before using Rituximab. Other examples include CD33 or CD22 or CD52 status. In these cases, a special minimal panel to identify the tumor cells and then their specific antigen status is devised by the pathologist

[Other Flow Cytometry Tests]

The following Flow Cytometry tests are often ordered directly by the clinician

CD34 Count. An evaluation of the number of CD34 cells in the specimen, used in the management of stem cell transplant patients. This is usually carried out on blood or an apheresis collection product but may occasionally be carried out on umbilical cord blood or bone marrow. In the case of apheresis samples, an interpretation of the results is rendered by the transfusion medicine team and a separate interpretation will not necessarily appear on the report

T&B Cell Subsets. As noted above, this analysis does NOT include review by the pathologist unless unusual phenotype cells are incidentally identified, nor does it include an interpretation. Numbers reported are the relative and absolute counts for CD3+ (T cells), CD3+ CD4+, CD3+ CD8+, CD4/CD8 ratio (calculated), CD16/56+ CD3- (NK cells), and CD19+ (B cells). There may also be circumstances where just T cell subsets are appropriate to order. These are reported as CD3+, CD3+CD4+, CD3+CD8+ cells, and the calculated CD4/CD8 ratio

Reticulated Platelets. Similar to a red cell reticulocyte count, the reticulated platelet assay measures the number of "young" platelets recently released from the marrow. It can be used to help distinguish hypoproductive thrombocytopenia from thrombocytopenia secondary to increased peripheral platelet destruction. For this purpose, the reticulated platelet percentage should always be interpreted in the context of the platelet count itself - for this reason, an interpretive report is issued with the reticulated platelet assay result

Updated: Sep-2007