WHO classification of hemato-lymphoid neoplasm has moved beyond morphology (FAB classification), to integrate clinical information, morphology, immunophenotyping by flow cytometry (FCM) or imunohistochemistry (IHC), cytogenetics, and molecular data. Commonly seen leukemias are acute myeloid leukemia (AML), acute lymphoid leukemia (ALL), chronic myeloid leukemias (CML) and chronic lymphoproliferative disorders (CLPD). Sub typing of acute leukemias and CLPDs is important, as treatment and prognosis is different. With the addition of cytochemistry to the morphologic assessment, most cases of acute leukemia can be appropriately designated as either AML or ALL. However, there remains a significant minority of cases that cannot be definitively diagnosed by these methods. In such cases we need immunophenotyping by flow cytometry (FCM) for a definite diagnosis. Similarly CLPD can also be diagnosed on a simple morphological evaluation of the peripheral blood smear. But for further characterization of CLPD, we need flow for further characterization. FCM has become a standard and a routine practice for the evaluation of lymphomas and leukemia in most diagnostic laboratories. It is considered second to morphology for a diagnosis of hemato-lymphoid neoplasms.

Immunophenotyping may be done by FCM or by IHC. FCM analysis is usually performed on blood, bone marrow specimens and other body fluids or tissues (lymph nodes). IHC is preferred on paraffin blocks, frozen sections and smears.

Commonest application of FCM in clinical practice is CD4/CD8 counts, immunophenotyping of leukemias and CD34 stem cell counts. Laboratories generally design their own panels for acute leukemias and CLPDs. Even for the same question, different cytometrist/pathologists might use different panels. There may be a significant difference in the number and the types of antibodies different laboratories use.

Applications of FCM
FCM and AML
In acute leukemia, the cornerstones of diagnosis are blast percentage, obtained by morphology, followed by cytochemistry, mainly myeloperoxidase (MPO) and non-specific esterase (NSE) stains) and lineage (myeloid versus lymphoid), which may be provided by FCM.

FCM studies are important in those cases of acute leukemia where blasts do not show Auer rods and are negative for MPO and NSE stains. Thus the subtypes like AML-MO, AML-M7 (may be NSE+), need FCM for a definitive diagnosis. Classical cases of AML do not require expensive FCM studies.

FCM for diagnosis of hematolymphoid malignancies is a very central element e.g., Identifying acute promyelocytic leukemia (APML) is important because it has a specific therapy. Having a classical morphology, the promyelocytes show a strong MPO positivity. FCM shows a myeloid leukemia (CD13, CD33 and CD117 positivity) along with HLADR negativity. However cytogenetics/FISH test is must for a confirmation and management of APML.

FCM and CML
FCM is not required for CML, unless the patient is progressing to accelerated phase or blast crisis.

FCM and ALL
ALL is one of the commonest leukemias in childhood. Most of the ALL are CALLA (CD10) positive ALL. All acute leukemia cases which do not show presence of Auer rods or are negative with MPO and NSE need flow evaluation for a definitive diagnosis and further characterization. Thus most of the childhood leukemias need FCM evaluation.

DNA index (DI) by FCM gives prognostic information in cases of ALL, showing that cases with a DI >1.12 (corresponding to 51 chromosomes, hyperploidy) have a superior outcome.

FCM and CLPDs
Peripheral blood smear might show monomorphic lymphocytosis with smudge cells, suggestive of CLL. FCM is done to separate CLL from other CLPDs, manly MCL and HCL.

Usually with a suspected lymphoma, we do a lymphoma panel. Second line of panel may be required in a few cases. For a B-cell lymphoma, 99 percent of the time flow cytometry will provide an answer. It is interesting to note that two of the common B-cell CLPDs (CLL and MCL) express CD5, a T-cell marker. Scoring system has been devised for the diagnosis of CLL. CLL is of two subtypes, depending upon their cell of origin. Poor prognostic group of CLL expresses CD38 and high levels of ZAP 70 protein.

Panels for Acute Leukemia at TMH
A) Primary panel :
B-cells - CD10, CD19, T-cells - CD3, CD4, CD7, CD8, Myeloid - CD13, CD33, CD117, Non-lineage - HLA-DR, CD34, TdT, Positive Control: CD45 (LCA), Negative Control: Isotype IgG1.

B) Secondary panel :
B-lineage specific - cytoCD22 / cytoCD79a, T-lineage specific - cytoCD3, Myeloid lineage specific - anti-MPO, Other Markers - CD41, CD61, Glycophorin A, CD56 and CD16.

Panels for CLPD at TMH
A) Positive Control : CD45 (LCA), Negative Control - Isotype IgG1

B) Primary CLPD panel : Mainly for chronic lymphocytic leukemia / follicular lymphoma - CD45, CD7, CD3, CD5, CD23, CD19, CD38, CD20, CD10, IgM, CD22, SmIg, K/L light chains, FMC7, CD79b (16 antibodies + controls) – This is the first panel for all cases of CLPDs and is decided on morphology of the smears.

C) Various secondary panels (depending upon morphology and primary panel) :

1. Hairy cell leukemia – CD19, CD25, CD103, CD11c, kappa and lambda light chains. When morphology is classical, than these six markers are done in addition to AP and TRAP.

2. Plasma cell dyscrasias – CD38, CD138 (in addition to CLPD panel)

3. T-cell chronic lymphoproliferative disorders – CD4, CD8, TCR a/b,g/d (in addition to CLPD panel).

4. LGL (NK) cell leukemia – CD16 and CD56 (in addition to CLPD panel).

FCM and PNH
Antibodies to CD55 and CD59 are specific for decay-accelerating factor and membrane-inhibitor of reactive lysis, respectively, and can be analyzed by FCM to make a definitive diagnosis of PNH. It is a extremely sensitive and specific method.

FCM and HD
FCM is unable to detect neoplastic cells of Hodgkin lymphoma.

FCM and Biphenotypic Leukemias

It means scores for myeloid and one of the lymphoid lineages, or score of T and B lymphoid lineage are > 2 points. Secondary panel (cytoCD22, cytoCD3 and Anti-MPO).

Table 6 : Scoring system (EGIL group) for acute biphenotypic leukemias (Bene et al)

Biphenotypic acute leukemia (EGIL) is defined when scores for the myeloid and one of the lymphoid lineages are > 2 points. Others follow St. Judes Criteria.\

Minimal residual disease (MRD) and FCM
Data are still being acquired as to what role detection of MRD will play in directing therapy, but there is already good information that it helps with long-term prognosis and survival, particularly for acute leukemias. FCM is a sensitive method to detect residual disease in CLPDs, but the clinical implications have not yet been established. At this point, even some high-volume flow centers don’t do MRD monitoring. We do not do it at TMH. It is preferred to have a dual laser FCM but we at TMH have a single laser and can at maximum study three colors. Most oncologists are not yet ready to act on information available after studying MRD.

Body Fluid Analysis and flow cytometry
Primary effusion lymphomas, pleural effusions and CSF involved by lymphoma are a few conditions where FCM may be used for immunophenotyping to differentiate benign from malignant conditions.

FNAC and FCM
In the setting of a busy cancer hospital, FCM on FNA samples may be a highly effective method of diagnosing and typing B cell lymphomas. FCM is of little use for T cell lymphomas or Hodgkin’s lymphomas. Rarely done at TMH, as open biopsy is preferred followed by IHC.

FCM and therapeutic uses
FCM can also be used to identify protein targets of therapeutic monoclonal antibodies, such as Rituximab (anti-CD20) and Mylotarg (anti-CD33), Alentuzimab (anti-CD52) as well as prognostic factors, such as ZAP-70/CD38 in CLL.

FCM versus IHC
FCM is more rapid and sensitive. We prefer FCM for peripheral blood/ BM and IHC for lymph nodes.

Referral laboratories and FCM
Pathologist signing out in a commercial laboratory should also look at histology, peripheral blood and bone marrow smears. It is dangerous to look at just one component without the others.

Samples are send to the tertiary laboratory for FCM but these lab might have their own problems like, how to select panels, ?cost restraints ?? secondary panels. They might send back percentage values without any interpretation. There is a real potential for misdiagnosing and missing low-frequency abnormal populations.

Conclusion
Based on the clinical information provided by the referring clinician, and a morphologic evaluation of the specimen, the hematopathologist will determine the specific immunophenotyping panel to be performed.  This panel is a carefully considered combination of antibodies, which allows for accurate assessment of specific cell populations within a specimen. The lymph nodes specimen is assessed with a panel predominantly focusing on T-cells and B-cells, whereas a bone marrow evaluation also includes markers for myeloid populations, depending on the clinical context. Thus an acute leukemia, CLPD or a more selective panel may be selected.  Most important four markers for CLPD are CD19, CD5, CD23 and CD10. Similarly most five important markers for acute leukemia will be CD3, CD19, CD10, CD13, CD33/CD117. Anti-MPO is preferred over other myeloid markers but staining is more tedious. Sample collection, transport and processing are important as pre analytical errors are extremely common.

References :
1. Bene MC, Bernier M, Casanovas RO, Castoldi G, Knapp W, Lanza F, Ludwig WD, Matutes E, Orfao A, Sperling C and van’t Veer MB for the European Group for the Immunological Classification of Leukemia (EGIL) (1998a). The reliability and specificity of c-kit for the diagnosis of acute leukemias. Blood 92, 596-599

2. Bain B, Barnett D, Linch D, Matutes E and Reilly JT (2002) Revised guidelines on immunophenotyping in acute and chronic lymphoproliferative disorders. Clin Lab Haem 24, 1-13.