Sitaram Ghogale

Evaluation of new markers for minimal residual disease monitoring in B-cell precursor acute lymphoblastic leukemia: CD73 and CD86 are the most relevant new markers to increase the efficacy of MRD 2016; 00B: 000–000

Multiparametric flow cytometry (MFC) is a popular technique for minimal residual disease (MRD) analysis. However, its applicability is still limited to 90% of B‐cell precursor acute lymphoblastic leukemia (BCPALL) due to two major issues, i.e. a proportion of cases do not express adequate leukemia associated immunophenotype (LAIPs) with currently used markers and drug‐induced antigen modulation. Hence, the incorporation of additional reliable markers is required for the further improvement of MFC‐based MRD evaluation. We studied the utility of new markers in improvising MFC‐based MRD detection in BCPALL.

A novel and easy FxCycle™ violet based flow cytometric method for simultaneous assessment of DNA ploidy and six-color immunophenotyping

Abnormal DNA ploidy is a valuable prognostic factor in many neoplasms, especially in hematological neoplasms like B‐cell acute lymphoblastic leukemia (B‐ALL) and multiple myeloma (MM). Current methods of flow‐cytometric (FC) DNA‐ploidy evaluation are either technically difficult or limited to three‐ to four‐color immunophenotyping and hence, challenging to evaluate DNA‐ploidy in minute tumor population with background rich of its normal counterpart cells and other hematopoietic cells. We standardized a novel sensitive and easy method of simultaneous evaluation of six‐ to seven‐color immunophenotyping and DNA‐ploidy using a dye–FxCycle Violet (FCV). Linearity, resolution, and coefficient of variation (CV) for FCV were studied using chicken erythrocyte nuclei. Ploidy results of FCV were compared with Propidium iodide (PI) in 20 samples and intra‐assay variation for FCV was studied. Using this six‐color immunophenotyping & FCV‐protocol DNA‐ploidy was determined in bone‐marrow samples from 124 B‐ALL & 50 MM patients. Dilution experiment was also conducted to determine the sensitivity in detection of aneuploidy in minute tumor population. FCV revealed high linearity and resolution in 450/50 channel. On comparison with PI, CV of Go/G1‐peak with FCV (mean‐CV 4.1%) was slightly higher than PI (mean‐CV 2.9%) but had complete agreement in ploidy results. Dilution experiment showed that aneuploidy could be accurately detected up to the limit of 0.01% tumor cells. Intra‐assay variation was very low with CV of 0.005%. In B‐ALL, hypodiploidy was noted in 4%, hyperdiploidy in 24%, near‐hyperdiploidy in 13% and remaining 59% were diploid. In MM, hypodiploidy was in 2%, hyperdiploidy in 58%, near‐hyperdiploidy in 8% and remaining 30% were diploid. FCV‐based DNA‐ploidy method is a sensitive and easy method for simultaneous evaluation of six‐color immunophenotyping and DNA analysis. It is useful in DNA‐ploidy evaluation of minute tumor population in cases like minimal residual disease and MM precursor conditions.

Comparison of platelet counts by CellDyn Sapphire (Abbot Diagnostics), LH750 (Beckman Coulter), ReaPanThrombo immunoplatelet method (ReaMetrix), and the international flow reference method, in thrombocytopenic blood samples

We compared the international flow reference method (IRM) platelet counts with those obtained from CellDyn Sapphire (impedance and optical counts), LH750 (impedance counts), and the flowcytometry based ReaPanThrombo Immunoplatelet method (ReaMetrix). We further evaluated the degree of agreement of above methods with the IRM at the transfusion thresholds of 10 × 109 l−1 and 20 × 109 l−1.

Evaluation of CD229 as a new alternative plasma cell gating marker in the flow cytometric immunophenotyping of monoclonal gammopathies: CD229, A NEW RELIABLE PLASMA CELL GATING MARKER

Current flow‐cytometric plasma cell (PC) gating is based on CD138, CD38, and CD45 expression. CD138 is known for variable expression and loss during storage and processing. Introduction of anti‐CD38 and anti‐CD138 monoclonal‐antibody therapies has limited the use of these markers during follow‐up. Hence, additional reliable PC‐gating markers are required. Recently, CD229 has been claimed as an alternative PC‐gating marker. However, these studies are limited to a small cohort of samples. We evaluated the utility of CD229 as a new PC‐gating marker in routine laboratory practice.

CD19 negative precursor B acute lymphoblastic leukemia (B-ALL)—Immunophenotypic challenges in diagnosis and monitoring: A study of three cases

CD19 is a B‐cell specific marker, expressed on all stages of B‐lymphocytes including plasma cells. It is widely used in the flow cytometric immunophenotyping (FCI) of B‐cell and plasma cell malignancies. The analysis approach of FCI for the diagnosis and monitoring of B‐cell acute lymphoblastic leukemia (B‐ALL) is totally based on the CD19‐based primary gating strategy and it would be challenging to study B‐ALL without CD19 expression. Since CD19 negative B‐ALL are extremely rare, we report three cases of B‐ALL with negative expression of CD19 and discussed its implication in the diagnosis, residual disease monitoring and future targeted therapy.

Morphological spectrum of leukemic mantle cell lymphoma.

BACKGROUND Leukemic involvement in mantle cell lymphoma (MCL) is common, and can be secondary to nodal or extranodal disease or can be de-novo. There is paucity of literature that describes the morphological spectrum. AIM This study was aimed at studying the morphological spectrum of leukemic MCL and to correlate the morphology with other features. MATERIALS AND METHODS Twenty six such cases diagnosed over a period of four years were studied. Peripheral blood and bone marrow aspiration smears stained with Wrights stain were examined by three hematopathologists. Immunophenotyping was done using multicolor flow cytometry. Fluorescence in situ hybridization (FISH) done in 12 cases showed t(11;14)(q13:q32). RESULTS Six cases had de-novo leukemic involvement; while 20 cases had secondary involvement. Morphologically, the cells were small (less than twice the size of red blood cell) or large. Small cell morphology in turn showed irregular nuclear border (n=13) or round nuclear contour (n=6). Large cells had blastic morphology (n=5) or had central prominent nucleoli resembling prolymhphocytes (n=2). Twenty cases showed characteristic immunophenotype of CD5+/CD19+/CD20+/FMC7+/CD10-/CD23- and light chain restrictions. Three cases expressed CD23 and two cases were negative for FMC7. Five out of 12 cases, where FISH was done, showed cytogenetic abnormalities in addition to t(11;14)(q13;q32). CONCLUSION Morphological spectrum of leukemic MCL ranges from small cells resembling chronic lymphocytic leukemia (CLL) or follicular lymphoma (FL) to large cell mimicking prolymphocytic leukemia (PLL) or acute leukemia. Large cell morphology was associated with more frequent additional cytogenetic abnormality as well as a poorer outcome.

Method for DNA Ploidy Analysis Along with Immunophenotyping for Rare Populations in a Sample using FxCycle Violet

The clinical use of flow cytometric DNA ploidy assay has been extended towards stratifying the risk of diseases, such as monoclonal gammopathies or B cell acute lymphoblastic leukemia, and to detect circulating tumor cells, both of which require detection of minute cell populations. This unit describes a protocol for determining DNA ploidy in fixed samples with simultaneous surface immunophenotyping. It is an easy method for simultaneous 6‐ to 8‐color immunophenotyping and DNA content analysis using FxCycle Violet (FCV; DAPI) dye. This protocol is a one‐step modification of routine multicolor immunophenotyping that includes surface staining followed by fixation and then DNA staining with FCV. It utilizes mature lymphocytes from the sample as an internal control for determination of DNA index. It is a sensitive method that allows DNA‐ploidy determination and cell cycle analysis in a rare tumor population as low as 100 events, as well as DNA ploidy determination in various subsets of hematopoietic cells in the same sample based on their immunophenotype.

Anaplastic lymphoma kinase-positive large B-cell lymphoma: Unusual clinical and immunophenotypic features

Anaplastic lymphoma kinase (ALK)-positive large B-cell lymphoma (ALK+ LBCL) is a rare subtype of diffuse large B-cell lymphoma. It is characterized by plasmacytic differentiation and cytoplasmic ALK positivity. Immunophenotype of ALK+ LBCL defines the terminally differentiated B-lineage cell characterized by the absence of B-cell antigens and expression of antigen associated with plasma cell differentiation. It is characterized by an aggressive behavior and poor response to standard chemotherapy. Advanced clinical stage and extranodal disease are associated with worse survival. We present a very rare case of ALK+ LBCL in a young immunocompetent lady who presented with multiple subcutaneous nodules and discuss the role of flow cytometry for prompt and accurate confirmation of the diagnosis.