Prashant Tembhare

Immunophenotypic profile of plasma cell leukemia: a retrospective study in a reference cancer center in India and review of literature.

BACKGROUNDnPlasma cell leukemia (PCL) is a rare but aggressive subtype of plasma cell dyscrasia. It is known to present with highly variable morphological features and may mimic with other lymphoid neoplasms. Multicolor flow cytometry (MFC) with availability of newer markers is highly useful in the diagnosis of the plasma cell leukemia. We present an immunophenotypic profile in ten cases of PCL along with their clinical and laboratory findings.nnnMATERIALS AND METHODSnWe retrospectively studied immunophenotypic profile of 10 cases of plasma cell leukemia (out of 4615 cases of hematolymphoid neoplasms) using five parameter, three color flow cytometric analysis. We also studied their clinical presentation and other laboratory findings.nnnRESULTSnCommon clinical features at presentation were weakness, bone pain, anemia, thrombocytopenia and osteolytic lesions. Plasma cell population was identified on strong expression of CD38 and co-expression of CD38 and CD138. CD56 was expressed in 44% cases. CD19 and CD20 were negative in all cases. Surface light chain restriction was seen in 50% cases and in remaining 50% cases revealed cytoplasmic light chain restriction. CD117 was expressed in one out of two cases studied.nnnCONCLUSIONSnMFC immunophenotyping is highly useful to differentiate Plasma cell leukemia from other chronic lymphoproliferative disorders with plasmacytoid morphology as well as from non-neoplastic reactive PC and co-expression of CD38 and CD138 is a best combination to identify the plasma cells by MFC.

Report of proceedings of the national meeting on "Guidelines for Immunophenotyping of Hematolymphoid Neoplasms by Flow Cytometry"

BACKGROUNDnImmunophenotyping of hematolymphoid neoplasms is being done in many laboratories in India. The first national meeting on Guidelines for Immunophenotyping of Hematolymphoid Neoplasms by Flow Cytometry was held on 14 March 2008 in Mumbai, India.nnnAIMnTo achieve uniformity in the laboratory practice regarding antibody panel selection in diagnosing hematolymphoid neoplasms.nnnSETTINGS AND DESIGNnMembers of the Inter-Laboratory Comparison Program (ILCP) group in Mumbai prepared a draft regarding immunophenotypic panel selection for acute leukemias (ALs) and chronic lymphoproliferative disorders (CLPDs), which was further circulated among national and international cytometrists, hematopathologists, and oncologists for their written inputs, suggestions, proposed modifications; as well as their indications, if any, of the recommendations not being acceptable. Practice-based questionnaire was circulated among all the participants.nnnRESULTSnConsensus was attained, and the panel recommended the use of a minimal screening panel, followed by a secondary directed panel. The aim of the minimal screening panel would be to provide a diagnosis of all commonly occurring hematolymphoid neoplasms without the need of additional antibodies in most cases.nnnCONCLUSIONnThus we could attain a consensus for our guidelines in selecting panels for ALs and CLPDs. The guideline is an attempt to formulate a minimal panel for immunophenotyping of hematolymphoid neoplasms. Laboratories are encouraged to add additional antibodies to the above panel to increase the sensitivity; however, they should refrain from immunophenotyping with fewer antibodies. This national guideline hopefully brings about uniformity and comparability in reporting of leukemia and lymphoma and bridges the divide between low-cost reporting and an accurate diagnosis.

Population pharmacokinetics of Reditux™, a biosimilar Rituximab, in diffuse large B-cell lymphoma

AbstractPurposenRituximab (MabThera™, Roche) is a chimeric IgG1 monoclonal antibody targeting the CD20 surface antigen on normal and neoplastic B cells. It revolutionized the treatment of non-Hodgkin’s lymphoma with superior progression-free and overall survival. However, its prohibitively high cost makes it inaccessible to majority of patients in developing countries. Reditux™ (Dr. Reddy’s Laboratories, India), a biosimilar, was introduced in India in 2007 at nearly half the price of the innovator. However, there is a dearth of data regarding the pharmacokinetics and efficacy of Reditux™.MethodsTwenty-one patients of diffuse large B-cell lymphoma on R-CHOP regimen were enrolled for the study. Reditux™ was administered as a slow intravenous infusion at a dose of 375xa0mg/m2 on day 1 of a 21-day cycle. Pharmacokinetic sampling was performed at pre-dose, post-infusion, 24, 48xa0h, 7 and 21xa0days. Rituximab levels were estimated by ELISA. Population pharmacokinetics was performed using NONMEM. In addition, B-cell count was determined at baseline and days 3 and 21 of the first cycle. Survival analysis was performed using Kaplan–Meier plots.ResultsThe volume of distribution of central compartment and clearance of Reditux™ were estimated at 0.95xa0L and 5.98xa0mL/h, respectively. No covariate effects were seen. B-cell count was completely depleted by day 3 and remained so on day 21. Overall survival was 84.6xa0% at a median follow-up of 36xa0months.ConclusionThe pharmacokinetic profile and B-cell response to Reditux™ are comparable with those reported for MabThera™. Thus, MabThera™ can be substituted with Reditux™ for the treatment of B-cell lymphomas.

Flow cytometric analysis of erythrocytes in paroxysmal nocturnal hemoglobinuria reveals superiority of CD59 as a diagnostic marker compared to CD55.

CONTEXTnParoxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal stem cell disorder characterized by complement-mediated hemolysis due to reduced expression of glycosyl phosphatidylinositol-anchored complement deactivating proteins such as CD55 and CD59 on RBC. Flow cytometric analysis of CD55 and CD59 expression by RBC is a reliable tool for the diagnosis of PNH.nnnAIMSnDetection and quantification of PNH clone and comparison of the relative role of CD55 and CD59 expression by RBC in the diagnosis of PNH.nnnMATERIALS AND METHODSnFlow cytometric analysis of RBC was performed in blood samples of 239 patients by direct immunofluorescence using monoclonal anti-CD55 and anti-CD59 antibodies. CD55 and CD59 expressions by RBC were compared in 54 cases in which PNH clones were detected.nnnRESULTSnOut of 54 cases, 85% and 72% revealed CD59 and CD55 negative populations, respectively. Various combinations of type II and III erythrocytes could be identified in all cases having CD59 deficient RBC. In contrast, distinct populations of CD55-deficient RBC were seen in only 33% cases. In the remaining (67%) cases, CD55 negative RBC caused sloping of the ascending limb of the histogram resulting in difficulties in interpretation. Fifteen percent cases had false CD55-deficient RBC and in 23% cases anti-CD55 antibody failed to identify PNH clones which were detected by CD59.nnnCONCLUSIONnCD59 is a better marker for the diagnosis of PNH. Although CD55 negativity supported the diagnosis of PNH in cases with CD59-deficient RBC, its role as an independent diagnostic marker for PNH is questionable due to its lower sensitivity and specificity.

Exflagellated microgametes of Plasmodium vivax in human peripheral blood: a case report and review of the literature.

Peripheral blood smear examination is the most specific as well as the most common test performed for the diagnosis of malaria. Schizonts, ring forms (trophozoites) and gametocytes are the stages of malarial parasite that are commonly seen in the peripheral blood smear of a patient. Here, we report an extremely rare case of a 40-year-old male patient who presented with Plasmodium vivax infection with multiple exflagellated microgametes in the peripheral blood smear with review of the literature. Exflagellation of microgametes in malarial parasites is only seen in the definitive host, mosquito, and is very unusual to see during the developmental phases in the intermediate host, human. It is important to recognize these exflagellated microgametes in the peripheral blood smear as they may lead to diagnostic confusion with organisms such as spirochetes and trypanosomes.

Hypergranular precursor B-cell acute lymphoblastic leukemia in a 16-year-old boy.

Presence of cytoplasmic granules in the blasts is a well known feature of myeloid leukemia. ALL presenting with the numerous cytoplasmic granules in blasts is a rarity and may be misdiagnosed as acute myeloid leukemia. We describe a rare case of hypergranular precursor B-cell acute lymphoblastic leukemia (ALL) in an adolescent male expressing CD10, CD19, CytoCD22, CD34, as well as CD13 and CD117. The blasts were cytochemically negative for myeloperoxidase (MPO), and acid phosphatase (ACP) but were positive for non-specific esterase (NSE). In centers where immunophenotypic panel is usually decided on the basis of morphology with limited antibodies may result in an erroneous typing of such rare diseases. Hence it is important to be aware of this rare entity and to confirm the lineage of acute leukemia by using a comprehensive panel of antibodies for immunophenotypic analysis.

Intracytoplasmic antigen study by flow cytometry in hematolymphoid neoplasm.

Flow cytometric detection of intracellular antigens has become a standard method in establishing proper leukemic cell lineage affiliation. It has a non-debatable contribution to the diagnosis of hematolymphoid neoplasm as well as in minimal residual disease. Combination of analysis of fluorescence labeling and light scatter properties of cells allows rapid and better determination of target cell antigens. Regarding the detection of intracellular antigens, standardization of the procedure remains, however, a real challenge. Detection of intracellular antigens by flow cytometry (FCM) requires effective fixation and permeabilization of the cell membrane. In the available literature, some reports describe methodologies to achieve satisfactory results for detection of either cytoplasmic or nuclear antigens; however, no methodological consensus has yet been achieved among the laboratories. This article is an attempt to describe different approaches to detect intracellular molecules by FCM.

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.

Expression of the IL-6 receptor alpha-chain (CD126) in normal and abnormal plasma cells in monoclonal gammopathy of undetermined significance and smoldering myeloma

Abstract IL-6 activity in normal plasma cells (nPCs) and abnormal plasma cells (aPCs) is CD126 (subunit of IL-6 receptor) dependent. We quantified CD126 expression on nPCs and aPCs in monoclonal gammopathy of undetermined significance (MGUS), smoldering myeloma (SMM), and multiple myeloma (MM). CD126 was detected on all nPCs and aPCs indicating that CD126 does not have diagnostic utility. CD126 expression was higher in aPCs than in nPCs in 85% SMM but only 41% MGUS and there was evidence that CD126 was higher in aPCs than nPCs in the SMM (pu2009=u2009.048) but not MGUS (pu2009=u2009.96) patients. There is also a greater association between nPC and aPC CD126 expression in low risk MGUS than observed in high risk MGUS and SMM, suggesting normal regulation of CD126 decreases with disease progression. Future studies need to elucidate the role of bone marrow milieu versus escape from normal CD126 regulation in malignant transformation of clonal plasma cells.

'Childhood systemic mastocytosis associated with t (8; 21) (q22; q22) acute myeloid leukemia'

Systemic mastocytosis (SM) with associated clonal nonmast cell lineage disease is seen in up to 20% cases of SM. SM is uncommon in the pediatric population. T (8; 21) (q22; q22) is a good prognostic factor in acute myeloid leukemia (AML). However, the presence of SM confers poor prognosis in t (8; 21) (q22; q22) associated AML. We report the case of a child with t (8; 21) (q22; q22) associated AML with SM and her minimal residual disease status over the course of her treatment. In our case, the abnormal mast cells, showing co-expression of CD25 and CD2, persisted even after the marrow showed no evidence of residual AML.