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CD229 has been found to be a useful plasma cell (PC) gating marker in multiple myeloma (MM). This study analyses the expression profile of CD229 on various bone marrow compartments namely, PC, non-PC and hematogones (HGs) using Multiparameter flow cytometry (MFC). Furthermore, it evaluates the ability of CD229 to delineate normal PC (NPC) from aberrant PC (APC) in measurable residual disease assessment (MRD) in MM.Bone marrow aspirates from patients diagnosed with MM (per standard IMWG criteria) were collected in EDTA and processed for MFC using a single tube 14-color antibody panel as per standard operating procedure.A total of 74 patients with a diagnosis of MM (26 treatment naïve and 48 on therapy) were evaluated. The expression of CD229 was homogenous on both the PC and HG compartments as compared to CD138 and CD38. On comparing the expression of individual markers, it was found to be statistically significant between PC, HGs and non-PC for all three markers (p 0.001). APC showed lower median expression of CD38 and higher median expression of CD138 and CD229 as compared to NPC and was found to be statistically significant for all markers (p 0.001). In terms of differential expression on NPC and APC; CD38 was found to be the most aberrantly expressed (70%; 52/74) followed by CD229 (7%; 5/74) and CD138 (5%; 4/74).CD229 can be used for the identification of PC and due to relatively homogenous expression; it can be used as a suitable marker for targeted therapies. However, precise discrimination of NPC from APC cannot be reliably achieved with CD229, limiting its utility as a useful marker of diagnostic relevance and MRD assessment in MM.

To gain insights into the idiosyncrasies of CD34 + enriched leukemic stem cells, we investigated the nature and extent of transcriptional heterogeneity by single-cell sequencing in pediatric AML. Whole transcriptome analysis of 28,029 AML single cells was performed using the nanowell cartridge-based barcoding technology. Integrated transcriptional analysis identified unique leukemic stem cell clusters of each patient and intra-patient heterogeneity was revealed by multiple LSC-enriched clusters differing in their cell cycle processes and BCL2 expression. All LSC-enriched clusters exhibited gene expression profile of dormancy and self-renewal. Upregulation of genes involved in non-coding RNA processing and ribonucleoprotein assembly were observed in LSC-enriched clusters relative to HSC. The genes involved in regulation of apoptotic processes, response to cytokine stimulus, and negative regulation of transcription were upregulated in LSC-enriched clusters as compared to the blasts. Validation of top altered genes in LSC-enriched clusters confirmed upregulation of TCF7L2, JUP, ARHGAP25, LPAR6, and PRDX1 genes, and serine/threonine kinases (STK24, STK26). Upregulation of LPAR6 showed trend towards MRD positive status (Odds ratio = 0.126; 95% CI = 0.0144-1.10; p = 0.067) and increased expression of STK26 significantly correlated with higher RFS (HR = 0.231; 95% CI = 0.0506-1.052; p = 0.04). Our findings addressed the inter- and intra-patient diversity within AML LSC and potential signaling and chemoresistance-associated targets that warrant investigation in larger cohort that may guide precision medicine in the near future.

CD123 is overexpressed in many hematologic malignancies and found to be useful in characterizing leukemic blasts of both acute myeloid leukemia (AML) and B-acute lymphoblastic leukemia (B-ALL). CD123 has been recently found to be a marker of leukemic stem cells, and its utility to measure residual disease and potential role in disease relapse is under evaluation.Herein, we have evaluated the expression of CD123 in 757 samples of acute leukemia including 479 treatment-naive and 278 follow-up samples and compared with post-induction morphologic complete remission and measurable residual disease (MRD) status. Multiparametric flow cytometry was used for assessment of CD123 expression and immunophenotypic characterization of leukemic blasts at diagnostic and MRD assessment time points.Using variable cutoffs of 5%, 10%, and 20% to define a case as CD123-positive, expression of CD123 was observed in 75.6%, 66.2%, and 50% of AML and 88.6%, 81.8%, and 75% of B-ALL, respectively. Of 11 patients, 7 (63.63%) had mixed phenotype acute leukemia, but none of the 12 patients with T-acute lymphoblastic leukemia showed positivity for CD123. CD123 expression at diagnosis was associated with post-induction MRD-positive status in both B-ALL (P .001) and AML (P = .001). We also evaluated the utility of CD123 as a leukemia-associated aberrant immunophenotype and found it to be useful in both patients with AML (baseline, 50.6%; follow-up, 53%) and B-ALL (baseline, 75%; follow-up, 73.07%).In conclusion, CD123 may be considered as a cardinal marker for residual disease assessment and response evaluation in AML and B-ALL.

Context A deeper understanding of the normal plasma cell (PC) compartment and changes in the immunophenotypic profile post-treatment is required to assess low levels of residual disease in multiple myeloma (MM) using multiparametric flow cytometry. Objective The expression profile of selective antigens on normal and aberrant plasma cells prior to and following anti-myeloma therapy was assessed to ascertain its relevance in MRD assessment. Design This is a prospective analysis of 702 patients with the diagnosis of MM (January 2015 to June 2020). Setting The study was conducted at a tertiary care cancer center. Patients A total of 702 cases (321 treatment naive; 321 post-therapy; 60 control samples) were evaluated. Sixty samples from non-MM staging marrow were assessed to study antigenic profiles of normal PC. Interventions Bone marrow samples were collected in EDTA and processed within 6 hours of collection per the consensus recommendation from the Euro-flow protocol using two tube 8-color/single tube 10-color panel. An aberrant PC cluster was defined by presence of >30 plasma cells with light chain restriction and/or aberrant immunophenotypes. Main Outcomes Measures 1. Expression profile of antigens on the polyclonal PC compartment; 2. Post-therapy immunomodulation in MM. Results Polyclonal PC were observed in 65.42% of treatment naive samples compared to 88.16% post-treatment samples (p=0.001). Frequency of aberrant expression of markers in polyclonal PC was higher at follow-up time point: CD45 (33% vs 38%; p=0.6), CD19 (19% vs 41%; p=0.001), CD56 (14% vs 41%; p Conclusions Flow cytometry-based MRD assessment in MM is challenged by wide immunophenotypic variation in non-neoplastic PCs and changes in immunophenotype after chemotherapy; thus, simultaneous analysis of multiple immunophenotypic markers, including light chain expression, is required for reliable MRD assessment.

Measurable residual disease (MRD) is an important parameter to predict outcome in B-cell acute lymphoblastic leukemia (B-ALL). Two different approaches have been used for the assessment of MRD by multiparametric flow cytometry that include the “Leukemia Associated Aberrant Immunophenotype (LAIP)” and “Difference from Normal (DFN)” approach. In this retrospective study, we analyzed 539 samples obtained from 281 patients of which 258 were paired samples and the remaining 23 samples were from post-induction time point only, to explore the utility of baseline immunophenotype (IPT) for MRD assessment. Single-tube 10-color panel was used both at diagnosis and MRD time points. Out of 281 patients, 31.67% (n = 89) were positive and 68.32% (n = 192) were negative for MRD. Among 258 paired diagnostic and follow-up samples, baseline IPT was required in only 9.31% (24/258) cases which included cases with hematogone pattern and isolated dim to negative CD10 expression patterns. Comparison of baseline IPT with post-induction MRD positive samples showed a change in expression of at least one antigen in 94.04% cases. Although the immunophenotypic change in expression of various antigens is frequent in post-induction samples of B-ALL, it does not adversely impact the MRD assessment. In conclusion, the baseline IPT is required in less than 10% of B-ALL, specifically those with hematogone pattern and/or dim to negative expression of CD10. Hence, a combination of DFN and LAIP approach is recommended for reliable MRD assessment.

Context The discriminative power of various antigens for delineating normal plasma cells (NPC) from aberrant plasma cells (APC) is variable; thus, understanding of the multiparametric flow cytometry (MFC)-based PC compartment is crucial for reliable PC characterization across various PC proliferative disorders (PCPD). Objective The expression profile and patterns of currently used immunophenotypic (IPT) markers were assessed for their ability to differentiate NPC from APC across the spectrum of PCPDs. Design This is a prospective analysis of patients diagnosed with PCPD (per standard IMWG criteria). Setting Tertiary care cancer center. Patients A total of 900 patients with diagnosis of PCPD were evaluated (MM: 724, PCL: 15, SMM: 12, MGUS: 42, Plasmacytoma: 54, Amyloidosis: 31, POEMS: 22). Interventions Bone marrow aspirate from cases (n=900) and controls (n=30) were processed per consensus guidelines from the Euro-flow protocol. An aberrant PC cluster was defined by >30 plasma cells with light chain expression beyond the range of control samples (0.26 to 3.08) and/or aberrant immunophenotypes (≥3). Main Outcomes Measures 1. Utility of cytoplasmic light chain restriction and 2. Most useful combination of markers to discriminate NPC from APC. Results All cases were segregated into three groups: cases with 1. only APC (n=181); 2. with both NPC and APC (n=574); or 3. only NPC (n=145). The PC compartment was solely characterized by light chain expression and IPT aberrancy in 36.22% (n=326) and 21.88% cases (n=197), respectively (p Conclusions Assessment of cytoplasmic light chain expression by MFC is essential for demonstrating clonality in cases with all normal or all aberrant PC. Pattern-based recognition is a useful parameter that identifies aberrant PC reliably in the majority of cases with co-existing NPC and APC, especially in setting of smaller clones of APC, which are likely to be missed by light chain assessment alone.

SummaryIntroduction The diagnosis of myelodysplastic syndrome (MDS) based on morphology is particularly difficult in low-grade MDS. Thus, the role of myeloid nuclear differentiation antigen (MNDA) and other flow cytometric (FCM) parameters in MDS was evaluated. Methods Bone marrow aspirates (BMA) collected from 52 patients with unexplained persistent cytopenias were divided into three groups: (i) proven MDS (n = 12) based on morphology and/or cytogenetics; (ii) suspected MDS (n = 6), noncontributory morphology, and cytogenetics; and (iii) non-MDS (n = 34). Sixteen control BMA were studied. Cases were analyzed for MNDA expression (on granulocytes, blasts, monocytes, and lymphocytes) and for seven quantitative parameters: CD34+ myeloblasts % in nucleated cells, CD34+ B-cell progenitor% in CD34+ cells, lymphocyte/myeloblast CD45 MFI ratio, granulocyte/lymphocyte SSC peak channel ratio and the proportion of CD34+ myeloblasts expressing CD15, CD11b, and CD56. A score of 1 was given to each parameter beyond the cutoff, and score ≥3 was considered FCM positive. Results MNDA expression on granulocytes and blasts was significantly lower in proven MDS and suspected MDS vs. non-MDS. Quantitative FCM parameters successfully distinguished MDS and suspected MDS from non-MDS. Conclusion MNDA expression is an independent marker for the evaluation of dyspoiesis and may be added to the standard panel for quantitative assessment by FCM.

Screening coagulation tests and assays for thrombosis and fibrinolysis were performed in 80 cases of malaria at presentation and during the course of the disease. Close correlation between the degree of thrombocytopenia (observed in >97% cases) and the presence hemorrhagic manifestations at presentation, and improvement in the platelet count in parallel with clinical recovery emphasised the role of platelets in the pathogenesis of coagulopathy in malaria. A potential selection bias resulting from inclusion of only patients admitted at a tertiary care hospital could explain the higher incidence (27.5%) of clinical bleeding observed in this study compared to that reported in the literature. Although a significant correlation between overt bleeding and abnormal PT/INR and APTT (observed in 20–37% cases) could not be demonstrated, a good correlation existed between normal screening coagulation tests and the absence of bleeding complications. Elevated D-Dimer and FDP levels in almost all cases (90%) of both types of malaria confirmed the high prevalence of disseminated intravascular coagulation and fibrinolysis. A correlation between rising D-Dimer levels and the incidence of bleeding was observed. Follow up studies in six cases with complications showed normalization of platelet counts and of screening coagulation assays with clinical recovery. D-Dimer and FDP levels however, remained elevated in most of these cases indicating the continuation of a smouldering coagulopathy even after full clinical recovery possibly due to the persistence of residual damage to the cells caused by the parasitic infection. Knowledge of this fact is important for avoiding unnecessary investigations and longer hospital stay in patients admitted with malaria.

T-cell antigens [CD5,CD1a,CD8] define early T-cell precursor acute lymphoblastic leukemia (ETP-ALL). To understand immature T-ALL of which ETP-ALL is part, we used these antigens to subcategorize non-ETP T-ALL for examining expression of myeloid/stem cell antigens (M/S) and clinical features. Using CD5 (+/-) to start categorization, we studied 69 routinely immunophenotyped patients with T-ALL. CD5(-) was a homogenous (CD8,CD1a)(-) M/S(+) ETP-ALL group (n = 9). CD5(+) cases were (CD8,CD1a)(-) pre-T-ALL (n = 22) or (CD8,CD1a)(+) (n = 38) thymic/cortical T-ALL; M/S(+) 20/22 (90.91%) in former and 22/38 (57.89%) in latter (P = 0.007). ETP- and pre-T-ALL together (CD1a(-) ,CD5(-/+) immature T-ALL group) were nearly always M/S(+) (29/31; 93.55%). In multivariate analysis, only ETP-ALL predicted poor overall survival (P = 0.02). We conclude (i) CD5 negativity in T-ALL almost always means ETP-ALL. CD1a and CD8 negativity, as much as CD5, marks immaturity in T-ALL, and the CD5(+/-) /CD1a(-) /CD8(-) immature T-ALL group needs further study to understand the biology of the T-ALL-myeloid interface. (ii) ETP-ALL patients may be pre-T-ALL if CD2(+) ; CD2(+) , conversely, CD5(-) /CD1a(-) /CD8(-) pre-T ALL patients are ETP-ALL. (iii) Immunophenotypic workup of T-ALL must not omit CD1a, CD5, CD8 and CD2, and positivity of antigens should preferably be defined as recommended for ETP-ALL, so that this entity can be better evaluated in future studies of immature T-ALL, a group to which ETP-ALL belongs. (iv) ETP-ALL has poor prognosis.