Search

The stability of the housekeeping gene (HKG) expression is an absolute prerequisite for accurate normalization of target gene expression in a quantitative real-time polymerase chain reaction (RQ-PCR). In RQ-PCR, the widely used normalization approach involves the standardization of target genes to the most stable HKG control genes. According to the recent literature, in different experimental conditions the HKGs exhibit either up or down-regulation and thus affecting the gene expression profiles of target genes which leads to erroneous results. This implies that it is very important to select the appropriate HKG and verify the expression stability of the HKG before quantification of the target gene.The present study aims to analyze six different HKGs for their expression profiles and stability in BCR-ABL1 negative cases and validate them in BCR-ABL1 positive cases, detected by multiplex reverse transcribed polymerase chain reaction (RT-PCR). Six commonly used reference genes (GAPDH, ABL1, RNA18S, ACTB, GUSB, and EEF2) were selected in this study. RQ-PCR was performed on 24 BCR-ABL1 negative cases and the outcomes were validated on 24 BCR-ABL1 positive cases. RefFinder™, a web-based composite software was used to check the stability of HKG genes by different algorithms and comprehensive ranking of each HKG gene in BCR-ABL1 negative cases and finally validated in BCR-ABL1 positive cases.It was found that RNA18S, ABL1 and GUSB are good stable HKG genes, which showed minimum variability in gene expression compared to GAPDH, EEF2, and ACTB, the most commonly used HKG.

For the detection of BCR-ABL1-like ALL cases, two methodologies, specifically Gene expression profiling (GEP) or Next-generation targeted sequencing (NGS) and TaqMan based low-density (TLDA) card, are being used. NGS is very costly and TLDA is not widely commercially available. In this study, we quantified the expression of 8 selected overexpressed genes in 536 B-ALL cases. We identified 26.67% (143/536) BCR-ABL1-like ALLs using hierarchical clustering and principal component analysis. BCR-ABL1-like ALL cases were significantly older at presentation (p = 0.036) and had male preponderance (p = 0.047) compared to BCR-ABL1-negative ALL cases. MRD-positivity and induction failure were more commonest in BCR-ABL1-like ALL cases (30.55 vs.19.35% in BCR-ABL1-negative ALL cases). Lastly, we built a PHi-RACE classifier (sensitivity = 95.2%, specificity= 83.7%, AUC= 0.927) using logistic regression to detect BCR-ABL1-like ALL cases promptly at diagnosis. This classifier is beneficial for hematologists in quick decision making at baseline to start tailored treatment regimes.

Background Platelet aggregation studies using conventional light transmission aggregometry (LTA) have several disadvantages and require strict pre-analytical measures for reliable results. We aimed to examine the utility of flow cytometric platelet aggregation (FCA) assay in detecting platelet function defects (PFDs) in patients with a history of bleeding symptoms. Methods Sixty-four participants (24 patients and 40 healthy controls) were included in this study. LTA and FCA assay were performed simultaneously in patients and healthy controls. In the FCA assay, two portions of platelets from the same individual were labeled separately with CD31-FITC and CD31-PE. After mixing and stimulation with agonists, the double- colored platelet aggregates were visualized using a flow cytometer. The results generated using the two techniques were compared and correlated. Results The patients' median age was 17 years (range, 3‒72 yr) with a male-to-female ratio of 1:1.7. There was substantial agreement between LTA and FCA assay in detecting a PFD (=0.792). Four patients showing a Glanzmann thrombasthenia-like pattern on LTA exhibited an abnormal FCA. A functional defect in collagen binding was detected on the FCA assay conducted in two immune thrombocytopenic patients with severe bleeding. Conclusion FCA assay can be used to identify functional defects in platelets, with potential applications in thrombocytopenic individuals. It also facilitates the diagnosis of inherited bleeding disorders with platelet defects.

The overexpression of cytokine receptor-like factor-2 (CRLF2) identified by anti-thymic stromal lymphopoietin receptor/TSLPR flow cytometry (FCM) has been reported as a screening tool for the identification of BCR-ABL1-like B-cell acute lymphoblastic leukemia/B-ALL with CRLF2 re-arrangement. TSLPR expression was studied prospectively in consecutive 478 B-ALLs (≤ 12 years (n = 244); 13–25 years (n = 129); > 25 years (n = 105)) and correlated with various hematological parameters and end-of-induction measurable residual disease (day 29; MRD ≥ 0.01% by 10-color FCM). TSLPR positivity in ≥ 10% leukemic cells was detected in 14.6% (n = 70) of B-ALLs. CRLF2 re-arrangement was detected in eight cases (11.4%) including P2RY8-CRLF2 (n = 6), and IgH-CRLF2 (n = 2) with a median TSLPR positivity of 48.8% and 99% leukemic cells, respectively. Recurrent gene fusions/RGF (BCR-ABL1 (17.1%); ETV6-RUNX1 (4.2%), TCF3-PBX1 (1.4%)), other BCR-ABL1-like chimeric gene fusions/CGFs (PDGFRB-rearrangement (2.9%), IgH-EPOR (1.4%)), CRLF2 extra-copies/hyperdiploidy (17.1%), and IgH translocation without a known partner (10%) were also detected in TSLPR-positive patients. CD20 positivity (52.9% vs 38.5%; p = 0.02) as well as iAMP21 (4.3% vs 0.5%; p = 0.004) was significantly more frequent in TSLPR-positive cases. TSLPR-positive patients did not show a significantly higher MRD, compared to TSLPR-negative cases (37% vs 33%). Increasing the threshold cut-off (from ≥ 10 to > 50% or > 74%) increased the specificity to 88% and 100% respectively in identifying CRLF2 translocation. TSLPR expression is not exclusive for CRLF2 translocations and can be seen with various other RGFs, necessitating their testing before its application in diagnostic algorithms. In patients with high TSLPR positivity (> 50%), the testing may be restricted to CRLF2 aberrancies, while patients with 10–50% TSLPR positivity need to be tested for both CRLF2- and non-CRLF2 BCR-ABL1-like CGFs.

OBJECTIVE Glucose-6-phosphate dehydrogenase (G6PD) deficiency, an X-linked recessive disorder, is the commonest erythrocytic enzymopathy worldwide. Reliable diagnosis and severity prediction in G6PD-deficient/heterozygous females remain challenging. A recently developed flow cytometric test for G6PD deficiency has shown promise in precisely identifying deficient females. This paper presents our experiences with this test in a subtropical setting and presents a modification in flow cytometric data acquisition strategy. METHODS The methaemoglobin reduction + ferryl Hb generation-based flow cytometric G6PD test was compared with the screening methaemoglobin reduction test (MRT) and confirmatory G6PD enzyme activity assay (EAA) in 20 G6PD-deficient males, 22 G6PD-heterozygous/deficient females and 20 controls. Stained cells were also assessed for bright/dim G6PD activity under a fluorescent microscope. RESULTS Flow cytometry separated and quantified %bright cells in heterozygous/deficient females, objectively classifying them into 6 normal (>85% bright cells), 14 intermediate (10-85%) and two G6PD-deficient (

Introduction The categorization of mature B cell neoplasms (MBN) infiltrating blood and bone marrow are met with difficulties. The inclusion of CD148 and CD180 in the routine flow cytometry/FCM panels has been suggested to refine the diagnosis. We studied the discriminatory ability of CD148 and CD180 median fluorescence intensity(MFI), CD148/CD180 ratio and their expression relative to T cells (CD148ab/T , CD180ab/T ), neutrophils (CD148ab/gr , CD180ab/gr ) and normal B cells (CD148ab/n , CD180ab/n ) in the differentiation of mature B cell neoplasms (MBN) especially non-chronic lymphocytic leukaemia (CLL). Methodology The flow cytometric (FCM) expression of CD148 and CD180 was studied prospectively in 102 patients (non-CLL; n = 72); diagnosed by a comprehensive panel of immunophenotypic and cytogenetic studies. The MFI and ratios were statistically compared across MBNs by Mann-Whitney U test. Cut-off values, sensitivity and specificity were calculated for significant parameters by receiver operator characteristic curve. Results CD180MFI > 4.35 showed 100% sensitivity and 90.9% specificity for a diagnosis of marginal zone lymphoma (MZL) while, CD148/180 > 5.15 was 100% specific and 81.8% sensitive for lymphoplasmacytic lymphoma. CD148ab/T (>4.3; 100% specificity, 83.4% sensitivity) and CD148ab/gr (>1.1; 100% sensitivity, 90% sensitivity) were useful for differentiating blastoid-mantle cell lymphoma/MCL from diffuse large B cell lymphoma; while CD148MFI (≥20.25), CD148ab/T (>3.35) and CD148ab/gr (>0.95) showed >90% specificity and sensitivity for distinguishing MCL from CLL. Pairwise analysis also showed a good discriminant function of various parameters for distinguishing SMZL from other MBNs like FL, MCL as well as CLL. Conclusions The current study shows an excellent utility of CD148MFI, CD180MFI, their ratio and relative expression levels in the subcategorization of immunophenotypically related MBNs.

Glanzmann thrombasthenia (GT) is an autosomal recessive platelet function disorder characterized by mucocutaneous bleeding as the most common clinical phenotype. Patients with GT have normal platelet counts, platelet morphology but reduced platelet aggregation in response to various agonists. Homozygosity or compound heterozygosity for variants in the ITGA2B/ITGB3 genes is the genetic basis for GT. Establishing a molecular diagnosis is definitive and is important for predictive testing. Using multi-gene panels is an accurate, faster, and cost-effective mode as compared to Sanger sequencing in large genes. We used a targeted resequencing based approach to identify pathogenic variants in eight cases in seven families. These variants were validated using Sanger sequencing in patients as well as family members and were predicted probably pathogenic using in-silico prediction tools. The variants include three missense (3/7 = 43%) (ITGA2B:c.1028 T > C, ITGA2B:c.1186G > A, ITGB3:c.1388G > C), two deletions (ITGA2B:c.559delG, ITGA2B:c.3092delT), one duplication (ITGA2B:c.1424_1427dupAGGT) and nonsense variant (ITGA2B:c.2578C > T, p.Gln860Ter). Except for one case which was compound heterozygous, the rest of the cases were homozygous. We found two novel variants that are reported for the first time in GT. The targeted resequencing based approach revealed varied genetic variants in North Indian patients, including two novels ones. The high yield of our panel indicates its suitability for usage in larger cohorts for the genetic diagnosis of GT patients. This approach is cost-effective and less cumbersome as compared to Sanger sequencing for these large size genes with multiple exons. The information so obtained is helpful in prenatal testing, carrier analysis, and genetic counseling.

Context: For the detection of BCR-ABL1-like ALL cases, two methodologies, specifically gene expression profiling (GEP) or next-generation targeted sequencing (NGS) with TaqMan based low-density (TLDA) card, are being used. NGS is very costly, and TLDA is not commercially available in India. We have built a binary logistic 'PHi-RACE’ classifier for rapid detection in Indian patients. Objective: BCR-ABL1-like ALL signature identification, followed by the creation of a PHi-RACE classifier using machine learning approach. Design: This study was conducted at PGIMER, a tertiary care center in India, for a period of 5 years (2017–2021). Flow cytometric immunophenotyping (FCM-IP) and multiplex RT-PCR were performed on 629 B-ALL cases to detect the positivity of BCR-ABL1 chimeric fusion transcripts. Further, 12 BCR-ABL1-positive cases were subjected to transcriptome profiling using Affymetrix microarray (U133 Plus 2.0 Array). A total of 536 B-ALL cases were subjected to GEP of 8 selected genes, followed by PHi-RACE classifier generation and validation (n=93). Patients: We examined 629 freshly diagnosed and treatment-naive B-ALL cases with complete laboratory workup. Results: Multiplex RT-PCR assay revealed BCR-ABL1 transcripts in 17.17% (108/629) of cases. Global transcriptome profiling of 12 BCR-ABL1 RNA transcripts revealed a total of 1574 differential expressed (DE) genes. DE genes were further filtered, and 45 genes with 10- to 86-fold change were identified. Based upon regression coefficient values, 8 best classifier genes were selected using penalized logistic regression. Out of 536 examined B-ALL cases, we identified 26.67% (143/536) BCR-ABL1-like ALLs using hierarchical clustering and principal component analysis. BCR-ABL1-like ALL cases were significantly older at presentation (p=0.036) and had male preponderance (p=0.047) compared to BCR-ABL1-negative ALL cases. Lastly, we built a PHi-RACE classifier (cut-off = 0.28, sensitivity = 95.2%, specificity= 83.7%, AUC= 0.927) using glm function in R, validated with 93 BCR-ABL1-negative ALL cases. Conclusions: We developed and validated a PHi-RACE classifier for the first time in a developing country. This predictive classifier is rapid, cost-effective [USD 42.00 (INR 3,000.00)/sample], with short turnaround time (4 hours), including testing and interpretation. This classifier is advantageous over other approaches for the prompt detection at baseline to start tailored treatment regimes in BCR-ABL1-like ALL cases.

Background: Aberrant phenotypes in acute leukemia have been reported with varying frequencies in independent studies and their association with prognostic factors is still a matter of debate. Aim: This study aims to identify the frequency of aberrant immunophenotypes in de novo acute myeloid leukemia (AML) and to evaluate their association with initial clinical and hematological features. Materials and Methods: A total of 181 patients of de novo AML were included during the time (July 2010–June 2012). The immunophenotype of all cases of AML was studied by using flow cytometry. Results: Aberrant lymphoid antigen expression was seen in 43.1% cases. Most frequent aberrant lymphoid antigen was CD7, seen in 26.5% cases. All French-American-British (FAB) subtypes except AML-M3 expressed aberrant lymphoid antigens. The expression was most common in AML-M4 in the current study. CD34 expression in AMLs was significantly associated with the expression of aberrant lymphoid antigens. Lymphoid antigen expression in adult AML was significantly associated with higher white blood cell (WBC) count (>50,000/mm3) and higher number of peripheral blasts (>70%). Conclusion: In summary, CD7 is the most common aberrant lymphoid antigen expressed in AML. FAB subtype AML-M3 is usually not associated with aberrant lymphoid antigen expression. AML cases with CD34 positivity are more likely to express aberrant lymphoid markers. The current study also supports that aberrant lymphoid antigen expression in adult AML is associated with adverse presenting hematological features (WBC count >50,000/mm3, peripheral blasts >70%). Pediatric Ly + AML cases are not associated with adverse presenting clinical and biological features.

Methyl green (MG), a conventional, low-cost histological stain, was used to design a flow cytometric cell-cycle/DNA-ploidy assay. On fluorometry, MG absorbed maximally at 633-nm, showed negligible fluorescence in free-state, but emitted brightly when bound to DNA. Optimal dye and cell concentrations for staining and effects of time and photobleaching on stained cells were determined for a lyse-permeabilize-stain protocol. Linearity of DNA-binding, coefficients-of-variation of G0/G1-peaks and minimal carryover were confirmed. Assay results correlated highly with a propidium iodide-based kit in 29 acute lymphoblastic leukemia specimens. The MG-based DNA-ploidy assay represented an accurate and inexpensive alternative to conventional PI-based assays.

Introduction A new provisional entity of "B-lymphoblastic leukaemia/lymphoma, BCR-ABL1-like" has been introduced in the 2017 revised edition of WHO classification of tumours of haematopoietic and lymphoid tissues. BCR-ABL1-like cases are negative for Ph chromosome or t(9:22)(q34;q11.2) translocation, do not express the fusion BCR-ABL1 RNA transcripts and proteins resulting from the Ph chromosome,and are characterized by similar gene expression profiles as that of BCR-ABL1-positive acute lymphoblastic leukemia (BCR-ABL1-positive ALL).There is no 'short-cut approach' for making an accurate diagnosis of BCR-ABL1-like ALL. Two approaches namely Gene expression profiling (GEP) or Next-generation sequencing (NGS) and TLDA (TaqMan low-density array) are used for the detection of BCR-ABL1-like ALL cases. NGS is very costly and data interpretation requires a lot of bioinformatics skills and TLDA is not commercially available in India. Aims We planned to study the whole transcriptome of BCR-ABL1-positive ALL cases using microarray GEP, followed by customizing targeted gene panel using nCounter NanoString technology, for the detection of BCR-ABL1-like cases. METHODS Flow cytometric immunophenotying (FCM-IP) and multiplex RT-PCR were performed on 200 B-ALL cases to detect BCR-ABL1 chimeric fusion transcripts. Further, 12 BCR-ABL1-positive cases were subjected to transcriptome profiling using Affymetrix microarray (Gene Chip Human Genome U133 Plus 2.0 Array). The results were analyzed using TAC 4.0 software. Finally, a targeted panel of 50 differentially expressed genes [including 5 Housekeeping genes (HKGs)] was constructed according to our microarray findings and previously published data (Harvey RC et al.ASH 2013). A total of 96 B-ALL cases (16 BCR-ABL1-positive cases & 80 BCR-ABL1-negative cases) were subjected to GEP using nCounter Platform. The results were analyzed using nSolver4.0 software. RESULTS In the study cohort of 200 adult B-ALL cases, BCR-ABL1 chimeric fusion transcripts were detected in 34% (b2a2 and b3a2=18.05% & e1a2=15.5%), as revealed by multiplex assay. Global transcriptome profiling of 12 BCR-ABL1 RNA transcripts revealed a total of 1574 as DE genes (460 genes in e1a2, 515 genes in b2a2 and 599 genes in b3a2). DE genes were further filtered through hierarchical clustering analysis and a total of 45 DE genes with 10- to -86-fold change were identified. These genes were further analyzed using nCounter NanoString. To further identify the best classifier genes, log2 normalized expression values were analyzed using penalized logistic regression. Based on previous literature and regression coefficient values, 15 genes were finally selected whose performance was individually analyzed using receiver operating characteristic curve (ROC) and area under the curve (AUC). Optimal thresholds for these genes were estimated as the values with maximum sensitivity and specificity. Out of 78 examined BCR-ABL1-negative cases, 33(42.30%) BCR-ABL1-negative cases were clustered together with 15 BCR-ABL1-positive cases and were attributed as BCR-ABL1-like ALL cases in principal component analysis. Further, we categorized CRLF2 in two categories; high CRLF2 cases 25/33 (75.75%) & low 8/33 (24.24%) in BCR-ABL1-like ALL cases. JAK2p.R683G mutation was screened in CRLF2 high cases and showed positivity in 19/24 (79.16%) by the Amplification Refractory Mutation (ARMS) PCR. In 25 cases, the average log fold change of -0.80 &-5.83 was seen in P2YR8 & CSF2RA respectively by qPCR. In CRLF2 low expressing cases, the average log fold change of 11 kinase genes showed -0.75 in CENPC, -0.66 FOXP1, -0.16 NUP153, 1.04 RCSD1, 1.50 PAX5, 1.12 FLT3, -5.65 EPOR, -4.03 ILR2B, -3.46 PDGRFB, -7.49 NTRK3 &-2.83 ZNF274 respectively. The average log fold change of IKZF1 in 80 BCR-ABL1-negative cases was found to be 1.07. DISCUSSION & CONCLUSION We have devised a method that includes 15 genes according to AUC/ROC for the detection of BCR-ABL1-like ALL cases, using nCounter NanoString technology for the first time in Indian patients. Furthermore, we are planning to validate this model in future, on 50 BCR-ABL1-positive and 150 BCR-ABL1-negative cases and devise a simple, efficient, cost-effective qPCR method. It is very important to detect BCR-ABL1-like ALL cases to start the desirable TKI therapy & aid in treatment stratification, prognostication, and improve the overall survival of these patients. Figure Disclosures No relevant conflicts of interest to declare.

Patients with paroxysmal nocturnal hemoglobinuria (PNH) may present with thrombosis at unusual sites, of which cerebral sinovenous thrombosis (CSVT) is one and screening for PNH is recommended in this condition. Though many patients diagnosed with PNH develop CSVT, it is unclear how many patients with PNH would present for the first time with thrombosis. We analysed the results of screening for PNH by flowcytometry in our patients with CSVT. The laboratory data of patients referred for thrombophilia and PNH testing in CSVT was examined to assess the frequency of PNH at presentation in these patients. FLAER and CD24 on granulocytes and FLAER and CD14 on monocytes respectively were used to screen the leucocytes for PNH by flowcytometry. The data for Protein C, S and Antithrombin deficiency, antiphospholipid antibodies and the Factor V Leiden mutation was examined and circumstantial risk factors were also assessed. Of the 180 cases of CSVT screened by flowcytometry for PNH, not a single case tested positive. Positivity for anti-phospholipid antibodies was the most common thrombophilic risk factor (5%). Pregnancy was the most common circumstantial risk factor. Our data on FLAER based flowcytometry in the North Indian population with CSVT suggests that PNH is not a common risk factor in our patients with thrombosis at this unusual site.

Background Thrombo-hemorrhagic complications cause significant morbidity and mortality in patients with polycythemia vera (PV). Objectives To assay and correlate inflammatory cytokines with the thrombotic risk in PV patients. Methods This prospective observational study was carried out at Postgraduate Institute of Medical Education and Research, Chandigarh, India over 18-months. The study enrolled 52 patients with PV (newly diagnosed = 28, follow-up = 24), and 20 age/sex-matched controls. Cytokine analysis for IL 1β, IL2, IL4, IL6, IL8, IL10, IL11, IL12/23p40, TNFα, and IFN-γ was performed on the peripheral blood (before treatment initiation for newly diagnosed cases, and after 7 days of stopping drugs for follow-up cases) by flow cytometry-based cytokine bead analysis (CBA) using CBA kits (BD™ biosciences, USA). Results were analyzed using SPSS Statistics 22.0. Results The mean age of patients was 51.9 ± 13 years. Levels of IL-6, IL-1β, IL-8, IL-11, IL-12/23p40 were significantly raised, however, TNF-α, and IFN-γ levels were significantly lower in the PV population as compared to controls. A significant correlation between the levels of IL-6, IL-2, and IL-8 with the overall risk of thrombosis in PV patients was observed. Conclusions PV patients display an aberrant pattern of plasma cytokine expression, the levels of which correlate with the thrombotic risk.

Treatment of multiple myeloma (MM) has evolved over decades with the introduction of novel therapeutic strategies. Response rates has significantly improved; however, there is a need for more sensitive techniques to study the residual disease other than conventional means. We evaluated the feasibility and utility of a two-tube six color multiparametric flow cytometry (MFC) assay for measurable residual disease (MRD) detection in MM patients on treatment.Pretitrated cocktails containing antibodies against CD38, CD138, CD45, CD19, CD56, CD81, CD27, and cytoplasmic kappa and lambda light chains were used in the combination of two tubes and were acquired on a flow cytometer. Limit of detection was determined through dilution and spiking experiments with a limit of 0.01%.Of the 62 patients screened, 58 patients were included in the final study cohort (day 100 postautologous stem cell transplant and at the end of induction chemotherapy). Twenty-eight patients (48%) revealed the presence of MRD in bone marrow on MFC (median = 0.12, range = 0.01-5.89%). Out of 28 MFC-MRD positive patients, only 16 patients showed M band on immunofixation-electrophoresis (IFE) (MRD+/IFE+, 57%), and rest of them were IFE negative (MRD+/IFE-, 42%). Patients with MRD positive status at the end of induction chemotherapy or day 100 posttransplant had an inferior overall survival (P = 0.009) and progression-free survival (P = 0.0002) than those with MRD negativity.We have demonstrated the impact of MRD testing in MM using MFC with a long follow-up data, suggesting its routine incorporation in monitoring the disease independent of the immunofixation status.

Danazol is an attenuated androgen and is used in the treatment of aplastic anemia (AA) in resource constraint settings. We chose to study the role of CD4+ CD25T-regs' percentages of 25 acquired idiopathic AA patients and 25 healthy controls who completed study protocol were analyzed by performing multicolor flowcytometry on peripheral blood samples.More than one-third (36%) of AA patients in our study received Danazol as monotherapy, whereas less than a third (32%) each received standard doses of IST with equine Anti Thymocyte Globulin (ATG) and Cyclosporine combination, or Cyclosporine and Danazol combination, respectively. Results showed that all AA patients had a significantly lower percentage of T-regs at the time of diagnosis when compared to healthy controls (p 0.0001), implicating their role in the pathophysiology. On treatment, all 25 patients showed a significant rise in the percentage of T-regs when compared to baseline (p 0.0001).The rise in T-regs' percentage was higher in patients treated with Danazol alone when compared to standard IST (ATG with Cyclosporine), or Cyclosporine with Danazol combinations (p = 0.585).We conclude that Danazol also leads to increase in T-regs in acquired idiopathic AA.

Introduction Patients of chronic myeloid leukemia (CML) eventually transform to blast crisis (BC) after a variable length of chronic phase (CP). With improvement in management protocols, many patients of CML-BC are expected to show remission after initial chemotherapy and a better survival. Hence, it becomes imperative to correctly classify the nature of BC for further course of management. Materials and methods This study retrospectively analyzed cases of CML-BC, over a period of 5 years, with comprehensive immunophenotyping done on flow cytometry. Results Fifteen cases of CML-BC with male-to-female ratio of 3:1 and mean age of 40.2 years were included in the study. Flow cytometry revealed 14 cases of myeloid and one case of lymphoid BC. One case each of megakaryocytic and myelomonocytic lineage were seen among the 14 cases of myeloid BC. Aberrant marker expression was a common occurrence. Myeloperoxidase cytochemical stain had shown positivity only in 3 of the 14 cases of myeloid BC. Conclusion Immunophenotyping is necessary for assigning specific lineage to blasts in patients of CML-BC and thus providing clinically useful information regarding the treatment protocols and prognosis of the patient. How to cite this article Narang V, Sachdeva MUS, Bose P, Varma N, Malhotra P, Varma S. Immunophenotyping in Chronic Myeloid Leukemia Blast Crisis: Looking beyond Morphology. J Postgrad Med Edu Res 2016;50(4):181-184

Background The literature on paroxysmal nocturnal hemoglobinuria (PNH) in aplastic anemia (AA) is largely focused on adults with few studies in children. Moreover, large studies are conspicuously absent from developing countries. Knowledge of the prevalence and utility of their detection is required before widespread use of PNH screening in pediatric AA in resource-limited settings. Methods We performed a retrospective audit over a period of 9 years to study the prevalence of PNH clones by flow cytometry (FCM) in children ≤12 years of age presenting with AA, and analyzed their response to immunosuppressant therapy. Results Nine (12.9%) out of 70 patients had PNH clones comprising >1% of the target cell population, including five patients (7.14%) with PNH clone size >10%. The clone size in monocytes ranged from 3.7% to 95.2% (median 21.1%) and in neutrophils from 1.6% to 87.6% (median 19.5%). Fluorescent aerolysin (FLAER)-based FCM screening significantly improved the detection of PNH clones compared to non-FLAER based screening techniques (18.4% vs. 6.25%). One child showed chronic intravascular hemolysis and another developed arterial stroke during the course of illness. None of our PNH-positive AA patients tested for chromosome breakage studies (n = 8) showed increased clastrogen-induced breakage. Conclusions A lower frequency but moderate/large-sized PNH clones were seen in our pediatric AA population, compared to western data. FLAER-based FCM screening significantly improved the detection of PNH clones. We recommend routine FLAER-based screening of PNH in pediatric AA patients. Pediatr Blood Cancer 2015; 9999:XX–XX © 2015 Wiley Periodicals, Inc.

The reasons for progression and autoimmune cytopenias (AIC) in chronic lymphocytic leukemia (CLL) are not entirely clear, with previous studies suggesting a role for regulatory T-cells (Treg). In this study we prospectively studied Treg (CD3 + CD4+ CD25highCD127low), interleukin-10 (IL-10) producing Treg and T-helper 17 (Th17) (CD3 + CD4+ IL-17+) cells in 40 treatment-naive patients with CLL. The percentage of Th17 and not Treg cells was significantly higher in the AIC cohort than in those without AIC (p < 0.0001). The Treg:Th17 ratio was skewed in favor of Th17 in the AIC cohort (p = 0.02). Th17 cells are responsible for AIC of CLL. Analysis of lymph-node aspirates showed that the percentage of Treg and IL-10 expression in Treg and not Th17 was significantly higher than in peripheral blood (p < 0.01). Treg cells play a major role in the microenvironment where disease progression occurs. This shows the importance of maintaining the Treg:Th17 equilibrium, for imbalance leads to CLL progression or AIC.

Background & objectives: Mixed-phenotype acute leukaemia (MPAL) is a rare neoplasm with no definite treatment protocols and a distinctly poor outcome. Advancement in polychromatic flow cytometry has made its identification easier. This prospective study was designed to identify cases of MPAL and study their clinical presentation and haematological profile in a tertiary care hospital in north India. Methods: Ethylenediaminetetraacetic acid (EDTA)-anticoagulated bone marrow aspirate samples of patients diagnosed as acute leukaemia (AL) on the basis of morphology were utilized for immunophenotyping. A comprehensive panel of fluorochrome-labelled monoclonal antibodies targeting myeloid, B-cell, T-cell and immaturity markers was utilized. The patients diagnosed to have MPAL, on the basis of the World Health Organization 2008 classification, were selected for further analyses. Results: There were 15 (2.99%) patients with MPAL of the total 501 cases of AL. Seven were children, all males and mean age of 5.08±3.88 yr. Eight were adults, male:female=6:2 and mean age of 21.43±5.74 yr. Eight were diagnosed as B/myeloid and seven were T/myeloid. No association was observed between age and immunophenotype of MPAL. On morphology, 11 were diagnosed as AML and four as ALL, and no specific morphology of blasts was predictive of a MPAL. Interpretation & conclusions: MPAL appeared to be a rare neoplasm (2.99% of AL cases). A comprehensive primary panel of monoclonal antibodies should be used to identify this neoplasm known to have a poor outcome.

The distinction of inherited bone marrow failure syndromes (IBMFS) from other acquired forms of aplastic anemia (AA) is very important for instituting proper therapy. However this is not often straight forward. A proper confirmatory genetic diagnosis is possible only in less than 50% of the patients even with the use of massive parallel sequencing (1). Moreover its application is still limited due to the cost and several logistical issues. Unfortunately, diethyl epoxy butane (DEB) or mitomycin C induced chromosome breakage studies (CBS) is the only test, which is widely employed, for the diagnosis of IBMFS in many resource-limited settings. This article is protected by copyright. All rights reserved.

To study the role of mid-induction (day 15) peripheral blood minimal residual disease (PB-MRD) detection in pediatric B- lineage acute lymphoblastic leukemia (B-ALL).Prospective.Tertiary-care center.40 consecutively-diagnosed treatment-naive, pediatric B-ALL patients.National Cancer Institute (NCI) standard risk patients were given three drug induction regimen comprising vincristine, L-asparginase and prednisolone; NCI high-risk patients were supplemented with daunorubicin.Day 15 PB-MRD and bone marrow MRD (BM-MRD) analyzed by six color flow cytometry.The sensitivity of day 15 PB-MRD to identify concurrent day 15 BM-MRD positivity was 64%, with 100% specificity. The positive and negative predictive values were 100% and 62.5%, respectively. PB-MRD was positive in 67% of relapsed patients.BM-MRD is a well-established prognostic factor in B-ALL. We suggest, day 15 PB-MRD could be considered as an early, minimally invasive and easily accessible MRD screening option.

Flow cytometry is the gold standard methodology for screening of paroxysmal nocturnal hemoglobinuria. In the last few years, proaerolysin conjugated with fluorescein (FLAER) has become an important component of antibody panel used for the detection of paroxysmal nocturnal hemoglobinuria (PNH) clone. This study aimed to compare PNH clone detection by flow cytometry in the pre-FLAER era versus the FLAER era. This was a retrospective analysis of 4 years and included 1004 individuals screened for PNH clone, either presenting as hemolytic anemia or as aplastic anemia. In the pre-FLAER time period, the RBCs and neutrophils were screened with antibodies against CD55 and CD59. With the introduction of FLAER, neutrophils were screened with FLAER/CD24/CD15 and monocytes with FLAER/CD14/CD33 combination. A comparative analysis was done for detection of PNH clone in aplastic anemia patients versus non-aplastic anemia patients, as well as between pre-FLAER and FLAER era. Out of a total of 1004 individuals, 59 (5.8 %) were detected to have PNH clone positivity. The frequency of PNH clone detected in aplastic anemia and non-aplastic anemia groups was 12.02 and 3.36 %, respectively. The detection rate of PNH clone increased from 4.5 % (32/711) in the pre-FLAER era to 9.2 % (27/293) with the introduction of FLAER. However, this increase could be attributed to increased detection of PNH clone in the aplastic anemia group, which showed a significant increase from 8.3 to 18.2 % after use of FLAER. In the non-aplastic group, PNH clone was detected with similar frequencies before and after use of FLAER (3.2 versus 3.8 %, respectively). Mean PNH clone size was lower in the aplastic anemia group when compared with the non-aplastic group. RBCs always showed a lower clone size than neutrophils. PNH clone on neutrophils and monocytes was however similar. Inclusion of FLAER increases the sensitivity of the test which is especially useful in picking up small PNH clones in patients of aplastic anemia.

Background Paroxysmal nocturnal hemoglobinuria (PNH) may present as cytopenia, hemolysis, or thrombosis at unusual sites including splanchnic vessels. Thrombosis of the portal veins and hepatic veins are associated with thrombophilic risk factors: deficiencies of protein C, protein S, and antithrombin, positivity for antiphospholipid antibodies, and factor V Leiden mutation. There is limited information regarding PNH presenting primarily as a thrombotic event. We prospectively screened 142 consecutive patients with intrabdominal thrombosis and 106 controls with fluorescently labeled inactive toxin aerolysin (FLAER)-based flowcytometry to assess the frequency of PNH as a thrombophilic risk factor in patients with intra-abdominal thrombosis. Methods Granulocytes of patients and controls were screened with CD 24 and FLAER and monocytes with CD 14 and FLAER. Dual negativity of >1% events in both lineages was interpreted as a positive PNH clone. Screening for thrombophilia risk factors was carried out. Results Two (1.4%) cases had large PNH clones. RBC also demonstrated the PNH defect. Thrombophilia risk factors were as follows: deficiency of protein S, protein C, and antithrombin in 13.4%, 4.9%, and 2.1%, respectively, and positivity for anti-beta-2 glycoprotein 1, anticardiolipin antibodies, and lupus anticoagulant in 9.2%, 1.4%, and 0.7%, respectively. Factor V Leiden mutation was seen in 1.4% patients. Conclusion PNH was uncommon in patients with intra-abdominal thrombosis in the ethnic Indian population. Despite low positivity, screening by flowcytometry for PNH is of value in this group of patients because it provides an opportunity to rapidly establish the diagnosis of this treatable disorder, which might otherwise be missed if the initial presentation is only thrombotic.

Regulatory T-cells (Tregs) have been shown to be important for the balance of autoimmunity and oncogenesis. Tregs have a protective role in autoimmune diseases and conversely promote oncogenesis. Chronic lymphocytic leukemia (CLL) is unique in being at the cross-roads of oncogenesis and autoimmunity. We studied Tregs, defined as CD4+CD25(high)CD127(low)FOXP3+, in 32 treatment-naive patients with CLL. Our study shows that patients with CLL had a higher absolute Treg count than the control group (p < 0.001). A progressive increase of Tregs was noted in advanced stages of the disease (p < 0.001). The increase in absolute Treg count is more significant than the increase in percentage Tregs. The absolute Treg count appears to be more important in disease pathogenesis. The absolute Treg count was significantly higher in those patients having autoimmune cytopenias. There was an inverse correlation between lymphocyte doubling time and absolute Treg count (p = 0.03). The absolute Treg count may be used as a prognostic marker in CLL.

Therapeutic advances in multiple myeloma (MM) incorporating the use of high-dose melphalan, novel therapeutic immunomodulatory agents, proteasome inhibitors and supporting autologous stem-cell transplantation (ASCT) have improved response rates and overall survival. The detection of minimal residual disease (MRD) is recognized as a sensitive and rapid approach to evaluate treatment efficacy as a tool for predicting patient outcomes and guiding therapeutic decisions. MRD analysis is reflected by many different techniques, however, multiparametric flow cytometry is a sensitive, feasible and adequate method for monitoring residual disease. Studies from India related to this context are lacking. In the present study, we compare MRD levels in patients of multiple myeloma after chemotherapy/ASCT assessed by multiparametric flow cytometry, with M band status, immunofixation (IFE) and percentage of plasma cells on bone marrow aspirate. Seventeen patients of multiple myeloma were included in the study over a duration of one year, (Male=13, Female=4) with mean age of 56.8 years (range 44-80 years). MRD was analyzed using a dual laser 6 color-flow cytometer in 9 patients of ASCT (day 100) and 8 patients on chemotherapy alone (post-induction). Pre-titrated cocktail of CD38, CD138, CD19, CD45, cytoplasmic Kappa light chain, cytoplasmic lambda light chain, CD81, CD27, CD28 CD200 and CD10 were used in 6-color combination of three tubes for MRD analysis. MRD was detectable in 5 patients, mean of 0.61% (range of 0.07 - 6.44%). M band and IFE were positive in 2 patients, each. Bone marrow plasma cells ranged from 0 to 22%. MRD levels did not show significant correlation with percentage of plasma cells in bone marrow aspirate, however it had an statistical agreement with presence or absence of serum M-band and IFE. Patients are on regular follow up for their clinical and hematological response. Disclosures No relevant conflicts of interest to declare.

Introduction Minimal residual disease (MRD) has emerged as an independent prognostic factor for patients of acute lymphoblastic leukemia (ALL). There is a strong correlation between MRD levels in bone marrow and the risk of relapse in childhood & adult leukemias 1, 2. Bone marrow MRD (BM-MRD) level of ≥ 0.01% is considered as positive and a mid-induction MRD of ≥ 1% is associated with high risk of relapse 3. Recently, the concept of peripheral blood MRD (PB-MRD), as a replacement for BM-MRD, has hit the lime light. In pediatric B-ALL, presence of PB-MRD is associated with a high relapse rate in comparison to cases which are PB-MRD negative 4, 5. This study was aimed to compare the levels of mid-induction (day 15) MRD levels in bone marrow and peripheral blood of pediatric B-ALL patients with a hypothesis that PB-MRD levels correlate with BM-MRD levels, and thus can predict BM-MRD levels for further management of the patient. Methods Forty newly diagnosed CD19+CD10+CD34+/- pediatric B-ALL patients under Vincristine, L-Asparaginase and Dexamethasone, were assessed for MRD levels on their paired day 15 PB & BM samples using six colour flow cytometry. With informed consent, both the samples were collected in EDTA vacutainers and lyse-stain-wash technique was used to prepare a single six colour tube comprising of SYTO 13/ CD34PE/ CD20PerCP/ CD19 PECy7/ CD10APC/ CD45APCH7 for each sample. The processed samples were run on BD FACS Canto II with acquisition of 1 million events or till the tubes were empty. Analysis was done using BD FACS Diva software and MRD of ≥ 0.01% was considered positive. Results Among 40 pairs of day 15 PB and BM samples, 25 (62.5%) were BM-MRD positive. Sixteen pairs (40%) had PB-MRD and BM-MRD co-positivity, 9 pairs (22.5%) had isolated BM-MRD positivity and 15 pairs (37.5%) were MRD negative in both PB and BM samples. In other words, among the 25 BM-MRD positive cases, simultaneous PB-MRD was positive in 16 patients (64%) and none of the samples had isolated PB-MRD positivity. Overall analysis of MRD positive cases showed a direct correlation between PB-MRD and BM-MRD (ρ = +0.684, p < 0.000) and BM-MRD levels were 7 times higher than the PB-MRD. In addition, ROC analysis with PB-MRD of ≥ 0.01% as a cut-off, revealed that, the most likelihood of PB-MRD being positive was when BM-MRD was ≥ 0.31%. Conclusions In contrast to the sparsely available literature, our study shows a significant correlation between PB & BM-MRD levels in day 15 paired samples of B-ALL cases. The MRD levels were 7 times higher in BM as compared to PB and PB-MRD was mostly positive with BM-MRD of ≥0.31%. In other words, day 15 PB-MRD positivity indirectly indicates that there is a minimum BM-MRD of 0.31%. Since literature reports prognostic significance of mid-induction BM-MRD at levels ≥1%, on day 15, an assessment of peripheral blood MRD alone, might yield clinically relevant prognostic information. A paired analysis at different time points might also establish a similar correlation as seen in the present study, eliminating the need of BM-MRD during further follow ups of the patient. This will help in avoiding an invasive procedure and improve patient compliance. References 1. Irving J, Jesson J, Virgo P, Case M, Minto L, Eyre L, et al. Establishment and validation of a standard protocol for the detection of minimal residual disease in B lineage childhood acute lymphoblastic leukemia by flow cytometry in a multi-center setting. haematologica. 2009;94(6):870-4. 2. Coustan-Smith E, Sancho J, Behm FG, Hancock ML, Razzouk BI, Ribeiro RC, et al. Prognostic importance of measuring early clearance of leukemic cells by flow cytometry in childhood acute lymphoblastic leukemia. Blood. 2002;100(1):52-8. 3. Basso G, Veltroni M, Valsecchi MG, Dworzak MN, Ratei R, Silvestri D, et al. Risk of relapse of childhood acute lymphoblastic leukemia is predicted by flow cytometric measurement of residual disease on day 15 bone marrow. Journal of Clinical Oncology. 2009;27(31):5168-74. 4. Elain CS, Sancho J, Michael LH, Bassem. Use of peripheral blood instead of bone marrow to monitor residual disease in children with acute lymphoblastic leukemia. Blood. 2002;100 (7):2399-402. 5. Brisco MJ, Sykes PJ, Hughes E, Dolman G, Neoh SH, Peng LM, et al. Monitoring minimal residual disease in peripheral blood in B lineage acute lymphoblastic leukaemia. British journal of haematology. 1997;99(2):314-9. Disclosures: No relevant conflicts of interest to declare.

Abstract 3864 Introduction: Chronic lymphocytic leukemia (CLL) originates from mature B lymphocytes which avoid death through the intercession of external signals, including T cells. There is improved understanding of the relationship between different cell types in the normal immune response and the dynamic cross talk between tumors and immune system regulating tumor growth. Cancer and autoimmunity are considered as anti-thesis of each other. Regulatory T cells (Tregs) are shown to have a protective role in autoimmune diseases and conversely known to promote oncogenesis. The exact role of Tregs in disease biology of CLL is still unclear. Aims and Objectives : to define the role of Tregs in clinical presentation, disease progression and autoimmune cytopenias in CLL. Materials and Methods : 32 treatment naive CLL patients and 10 normal healthy volunteers were evaluated in this prospective study. Investigations included clinical examination, biochemical tests, complete blood counts, bone marrow examinaton and multicolor flowcytometry (FCM). More stringently defined specific markers (CD4+CD25highCD127low FoxP3+) were used to enumerate Tregs. Routine CLL panel for diagnosis and prognosis included 17 monoclonal antibodies (MoAbs) labeled with fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC) or peridinin chlorophyll protein (PerCP). FCM analysis was performed on FACS Calibur flowcytometer (BD Biosciences) using CellQuest® software. Diagnosis of autoimmune hemolytic anemia (AIHA), autoimmune thrombocytopenia (ITP) and pure red cell aplasia (PRCA) was made according to standard criteria. Results : Study group included 20 male and 12 female CLL patients. The mean age was 64.2 years; with 8/32 (25%) patients being 0.05) from stages A and B (early CLL) to stage C (advanced CLL), increase in mean ALC was 172% (p Disclosures: No relevant conflicts of interest to declare.