A surrogate molecular approach for the detection of Philadelphia chromosome-like B-acute lymphoblastic leukemia.

BACKGROUND: Philadelphia chromosome (Ph)-like B-acute lymphoblastic leukemia (B-ALL) is a clinically significant, high-risk genetic subtype of B-ALL cases. There are few data on the incidence, characterization, and treatment outcomes of Ph-like ALL cases from low- and middle-income countries. There is a pressing need to establish a well-organized/cost-effective approach for identifying Ph-like ALL instances. METHODS: Multiplex reverse transcriptase polymerase chain reaction, nCounter NanoString, and fluorescence in situ hybridization were used to detect and characterize Ph-like ALL cases among recurrent genetic abnormalities (RGA)neg B-ALL cases. At the end of induction therapy, flow cytometry-minimal residual disease (MRD) assay was used to quantify MRD positivity in Ph-like ALL cases. RESULTS: Of 130 newly diagnosed B-ALL cases, 25% (BCR::ABL1), 4% (ETV6::RUNX1), 5% (TCF3::PBX1), 2% (KM2TA::AFF1), and 65% RGAneg B-ALL cases were revealed by multiplex reverse transcriptase polymerase chain reaction. Among RGAneg B-ALL cases, 24% Ph-like ALL cases using nCounter NanoString were identified, with 48% CRLF2high cases with 45% CRLF2::P2RY8 and 18% CRLF2::IGH rearrangements(∼r) revealed by fluorescence in situ hybridization. In 52% of CRLF2low cases, 17% ABL1 and JAK2∼r 8% EPOR::IGH & PDGRFB∼r were identified. Ph-like ALL cases had higher total leukocyte count (p < .05), male preponderance (p < .05), and high MRD-positivity/induction failure compared with RGAneg B-ALL cases. Furthermore, in Ph-like ALL cases, 11 significant genes using quantitative polymerase chain reaction were identified and validated. CRLF2, IGJ, CEACAM6, MUC4, SPATS2L and NRXN3 genes were overexpressed and show statistical significance (p < .05) in Ph-like ALL cases. CONCLUSIONS: This study showed the high incidence of Ph-like ALL cases with kinase activating alterations and treatment outcomes from low- and middle-income region. Furthermore, a surrogate cost-effective multiplex panel of 11 overexpressed genes for the prompt detection of Ph-like ALL cases is proposed. PLAIN LANGUAGE SUMMARY: Identification of recurrent gene abnormalities (RGA)neg B-acute lymphoblastic leukemia (B-ALL) cases using multiplex-reverse transcriptase polymerase chain reaction. Identification and characterization of Philadelphia (Ph)-like ALL cases using nCounter NanoString gene expression profiling and fluorescence in situ hybridization. Furthermore, Ph-like ALL cases were characterized according to CRLF2 expression and kinase-activating genomic alterations. Minimal residual disease of Ph-like ALL cases were quantified using flow cytometry-minimal residual disease assay. A surrogate molecular approach was established to detect Ph-like ALL cases from low- and middle-income countries.

Aberrant baseline cytokine profile in patients with newly diagnosed acquired aplastic anaemia correlates with disease severity and the treatment response.

BACKGROUND: Immune dysregulation is crucial in the pathogenesis of acquired aplastic anaemia (aAA). There is paucity of data regarding correlation of baseline cytokine profile with treatment response in aAA. OBJECTIVE: Present prospective case-control study aimed to correlate the baseline cytokines in patients with aAA with the treatment response. METHODS: Fifty-one patients with newly-diagnosed aAA > 13 years of either sex were enrolled over 1.5 years. Twenty age-and sex-matched healthy controls (HC) were also included. The cytokine profile (IL-2, 4, 6, 8, 10, 17, IFN-γ and TNF-α) in the peripheral blood plasma of aAA patients was performed at the baseline using cytometric bead analysis. The cytokine levels were compared with HC and correlated with response to immunosuppressive therapy (IST) at 3-months. RESULTS: The median age of cases was 29 years (range,13-74). The cases had higher mean levels of IL2 (p = 0.326), IL4 (p = 0.038), IL6 (p = 0.000), IL10 (p = 0.002), TNF-α (p = 0.302), IFN-γ (p = 0.569) and IL-17 (p = 0.284) than the HC. The baseline levels of all the cytokines were higher (statistically non-significant) among responders (n = 13) than the non-responders (n = 14) to IST. CONCLUSIONS: Baseline cytokine profile in patients with aAA might predict response to the IST. Larger studies are needed to validate our results.

Genomic and proteomic characterization of Philadelphia-like B-lineage acute lymphoblastic leukemia: A report of Indian patients.

BACKGROUND: The gold standard for the identification of Philadelphia (Ph)-like acute lymphoblastic leukemia (ALL) patients is gene expression profiling. Because of its diverse nature, its identification is extremely difficult and expensive. On the genomic and proteomic landscape of Ph-like ALL patients, there is a paucity of published literature from developing countries. METHODS: The authors used digital barcoded nCounter NanoString gene expression profiling for its detection, followed by molecular and proteomic characterization using fluorescence in situ hybridization and liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: The authors found 32.05% Ph-like ALL patients and their median age at presentation was considerably higher than Ph-negative ALL cases (p = .0306). Furthermore, we identified 20% CRLF2 overexpressed cases having 8.33% CRLF2-IGH translocation with concomitant R683S mutation and 8.33% CRLF2-P2RY8 translocation. In 80% of CRLF2 downregulated cases, we identified 10% as having JAK2 rearrangement. Minimal residual disease-positivity was more common in Ph-like ALL cases (55.55% vs. 25% in Ph-negative ALL cases). Immunoglobulin J chain (Jchain), small nuclear ribonucleoprotein SmD1 (SNRPD1), immunoglobulin κ constant (IGKC), NADH dehydrogenase (ubiquinone) 1 α subcomplex subunit 2 (NDUFA2), histone H2AX (H2AFX), charged multivesicular body protein 4b (CHMP4B), and carbonyl reductase (NADPH) (CBR1) proteins were identified to be substantially expressed in Ph-like ALL patients, using LC-MS/MS. Gene enrichment analysis indicated that involvement of spliceosomal mediated messenger RNA splicing pathway and four microRNAs was statistically significant in Ph-like ALL patients. CONCLUSIONS: For the first time, we have described incidence, molecular, and proteomic characterization of Ph-like ALL, in developing nations. PLAIN LANGUAGE SUMMARY: In developing countries, detecting Philadelphia (Ph)-like B-lineage acute lymphoblastic leukemia is complicated and challenging due to its diverse genetic landscape. There is no well-defined and cost-effective methodology for its detection. The incidence of this high-risk subtype is very high in adult cases, and there is an urgent need for its accurate detection. We have developed an online PHi-RACE classifier for its rapid detection, followed by delineating the genomic and proteomic landscape of Ph-like acute lymphoblastic leukemias for the first time in Indian patients.

'Evaluation of adverse prognostic gene alterations & MRD positivity in BCR::ABL1-like B-lineage acute lymphoblastic leukaemia patients, in a resource-constrained setting.

BACKGROUND: Early detection of BCR::ABL1-like ALL could impact treatment management and improve the overall survival/outcome. BCR::ABL1-like ALL cases are characterised by diverse genetic alterations activating cytokine receptors and kinase signalling. Its detection is still an unmet need in low-middle-income countries due to the unavailability of a patented TLDA assay. METHODS: This study's rationale is to identify BCR::ABL1-like ALLs using the PHi-RACE classifier, followed by the characterisation of underlying adverse genetic alterations in recurrent gene abnormalities negative (RGAneg) B-ALLs (n = 108). RESULTS: We identified 34.25% (37/108) BCR::ABL1-like ALLs using PHi-RACE classifier, characterised by TSLPR/CRLF2 expression (11.58%), IKZF1 (Δ4-7) deletion (18.9%) and chimeric gene fusions (34.61%). In overexpressed TSLPR/CRLF2 BCR::ABL1-like ALLs, we identified 33.33% (1/3) CRLF2::IGH and 33.33% (1/3) EPOR::IGH rearrangements with concomitant JAK2 mutation R683S (50%). We identified 18.91% CD13 (P = 0.02) and 27.02% CD33 (P = 0.05) aberrant myeloid markers positivity, which was significantly higher in BCR::ABL1-like ALLs compared to non-BCR::ABL1-like ALLs. MRD positivity was considerably higher (40% in BCR::ABL1-like vs. 19.29% in non-BCR::ABL1-like ALLs). CONCLUSIONS: With this practical approach, we reported a high incidence of BCR::ABL1-like ALLs, and a lower frequency of CRLF2 alteration & associated CGFs. Recognising this entity, early at diagnosis is crucial to optimise personalised treatment strategies.

Characteristics and outcome of infectious complications after autologous hematopoietic cell transplantation in multiple myeloma patients.

BACKGROUND: Infections are a significant cause of morbidity and mortality after autologous hematopoietic cell transplantation (AHCT) in multiple myeloma (MM) patients. There has been a rapid advancement and evolution in MM treatment landscape in the last decade. There is limited information on post-AHCT infectious complications among MM patients with or without levofloxacin prophylaxis from developing countries. MATERIALS AND METHODS: We performed a retrospective study to explore the incidence, pattern, and clinical outcome of infections following AHCT in MM patients from 2010 to 2019 at our center. Patient-specific, disease-specific, and transplant-specific details were retrieved from the case files. The characteristics of infectious complications (site, intensity, organism, treatment, and outcomes) were analyzed. All patients who underwent transplantation from 2010 to 2016 received levofloxacin antibiotic prophylaxis. Common terminology criteria for adverse events (CTCAE) criteria (v5.0) were used for the grading of infections and regimen-related toxicity. International Myeloma Working Group updated criteria were used for the assessment of disease response before transplant and at day +100. RESULTS: Ninety-five consecutive patients with newly diagnosed multiple myeloma (NDMM) (n = 85), RRMM (n = 7), plasma cell leukemia (n = 2), and Polyneuropathy, Orgaomegaly, Endocrinopathy, Monoclonal gammopathy, skin abnormalities (POEMS) syndrome (n = 1) underwent AHCT during the study period. Their median age was 55 years (range 33-68); 55.8% were males. Immunoglobulin IgG kappa was the most common monoclonal protein (32.6%), International Staging System stage III disease was present in 45.3%, and 84.2% of patients achieved more than very good partial response before AHCT. The median time from diagnosis to AHCT was 10 months (range 4-144). Eighty-nine patients (93.7%) developed fever after AHCT. Fever of unknown focus, microbiologically confirmed infections, and clinically suspected infections were found in 50.5%, 37.9%, and 5.3% of patients, respectively. Clostridiodes difficile-associated diarrhea was observed in eight patients (8.4%). Neutrophil and platelet engraftment occurred after a median of 11 days (range 9-14) and 12 days (range 9-23), respectively. The median duration of hospital stay was 16 days (range 9-29). Only two patients (2.1%) required readmission for infections within 100 days of AHCT. Transplant-related mortality (TRM) in the study population was 4.2% (n = 4). The levofloxacin prophylaxis group (n = 32, 33.7%) had earlier neutrophil engraftment (day +10 vs. day +11) and platelet engraftment (day +11 vs. day +12), but time to fever onset, duration of fever, hospital stay, TRM, and day +100 readmission rates were not significantly different from those of patients without levofloxacin prophylaxis. There was no significant difference in the spectrum of infections between patients with and without levofloxacin prophylaxis. The overall survival and progression-free survival of the study population at 5 years were 72.7% and 64.8%, respectively. CONCLUSION: This study shows that the incidence of infections and TRM are higher in MM patients from lower-middle income countries after AHCT than in those from developed countries. The majority of such patients lack clinical localization and microbiological proof of infection. There was no significant difference in the spectrum of infections and their outcomes in patients with and without levofloxacin prophylaxis.

A Novel CEBPE Variant Causes Severe Infections and Profound Neutropenia.

PURPOSE: Specific granule deficiency (SGD) is a rare inborn error of immunity resulting from loss-of-function variants in CEBPE gene (encoding for transcription factor C/EBPε). Although this genetic etiology has been known for over two decades, only a few patients with CEBPE variant-proven SGD (type I) have been reported. Herein, we describe two siblings with a novel homozygous CEBPE deletion who were noted to have profound neutropenia on initial evaluation. We aimed to evaluate the immunohematological consequences of this novel variant, including profound neutropenia. METHODS: Light scatter characteristics of granulocytes were examined on various automated hematology analyzers. Phagocyte immunophenotype, reactive oxygen species generation, and Toll-like receptor (TLR) signaling were assessed using flow cytometry. Relative expression of genes encoding various granule proteins was studied using RT-PCR. Western blot analysis and luciferase reporter assay were performed to explore variant C/EBPε expression and function. RESULTS: Severe infections occurred in both siblings. Analysis of granulocyte light scatter plots revealed automated hematology analyzers can provide anomalously low neutrophil counts due to abnormal neutrophil morphology. Neutrophils displayed absence/marked reduction of CD15/CD16 expression and overexpression (in a subset) of CD14/CD64. Three distinct populations of phagocytes with different oxidase activities were observed. Impaired shedding of CD62-ligand was noted on stimulation with TLR-4, TLR-2/6, and TLR-7/8 agonists. We demonstrated the variant C/EBPε to be functionally deficient. CONCLUSION: Homozygous c.655_665del variant in CEBPE causes SGD. Anomalous automated neutrophil counts may be reported in patients with SGD type I. Aberrant TLR signaling might be an additional pathogenetic mechanism underlying immunodeficiency in SGD type I.

Identification and validation of suitable housekeeping genes for gene expression studies in BCR-ABL1 positive B-lineage acute lymphoblastic leukemia.

BACKGROUND: 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. METHODS AND RESULTS: 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. CONCLUSIONS: 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.

Aberrant expression of cytokines in polycythemia vera correlate with the risk of thrombosis.

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 1ß, 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-1ß, 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.

Evaluation of a flow cytometric test for G6PD-deficient erythrocytes.

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 (<10% bright cells). Concordance with MRT was 89% (55/62 cases) and with EAA was 77% (48/62 cases). Fluorometrically predicted violet laser excitation (405-nm) with signal acquisition in the 425-475 nm region was a technical advancement noted for the first time in this paper. CONCLUSION: Flow cytometry/fluorescence microscopy represent technically straightforward methods for the detection and quantification of G6PD-deficient erythrocytes. Based on our results, we recommend their application as a first-line investigation to screen females who are prescribed an oxidant drug like primaquine or dapsone.

Hematological characteristics, cytogenetic features, and post-induction measurable residual disease in thymic stromal lymphopoietin receptor (TSLPR) overexpressed B-cell acute lymphoblastic leukemia in an Indian cohort.

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.

An Evaluation of a Fluorescence In Situ Hybridization Strategy Using Air-dried Blood and Bone-marrow Smears in the Risk Stratification of Pediatric B-Lineage Acute Lymphoblastic Leukemia in Resource-limited Settings.

Cytogenetic abnormalities (CAs), one of the strongest influencers of therapeutic outcome in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL), can be identified by different techniques. Despite several technological advances, many centers with resource-limited settings continue to use either reverse-transcriptase polymerase chain reaction (RT-PCR) and/or fluorescence in situ hybridization (FISH) to identify prognostically relevant CAs. We evaluated a simple and cost-effective triple-probe FISH strategy on air-dried blood and bone-marrow smears and compared its performance with a multiplex RT-PCR-based approach in the prognostication of pediatric BCP-ALL patients. Three hundred twenty BCP-ALL patients were tested prospectively and in parallel by FISH on air-dried blood or bone-marrow smears and RT-PCR. The FISH strategy correctly diagnosed all genetic abnormalities identified by RT-PCR. Prognostically relevant genetic abnormalities were missed by RT-PCR in 24 (8.1%) patients. In another 20 children (6%), with samples inadequate for RT-PCR testing (dry taps or due to poor sample quality), a successful FISH testing could be performed on bone-marrow aspirate or trephine-imprint smears. In addition, FISH detected ploidy changes, which could be confirmed by FxCycle Violet-based flow-cytometry. FISH testing on air-dried smears identified more prognostically relevant CAs, provided information on the ploidy status, and could be successfully performed in children with difficulty in bone-marrow sampling.

Deciphering molecular heterogeneity of Indian families with hereditary spherocytosis using targeted next-generation sequencing: First South Asian study.

Defects in various erythrocyte membrane proteins genes (ankyrin, band-3, ß- and α-spectrin and protein 4·2) can cause hereditary spherocytosis (HS). This molecular heterogeneity of HS, together with co-inherited genetic modifiers, results in marked phenotypic variability among patients. We studied the molecular spectrum and genotype-phenotype correlations in 73 families (with 113 patients) with HS. Deleterious variants including nonsense (42%), deletions (18%), splice site (20%), missense (10%) and duplication/insertion (10%) were found in 47 patients. The variants detected included sporadic and dominantly-inherited defects in ANK1 (53·2%), SPTB (36·2%) and SLC4A1 (4·2%). Compound heterozygous variants in SPTA1 (6·4%) showed autosomal recessive inheritance. Alpha-spectrin variants were associated with severe anaemia and splenectomy alleviated symptoms. Co-inherited glucose-6-phosphate dehydrogenase (G6PD) deficiency was found in 15%. G6PD variants (n = 5) led to greater transfusion requirements (1-8 times) in males with HS. Homozygosity (41%) for the promoter variant of UGT1A1 (Gilbert syndrome) led to a significantly higher mean bilirubin level (126·54 µmol/l) with a higher frequency of cholelithiasis (30%) (P < 0·001). This first-ever south Asian study on the molecular spectrum of HS found ANK1 and SPTB genes variants to be the commonest with inheritance being sporadic/dominant. Next-generation sequencing provided a relatively sensitive and rapid tool for molecular diagnosis with a diagnostic yield of 64·4%.

Variables affecting the presence of mesenchymal stromal cells in peripheral blood and their relationship with apheresis products.

Mesenchymal stromal cells (MSCs) demonstrate the properties of self-renewal, multipotentiality, and immunosuppression, which are responsible for their widespread clinical applications in tissue repair and regeneration. MSCs have been isolated from bone marrow, adipose tissue, and cord blood using culture in specialised media. Their presence in peripheral blood (PB) is debatable. We studied the presence of MSCs at baseline (PB) and following mobilisation with growth factors [PB and apheresis product (AP)] in patients undergoing autologous stem cell transplantation and healthy donors using flow cytometry. We conclude that both mobilised PB and AP are potential sources of MSCs. Given their small numbers in PB/AP, clinical use is feasible following ex-vivo expansion. Variables affecting the presence of MSCs in PB and AP are also discussed.

A flow cytometric cell-cycle assay using methyl green.

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.

The utility of the basophil activation test in differentiating asthmatic subjects with and without allergic bronchopulmonary aspergillosis.

BACKGROUND: Asthma with Aspergillus sensitisation (ASA) and allergic bronchopulmonary aspergillosis (ABPA) are distinct clinical conditions, with different management strategies. OBJECTIVE: To determine the utility of the basophil activation test (BAT) in differentiating between asthmatic subjects with and without ABPA. METHODS: We performed the BAT using flow cytometry for the basophil activation markers CD63, CD193, and CD203c in consecutive subjects with unsensitised asthma (UA), ASA and ABPA. The BAT was performed unstimulated (baseline) and after stimulation with peanut and Aspergillus fumigatus antigens. Stimulation indices (SI) for CD63, CD193 and CD203c were compared between the study groups. RESULTS: We enrolled 82 (UA [n = 25], ASA [n = 25], ABPA [n = 32]) subjects. Only those subjects without peanut sensitisation on serological testing were included in further analyses (n = 50). The receiver operating characteristic analysis of SI for CD63, CD193 and CD203c for the diagnosis of ABPA vs other asthmatics (UA and ASA) showed an area under the curve of 0.577, 0.317 and 0.625, respectively. The SI CD203c at a cut-off of 1.2 (sensitivity 50.0%, specificity 88.9%) and the SI CD63 at a cut-off of 1.3 (sensitivity 42.9%, specificity 91.7%) also had limited utility for the diagnosis of ABPA. CONCLUSION: The BAT cannot be used as a diagnostic test in distinguishing ABPA complicating asthma from ASA and UA.

Genetic basis of unexplained erythrocytosis in Indian patients.

OBJECTIVE: To evaluate the spectrum of genetic defects in Indian patients with unexplained erythrocytosis. METHODS: Fifteen families (18 patients) with unexplained erythrocytosis were enrolled after excluding polycythemia vera and secondary erythrocytosis. Focused Sanger sequencing from genomic DNA was performed for EPOR (exon 8), VHL (exons 2-3), EGLN1 (exons 2-5), EPAS1 (exon 12), and all exons of HBB, HBA1, and HBA2 genes. RESULTS: Eleven of the 18 patients (including two pairs of brothers) had Chuvash polycythemia, that is, homozygosity for VHL:c.598C > T (p.Arg200Trp). Three patients (two of whom were brothers) had HBB mutations associated with increased oxygen-affinity hemoglobin-one had a heterozygous Hb McKees Rocks HBB:c.438T > A (p.Tyr146*), and two brothers showed heterozygous Hb Rainier HBB:c.437A > G (p.Tyr146Cys). No pathogenic variants were found in the remaining four cases. CONCLUSION: A gene-by-gene Sanger sequencing approach could determine a genetic basis for erythrocytosis in 11 of the 15 (73%) Indian families, with homozygous VHL:c.598C > T (p.Arg200Trp) being the commonest pathogenic variant. This first study from the Indian subcontinent provides a rationale for analyzing this variant in patients with suspected congenital erythrocytosis from this region. Rare first occurrences of Hb McKees Rocks and Hb Rainier in Indians are also being reported.

Childhood Blastic Plasmacytoid Dendritic Cell Neoplasm Mimicking Acute Rheumatic Fever: Report of an Unusual Clinical Presentation and Review of Literature.

BACKGROUND: Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is very rarely diagnosed in children with less than 50 cases in the literature. OBSERVATION: We report a case of childhood BPDCN who mimicked acute rheumatic fever at presentation. Majority of the reported childhood BPDCN received acute lymphoblastic leukemia-like chemotherapy with/without stem cell therapy, whereas those who received acute myeloid leukemia-like therapy predominantly succumbed to disease or sepsis. Overall 68% of the patients were alive and achieved complete remission with an overall prognosis slightly better in children compared with adults. CONCLUSIONS: The case is reported due to its unique unusual clinical presentation and its rarity in pediatric population.

Haematological profile of patients with mixed-phenotype acute leukaemia from a tertiary care centre of north India.

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.