A Pilot Study to Determine the Utility of Automated Tissue Dissociator for Flowcytometry Based Evaluation of Hematolymphoid Tumor Tissue Biopsies.

Routine diagnostic biopsy tissue processing, conventional histology/immunohistochemistry (IHC) method is a multi-step and time consuming practice. With the advanced tissue dissociation protocols and panel designing, flow cytometric immunophenotyping (FCI) can be performed on diagnostic hematolymphoid tissue samples using single cell suspensions that economize steps and the time taken. Diagnostic tissue samples from lymph node, mediastinal mass, testicular biopsies and similar sites were dissociated using gentle MACS Octo-dissociator and FCI was performed thereafter. Oral tissue biopsy samples were also processed as a validation set for the protocol. 21 prospective tissue biopsy samples with suspected involvement by a known hematolymphoid neoplasm were processed and evaluated. These included B lymphoblastic lymphoma (n = 12), T lymphoblastic lymphomas (n = 3), Burkitts lymphoma (n = 3) and one case each of granulocytic sarcoma, Hodgkin lymphoma and granulomatous disease. Tissue FCI and IHC were found concordant with identified profile FCI obtained from blood/bone marrow analyses. FCI can produce a highly sensitive and reliable report, within hours, by processing fresh tumor tissue samples from suspected hematolymphoid malignancies. This method can be considered as an effective adjunct to IHC and can be applicable in routine clinical diagnostics, especially in cases that needs quick diagnosis and immediate clinical treatment.

Spectrum and Immunophenotypic Profile of Acute Leukemia: A Tertiary Center Flow Cytometry Experience.

BACKGROUND: For diagnosis, sub-categorization and follow up of Acute Leukemia (AL), phenotypic analysis using flow cytometry is mandatory. MATERIAL AND METHODS: We retrospectively analyzed immunophenotypic data along with cytogenetics/molecular genetics data (wherever available) from 631 consecutive cases of AL diagnosed at our flow cytometry laboratory from January 2014 to August 2017. RESULTS: Of the total 631 cases, 52.9% (n=334) were acute lymphoblastic leukemia (ALL), 43.9% (n=277) acute myeloid leukemia (AML), 2.2% (n=14) mixed phenotypic acute leukemia (MPAL), 0.5% (n=3) acute undifferentiated leukemia (AUL) and 0.5% (n=3) chronic myeloid leukemia in blast crisis (CML-BC). ALL cases comprised of 81.7% (n=273/334) B-cell ALLs (95.2%, n=260/273 common B-ALLs and 4.8%, n=13/273 Pro B-ALLs). CD13 was the commonest cross lineage antigen, expressed in B-ALL (25.6%, n=70/273), followed by CD33 (17.9%, n=49) and combined CD13/CD33 (11.3%, n=31/273) expression. T-ALLs constituted 18.3% (n=61/334) of total ALLs and included 27.9% (n=17/61) cortical T- ALLs. CD13 was commonest (32.7%, n=20/61) aberrantly expressed antigen in T-ALLs, followed by CD117 (19.1%, n=9/47). AML cases included 32.1% (n=89/277) AML with recurrent genetic abnormalities, 9.0% (n=25/277) with FLT3/NPM1c mutation and 58.9% (n=163/277) AML NOS including 14.7% (n=24/163) AML M4/M5, 1.8% (n=3/163) AML M6 and 3.7% (n=6/163) AML M7. In AMLs, CD19 aberrancy was the most common (20.2%, n=56/277) followed by CD56 (15.8%, n=42/265). CONCLUSIONS: In this study, we document the spectrum, correlate the immunophenotype with genetic data of all leukemias, especially concerning T-ALL where the data from India is scarce.

FxCycle™ Based Ploidy Correlates with Cytogenetic Ploidy in B-Cell Acute Lymphoblastic Leukemia and Is Able to Detect the Aneuploid Minimal Residual Disease Clone.

BACKGROUND: Flow cytometry (FCM) is a simple, sensitive, and specific technique that can potentially determine DNA ploidy in B-cell precursor ALL (BCP-ALL) and is complementary to cytogenetics. METHODS: A prospective FCM DNA ploidy analysis using FxCycle™ Violet (assay sensitivity 0.01%) was done in 125 consecutive new cases of BCP-ALL (90 cases <15 years of age) and compared with corresponding cytogenetic ploidy (karyotyping and/or FISH) data wherever available. This assay was also subsequently evaluated for detection of residual aneuploid clone in few BCP-ALL cases. RESULTS: Of the total 125 BCP-ALL cases evaluated, flow ploidy analysis revealed diploidy (DI 0.96-1.05) in 44.8% (n = 56), low-hyperdiploidy (DI 1.06 to 1.15) in 13.6% (n = 17), high-hyperdiploidy (DI 1.16-1.39) in 32.8% (n = 41) and near-tetraploidy (DI ≥ 1.80) in 2.4% (n = 3) cases. The high risk sub-group of low-hypodiploidy (DI 0.70 to 0.88)/near-triploidy (DI 1.40 to 1.79) constituted 5.6% (n = 7) cases while there was only one case with haploidy (DI 0.58). Overall, high concordance of 90.4% (n = 113) was noted between the combined cytogenetics ploidy and FCM ploidy. Of the total discordant cases (n = 12), the maximum discordance was seen in the low-hyperdiploid DI subgroup (n = 10), which included seven cases with low DNA index high hyperdiploidy (LDI-HHD). FCM DNA ploidy assay was able to detect the residual clone in all six MRD positive aneuploid cases evaluated. CONCLUSIONS: FxCycle™ based DNA ploidy ascertains strong correlation with cytogenetic profiles and yields complementary information that can be used by the cytogenetics laboratories or otherwise. © 2019 International Clinical Cytometry Society.

Mixed-phenotypic acute leukemia series from tertiary care center.

INTRODUCTION: Mixed-phenotype acute leukemias (MPALs) are a heterogeneous group of rare leukemias constituting approximately 2%-5% of all leukemias, in which assigning a single lineage of origin is not possible. They are diagnosed by either the presence of antigens of more than one lineage or by the presence of dual population of blasts belonging to two or more lineages. We highlight the clinicopathological, immunophenotype, and genetic data of a cohort (n = 14) of patients diagnosed and treated at our center. MATERIALS AND METHODS: We retrospectively analyzed consecutive cases of MPAL diagnosed in our flow cytometry laboratory from May 2012 to August 2015. These cases were diagnosed based on immunophenotyping of peripheral blood/bone marrow aspirates and morphology/genetics wherever available as per the World Health Organization (WHO) 2008 guideline. RESULTS: Among 628 consecutive acute leukemia (AL) cases diagnosed and evaluated during this period, we identified 14 (2.2%) patients with MPAL fulfilling WHO 2008/EGIL criteria for immunological characterizing of AL criteria. Majority of these were males (n = 8, male:female ratio 1.3:1) and adults (n = 11, 78.5%). The median age of this cohort was 41 years (range 2-80). These cases were further classified as: B/myeloid (n = 9), T/myeloid (n = 4), and B/T MPAL (n = 1). Cytogenetics was available in 12 out of 14 cases, out of which, three cases had normal karyotype, three with t(9;22)(q34;q11), and two cases with complex karyotype. We also came across a rare case of B + T lymphoid MPAL who had mixed-lineage leukemia gene t(v; 11q23) rearrangement. CONCLUSION: MPAL is a complex entity with heterogeneous clinical, immunophenotypic, cytogenetic, and molecular features. Multiparametric flowcytometry by using comprehensive antibody panels is a valuable tool for diagnosis. Subsequent cytogenetic and molecular analysis for further prognostic stratification and treatment modalities are important.

Possible involvement of glutamic and/or aspartic acid residue(s) and requirement of mitochondrial integrity for the protective effect of creatine against inhibition of cardiac mitochondrial respiration by methylglyoxal.

We had previously shown that creatine exerted a protective effect against inhibition of cardiac mitochondrial respiration by methylglyoxal (SinhaRoy S, Biswas S, Ray M, Ray S. Biochem J 372: 661-669,2003). In the present study, we have investigated the mechanism of this protective effect by specific amino acid modifying reagent and by several compounds, which are structurally related to creatine. The results show that the compounds, which contain guanidine group such as arginine and guanidinopropionic acid, exert a protective effect, which is quantitatively similar to creatine. This result suggests the presence of carboxylic acid(s) such as glutamic and/or aspartic acid(s) in the creatine-binding site, which has been further supported by experiments with N-ethyl-5-phenyl isoxazolium-3'-sulfonate a reagent known to modify these amino acids. Both polarographic and spectrophotometric assays were performed with NADH as respiratory substrate by using a) submitochondrial particles by sonication, b) freeze-thawed mitochondria and c) mitochondria permeabilized by alamethicin treatment. The results of these studies as compared to that of intact mitochondria indicate that structural integrity of mitochondria is essential for the protective effect of creatine.