Monocyte HLADR and Immune Dysregulation Index as Biomarkers for COVID-19 Severity and Mortality.

Immune dysregulation in COVID-19 is the major causal factor associated with disease progression and mortality. Role of monocyte HLA-DR (mHLA-DR), neutrophil CD64 (nCD64) and Immune dysregulation index (IDI) were studied in COVID-19 patients for assessing severity and outcome. Results were compared with other laboratory parameters. Antibody bound per cell for mHLA-DR, nCD64 and IDI were measured in 100 COVID-19 patients by flow cytometry within 12 h of hospital admission. Thirty healthy controls (HC) were included. Clinical and laboratory parameters like C - reactive protein (CRP), Procalcitonin (PCT), Absolute Lymphocyte count (ALC), Absolute Neutrophil count (ANC) and Neutrophil to Lymphocyte ratio (NLR) were recorded. Patients were followed up until recovery with discharge or death. Parameters from 54 mild (MCOV-19), 46 severe (SCOV-19) and 30 HC were analysed. mHLA-DR revealed significant and graded down regulation in MCOV-19 and SCOV-19 as compared to HC whereas IDI was lowest in HC with increasing values in MCOV-19 and SCOV-19. For diagnostic discrimination of MCOV-19 and SCOV-19, IDI revealed highest AUC (0.99). All three immune parameters revealed significant difference between survivors (n = 78) and non-survivors (n = 22). mHLA-DR < 7010 and IDI > 12 had significant association with mortality. Four best performing parameters to identify patients with SCOV-19 at higher risk of mortality were IDI, NLR, ALC and PCT. mHLA-DR and IDI, in addition to NLR and ALC at admission and during hospital stay can be utilized for patient triaging, monitoring, early intervention, and mortality prediction. IDI reported for the first time in this study, appears most promising. Immune monitoring of 'in hospital' cases may provide optimized treatment options. Supplementary Information: The online version contains supplementary material available at 10.1007/s12291-022-01087-z.

Revealing and IgG4 analysis to factor VIII in haemophilia-A patients with and without inhibitors.

INTRODUCTION: The acquisition of factor VIII inhibitors poses major management challenges for haemophilia A (HA) patients. Most (Factor VIII) inhibitors are immunoglobulin G4 (IgG4) and G1 (IgG1) subclasses, with IgG4 being the most prevalent. The Nijmegen Bethesda Assay (NBA) was used to quantify inhibitors. However, the requirement for a large sample volume, accompanying costs, and required technical expertise complicate NBA, particularly in developing countries. ELISA-based screening proved to be more viable in a resource-constrained scenario. AIM: This study aimed to standardise and evaluate an in-house IgG4 ELISA for the detection of haemophilia A inhibitors. METHODS: This study enrolled thirty HA patients with inhibitors, thirty three HA without inhibitors, and 33 healthy controls. Standardisation of in-house IgG4 ELISA was performed. The checkerboard method was employed to optimise plasma-derived Factor VIII concentrations (HEMOFIL M Baxalta US Inc.), sample dilutions, and anti-human IgG4-HRP conjugate (Southern Biotechnology, USA). The samples were evaluated three times, and the mean optical density (OD) was used to determine the cutoff. RESULTS: Using a cutoff OD (mean±2SD) of 0.502 in our in-house ELISA, we could differentiate healthy controls and HA without inhibitors from HA with inhibitors with 93.3 % sensitivity, 97.0 % specificity, 97 % NPV, and 93.3 % PPV, respectively. However, the accuracy was 95.83 %. The two-way mixed-effects model, interclass correlation (ICC) derived by Cronbach's Alpha was 0.912 (p = 0.001) and close to perfect agreement. CONCLUSIONS: IgG4 ELISA is an effective method for detecting neutralising or functionally significant FVIII inhibitors, particularly in resource-constrained settings, following which patients may be referred to referral laboratories for quantification of inhibitors.

Evaluation of Diagnostic Efficacy of Novel Red Blood Cell Parameters as Potential Screening Test for Detecting Latent Iron Deficiency in Blood Donors.

Iron deficiency anemia (IDA) forms a major share of global burden of anemia. Frequent blood donation is a common iatrogenic cause of iron insufficiency in healthy adults. Serum iron and hemoglobin levels are normal despite low serum ferritin levels, referred to as latent iron deficiency (LID). Aim of the present study was to evaluate the role of novel RBC parameters-percentage of hypochromic RBCs (%HPO), percentage of microcytic RBCs (%MIC), and haemoglobin content of reticulocytes (MCHr) of Abbott Alinity autoanalyzer as indicators of latent iron deficiency in blood donors. 260 consenting and eligible blood donors were included in the study. Complete blood counts including new RBC parameters on Abbott Alinity autoanalyzer and serum iron profile were measured for all donors. Donors were categorized into LID and No LID based on Ferritin and Transferrin saturation (TSAT). Serum transferrin receptors (sTfR) were studied in a subset of samples [LID (n = 46), No LID (n = 18) and IDA (n = 27)]. Statistical analyses was done on IBM SPSS version 22. Among 260 donors, 56 (21.5%) were found to have LID. The difference in mean values for % HPO, % MIC, and MCHr were not found to be statistically significant in LID and No LID groups. sTfR results between LID, No LID and IDA sub-groups revealed significant difference. This study does not support the role of % HPO, % MIC and MCHr measured on Abott Alinity analyzer, as potential screening parameters for LID amongst blood donors. STfr was more informative in this regard. Further research on much larger sample size is required to confirm these findings. Supplementary Information: The online version contains supplementary material available at 10.1007/s12288-023-01683-w.

Immunophenotypic characterization of normal and abnormal plasma cells in bone marrow of newly diagnosed multiple myeloma patients.

Background: Identification of plasma cells into abnormal (APC) and normal (NPC) compartments is of utmost importance in flow cytometric (FC) analysis of multiple myeloma (MM) and related plasma cell dyscrasias for diagnosis, prognosis, and follow-up. No single phenotypic marker is sufficient to distinguish NPC from APC. Materials and Methods: 43 newly diagnosed cases of MM and 13 controls were included in the study. Bone marrow (BM) samples from the 2nd pass were processed on the same day with antibodies against CD38, CD138, CD19, CD81, CD45, CD117, CD200, CD56, cytoKappa, and cytoLambda in a 4-color experiment with CD38 and CD138 as gating antibodies. Results: Mean APC% in cases was 96.5%. The expected Immunophenotype (IP) of APC which is CD19-/56+/45-/81-/117+/200+ was found in only 13/43 MM cases. In 30/43 cases, APC revealed deviation from expected IP either for single or a combination of markers. Sensitivity for APC detection was highest for CD19 (95.2%) followed by CD56 (90.4%) and CD81 (83.7%). Specificity was highest for CD19 (100%), CD56 (100%), and CD81 (100%) followed by CD117 (92.3%). Combination of markers with maximum sensitivity to detect APC (97.6%) was CD81- or CD19- and CD200+ or CD56+ (two markers); and for NPC (92.3%) was CD81+ and CD19+ and CD56- (three markers). Conclusion: Plasma cell IP can be highly variable with multiple minor subpopulations in both cases and normal controls. CD 19 and CD56 are highly informative markers for a 4-color experiment. Assessment of multiple markers in an 8-10 color experiment is more informative but the lack of advanced flow cytometers should not limit the use of FC in a 4-color approach. Our results emphasize that even basic equipment with limited fluorochrome can provide meaningful information if used appropriately.

Clinicopathological parameters influencing inhibitor development in patients with hemophilia A receiving on-demand therapy.

BACKGROUND: Development of inhibitors to transfused factor VIII in patients with hemophilia A continues to be a challenge for professionals involved in hemophilia care. The majority of patients in India receive 'on-demand' rather than prophylactic therapy. The present study was done to assess the prevalence of factor VIII inhibitors in patients with hemophilia A (PWHA) receiving 'on-demand' therapy in a North Indian population and to study the clinicopathological parameters influencing the development of inhibitors. METHODS: The study group comprised of 300 PWHA. Detailed clinical parameters, treatment history, bleeding profile including family history were recorded. Diagnosis of hemophilia A was confirmed by relevant coagulation tests. Inhibitors were screened using mixing based studies followed by quantification by Bethesda assay and Nijmegen modified Bethesda assay. Samples were collected from five cities in North India where a free supply of factor VIII was available and effectively used in three of these cities. RESULTS: In the 300 PWHA, disease phenotype was severe in 219 (73%), moderate in 62 (20.67%) and mild in 19 (6.34%), based on the factor VIII bioassay. Inhibitor prevalence was 9.6% (n = 29) and seen only in the severe phenotype. Inhibitor titers ranged from 0.8 to 108.8 BU/ml. A total of 12 PWHA had low and 17 had high titers. Correlation of various clinicopathological parameters in inhibitor-positive versus negative PWHA showed significant correlation with age at onset of disease, severity of disease, age at first exposure to treatment, annual factor intake (IU/kg/year), intense treatment episodes and bleeding manifestations like central nervous system bleed and hematuria. The total study sample had blood group B in 33.34% PWHA, followed by O (27.34%), A (24.34%) and AB (15%), however, in inhibitor-positive samples, significant inhibitor formation was associated with the ABO subtype A (19/29, 65.51%). CONCLUSIONS: Factor VIII inhibitor prevalence in PWHA receiving 'on-demand' therapy was 9.6%. Clinicopathological correlates of inhibitor development in such PWHA have been analyzed in this novel study.

Flow cytometric detection of gamma-H2AX to evaluate DNA damage by low dose diagnostic irradiation.

INTRODUCTION: γH2AX assay has been used for DNA damage assessment at higher doses of radiation exposure. Its expression has not been studied in cases with diagnostic low dose radiation exposure. Concerns have been raised about the after-effects of radiation in diagnostic procedures like Computed Tomography (CT) scan, Angiography etc especially when such scans are repeated within short span of time. The purpose of the present study was to assess immediate DNA damage after exposure to low level of ionizing radiation by the flow cytometric method of gamma-H2AX. MATERIAL AND METHODS: Study sample includes total 60, cases and controls with two groups Group I-Normal controls (n = 15); Group II-Low dose, further divided in three groups: Group IIA-single CT scan (n = 15); Group IIB-Multiple CT scans (n = 15); and Group IIC-angiography single exposure (n = 15). For Low dose group blood was collected within 1 h after exposure in EDTA vaccutainers and immediately kept on ice. Lymphocytes were isolated and were fixed in 80% chilled ethanol and stored at -20 °C till further analysis. The H2AX assay was done and 10,000 cells were analysed for gamma H2AX positivity in flowcytometer. RESULTS: Significant gamma-H2AX positivity was found in cases versus control, the most significant DNA damage amongst cases was observed in cases with multiple CT scans. CONCLUSION: The exposure to multiple CT scans causes more double strand breaks as compared to single scan. DNA damage can be studied by flow cytometric analysis of gamma-H2AX in human peripheral lymphocytes.

Blastic plasmacytoid dendritic cell neoplasm presenting as leukemia without cutaneous lesion.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare hematologic malignancy, recognized as a distinct entity in the WHO 2008 classification of hematolymphoid neoplasm. Described for the first time in 1994 as CD4+ cutaneous lymphoma with high expression of CD56, BPDCN has been known previously with various names such as blastic natural killer (NK) leukemia/lymphoma, agranular CD4+ CD56+ hematodermic neoplasm, and agranular CD4+ NK cell leukemia. This disease usually presents with cutaneous involvement as the first manifestation, with subsequent or simultaneous spread to bone marrow and peripheral blood. Leukemia as the first presenting symptom without any cutaneous involvement is a rare finding and can masquerade as acute undifferentiated leukemia. We present here such a case of a 59-year-old male who presented as leukemia without any cutaneous lesion but subsequently developed a scalp nodule.

EpCAM-based Flow Cytometric Detection of Circulating Tumor Cells in Gallbladder Carcinoma Cases

Purpose: Liquid biopsy has entered the arena of cancer diagnostics in the past decade and detection of circulating tumor cells (CTC) is one diagnostic component. CTCs in gallbladder cancer (GBC) have hitherto not been comprehensively analysed. Methods and Results: The current study focused on the diagnostic role of CTCs in 27 cases of treatment-naive GBC and 6 normal controls as well as 6 cases of cholecystitis. An EasySep kit featuring negative immunomagnetic bead separation and flow cytometric detection of EpCAM positive and CD45 negative cells revealed CTCs in 25 of the 27 cases. At a cut-off point of ≥1, the CTC count discriminated GBC from controls with a sensitivity, specificity and diagnostic accuracy of 92.6%, 91.7% and 92.3%, respectively. CTC levels in turn correlated significantly with clinico-pathological parameters of cases in terms of known prognostic indicators, with significant diagnostic potential at a cut-off point of >4, to discriminate disease stage I and II vs. III and IV GBC. With a cut-off of >3, the CTC count discriminated tumor stages I and II vs. III and IV and at >6 CTCs could discriminate metastatic vs. non metastatic GBCs with a sensitivity, specificity and diagnostic accuracy of 55. 6%, 100.0% and 85.2, respectively. A review of CTC in pancreatico-biliary malignancies is included. Conclusion: Detection and quantification of CTCs may serve as a non-invasive biomarker for GBC diagnosis in correlation with radiological studies.

CD20 negative primary diffuse large B cell lymphoma of breast: Role of Pax-5.

Pax-5 is a B cell marker, the expression of which is detectable in as early as the pro B stage, and subsequently, in all further stages of B cell development except the plasma cells. Malignant lymphomas of breast are uncommon and occur as either primary or secondary lesions. Primary lymphoma is a rare disorder of breast and constitutes less than 0.6% of all breast malignancies and 2.2% of extranodal lymphomas. We report an unusual case of CD20 negative Pax-5 positive primary diffuse large B cell lymphoma (DLBCL) of breast. The case highlights the diagnostic challenge posed by extranodal CD20 negative DLBCL. Pax-5 immunohistochemistry has diagnostic benefit as a B-cell marker in the work-up of undifferentiated malignant neoplasms. Although it is available for nearly a decade now, it is not widely used. Pax-5 is a valuable addition to the armamentarium of markers currently available for lymphoma subtyping.

Gliosarcoma with leiomyomatous differentiation: A case report with an emphasis on histogenesis.

Gliosarcoma is an uncommon high-grade tumor which constitutes about 2% of all glioblastomas. These tumors have the histological hallmark of a biphasic pattern of high-grade glial and sarcomatous components. The histogenesis of these tumors is controversial. We report a case of primary gliosarcoma in an adult male with leiomyomatous differentiation and discuss the histogenesis as it appears in our case. Primary gliosarcomas of the brain are clinically challenging with a poor clinical outcome.

Penile neurilemmoma: Utility of fine-needle aspiration cytology in diagnosis of a rare entity.

Subcutaneous lesions in the penis are of rare occurrence and encompass benign as well as malignant tumors. These include lipomas, leiomyomas, neurilemmomas and their malignant counterparts. A surgical excision at this site carries the risk of postoperative penile curvature and erectile dysfunction. We report a rare case of penile neurilemmoma which presented as a subcutaneous nodule on the dorsal surface of the penis. A fine-needle aspiration was performed which aided in the preoperative diagnosis and guided the extent of excision. We report this case to highlight the importance of needle aspiration as a simple outdoor procedure for penile lesions which can aid surgical approach and postoperative outcome.