Prevalence of anti-HLA antibodies in COVID-19 convalescent plasma donors: an Indian experience.

BACKGROUND: COVID-19 convalescent plasma is one of the experimental therapies used widely in moderately sick COVID-19 patients. However, there are a few risks involved in plasma transfusion; notably, transfusion-related acute lung injury (TRALI) caused by antibodies against human leukocyte antigens (HLA). This study was designed to assess the prevalence of anti-HLA antibodies in convalescent plasma donors using the single antigen bead method. STUDY DESIGN AND METHODS: This was a hospital-based observational study of consecutive plasma donors. A total of 252 samples were screened for anti-HLA Class I and Class II antibodies using the microbead assay with the identification of anti-HLA Ab in positive samples being performed using a single antigen bead assay. Luminex-based normalized background cutoff ratios of 10.8 for Class I and 6.9 for Class II and mean fluorescence intensity cutoffs of 2500 for Class I and 1500 for Class II were used for screening and the single bead assay, respectively. RESULTS: Of 252 screened samples, 28 (11.1 %) were positive for Class I, Class II or both Class I and Class II anti-HLA antibodies in donors with no history of a previous immunizing event. Moreover, 20/252 (7.9%) donors without any history of prior immunization had specific anti-HLA antibodies of Class I or Class II or both by the single bead assay. CONCLUSIONS: The high prevalence of anti-HLA antibodies in our cohort of donors raises an urgent and immediate need for anti-HLA antibody screening in all convalescent plasma donors for safe therapy of COVID-19 patients.

Prevalence of unexpected red blood cell antibodies in pregnant women and follow-up of pregnancy outcome in pregnant women treated with intra-uterine transfusion.

BACKGROUND: For the management of hemolytic disease of the fetus and newborn (HDFN), it is important to detect unexpected red cell antibody in pregnant women. We assessed the prevalence of unexpected red cell antibodies in consecutive pregnant women attending antenatal clinic (ANC). More importantly, cases with unexpected antibody causing severe anemia were followed-up for intervention (Intra-uterine transfusion {IUT}) and outcome of pregnancy (still-birth/live-healthy). AIMS AND OBJECTIVES: The study was conducted with an objective to find the prevalence of unexpected RBC antibodies in pregnant women, their specificity and to do the follow-up for IUT and outcome of pregnancy (still-birth, live-birth) in antibody positive women. MATERIALS AND METHODS: This was a prospective study from January 2021 to May 2022 at two tertiary care centres. All antenatal samples received by the laboratory were screened for unexpected red cell antibody. Whenever antibody screen was positive, antibody identification was performed. Patients, positive for unexpected antibody and anemia were followed up for any transfusion-based intervention and outcome of pregnancy. RESULTS: A total of 539 consecutive samples were worked up and among these, 10 samples (1.85%) were found to be antibody positive. The antibodies identified were Anti-D (n=6), anti-Leb (n=1), anti-M (n=1), anti-C (n=1) and anti-E (n=1).The prevalence of unexpected antibodies in Rh positive and Rh negative pregnant women was 0.83% and 10.9% respectively. Follow-up was done for all 10 cases with unexpected antibody and anemia was monitored by MCA PSV (middle cerebral artery peak systolic velocity).Two women developed severe anemia thus requiring single intrauterine transfusion (at 26 weeks and 28 weeks respectively) each, for correction of anemia. In both these cases, healthy male child was delivered. At 3-month follow-up both children were alive and healthy. CONCLUSION: The study found prevalence of unexpected RBC antibodies in pregnant women as 1.85%. The study also underlined importance of transfusion-based interventions contributing to successful outcome in couple of cases with severe anemia.

'Old is gold' does conventional test tube method still reign supreme? An immuno-haematological survey of anti-D detection and titration in ante-natal cases among major hospitals across India.

INTRODUCTION: Anti-D detection and titration plays a major role in RhD negative antenatal cases both, for monitoring maternal as well as fetal status as well as initiation of early therapeutic interventions, such as intra-uterine transfusions (IUT) to improve maternal as well as fetal morbidity and mortality and reduce the adverse effects of haemolytic disease of fetus and newborn (HDFN). We conducted a survey focusing on the policies and procedures of anti-D detection and titration among major tertiary care centres across India. METHODOLOGY: The survey was drafted by a working group of transfusion medicine and immunohematology specialists from six different centres in India. Data were obtained via the use of an online questionnaire. RESULTS: Results were categorised into four categories, Hospital information, immuno-haematological testing methodology, clinical significance of anti-D testing and the role of transfusion medicine specialists. The survey highlighted the modalities as well as the methodologies of anti-D detection and titration in antenatal women across different major tertiary care centres in India. CONCLUSION: This survey provided a unique snapshot of the prevalent methodologies being employed by major tertiary care centres across the country for detection and titration of anti-D levels as well as the important role it plays in the therapy of affected antenatal women to minimise adverse effects on the fetus.

A patient with anti-f in India identified by extensive immunohematologic workup.

Anti-f is produced by exposure to the compound antigen ce (f) on red blood cells (RBCs), expressed when both c and e are present on the same protein (cis position). Although anti-f was discovered in 1953, there are few cases reported worldwide because the presence of anti-f is often masked by anti-c or anti-e and is not generally found as a single antibody. In the present case, anti-f was identified by using three-cell screening and 11-cell identification panels. The identification of anti-f was further supported by additional testing, including (1) Rh antigen typing; (2) antibody identification panels (enzyme-treated panel [ficin] and an in-house-constructed Rh panel); (3) look-back and phenotyping of donor RBC units, which were responsible for alloimmunization; and (4) molecular testing of the patient's RBCs.

Determination of optimum levels of binding antibody units (BAU) of new quantitative chemiluminescent immuno-assay (CLIA) in COVID-19 vaccinated volunteer blood donors.

BACKGROUND: For assessment of COVID-19 vaccine efficacy, neutralization activity of anti-SARS-CoV-2 antibody is measured. This study was undertaken to determine optimum levels of binding antibody units (BAU/ml) in new quantitative chemiluminescent assay (CLIA) that corresponded to neutralizing potential (30% inhibition) of sVNT assay. METHODS: Ninety-one blood samples were analyzed by CLIA and sVNT assays. Test samples (n = 75) were collected from blood donors post-2nd vaccination dose, while control samples (n = 16) were archived pre-COVID donor samples. Correlation between CLIA and sVNT was calculated and receiver operating characteristic (ROC) curve was drawn and analyzed. RESULTS: Results indicated excellent correlation between 57.5 BAU/ml on CLIA and 30%inhibition on sVNT assay. ROC curve analysis revealed that the area under the curve (AUC) was 0.971. DISCUSSION: The present study determined that 57.5 BAU/ml on CLIA corresponded to 30% inhibition on sVNT assay. Periodic quantitative analysis.

Evaluation of new haematology analyser, XN-31, for malaria detection in blood donors: A single-centre study from India.

BACKGROUND AND OBJECTIVES: Malaria continues to be a significant public health concern in India, with several regions experiencing endemicity and sporadic outbreaks. The prevalence of malaria in blood donors, in India, varies between 0.02% and 0.07%. Common techniques to screen for malaria, in blood donors and patients, include microscopic smear examination and rapid diagnostic tests (RDTs) based on antigen detection. The aim of this study was to evaluate a new fully automated analyser, XN-31, for malaria detection, as compared with current practice of using RDT. MATERIALS AND METHODS: Cross-sectional analytical study was conducted to evaluate clinical sensitivity and specificity of new automated analyser XN-31 among blood donors' samples and clinical samples (patients with suspicion of malaria) from outpatient clinic collected over between July 2021 and October 2022. No additional sample was drawn from blood donor or patient. All blood donors and patients' samples were processed by malaria rapid diagnostic test, thick-smear microscopy (MIC) and the haematology analyser XN-31. Any donor blood unit incriminated for malaria was discarded. Laboratory diagnosis using MIC was considered the 'gold standard' in the present study. Clinical sensitivity and specificity of XN-31 were compared with the gold standard. RESULTS: Fife thousand and five donor samples and 82 diagnostic samples were evaluated. While the clinical sensitivity and specificity for donor samples were 100%, they were 72.7% and 100% for diagnostic samples. CONCLUSION: Automated haematology analysers represent a promising solution, as they can deliver speedy and sensitive donor malaria screening assessments. This method also has the potential to be used for pre-transfusion malaria screening along with haemoglobin estimation.

I-JAMM-(I): A survey providing an insight into the practices of isoagglutinin titration in ABO incompatible kidney and liver transplantation.

BACKGROUND AND OBJECTIVES: ABO-incompatible transplantations allow patients to receive timely transplants. Isoagglutinin titration to ascertain levels of incompatible antibodies in the recipient is important in determining patient selection and transplant survivability. To find out the prevalent trends in India, the largest, first of its kind survey was carried out among the transplant centers regarding their practices in isoagglutinin titration. METHODS: The survey was drafted by a working group of Transfusion and Transplant Immunology specialists from six different centers. Data was obtained via the use of an online questionnaire. RESULTS: Results were categorized into four categories, Hospital information, Titration methodology, Role of transfusion specialists and cut-off titers. Most centers had a well-established solid-organ transplant program with considerable number of ABO-incompatible transplantations. Most centers performed isoagglutinin titration in Transfusion Medicine department. Column Agglutination Technique (CAT) was the most common method, using EDTA blood samples and freshly-prepared in-house pooled cells. Most centers had a turn-around time of less than 12 h. While the policy for ascertaining baseline and threshold titers is well-defined in ABO-incompatible renal transplants, variations from center to center still exist for ABO-incompatible liver transplants. Most centers required a Transfusion Medicine consultation for the patients before such transplants. CONCLUSION: With increasing ABO-incompatible kidney and liver transplants across the country, the role of Transfusion medicine specialists has become vital in pre-conditioning regimes enabling the viability and success of such transplants. This was a unique survey that provided a snapshot of current trends and practices of isoagglutinin titration for ABO-incompatible transplants in India.

Anti-G with Concomitant Anti-D and Anti-C in a Pre-natal Patient, in India: Multi-disciplinary Approach to Diagnosis and Management.

'G' antigen belongs to the Rh family and it was first described by Allen and Tippet in 1958. Various anti-D, anti-C, and anti-G antibody combinations can be found in patients. Ruling out the presence of anti-D is important for administering RhIg prophylaxis in RhD-negative pregnant women to prevent hemolytic disease of fetus and newborn (HDFN). RhIg prophylaxis is not indicated in the presence of an anti-D antibody. Time-to-time monitoring and follow-up of cases of RhD-negative pregnant women with a multi-disciplinary approach including an obstetrician, neonatologist, and transfusion medicine specialist helps diagnose, manage, and monitor HDFN in such cases. This case report emphasizes the need for proper antibody identification (anti-G) and managing HDFN (with intrauterine transfusions and exchange transfusion) during the perinatal period.

Clinical Outcomes of Autologous Hematopoietic Stem Cell Transplant in Multiple Myeloma Patients: A 5-year Experience from a Single Centre in North India.

Swati PabbiIntroduction Multiple myeloma (MM) forms a significant proportion of hematological malignancies. Autologous transplantation continues to be an effective consolidation strategy in resource-restricted settings such as India. Objectives The main objective of the study was to analyze the clinical outcomes of autologous hematopoietic stem cell transplant (HSCT) in MM patients in a single tertiary care center in north India over a period of 5 years. Materials and Methods This retrospective observational study was conducted in a tertiary care center in north India. Data of all MM patients who underwent HSCT between January 2014, and December 2018, were analyzed. The outcome of HSCT was investigated in terms of transplant-related mortality (TRM), progression-free survival (PFS), overall survival (OS), and relapse. PFS and OS were calculated by Kaplan-Meier method and differences between the groups were tested for statistical significance using the two-tailed log-rank test. Life-table method was used for the estimation of survival rate at 1, 3, 5, and 6 years. Results Patient characteristics and survival post-transplant was similar to other published Indian studies. In total, 378 patients were diagnosed with MM in our hospital between 2014 and 2018. One hundred ninety-three patients were found to be eligible for autologous HSCT, out of which 52 ended up having a transplant giving us a high percentage (26.9%) of patients receiving a transplant in our setting. Transplant-related mortality (TRM) was nil in the present study. The mean PFS and OS were 62.8 and 70.1 months, respectively. The mean PFS and OS rates at 5 years were 75.3% and 84.2%, respectively. The average cost estimate of HSCT in our setting was 7.2 lakh Indian national rupees. Conclusion Autologous HSCT is a safe procedure with nil 100-day mortality in present series. Moreover, considering the cost of novel agents, autologous transplant remains a cost-effective way for prolonging remission and time-to-next treatment in India.

Low-MFI (median fluorescence intensity) pre-transplant DSA (donor specific antibodies) leading to anamnestic antibody mediated rejection in live-related donor kidney transplantation.

"In solid organ transplantation, the compatibility between recipient and donor relies on testing prior to transplantation as a major determinant for the successful transplant outcomes. This compatibility testing depends on the detection of donor-specific antibodies (DSAs) present in the recipient. Indeed, sensitized transplant candidates are at higher risk of allograft rejection and graft loss compared to non-sensitized individuals. Most of the laboratories in India have adopted test algorithms for the appropriate risk stratification of transplants, namely: 1) donor cell-based flow-cytometric cross-match (FCXM) assay with patient's serum to detect DSAs; 2) HLA-coated beads to detect anti-HLA antibodies; and 3) complement-dependent cytotoxicity crossmatch (CDCXM) with donor cells to detect cytotoxic antibodies. In the risk stratification strategy, laboratories generally accept a DSA median fluorescence index (MFI) of 1000 MFI or lower MFI (low-MFI) as a negative value and clear the patient for the transplant. We present two cases of live-related donor kidney transplants (LDKTs) with low-MFI pre-transplant DSA values who experienced an early acute antibody-mediated rejection (ABMR) as a result of an anamnestic antibody response by DSA against HLA class II antibodies. These results were confirmed by retesting of both pre-transplant and post-transplant archived sera from patients and freshly obtained donor cells. Our examples indicate a possible ABMR in patients with low MFI pre-transplant DSA. Reclassification of low vs. high-risk may be appropriate for sensitized patients with low-MFI DSA."

Patient outcome in antibody-positive systemic vasculitis treated with therapeutic plasma exchange.

BACKGROUND: Therapeutic plasma exchange (TPE) has been advocated as an adjunct to steroids and cytotoxic drugs in treating patients suffering from vasculitis and presenting with active disease, but we still have insufficient evidence on its effectiveness in improving the clinical response, especially in India. This study was planned to study the clinical outcome in severe vasculitic presentations treated with TPE as an adjunctive therapy. MATERIALS AND METHODS: A retrospective analysis of TPE procedures performed from July 2013 to July 2017 in the department of transfusion medicine at a large tertiary care hospital was done. All consecutive patients admitted with new diagnosis of systemic vasculitis presenting with active disease and severe presentations such as advanced renal failure or severe respiratory abnormalities or life-threatening vasculitis affecting the gastrointestinal tract, neurological and musculoskeletal system; who needed TPE for removal of preformed antibodies, were included in the study. RESULTS: There were a total of 31 patients in whom TPE was performed for severe systemic vasculitis; 26 adults and five pediatric. Six patients tested positive for perinuclear fluorescence, 13 for cytoplasmic fluorescence (cANCA), two for atypical antineutrophil cytoplasmic autoantibody, seven for anti-glomerular basement membrane antibodies, two for antinuclear antibodies (ANA), and one patient tested positive for ANA as well as cANCA before the augmentation of TPE. Out of 31, seven patients showed no clinical improvement and succumbed to the disease. At the end of desired number of procedures, 19 tested negative and five tested weak positive for their respective antibodies. CONCLUSION: Favorable clinical outcomes were observed with TPE in patients with antibody-positive systemic vasculitis.

Comparison of Two Different Serological Viral Marker Testing Assays for Screening of Apheresis Donors: Which Assay Provides Optimum Safety for Transfusion?

While whole blood testing has evolved over the years, viral marker testing for plateletpheresis donors is still performed by Rapid Diagnostic Tests (RDT). Aim of this study was to compare diagnostic accuracy of RDT and Chemiluminescence Immunoassay (CLIA) in serological testing for HBsAg, anti-HCV and anti-HIV antibodies. A prospective, analytical study was conducted in the department of Transfusion Medicine at a tertiary healthcare center in India between September 2016 and August 2018. Samples were simultaneously tested by CLIA, RDT and a confirmatory test. Sensitivity, specificity, negative and positive predictive values and mean time taken to report results were calculated. A total of 102 (1.48%) of the 6883 samples were found to be reactive by either or both the assays. A total of 74 (1.08%) samples were HBsAg reactive, 23 (0.33%) were reactive for anti-HCV antibodies and 5 (0.07%) were reactive for anti-HIV I and II antibodies. A combined sero-prevalence of 1.05% (72) was observed; 0.78% (54) for HBsAg, 0.26% (18) for anti-HCV antibodies and none for anti-HIV I and II antibodies. Four (3.85%) reactive samples were missed by RDT and therefore sensitivity of RDT was quite less as compared to CLIA. RDT and CLIA both were found to have a statistically significant shorter turnaround time than confirmatory tests. There is increasing need to develop a safe donor screening strategy for plateletpheresis. CLIA offers an excellent alterative to RDT for viral marker testing in terms of sensitivity.

Real-world data on renal transplantations from a tertiary-care hospital in North India, in context of Indian regulatory act-transplantation of human organs and tissues act (THOTA): A retrospective analysis.

INTRODUCTION: Renal transplantation is the treatment of choice for patients suffering from end stage renal disease (ESRD). Indian regulations defined under Transplantation of Human Organs and Tissues Act (THOTA), 2014 restricts organ donations to near-related living donors to curb any malpractices like 'paid donors' in living-donor kidney transplantation (LDKT). The aim of our study was to look at real-world data of donor-recipient pairs and to identify relationship of donors (with their respective patients) and the common (or uncommon) DNA profiling methods used for supporting "claimed relationship" in accordance with the regulations. MATERIAL AND METHODS: The donors were categorized and grouped into near-related donor, donors other than near-related donors, swap donors and deceased donors. Claimed relationship was confirmed, commonly by HLA typing, using SSOP method. In few cases, which were uncommon (and infrequent), autosomal DNA analysis, mitochondrial DNA analysis and Y-STR DNA analysis were performed to support the claimed relationship. Data collected included age, gender, relationship, DNA profiling test method. RESULTS: Among the 514 donor-recipient pairs evaluated, numbers of female donors out-numbered male donors. The decreasing order of relationships in near-related donor group were wife>mother>father>sister>son>brother>husband> daughter>grandmother. 11.9% of donors were in the category of donors other than near-related donors. In 97.86% cases, the claimed relationship was supported by HLA typing and in just 2.1% cases autosomal DNA analysis>mitochondrial DNA analysis> Y-STR DNA analysis, in this order, were performed to establish relationship. CONCLUSION: This study brought out gender disparity with women out-numbering men as donors. Among recipients, access to renal transplant was largely restricted to men. As far as relationship of donors to recipients was concerned, mostly near-related family members, like wife, were donors and claimed relationship was almost always (99%) was corroborated by HLA typing.

Implementation of an international barcode labeling standard, International Society of Blood Transfusion 128, and its integration with local regulations at a blood center in India: Step-by-step journey.

The International Society of Blood Transfusion (ISBT) 128 is an internationally endorsed, electronically readable labeling standard that provides a convenient and accurate means of identification, traceability, publication, and storage of information for blood and blood products. The authors' center recently registered with the International Council for Commonality in Blood Banking Automation (ICCBBA) and progressed to ISBT 128 labeling standard. This manuscript was written with the objective of sharing the authors' experience with respect to the implementation of ISBT 128 standards for whole blood donations and integration of ISBT 128 standards with Indian licensing regulations. The authors explore the process of implementation of ISBT 128 standards through a step-by-step journey that included facility registration with International Council for Commonality in Blood Banking Automation (ICCBBA), allotment of facility identification number, development of four-quadrant label for blood components, and integration of local regulatory requirements in the final "composite" label. Acknowledging the lack of any published report from India on ISBT 128 standards implementation, the authors wish to attempt help their peers in understanding and implementation of this global standard at their respective facilities.

Analytical and clinical performance evaluation of enhanced chemiluminescence-based fourth-generation HIV combo assay: Report from tertiary health-care setup in North India.

INTRODUCTION: HIV fourth-generation assay, designed for the detection of HIV p24 antigen along with anti-HIV antibodies of both immunoglobulin M and immunoglobulin G type against HIV 1 and HIV 2 viral antigens, have helped in the early detection of HIV infection and supports in minimizing the transmission risk in the acute phase of infection. The objective of this study was to evaluate the analytical and clinical performance of HIV fourth-generation assay based on enhanced chemiluminescence technology. MATERIALS AND METHODS: The analytical performance of the assay was evaluated in terms of accuracy, precision, limit of detection, type of sample (serum vs. plasma), cross-reactivity (with other transfusion transmissible infections markers), and interference (with endogenous substances). Proficiency control material included kit-controls, archived known positive donor samples, third-party controls, and World Health Organization (WHO)/National Institute for Biological Standards and Controls (NIBSC, MHRA, UK) controls. The clinical performance was evaluated using routine donor and patient samples received during the study period. RESULTS: HIV fourth-generation assay showed reliable and reproducible results measured in terms of coefficient of variation % with kit-controls, archived known positive donor samples, third-party controls, and WHO international standards for anti-HIV 1 and 2 antibodies, HIV1 p24 antigens and HIV2 p26 antigen controls. The analytical sensitivity of the HIV fourth-generation assay was found to be 0.1 IU/mL of HIV1 p24 antigen control and there was no cross-reactivity or interference observed. In the clinical performance of the assay, HIV fourth-generation assay showed reliable performance in both donor and patient samples. CONCLUSION: HIV fourth-generation assay meets the requirements for its use as a screening assay for HIV infection based on the analytical and clinical performance of the assay.

Correlation of various methods of hematopoietic progenitor cell estimation with standard flowcytometric CD34 enumeration.

BACKGROUND AND OBJECTIVES: Enumeration of hematopoietic progenitor cell (HPC) is vital to decide the time to initiate harvest (TTIH) and adequacy of harvest dose (AOHD). Standard of care used for HPC enumeration is flowcytometric CD34+ enumeration, but it is expensive, time-consuming and requires skilled staff to perform the test. Alternatively, HPC-count by advanced automated cell analyzer is cheaper, quicker, and easy-to-perform test. Our objective was to find a correlation of HPC count with CD34+ enumeration in leukapheresis. MATERIALS AND METHODS: An observational, prospective study was conducted in the year 2018-2019. A total of 126 samples were included in the study, the peripheral blood (PB) group comprised of 42samples and apheresis group of 84 samples. The samples were simultaneously tested for CD34+ expression and complete blood count which included the HPC count, white blood cells (WBC) count and multinational corporation (MNC) count and correlation analysis was performed with CD34+ flowcytometric count. The cut-off of PB HPC count for the target dose of 5 × 106 CD34+ cells/kg was established using Receiver Operator Curve. RESULTS: The correlation coefficient (r) of HPC with CD34+ count was 0.617 and 0.699 for PB group and apheresis group sample respectively, which was statistically significant. The correlation with MNC and WBC count was not very significant. A cut-off value of PB HPC was established to be 66 HPC/µl with a positive predictive value of 94.12%. The cost of CD34 + flow cytometric enumeration was six times that of HPC enumeration by analyzer. CONCLUSION: The HPC count is a cheaper, rapid and easy test and can be clinically applied to predict TTIH and AOHD but requires more studies to validate its efficacy in clinical use.

Assessment of dengue virus inactivation in random donor platelets using amotosalen and ultraviolet A illumination.

OBJECTIVES: The study objective was evaluation of amotosalen and ultraviolet A (UVA) illumination-based inactivation of dengue virus (DENV) in blood platelets. MATERIALS AND METHODS: Whole blood was collected from healthy donors and platelet concentrates were prepared at a tertiary care hospital in Gurugram, India. Platelet units collected from five blood group matched individuals were pooled and spiked with DENV. The spiked platelet units were subjected to amotosalen treatment followed by UVA illumination, to evaluate the efficiency of this method for viral inactivation. The treated platelet units were evaluated for the presence of infectious DENV. Amotosalen levels were quantified in the treated samples using high-performance liquid chromatography. RESULTS: The presence of replicating DENV was not observed in spiked platelet units treated with amotosalen and UVA illumination, whereas untreated units contained actively replicating DENV. Amotosalen levels were found to be in the permissible range after photochemical inactivation. CONCLUSIONS: Amotosalen/UVA pathogen inactivation treatment showed efficient inactivation of DENV in platelet components. Therefore, it seems to be a promising method for mitigating the risk of dengue transmission through transfusion of potentially contaminated platelet components in dengue-endemic countries such as India.

Diagnostic value of different interleukins and procalcitonin in critically ill patients admitted with suspected sepsis.

BACKGROUND: : Many biomarkers have now been studied such as C-reactive Protein (CRP), procalcitonin (PCT), etc., and are widely used for the diagnosis of sepsis in clinical practice which may determine the appropriate antibiotic treatment. A flowcytometric cytokine bead array (CBA) assay has now been used to determine multiple interleukins (IL), simultaneously. The aim of this study was to determine the cytokine (IL2, IL4, IL6, IL10, TNFα, INFγ, and IL17) profiles of interleukins in plasma of sepsis patients by using multiplex Flowcytometric CBA array assay. MATERIALS AND METHOD: s: A total of 99 consecutive patients admitted with the suspected sepsis were studied. PCT concentrations were measured by using the enzyme-linked fluorescent immunoassay (ELFA) technique and flow cytometry-based BD™ CBA Cytokine Kit was used to evaluate levels of 7 cytokines [IL-2, IL-4, IL-6, IL-10, Tumour Necrosis Factor (TNF), Interferon- γ (IFN-γ), and IL-17A]. RESULTS: Microbiologically defined infection (MDI) demonstrated a positive culture report in 79/99 (79.7%) of patients. The IL6 [1873.7 (4-5000)] and IL10 [(154.7 (0-1764)] levels were significantly higher in septic patients than those in the negative MDI IL6 [901 (4-5000)] and IL10 [110.4 (4-1372)] levels. The AUROC value of IL6 [0.66 (0.53-0.79)] was found to be the highest among all followed by IL10 [0.65 (0.51-0.79)], IFNγ [0.63 (0.51-0.77)], PCT [0.61 (0.48-0.75)], and TNFα [0.55 (0.42-0.69)]. CONCLUSION: Our study suggests that that IL6 is substantially more economical and can reduce the investigation cost to half as compared with the procalcitonin assay.

Laboratory Diagnosis of ANCA-Associated Vasculitis (AAV) Using a Combination of Immunofluorescence Test (IIFT) and Line Immunoassay (LIA): Single-Centre Report From India.

INTRODUCTION: Anti-neutrophil cytoplasmic autoantibodies (ANCA) associated vasculitis (AAV) is a small vessel vasculitis with insufficient epidemiological estimates in India. We aimed to determine demographic, clinical features, and laboratory diagnosis of AAV patients presenting to a large tertiary care centre in India. MATERIAL AND METHODS: 1289 patient samples were screened for ANCA by indirect immunofluorescence test (IIFT) and confirmation of ANCA target antigens was done by line immunoassay. Association between IIFT and LIA was determined in AAV. RESULTS: By IIFT, ANCA was detected in 13.0% (168 out of 1289), of which 23.8% (40/168) were positive with P-ANCA pattern, 25.0% (42/168) were positive with C-ANCA and 47.6% (80/168) showed an atypical pattern. On evaluation with a line immunoassay, 6.7% (86/1289) were positive out of which 52.3% (45/86), 41.9% (36/86), 8.8% (6/86) were positive for anti-MPO, anti-PR3, and anti-GBM respectively. In eosinophilic granulomatosis with polyangiitis (EGPA) 87.5% (7/8), and microscopic polyangiitis (MPA/RLV) 91.3% (21/23), anti-MPO was the predominantly observed antibody. In granulomatosis with polyangiitis (GPA) anti-PR3 antibody was predominant in 87.5% (28/32) cases. Out of 168 IIF positive samples 8, 32, and 23 cases of EGPA, GPA, and MPA/RLV were observed respectively. CONCLUSIONS: The primary aim of the study was to provide single-centre data to determine the laboratory diagnosis of AAV. A combination of IIFT and LIA was found to be an optimum testing strategy for the laboratory diagnosis of AAV.

Development and validation of novel kit for quantification of SARS-CoV-2 antibodies on clinical samples.

Since the pandemic occurred due to the emergence of SARS-CoV-2, there has always been a demand for a simple and sensitive diagnostic kit for detection of SARS-Cov-2 infection. In January 2020, WHO approved the Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) for detecting the presence of Covid-19 genetic material in individuals. Till date many diagnostic kits have arrived in the market for quantification of SARS-CoV-2 antibodies. In spite of being the gold standard method of Covid-19 detection, there are some drawbacks associated with RT-PCR which leads to false-negative results. Hence, in order to fulfil the need for an antibody testing kit for evaluating seroconversion and immunity acquisition in the population, an efficient, highly specific and sensitive assay, Chimera Soochak, an enzyme-linked immunoassay (ELISA) Kit has been developed. It works on the principle of detecting IgG antibodies developed specifically against the S1-RBD by employing a recombinant strain of S1-RBD produced in the HEK293 cell line. The developed kit was validated using different modes and methods to attain the utmost confidence on the samples collected from patients. The validation methodology included, validation with known samples, blind study, third-party validation, validation using WHO Reference Panel and comparison with FDA approved Surrogate virus neutralization kit. The kit was found successful in detecting IgG against the S1-RBD of SARS-CoV-2. The kit had been validated on multiple parameters. A total of 900 samples had been tested by using this kit and it has exhibited the sensitivity, specificity and accuracy for all the above-mentioned parameters.