BackgroundGiven the physiological hemostasis changes during pregnancy, limited data exists on COVID-19-induced inflammatory response and hemostasis alterations in pregnant women.ObjectivesTo test a comprehensive set of hemostasis and inflammatory cytokines in pregnancies with/without COVID-19 and correlate results with maternal and perinatal outcomes.Patients/methodsIn this observational case-control study, 100 women with acute COVID-19 at 24-40 gestational weeks (COVID-19+ group), and 100 healthy, age- and gestational week-matched, SARS-CoV-2 negative pregnant women (32 with proven recovery of COVID-19) were enrolled. All women were outpatients with mild/no symptoms at admission. Detailed hemostasis (fibrinogen, FVIII, FXIII, VWF, plasminogen, α2-plasmin inhibitor, PAI-1, thrombin generation, clot lysis, D-dimer) and inflammatory cytokine/chemokine panels were performed. Clinical parameters of pregnancy, labor and postpartum period were registered.ResultsCOVID-19+ women exhibited significantly lower FVIII, FXIII, plasminogen, higher VWF levels, decreased peak thrombin and enhanced clot lysis vs. controls. Despite mild/no symptoms, significantly elevated cytokine levels, including IL-6, INF-γ, MCP-1, and IL-18 were observed in COVID-19+ pregnancies, associated with distinct hemostasis alterations. Admission IL-1β, and IL-33 were significantly lower, while IL-18 was significantly higher in cases when COVID-19 became more severe, along with significantly decreased FVIII, FXIII and plasminogen. In the COVID-19+ group, postpartum hemorrhage (PPH) developed in 4 cases, associated with significantly reduced plasminogen, α2-plasmin inhibitor, and increased IL-8, IL-17A, IL-23 levels.ConclusionIn third trimester mild/asymptomatic COVID-19+ pregnancies, marked inflammatory cytokine changes, hemostasis alterations and enhanced fibrinolysis were found. A potential link between inflammation and PPH in the context of COVID-19 warrants further research.

ABCB1/MDR-1/P-glycoprotein (Pgp) is an ABC transporter responsible for cancer cell multi-drug resistance. It is expressed in cytotoxic T lymphocytes (CTL). Eliminating sensitive cancer cells during high-dose chemotherapy can also damage immune cells. Our study aimed to assess which maturing human CD8 + CTL memory subsets may be affected based on their Pgp protein expression. In an in vitro CTL differentiation model system, we tracked the maturation of naive, effector, and memory cells and the expression of Pgp. This system involves co-culturing blood lymphocytes with proliferation-inhibited JY antigen-presenting B-lymphoblastoid cells expressing HLA-I A2. These JY-primed maturing CTLs were TCR-activated using beads, and the effect of the maturation-modifying JAK1/2 inhibitor ruxolitinib was examined. Multidimensional analysis identified six major CTL subsets: naive, young memory (Tym), stem cell memory (Tscm), central memory (Tcm), effector memory (Tem), and effectors (Te). These subsets were further divided into thirteen specific subsets: TymCD127 + , TymCD127-, Tscm, TcmCD95 + , TcmCD73 +CD95 + , TcmCD95+CD127 + , TcmPD1 + , TemCD95 + , TemraCD127 + , TemraCD127-, TeCD95 + , and TeCD73 +CD95 + . Pgp expression was detectable in naïve cells and dynamically changed across the thirteen identified subsets. Increased Pgp was detected in young memory T cells and in Tscm, TcmCD95 + , and TcmPD1 + human CTL subsets. Unlike other transiently appearing memory cells, the number of cells in these core Pgp-expressing memory subsets stabilized by the end of the contraction phase. Ruxolitinib treatment downregulated effector T-cell polarization while upregulating small memory subsets expressing Pgp. In conclusion, activation increased Pgp expression, whereas ruxolitinib treatment preserved small early and late memory subset core that primarily expressed Pgp.

Mucosal-associated invariant T (MAIT) cells, a subset of Vα7.2+ T cells, are a crucial link between innate and adaptive immunity, responding to various stimuli through TCR-dependent and independent pathways. We investigated the responses of MAIT cells and Vα7.2+/CD161− T cells to different stimuli and evaluated the effects of Cyclosporin A (CsA) and Vitamin D3 (VitD). Peripheral blood mononuclear cells (PBMCs) from healthy donors were stimulated with various agents (PMA/Ionomycin, 5-OP-RU, 5-OP-RU/IL-12/IL-33) with or without CsA and VitD. Flow cytometric analysis assessed surface markers and intracellular cytokine production. Under steady-state conditions, MAIT cells displayed elevated expression of CCR6 and IL-13. They showed upregulated activation and exhaustion markers after activation, producing IFNγ, TNFα, and TNFα/GzB. CsA significantly inhibited MAIT cell activation and cytokine production. Conversely, Vα7.2+/CD161− T cells exhibited distinct responses, showing negligible responses to 5-OP-RU ligand but increased cytokine production upon PMA stimulation. Our study underscores the distinct nature of MAIT cells compared to Vα7.2+/CD161− T cells, which resemble conventional T cells. CsA emerges as a potent immunosuppressive agent, inhibiting proinflammatory cytokine production in MAIT cells. At the same time, VitD supports MAIT cell activation and IL-13 production, shedding light on potential therapeutic avenues for immune modulation.

As the association of human leukocyte antigen B27 (HLA-B27) with spondylarthropathies is widely known, HLA-B27 antigen expression is frequently identified using flow cytometric or other techniques. Because of the possibility of cross-reaction with off target antigens, such as HLA-B7, each flow cytometric technique applies a "gray zone" reserved for equivocal findings. Our aim was to use machine learning (ML) methods to classify such equivocal data as positive or negative. Equivocal samples (n = 99) were selected from samples submitted to our institution for clinical evaluation by HLA-B27 antigen testing. Samples were analyzed by flow cytometry and polymerase chain reaction. Features of histograms generated by flow cytometry were used to train and validate ML methods for classification as logistic regression (LR), decision tree (DT), random forest (RF) and light gradient boost method (GBM). All evaluated ML algorithms performed well, with high accuracy, sensitivity, specificity, as well as negative and positive predictive values. Although, gradient boost approaches are proposed as high performance methods; nevertheless, their effectiveness may be lower for smaller sample sizes. On our relatively smaller sample set, the random forest algorithm performed best (AUC: 0.92), but there was no statistically significant difference between the ML algorithms used. AUC values for light GBM, DT, and LR were 0.88, 0.89, 0.89, respectively. Implementing these algorithms into the process of HLA-B27 testing can reduce the number of uncertain, false negative or false positive cases, especially in laboratories where no genetic testing is available.

BackgroundSARS-CoV-2 infection during pregnancy increases the risk of severe obstetrical complications. Detailed evaluation of COVID-19-associated coagulopathy in a pregnancy with stillbirth hasn’t been described so far. Besides knowledge gaps in the pathomechanism leading to stillbirth in COVID-19 pregnancies, currently, no prognostic biomarker is available to identify pregnant patients who are at imminent risk of COVID-19-associated maternal and fetal complications, requiring immediate medical attention.CaseHere we report the case of a 28-year-old SARS-CoV-2 infected pregnant patient, admitted to our hospital at 28 weeks of gestation with intrauterine fetal loss. The presence of SARS-CoV-2 placentitis was confirmed by immunohistological evaluation of the placenta. She had only mild upper respiratory symptoms and her vital signs were within reference throughout labor and postpartum. The stillborn infant was delivered per vias naturales. Fibrinogen concentrate was administered before and after labor due to markedly decreased fibrinogen levels (1.49 g/l) at admission and excessive bleeding during and after delivery. Although coagulation screening tests were not alarming at admission, the balance of hemostasis was strikingly distorted in the patient. As compared to healthy age- and gestational age-matched pregnant controls, increased D-dimer, low FVIII activity, low FXIII level, marked hypocoagulability as demonstrated by the thrombin generation assay, together with shortened clot lysis and decreased levels of fibrinolytic proteins were observed. These alterations most likely have contributed to the increased bleeding observed during labor and in the early postpartum period. Interestingly, at the same time, only moderately altered inflammatory cytokine levels were found at admission. Serum ACE2 activity did not differ in the patient from that of age- and gestational age-matched healthy controls, suggesting that despite previous speculations in the literature, ACE2 may not be used as a potential biomarker for the prediction of COVID-19 placentitis and threatening fetal loss in SARS-CoV-2-infected pregnancies.ConclusionsAlthough based on this case report no prognostic biomarker could be identified for use in pregnant patients with imminent risk of fetal loss associated with COVID-19 placentitis, the above-described hemostasis alterations warrant awareness of postpartum hemorrhagic complications and could be helpful to identify patients requiring intensified medical attention.

This study investigates the roles of mucosal-associated invariant T (MAIT) cells and Vα7.2+/CD161− T cells in skin diseases, focusing on atopic dermatitis. MAIT cells, crucial for bridging innate and adaptive immunity, were analyzed alongside Vα7.2+/CD161− T cells in peripheral blood samples from 14 atopic dermatitis patients and 10 healthy controls. Flow cytometry and machine learning algorithms were employed for a comprehensive analysis. The results indicate a significant decrease in MAIT cells and CD69 subsets in atopic dermatitis, coupled with elevated CD38 and polyfunctional MAIT cells producing TNFα and Granzyme B (TNFα+/GzB+). Vα7.2+/CD161− T cells in atopic dermatitis exhibited a decrease in CD8 and IFNγ-producing subsets but an increase in CD38 activated and IL-22-producing subsets. These results highlight the distinctive features of MAIT cells and Vα7.2+/CD161− T cells and their different roles in the pathogenesis of atopic dermatitis and provide insights into their potential roles in immune-mediated skin diseases.

Introduction: India has a huge disease burden of thalassemia major with an estimated 40 million carriers and over a million thalassemia major patients. Very few patients are optimally treated, and the standard of care “hematopoietic stem cell transplant” (HSCT) is out of reach for most patients and their families. The cost of HSCT is significant, and a substantial proportion of it goes to human leukocyte antigen (HLA) testing of family members (HLA screening) in hope of getting a matched related donor (MRD) for HSCT. The aim of this study was to establish that a new proposed testing algorithm of HLA typing would be more cost-effective as compared to the conventional HLA screening within MRD families for possible HSCT. Material and Methods: Buccal swab samples of 177 thalassemia patients and their prospective family donors (232) were collected. Using a new HLA testing algorithm, samples were tested for HLA typing in a sequential manner (first HLA-B, then HLA-A, and finally HLA-DR) using the sequence-specific oligonucleotide probe method on the Luminex platform. Results: The new sequential HLA-A, HLA-B, and HLA-DRB1 testing algorithm showed a 49.1% reduction in cost compared to the conventional HLA testing algorithm. Furthermore, 40 patients (22.59%) were found to have HLA-MRD within the family among other samples that were tested. Conclusion: The new HLA testing algorithm proposed in the present study for identifying MRD for HSCT resulted in a substantial reduction in the cost of HSCT workup.

Unconventional T cells show distinct and unique features during antigen recognition as well as other immune responses. Their decrease in frequency is associated with various autoimmune disorders, allergy, inflammation, and cancer. The landscape frequency of the unconventional T cells altogether (iNKT, γδ T, and MAIT) is largely unestablished leading to various challenges affecting diagnosis and research in this field. In this study, we have established the age group–wise frequency of iNKT, γδ T, and MAIT cells altogether on a total of 203 healthy adult samples of the Caucasian population. The results revealed that iNKT cells were 0.095%, γδ T cells were 2.175%, and MAIT cells were 2.99% of the total T cell population. γδ and MAIT cell frequency is higher in younger age groups than elderly; however, there is no statistically significant difference in the frequency of iNKT cells. Furthermore, γδ and MAIT cells were negatively correlating with age, supporting immunosenescence, unlike iNKT cells. Our finding could be used for further age-wise investigation of various pathological conditions such as cancer and their prognosis, autoimmune diseases and their pathogenicity.

Background: The understanding of transplant acceptance or rejection derives directly from knowing human leukocyte antigen (HLA) involved in the immune response. The HLA of the major histocompatibility complex contains a numerous family of genes located on the short arm of the chromosome number 6 and divided majorly into two classes, Class I (HLA-A, B, and C) and Class II (HLA-DR, DQ, and DP). The detection and detailed information of these histocompatibility genes are essential for the better outcome of organ transplants. Aims and Objectives: The aim of the present study is to analyze the frequencies of HLA-A, B, and DRB1 in chronic kidney disease (CKD) patients and their prospective donors as well as to see the prevalence of heterogenicity. Materials and Methods: A total of 264 patients diagnosed with CKD and 333 potential donors were studied retrospectively in the present study. All these cases were analyzed for HLA-A, B, and DRB1 loci typing by PCR-SSOP method based on the Luminex platform. Results: We identified 15 different alleles of HLA-A, 29 of HLA-B, and 13 of HLA-DRB1 amongst studied samples. Out of these identified HLA alleles, HLA-A*02, HLA-B*35, and HLA-DRB1*15 were more frequent as compared to others. The results showed significant heterogenicity in the identified HLA-A, B, and DRB1alleles. Conclusion: The result highlights the diversity of HLA-A, B, and DRB1 alleles and is valuable as a reference for organ transplantations. Further, these findings can also guide in better donor selection and hence improved transplant outcome. In addition, this information could be a starting point for the development of an indigenous transplant assay kit.

This study tested the hypothesis of gender bias in frequency of unconventional T cells. Unconventional T cells exist as minor subsets of T cells in peripheral blood. Despite their low number, they play a crucial role in various immune-mediated diseases such as inflammation, autoimmunity, allergy, and cancer. Gender-based frequency of these cells altogether on large number of healthy individuals are unestablished creating hurdles to manifest association with various immune-mediated pathologic conditions. In this study, we used a multicolor flow cytometric panel to identify iNKT cells, γδ T cells, and MAIT cells altogether in the peripheral blood samples of 93 healthy adult males and 109 healthy adult females from the Caucasian population. The results revealed iNKT cell median value (% T cells) in females was higher: 0.114% ranging from 0.011 to 3.84%, than males: 0.076% ( p value 0.0292), ranging from 0.007 to 0.816% and found to be negatively correlated with age in females ( p value 0.0047). However, γδ T cell median value in males was higher: 2.52% ranging from 0.31 to 16.09%, than females: 1.79% ( p value 0.0155), ranging from 0.078 to 12.49% and each gender was negatively correlated with age (male p value 0.0003 and female p value 0.0007). MAIT cell median values were 3.04% ranging from 0.11 to 10.75% in males and 2.67% ranging from 0.2 to 18.36% in females. MAIT cells did not show any statistically significant difference between genders and found to be negatively correlated with age ( p value < 0.0001). Our results could be used for further gender-wise investigations of various pathologic conditions such as cancer and their prognosis, autoimmune diseases, allergies, and their pathogenicity.

BACKGROUND: Human leukocyte antigen (HLA) is a major determinant in deciding upon solid organ histocompatibility. Donor-specific anti-HLA antibodies (Donor-specific anti-HLA antibodies (DSAs)) are always a contraindication for solid organ transplantation, and identification of DSA becomes very crucial before transplantation to provide long-term graft survival. For identification of DSA, usually, either cell-based or HLA bead-based assay is being used in laboratories. However, both cell-based and bead-based assays have certain limitations. One such common limitation is “prozone effect,” which can give false-negative results. Here, we would like to present a small pilot study to analyze the effect of the prozone phenomenon in the cell-based and HLA bead-based assays and its utility in histocompatibility testing. MATERIALS AND METHODS: In a series of four experiments, cell-based assay, flow cytometric cross-match (FCXM), and HLA bead-based flow cytometric panel reactive antibodies (PRAs) were performed. Single-antigen bead (SAB) testing was conducted as a first experiment on four known positives samples for anti-HLA antibody-antibodies. In the second experiment, these four samples were pooled together (called pooled sera in the text) and tested for FCXM and PRA. In the third experiment, known commercially available positive control sera were mixed with pooled positive sera (positive control sera + pooled sera) to prepare, what we have called “positive concoction” in the text. In the fourth experiment, the positive concoction was diluted serially (1:2, 1:4, 1:8, and 1:16) and FCXM and PRA were performed again to analyze and compare the prozone effect. RESULTS: Pooled sera did not have the expected median fluorescence intensity (MFI) values in FCXM assay, whereas the PRA was showing >90% positivity. In positive concoction, the MFI of FCXM assay was observed to be declining; however, PRA values remained almost constant. Dilutions of the pooled sera showed that MFI values of FCXM assays were increased suddenly after dilution. The highest MFI values were observed in 1:4 dilution of the sera, and then, it declined gradually, but the PRA values remained almost constant even after serial dilutions. CONCLUSION: In our experimental findings, it was clear that cell-based assay (FCXM) was more severely affected by the prozone, whereas solid-phase (flow PRA) assay remained resistant to prozone.

Introduction: In India, 90% kidneys for transplantation are obtained from living donor while only 10% come from deceased donors. Since the rate of living organ donors is high, it therefore leads to the problem of organ trafficking.To minimize the chances of organ trafficking, the Transplantation of Human Organ Act (THOA) 2014 was enacted in India that makes it mandatory to prove the relationship between patient and donor by DNA testing. The present study was undertaken to evaluate the degree of matching between maternally related patients and donors, performed using mitochondrial DNA (mtDNA).Methods: After taking an informed consent, a total of 84 subjects were recruited in the study, 42 kidney transplant recipients and 42 their corresponding donors. An attempt was made to establish and confirm the claimed relationship betweenrecipient and donor using mtDNA analysis. Results: Out of the total 42 cases, mtDNA analysis supported the claimed relationship in 33 (78.57%) cases, whereas in 9 (21.42%) cases claimed relationship could not be supported. Conclusion: mtDNA can be used as valuable tool to support the claimed relationships of maternal lineage. It is important that more and more organ transplant physicians, surgeons and committees are made aware of this diagnostic modality.

Background and ObjectivesTimely detection of human leucocyte antigen (HLA) antibodies plays a key part in transplant outcome. Conventionally, several cell‐based assays are available for detection of HLA antibodies upgrading to virtual crossmatch in which single‐antigen bead (SAB) assay is widely used for characterization of pre‐formed HLA donor‐specific antibodies (DSAs). The main objective here is to find the prevalence and specificity of anti‐HLA antibodies in Indian population using Luminex single‐antigen (LSA) bead assay.Materials and MethodsA total of 179 patients are included in the study for analysing anti‐HLA antibodies by Luminex bead assays that comprise a panel of beads coated with purified HLA antigens (both class I and class II).ResultsHLA‐A*24:02, HLA‐A*02:02, HLA‐B*15:02 and HLA‐B*27:08 and HLA‐C*07:01, HLA‐C*07:02 and HLA‐C*06:02 are frequently identified HLA antibodies against class I, whereas HLA‐DRB1*09:01, HLA‐DRB3*01:01, HLA‐DQA1*01:02, HLA‐DQB1*06:04, HLA‐DQA1*05:01, HLA‐DQB1*03:01, HLA‐DPA1*02:01, HLA‐DPB1*14:01, HLA‐DPA1*01:03 and HLA‐DPB1*18:01 are most common anti‐HLA antibodies against class II.ConclusionsTo conclude, this study helps to select the population‐specific best possible donor and hence improve transplant outcome to decrease risk of antibody‐mediated rejection.