Immune Checkpoint Pathway Expression in Lymphocyte Subpopulations in Patients with Common Variable Immunodeficiency and Chronic Lymphocytic Leukemia.

This study aims to gain a deeper understanding of chronic lymphocytic leukemia (CLL) and common variable immunodeficiency (CVID) by studying immune cells and specific immune checkpoint signaling pathways. The analysis of the percentage of selected immune points and their ligands (PD-1/PD-L1, CTLA-4/CD86, and CD200R/CD200) on peripheral blood lymphocyte subpopulations was performed using flow cytometry, and additional analyses determining the serum concentration of the above-mentioned molecules were performed using enzyme immunoassay tests. The obtained results indicate several significant changes in the percentage of almost all tested molecules on selected subpopulations of T and B lymphocytes in both CVID and CLL patients in relation to healthy volunteers and between the disease subunits themselves. The results obtained were also supported by the analysis of the serum concentration of soluble molecules tested. By uncovering valuable insights, we hope to enhance our comprehension and management of these conditions, considering both immunodeficiencies and hematological malignancies. Understanding the role of these signaling pathways in disease development and progression may lead to the development of modern, personalized diagnostic and therapeutic strategies. Ultimately, this knowledge may enable the monitoring of the immune system in patients with CVID and CLL, paving the way for improved patient care in the future.

What's with the boys? Lower birth weight in boys from HPA-1a alloimmunized pregnancies - New insights from a large prospective screening study in Poland.

Fetomaternal incompatibility in human platelet antigens (HPAs) can cause maternal alloimmunization, which in turn may lead to thrombocytopenia with or without intracranial hemorrhage (ICH) in the fetus or newborn. Retrospective studies suggest that boys from alloimmunized mothers may have higher risk of ICH and lower birth weight than girls. The objective of this study was to assess how maternal HPA-1a alloimmunization, sex of the neonate and birth weight relates in a large prospective cohort. Through a national screening study in Poland (PREVFNAIT) involving HPA-1 typing of 24,259 pregnant women during 2013-2017, 606 HPA-1a negative pregnant women and their offspring were identified and included. Various multivariate models were used to assess if and how maternal HPA-1a alloimmunization status was associated with birth weight and risk of having a small for gestational age (SGA) neonate, and if and how sex of the neonate mattered. Most immunized pregnancies had male fetuses (69 %). Women carrying a male fetus had increased likelihood of having an SGA newborn if they were HPA-1a alloimmunized compared to non-immunized mothers. Increasing maternal anti-HPA-1a antibody levels were significantly associated with reduced birth weight and SGA risk among male-fetus pregnancies, but not if the fetus was female. In conclusion, anti-HPA-1a antibodies in a male fetus pregnancy is associated with increased risk of SGA and lower birth weight, especially if the antibody level is high. Sex of the fetus may therefore be considered as a new clinical predictor of more severe FNAIT neonatal outcome.

Could Immune Checkpoint Disorders and EBV Reactivation Be Connected in the Development of Hematological Malignancies in Immunodeficient Patients?

Primary immunodeficiencies (PIDs) and secondary immunodeficiencies (SIDs) are characterized by compromised immune function, rendering individuals susceptible to infections and potentially influencing cancer development. Epstein-Barr virus (EBV), a widespread herpesvirus, has been linked to cancer, particularly in those with weakened immune systems. This study aims to compare selected immune parameters, focusing on immune checkpoint molecules (PD-1/PD-L1, CTLA-4/CD86, CD200R/CD200), and EBV reactivation in patients with chronic lymphocytic leukemia (CLL, a representative of SIDs) and common variable immunodeficiency (CVID, a representative of PIDs). We performed a correlation analysis involving patients diagnosed with CLL, CVID, and a healthy control group. EBV reactivation was assessed using specific antibody serology and viral load quantification. Peripheral blood morphology, biochemistry, and immunophenotyping were performed, with emphasis on T and B lymphocytes expressing immune checkpoints and their serum concentrations. Our findings revealed elevated EBV reactivation markers in both CLL and CVID patients compared with healthy controls, indicating increased viral activity in immunodeficient individuals. Furthermore, immune checkpoint expression analysis demonstrated significantly altered percentages of T and B lymphocytes expressing PD-1/PD-L1, CTLA-4/CD86, and CD200R/CD200 in CLL and CVID patients. This suggests a potential interplay between immune checkpoint dysregulation and EBV reactivation in the context of immunodeficiency. In conclusion, our study underscores the intricate relationship between immune dysfunction, EBV reactivation, and immune checkpoint modulation in the context of immunodeficiency-associated cancers. The altered expression of immune checkpoints, along with heightened EBV reactivation, suggests a potential mechanism for immune evasion and tumor progression. These findings provide insights into the complex interactions that contribute to cancer development in immunocompromised individuals, shedding light on potential therapeutic targets for improved management and treatment outcomes. Further investigations are warranted to elucidate the underlying mechanisms and to explore potential interventions to mitigate cancer risk in these patient populations.

A high prevalence of neutrophil-specific antibodies in ELANE-mutated severe congenital neutropenia.

An assay for neutrophil-specific antibodies is frequently used in the workup of chronic severe neutropenia and is suggestive of autoimmune, or sporadically alloimmune neutropenia, rather than severe congenital neutropenia (SCN). We analyzed a neutropenia consortium database for the outcomes of antibody testing initiated before receiving genetic diagnosis in Polish SCN cohort. Test results, performed in a single reference laboratory, were available for 14 patients with ELANE-mutated SCN or cyclic neutropenia, and were frequently positive (36%). We note that the trigger for genetic studies in severe neutropenia should not be affected by antibody-positivity and should be clinically driven.

Human Intramuscular Hyperimmune Gamma Globulin (hIHGG) Anti-SARS-CoV-2-Characteristics of Intermediates and Final Product.

This study aims to characterize the intermediates, and the final product (FP) obtained during the production of human intramuscular hyperimmune gamma globulin anti-SARS-CoV-2 (hIHGG anti-SARS-CoV-2) and to determine its stability. Material and methods: hIHGG anti-SARS-CoV-2 was fractionated from 270 convalescent plasma donations with the Cohn method. Prior to fractionation, the plasma was inactivated (Theraflex MB Plasma). Samples were defined using enzyme immunoassays (EIA) for anti-S1, anti-RBD S1, and anti-N antibodies, and neutralization assays with SARS-CoV-2 (VN) and pseudoviruses (PVN, decorated with SARS-CoV-2 S protein). Results were expressed as a titer (EIA) or 50% of the neutralization titer (IC50) estimated in a four-parameter nonlinear regression model. Results: Concentration of anti-S1 antibodies in plasma was similar before and after inactivation. Following fractionation, the anti-S1, anti-RBD, and anti-N (total tests) titers in FP were concentrated approximately 15-fold from 1:4 to 1:63 (1800 BAU/mL), 7-fold from 1:111 to 1:802 and from 1:13 to 1:88, respectively. During production, the IgA (anti-S1) antibody titer was reduced to an undetectable level and the IgM (anti-RBD) titer from 1:115 to 1:24. The neutralizing antibodies (nAb) titer increased in both VN (from 1:40 to 1:160) and PVN (IC50 from 63 to 313). The concentration of specific IgG in the FP did not change significantly for 14 months. Conclusions: The hIHGG anti-SARS-CoV-2 was stable, with concentration up to approximately 15-fold nAb compared to the source plasma pool.

Noninvasive diagnostics of fetal KEL*01.01 allele from maternal plasma of immunized women using digital PCR protocols.

Allo-antibodies produced by K-negative pregnant women against a fetal K antigen from the Kell blood group system may cause hemolytic disease of the fetus and newborn (HDFN). Predicting the fetal K antigen using noninvasive prenatal testing (NIPT) is important for decisions concerning management of pregnancies. Digital and droplet digital PCR techniques permit the detection of fetal single nucleotide variant with a higher specificity and sensitivity than real-time polymerase chain reaction (PCR). AIM: The aim was to evaluate and compare protocols for fetal KEL*01.01 genotyping using different assays and digital PCR platforms. METHODS: DNA isolated from 59 pregnant women (9-39 weeks of gestation, 49 with anti-K) was tested using home-made and custom-ordered KEL*01.01/KEL*02 assays with Droplet Digital™ and QuantStudio™3D. The results were compared with fetal/neonatal genotypes/phenotypes. RESULTS: Fetal KEL*01.01 results using all tested protocols were concordant with fetal/neonatal KEL*01.01 genotypes/phenotypes. None of the tested combinations of assays or digital PCR platforms gave false KEL*01.01-negative results, but inconclusive KEL*01.01 reads were observed in all tested protocols. For 36 cases compared using two digital PCR platforms and assays, there were not statistically significant differences in a level of fetal KEL*01.01 fraction (p < .72). CONCLUSION: Independent of the applied dPCR and ddPCR platforms and KEL*01.01 assays, prediction of the fetal KEL*01.01 is highly reliable. Before implementation in routine practice further validation of the KEL*01.01 protocol with a larger group of pregnant women should be performed.

Prediction of fetal blood group antigens from maternal plasma using Ion AmpliSeq HD technology.

BACKGROUND: Fetal blood group (BG) and platelet (HPA) antigens may trigger maternal immunization, causing a fetal disease. Noninvasive prenatal diagnostics (NIPT) predicts fetal genotype, identifying pregnancies with no risk. All current techniques detect fetal antigen alleles with unspecific background and without estimation of fetal fraction, thus new protocols for detection of fetal BG/HPA alleles with ultrahigh sensitivity still need to be tested to improve NIPT. AIM: To design NIPT of clinically important antigens using Ion AmpliSeq HD technology. METHODS: Plasma DNA from 36 pregnant women (9-33 week of gestation, 24 immunized with anti-HPA-1a,-3b,-15a, -K, or -D+C+S), with known BG/HPA genotypes of their neonates/partners, was tested on Ion S5 System using the Ion AmpliSeq HD designer custom gene panel. NGS contained 25 rs-targets encoding relevant BG/HPA antigens and 10 markers. RESULTS: Using the NGS protocol, 76 out of 85 differences in fetal/maternal BG/HPA genotypes were determined in concentration above 2% fetal paternally inherited allele chimerism. The level of unspecific reads for BG/HPA alleles was below 0.87%. In 24 immunized women NGS revealed feto-maternal incompatibility in 11 cases (from 2.44% to 7.41%) and excluded in 10 (<0.05%), three cases had inconclusive results (1.79%, 0.19%, 0.11%). The presence of fetal DNA was confirmed in each case by detecting markers with at least 2% chimerism. CONCLUSION: The use of Ion AmpliSeq HD technology improves the prediction of feto-maternal incompatibility, increasing the sensitivity of BG/HPA NIPT and serving confirmation of the fetal DNA at the same workflow.

Recommendation for validation and quality assurance of non-invasive prenatal testing for foetal blood groups and implications for IVD risk classification according to EU regulations.

BACKGROUND AND OBJECTIVES: Non-invasive assays for predicting foetal blood group status in pregnancy serve as valuable clinical tools in the management of pregnancies at risk of detrimental consequences due to blood group antigen incompatibility. To secure clinical applicability, assays for non-invasive prenatal testing of foetal blood groups need to follow strict rules for validation and quality assurance. Here, we present a multi-national position paper with specific recommendations for validation and quality assurance for such assays and discuss their risk classification according to EU regulations. MATERIALS AND METHODS: We reviewed the literature covering validation for in-vitro diagnostic (IVD) assays in general and for non-invasive foetal RHD genotyping in particular. Recommendations were based on the result of discussions between co-authors. RESULTS: In relation to Annex VIII of the In-Vitro-Diagnostic Medical Device Regulation 2017/746 of the European Parliament and the Council, assays for non-invasive prenatal testing of foetal blood groups are risk class D devices. In our opinion, screening for targeted anti-D prophylaxis for non-immunized RhD negative women should be placed under risk class C. To ensure high quality of non-invasive foetal blood group assays within and beyond the European Union, we present specific recommendations for validation and quality assurance in terms of analytical detection limit, range and linearity, precision, robustness, pre-analytics and use of controls in routine testing. With respect to immunized women, different requirements for validation and IVD risk classification are discussed. CONCLUSION: These recommendations should be followed to ensure appropriate assay performance and applicability for clinical use of both commercial and in-house assays.

Platelet alloimmunization is associated with low grade chronic histiocytic intervillositis - A new link to a rare placental lesion?

INTRODUCTION: Maternal alloimmunization against human platelet antigen (HPA)-1a has been implied to mediate both reduced birth weight and chronic placental inflammation. Fetal growth restriction is associated with different types of chronic inflammation in the placenta, mainly chronic histiocytic intervillositis and chronic villitis. The aim of this prospective study was to do a systematic examination of placentas from HPA-1a alloimmunized pregnancies, with focus on the histopathological and immunohistochemical diagnosis of variants of chronic inflammation. MATERIAL AND METHODS: In a Polish-Norwegian study, 48 placentas were examined. The histopathology of placentas from 27 HPA-1a immunized women was compared with 21 placentas from non-immunized HPA-1a negative women (controls). In the group of alloimmunized women, ten received antenatal intravenous immunoglobulin G (IVIg). Tissue sections from formalin fixed paraffin embedded placental tissue were stained with hematoxylin and eosin and microscopically examined with focus on various types of chronic placental inflammations. RESULTS: Chronic histiocytic intervillositis was observed in 40.7% of placentas from HPA-1a alloimmunized pregnancies, compared to none in the control group (p = 0.001). Chronic villitis of unknown etiology was more frequently found in the alloimmunized group, however this difference was not statistically significant. Maternal administration of IVIg did not seem to protect against chronic inflammatory lesions. DISCUSSION: Placentas with detectable maternal anti-HPA-1a antibodies are associated with highly increased risk of low-grade chronic histiocytic intervillositis.

Prediction of fetal blood group and platelet antigens from maternal plasma using next-generation sequencing.

BACKGROUND: Fetuses whose mothers have produced antibodies to red blood cell (RBC) or platelet antigens are at risk of being affected by hemolytic disease or alloimmune thrombocytopenia, respectively, only if they inherit the incompatible antigen. Noninvasive diagnosis of the fetal antigen is employed for management of immunized pregnancies, but the specific detection of SNPs, encoding the majority of antigens, in maternal plasma is still a challenge. We applied targeted next-generation sequencing (NGS) to predict the fetal antigen based on the detection of fetomaternal chimerism. METHODS AND MATERIALS: The DNA of 13 pregnant women (with anti-K [3] anti-k [1], anti-Fya [1], anti-D + C + Jka [1], anti-D + E + K [1], anti-HPA-1a [1], anti-HPA-3b [1], anti-HPA-5b [1], and nonimmunized [3]) was sequenced using primers for regions encoding RhD, RhC, Rhc, RhE/e, K/k, Fya/b, Jka/b, MN, Ss, and HPA-1, 2, 3, 5, 15, 4 X-polymorphisms on the Ion Torrent Personal Genome Machine (PGM) System (Thermo Fisher Scientific, Inc., Waltham, MA, USA). RESULTS: NGS results were in agreement with the phenotype/genotype of women and their neonates (except for the unsuccessful detection of MN and RhC). NGS determined fetal allele chimerism for K, k, Fya, Fyb, Jka, Jkb, S, RhE (from 0.42% to 6.08%); RhD, Rhc (100%); HPA-1a, -2b, -3a, 3b, -5b, -15a, 15b (from 0.23% to 4.11%). NGS revealed fetal chimerism for incompatible antigens (from 0.7% to 4.8%) in 7 immunized cases, excluded in 3 (with anti-K, anti-Fya , anti-HPA-3b). CONCLUSION: The designed NGS predicts the fetal RBC and platelet antigen status universally in cases with various clinically significant antibodies as well as providing confirmation of the presence of fetal DNA. However, some improvement of the unsuccessful primers is required.

Noninvasive prenatal HPA-1 typing in HPA-1a negative pregnancies selected in the Polish PREVFNAIT screening program.

BACKGROUND: Anti-HPA-1a alloantibodies in HPA-1a negative mothers can lead to fetal/neonatal alloimmune thrombocytopenia (FNAIT). Noninvasive prenatal testing (NIPT) of HPA-1a determines fetuses at risk and the course of maternal antenatal treatment. STUDY DESIGN AND METHODS: The aim was to develop and validate HPA-1a NIPT by real-time polymerase chain reaction (PCR) or next-generation sequencing (NGS) for a high-throughput screening setting. DNA from 328 plasma samples of 299 HPA-1a negative pregnant women was examined for HPA-1a by real-time PCR and in two cases also by NGS (Ion Torrent). The results were compared with neonatal HPA-1a genotyping in 281 cases. RESULTS: HPA-1a NIPT was negative in 44 of 51 HPA-1a negative fetuses, inconclusive in five, and false positive in two. In 228 of 229 HPA-1a positive fetuses, the NIPT results were positive (mean threshold cycle 36.0 ± 1.7) and inconclusive in one. In 22 cases with HPA-1a positive fetuses analyzed twice, the sensitivity of HPA-1a detection was significantly higher at 28 weeks compared with 16 to 20 weeks. NGS efficiently detected the ITGB3 coding HPA-1a/b (1% and 5% fetal HPA-1a reads). CONCLUSION: Real-time PCR is reliable to predict the fetal HPA-1a positive genotype in a screening study, but false-positive results are reported in 4%, with unnecessary prenatal treatment if anti-HPA-1a is detected.

Identification and follow-up of pregnant women with platelet-type human platelet antigen (HPA)-1bb alloimmunized with fetal HPA-1a.

INTRODUCTION: Pregnant women negative for human platelet antigen 1a (HPA-1a) are at risk of alloimmunization with fetal HPA-1a antigen inherited from the father, and their offspring may develop fetal and neonatal alloimmune thrombocytopenia (FNAIT). The aim of this study was to analyze the frequency of HPA-1a alloimmunization in pregnant Polish women, the feasibility of using maternal platelets for intrauterine transfusions in women subjected to diagnostic fetal blood sampling (FBS) and to discuss potential consequences of alloimmunization. MATERIAL AND METHODS: Fifteen thousand two hundred and four pregnant women were typed for HPA-1a; HPA-1a negative were screened for anti-HPA-1a. Alloimmunized women received specialist perinatology care; some of them were subjected to FBS, followed by transfusion of HPA-1a negative platelet concentrates (PC) prepared from maternal blood. RESULTS: Three hundred seventy-three (2.5%) women were HPA-1a negative, and 32 (8.6%) tested positively for anti-HPA-1a. Antibodies were detected in 22 women during pregnancy. Diagnostic FBS followed by PC transfusion was performed in 14 woman, who were platelet donors for their 16 unborn babies. Blood donations were tolerated well by the patients, and also intrauterine platelet transfusions were uneventful. Pharmacotherapy with intravenous immunoglobulins was implemented in 11/22 patients. CONCLUSIONS: HPA-1a negative women (ca. 2.5% of all pregnant patients) are at risk of alloimmunization with HPA-1a antigen and developing FNAIT. Alloimmunized women can be donors of platelets for their offspring providing removal of antibodies from PC. Owing to potential complications, special care should be taken if an alloimmunized woman was qualified as a blood or stem cell recipient.

A preliminary evaluation of next-generation sequencing as a screening tool for targeted genotyping of erythrocyte and platelet antigens in blood donors.

BACKGROUND: Matching the compatibility of donor blood with the recipient's antigens prevents alloimmunisation. Next-generation sequencing (NGS) technology is a promising method for extensive blood group and platelet antigen genotyping of blood donors. It circumvents the limitations of detecting known alleles based on predefined polymorphisms and enables targeted sequencing on a massive scale. The aim of this study was to evaluate the NGS AmpliSeq application on the Ion Torrent platform as a screening tool for genotyping blood donors' erythrocyte/platelet antigens. MATERIALS AND METHODS: Primers for regions encoding antigens RhD (exons 5, 7), Rhc, RhE/e, Fya/b, Jka/b, M/N, S/s, HPA-1, 2, 3, 5, 15 were designed with Ion AmpliSeq Designer with manual inclusion of RHCE*C primers. DNA libraries of 57 regular blood donors with determined phenotype/genotype (prepared using the Ion AmpliSeq Library Kit and 14 primer pairs) were sequenced on the Ion Torrent PGM using 316v2 chips and 200 bp chemistry. RESULTS: Sequencing was successful in all but the MN and HPA-5 regions. Mean sequencing coverage in one experiment was 4,606 reads, except for the RHCE*C region (mean 568 reads). NGS results agreed with the known phenotype/genotype of donors except in one phenotypically Fy(a+b-) case in whom FY*A/FY*B alleles were found. Reading rates for homozygotes were 97-100%, while they were around 50% for heterozygotes. NGS of RHD regions led to identification of mutations in two RhD negative donors. DISCUSSION: NGS can be performed as a screening test to determine erythrocyte/platelet antigens in blood donors. This method allowed testing of 48 donors for 14 features (200 bp long) with the depth of a few thousand reads simultaneously, and the estimation of natural chimerism or hemi/homozygotic status. NGS screening can be adjusted to the genetic background of a given tested population.

Recent advances in understanding the clinical relevance of antiplatelet alloantibodies.

Alloimmunization to human platelet antigens (HPAs) may occur either during pregnancy, when a HPA­negative mother gives birth to a newborn who inherits HPAs from the father, or following blood transfusion or stem cell transplantation. Antiplatelet alloantibodies do not cause thrombocytopenia in a patient, but their detection must always be recorded in medical records because they may induce fetal and neonatal alloimmune thrombocytopenia in present and all subsequent pregnancies, platelet refractoriness, posttransfusion purpura, or prolonged thrombocytopenia with engraftment failure after stem cell transplantation. Passive transfer of platelet alloantibodies through transfused blood components may trigger thrombocytopenia and severe posttransfusion reactions in the recipient. In a Caucasian population, such clinical outcome of platelet alloimmunization is mostly due to anti­HPA­1a antibodies, less frequently to anti­HPA­5b, anti­HPA­1b, and others. Information on anti­HPA alloantibodies is crucial for the prevention and treatment of their consequences.

Compound heterozygosity of two novel RHAG alleles leads to a considerable disruption of the Rh complex.

BACKGROUND: The Rhesus (Rh) complex consists of a core comprising the Rh proteins (RhD/RhCE) and the Rh-associated glycoprotein (RhAG) with accessory chains (GPB, LW, CD47). Molecular defects of the RHAG gene may cause a regulator Rhnull phenotype without Rh antigen expression or a Rhmod phenotype with decreased Rh antigen expression. STUDY DESIGN AND METHODS: Blood samples of a donor with strongly diminished Rh antigens and five family members were analyzed by serological phenotyping, flow cytometry, molecular testing, and gene expression analysis of Rh complex candidate genes. RESULTS: RHAG sequencing identified a missense mutation, c.241G>C (p.Gly81Arg) and a splice site mutation, c.640 + 3del14, among the cohort. Compound heterozygosity of these novel alleles identified in the propositus and two siblings gave rise to a strongly diminished expression of RhAG, Rh, and CD47 antigens on the RBC surface. CONCLUSION: The Rhmod phenotype was caused by a novel RHAG splice site mutation in association with a non-functional allele. The primary depression of RhAG is most likely due to posttranslational events that affect the interaction and processing of the RhAG glycoprotein and gave rise to a secondary depression of RhD, RhCE, and CD47, the major members of the Rh complex.

Fetal/Neonatal Alloimmune Thrombocytopenia: Pathogenesis, Diagnostics and Prevention.

Fetal/neonatal alloimmune thrombocytopenia (FNAIT) is a relatively rare condition (1/1000-1/2000) that was granted orphan status by the European Medicines Agency in 2011. Clinical consequences of FNAIT, however, may be severe. A thrombocytopenic fetus or new-born is at risk of intracranial hemorrhage that may result in lifelong disability or death. Preventing such bleeding is thus vital and requires a solution. Anti-HPA1a antibodies are the most frequent cause of FNAIT in Caucasians. Its pathogenesis is similar to hemolytic disease of the newborn (HDN) due to anti-RhD antibodies, but is characterized by platelet destruction and is more often observed in the first pregnancy. In 75 % of these women, alloimmunization by HPA-1a antigens, however, occurs at delivery, which enables development of antibody-mediated immune suppression to prevent maternal immunization. As for HDN, the recurrence rate of FNAIT is high. For advancing diagnostic efforts and treatment, it is thereby crucial to understand the pathogenesis of FNAIT, including cellular immunity involvement. This review presents the current knowledge on FNAIT. Also described is a program for HPA-1a screening in identifying HPA-1a negative pregnant women at risk of immunization. This program is now performed at the Institute of Hematology and Transfusion Medicine in cooperation with the Department of Obstetrics and Gynecology of the Medical Centre of Postgraduate Education in Warsaw as well as the UiT The Arctic University of Norway.