Kumánovics, Attila

Context.— Transporting specimens can expose them to uncontrolled environmental factors. Whole blood viability is a commonly used preanalytic marker of quality for analysis of white blood cells. Objective.— To evaluate whole blood viability as a marker for transportation and specimen integrity for lymphocyte subset evaluation. Design.— More than 9000 whole blood specimens, transported during a 4-year period, and their lymphocyte subset evaluation results were analyzed retrospectively. Viability was measured by using Cellaca MX imaging cytometer, and lymphocyte and lymphocyte subset counts were determined with flow cytometry. Specimen cell viability results were stratified based on different transportation factors to discern their potential effects. Lymphocyte subset evaluation results from specimens with lower viability were compared with those from optimal viability specimens. Results.— Cell viability of transported specimens exhibited seasonal variation, decreasing during the warm summer months (median, 77.0% versus 83.4% in other seasons). The decline in viability negatively correlated with ambient temperature during transportation ( r = −0.7876). Longer transportation time (>60 hours) also caused a decrease in viability (median, 64.0%). While we observed a decrease in absolute cell counts for CD45 + lymphocytes, CD3 + T cells, CD8 + T cells, and CD19 + B cells in specimens with low viability (50%–80%), the differences were well within the assay’s total allowable error. Conclusions.— Control of preanalytic factors is critical for laboratory testing. Whole blood viability is a sensitive indicator of transport conditions and time, but it is not a specific predictor of quality for the main lymphocyte populations in cellular immunology testing.

This study demonstrates successful hematopoietic stem cell transplant of CD40L deficiency using an asymptomatic carrier mother, leading to sustained 32% CD40L expression, normal recovery of T and B cell development and diversity, and expected growth and development 7.5 years post-transplant.

Abstract Regulation of gene expression is central to the development of immune cells and their ability to respond to infection. As part of a clinical evaluation, we identified two sisters with recurrent infections, hypogammaglobulinemia, and memory B cell deficiency, diagnosed as common variable immunodeficiency. Whole exome sequencing identified a heterozygous variant (Leu50Ser, L50S) in a conserved region of EZH2, the catalytic subunit of the epigenetic gene repressor Polycomb Repressive Complex 2 (PRC2). EZH2-catalyzed histone H3 lysine 27 methylation (H3K27me) in bulk was not overall significantly disrupted by this variant, in patient samples or cell lines expressing EZH2-L50S. EZH2-L50S protein is expressed similar to wild-type and can form PRC2. However, we find that specific genomic regions that normally have high wild-type levels of H3K27me3 are deficient in the L50S context, particularly around gene promoters. EZH2-L50S is still recruited to these sites, but is not as active. Using recombinant purified PRC2, we determine that L50S affects methylation of nucleosomes and disrupts allosteric stimulation that normally amplifies H3K27me3, consistent with the location of L50 in the allosteric regulatory region of PRC2. Thus, variation of EZH2 L50, occurring at low frequency in the population may interfere with normal B cell gene expression patterns, contributing to immunodeficiency. This study has implications for genetic variation in PRC2 in the general population.

Abstract Description Common variable immunodeficiency (CVID) is a common inborn error of immunity, but a monogenic cause is rare. Biallelic loss-of-function pathogenic variants in the gene encoding BAFF-Receptor (TNFRSF13C) is one such cause. A common missense variant in BAFF-R, p.Pro21Arg, was proposed as contributing to the pathogenesis of non-monogenic CVID by Peiper et al. 2014 with an allelic frequency of 6.7% in controls versus 10.2% in CVID patients (OR 1.57, p = 0.0026) and Block et al. 2023 with an 11.36% allele frequency in CVID when compared to the Non-Finnish European population in the gnomAD database (OR = 1.316, p = 0.0145) and to an internal cohort of controls (OR = 1.316, p = 0.0458). Exact population composition matching is critical, as the frequency of this variant is highly variable even in Europe. We found the allelic frequency of BAFF-R p.Arg21Pro to be 10% from 90 non-familial CVID patients from the University of Utah Health Sciences Center, similar to Pieper et al. (10.2%) and Block et al. (11.36%), but comparing our Utah CVID patients to the 1000 Genomes project Utah Residents (CEU) with Northern and Western European Ancestry, which matches our CVID cohort (9.6% allelic frequency); we obtained an OR of 1.0468 (95% CI: 0.5309 to 2.0638; p = 0.8950). For further studies on BAFF-R p.Pro21Arg, we recommend closely matching the reference populations, and not using a broad European or other control populations. Topic Categories Translational and Interventional Immunology (TI)

Abstract Background Lymphocyte function and immune competence can be measured by proliferative response to stimuli and is useful in several clinical contexts including primary (or genetic) and secondary immunodeficiencies (e.g., immunosuppression following transplantation). Proliferation assays have traditionally been prepared manually due to the difficulty in maintaining the sterile environment required to prevent bacterial and fungal impact on cell growth. On most automated liquid handlers, the sample and reagents are placed inside a cabinet whose inner surfaces include rails, pullies, pumps, and circuitry that face the sample and are not amenable to sterilization. The Andrew+ (Waters, MA) is a small surface cleanable liquid handler with all machinery enclosed. We demonstrate here the use of an Andrew+ placed in a biological safety cabinet (BSC) to perform sterile assay setup. Methods Peripheral blood monocytes (PBMCs) from five individuals were manually generated via buffy coat isolation, then brought to standardized concentration. An Andrew+ was placed in a 4’ BSC and all surfaces disinfected with Oxivir Tb (Diversey, SC). The Andrew+ prepared three serial dilutions of two stimulant reagents (phytohemagglutinin (PHA) and pokeweed (PWM)), then dispensed stimulant and control reagents horizontally and PBMCs vertically to a 48 well plate. This process was duplicated manually from the same specimens to create a reference plate. The plates were incubated at 37°C and 5% CO2 for three days, then pulsed with EdU (Thermo Fisher, MA) and incubated for one additional day. Cells were harvested and counted via flow cytometry. Overall time and hands-on time were measured. Results A total of five samples were tested inside a using both manual and automation setup under eight conditions including with isotype control, media without stimulant, three concentrations of PHA, and three concentrations of PWM. The percent proliferation for various cell types as induced by each stimulant was calculated from the difference of the highest stimulant signal from the background (media-only) signal. Comparing the automated and manual method results showed a slope of 1.0217 and R2 of 0.9966. For a simulated run of 6 samples, technologist time for automated steps was under 8 minutes with a total time of 56 minutes. When these steps were performed manually, the time was 30 minutes. Automation increased total time by 26 minutes but saved 22 minutes of technologist hands-on time. Conclusion The Andrew+ was able to perform sterile setup of a cell culture assay. While slower overall than manual processing, technologist time was reduced. Ergonomic, inter-tech variability, and sample transcription error issues were subjectively observed to be less likely. The Andrew+ can work with up to four “dominos” (reagent or tip blocks) in width and up to three dominos in depth. Non-custom, standard depth BSCs from a variety of manufacturers (Labconco, Nuaire, ThermoFisher) have room for one domino in depth so, except for timings performed in this evaluation, all steps were broken down to be performed with a max of four dominos. Efficient sterile use of the system would require an extended depth BSC.

Background Bruton tyrosine kinase (BTK) is a protein-coding gene crucial for B cell development. It is found on Xq21.3-Xq22 of the long arm of the X chromosome and belongs to the Tec family of cytoplasmic protein tyrosine kinases consisting of 5 structural domains: PH, TH, Src3, Src2, and TK. Pathogenic variants in the BTK gene can fully or partially block B cell development. BTK has been linked to various antibody deficiencies beyond agammaglobulinemia, including CVID. In hypomorphic BTK variants, there are detectable B cells in the periphery that may result in variable levels of serum immunoglobulins and clinical phenotype. Objective Investigate the impact of a missense pathogenic hypomorphic BTK variant (p.Arg525Gln) observed in a large kindred. Results We plan to assess by flow cytometry BTK protein expression in specific B cell subsets in hypomorphic XLA patients in this kindred. The flow cytometry revealed a uniformly reduced BTK expression in B cells (B cells evaluation is limited by low cell counts). BTK expression was decreased with an unexpected bimodal distribution in monocytes. Since both patients were male, XXY syndrome was initially considered but deemed less likely. The unexpected monocyte expression pattern in the current patient suggests possible somatic mosaicism or other genetic factors, or the two peaks are related to BTK protein degradation, and this may differ in specific cellular compartments or change with age, warranting further investigation. Conclusions This highlights the challenges of diagnosing hypomorphic XLA, especially in patients with detectable B cell counts and atypical BTK expression. Flow cytometric analysis of two patients with BTK mutations identified bimodal mosaic patterns of BTK expression in monocytes. This bimodal expression pattern may reflect cellular mosaicism like in mothers who are obligate carriers of XLA. BTK biology with hypomorphic variants remains incompletely understood, particularly how these variants affect B cell development and result in milder antibody deficiency phenotypes, warranting further investigation. This emphasizes the importance of genetic screening in kindreds with XLA and the need for increased awareness of partial XLA among healthcare providers to ensure timely diagnosis and treatment.

Inositol 1,4,5-trisphosphate (IP3) receptor type 1 ( ITPR1 ), 2 ( ITPR2 ), and 3 ( ITPR3 ) encode the IP3 receptor (IP3R), a key player in intracellular calcium release. In four unrelated patients, we report that an identical ITPR3 de novo variant—NM_002224.3:c.7570C>T, p.Arg2524Cys—causes, through a dominant-negative effect, a complex multisystemic disorder with immunodeficiency. This leads to defective calcium homeostasis, mitochondrial malfunction, CD4 + lymphopenia, a quasi-absence of naïve CD4 + and CD8 + cells, an increase in memory cells, and a distinct TCR repertoire. The calcium defect was recapitulated in Jurkat knock-in. Site-directed mutagenesis displayed the exquisite sensitivity of Arg 2524 to any amino acid change. Despite the fact that all patients had severe immunodeficiency, they also displayed variable multisystemic involvements, including ectodermal dysplasia, Charcot-Marie-Tooth disease, short stature, and bone marrow failure. In conclusion, unlike previously reported ITPR1-3 deficiencies leading to narrow, mainly neurological phenotypes, a recurrent dominant ITPR3 variant leads to a multisystemic disease, defining a unique role for IP3R3 in the tetrameric IP3R complex.

This study aimed to evaluate and compare the performance of three anti-S and one anti-N assays that were available to the project in detecting antibody levels after three commonly used SARS-CoV-2 vaccines (Pfizer, Moderna, and Johnson & Johnson). It also aimed to assess the association of age, sex, race, ethnicity, vaccine timing, and vaccine side effects on antibody levels in a cohort of 827 individuals. In September 2021, 698 vaccinated individuals donated blood samples as part of the Association for Diagnostics & Laboratory Medicine (ADLM) COVID-19 Immunity Study. These individuals also participated in a comprehensive survey covering demographic information, vaccination status, and associated side effects. Additionally, 305 age- and gender-matched samples were obtained from the ADLM 2015 sample bank as pre-COVID-19-negative samples. All these samples underwent antibody level analysis using three anti-S assays, namely Beckman Access SARS-CoV-2 IgG (Beckman assay), Ortho Clinical Diagnostics VITROS Anti-SARS-CoV-2 IgG (Ortho assay), Siemens ADVIA Centaur SARS-CoV-2 IgG (Siemens assay), and one anti-N antibody assay: Bio-Rad Platelia SARS-CoV-2 Total Ab assay (BioRad assay). A total of 827 samples (580 COVID-19 samples and 247 pre-COVID-19 samples) received results for all four assays and underwent further analysis. Beckman, Ortho, and Siemens anti-S assays showed an overall sensitivity of 99.5%, 97.6%, and 96.9%, and specificity of 90%, 100%, and 99.6%, respectively. All three assays indicated 100% sensitivity for individuals who received the Moderna vaccine and boosters, and over 99% sensitivity for the Pfizer vaccine. Sensitivities varied from 70.4% (Siemens), 81.5% (Ortho), and 96.3% (Beckman) for individuals who received the Johnson & Johnson vaccine. BioRad anti-N assays demonstrated 46.2% sensitivity and 99.25% specificity based on results from individuals with self-reported infection. The highest median anti-S antibody levels were measured in individuals who received the Moderna vaccine, followed by Pfizer and then Johnson & Johnson vaccines. Higher anti-S antibody levels were significantly associated with younger age and closer proximity to the last vaccine dose but were not associated with gender, race, or ethnicity. Participants with higher anti-S levels experienced significantly more side effects as well as more severe side effects (e.g., muscle pain, chills, fever, and moderate limitations) (p < 0.05). Anti-N antibody levels only indicated a significant correlation with headache. This study indicated performance variations among different anti-S assays, both among themselves and when analyzing individuals with different SARS-CoV-2 vaccines. Caution should be exercised when conducting large-scale studies to ensure that the same platform and/or assays are used for the most effective interpretation of the data.

Abstract In 2021, the Association for Diagnostics & Laboratory Medicine (ADLM) (formerly the American Association for Clinical Chemistry [AACC]) developed a scientific study that aimed to contribute to the understanding of SARS-CoV-2 immunity during the evolving course of the pandemic. This study was led by a group of expert member volunteers and resulted in survey data from 975 individuals and blood collection from 698 of those participants. This paper describes the formulation and execution of this large-scale scientific study, encompassing best practices and insights gained throughout the endeavor.