anti-B19 IgG Research Articles

Erythema nodosum manifestation of Parvovirus B19-associated reactive arthritis.

Parvovirus B19 virus-mediated viral inflammation and immune-complex deposition generate mainly short-term manifestations in the affected individuals. The objective of this study was to determine Parvovirus B19 infection in rheumatoid arthritis (RA) patients. The study employed 50 patients diagnosed with RA and 30 healthy individuals. Blood samples were collected from both groups. The blood samples were screened for Parvovirus B19 infection using polymerase chain reaction to detect B19 DNA and enzyme-linked immunosorbent assay to detect anti-B19 IgM and IgG. 17 (34%) of 50 patients tested positive for parvovirus B19 DNA. In contrast, the mortality rate in the control group was significantly lower (6.7%; p = 0.005). Anti-B19 IgG antibody levels differed significantly with patients and control (p = 0.007), whereas anti-B19 IgM Ab levels did not (p = 0.6). There was a significant correlation between viremia B19 and all measured parameters. Parvovirus-affected patients had significantly higher CRP and ESR, elevated DAS28 scores, and more joint pain compared to parvovirus (-) patients. Anti-CCP and RF values were significantly high in parvovirus (+) patients. Joint erosion was also prevalent in patients who tested positive for parvovirus. The findings of this study suggest that infection with parvovirus in patients with RA, and a possible role of this viral infection in the pathogenesis of RA may contribute to the pathogenesis of RA.

Relative Quantification for Obtaining the IgG Blood Group Antibodies in Plasma by the Absorption and Elution Test

To establish the absorption and elution test for relatively quantitive obtaining anti-A and anti-B blood group IgG antibodies in the plasma of O-type RhD-positive pregnant women. 95 cases of the O-type RhD-positive pregnant women plasma samples were randomly selected for obtaining the IgG antibodies of anti-A and anti-B blood group, with absorption test under 37 ℃ and elution test under 56 ℃, and the IgG anti-A and anti-B antibody titers of plasma and elution were determined by the microcolumn gel anti-human globulin test. The differences and correlation between the titers of IgG antibodies in the eluent and plasma were compared and analyzed. After a logarithmic transformation (Log2), there was no statistically difference between IgG antibody anti-A difference value and anti-B difference value in the eluent and plasma (P >0.05). The titer of IgG antibody in the eluent was positively correlated with the titer of IgG antibody in the plasma (r =0.914). The linear equation for IgG antibody titers fitted by a scatter plot between the eluent and plasma was Y=-3.55+0.96X. The absorption and elution test can be used to obtain the anti-A and anti-B IgG antibodies in the plasma of O-type RhD-positive pregnant women, whose plasma origin IgG titer is greater than 8. Meanwhile, the acquisition of anti-A antibodies was as effective as anti-B antibodies at the same time, and the antibodies obtained are positive proportional to their respective concentrations in the plasma.

A small proportion of Zebu genetic background in crossbred calves may not be enough to improve resistance against natural bovine Babesia spp. infections

The main objective of cattle breeders in tropical and subtropical regions is to acquire animals with taurine-productive traits adapted to the broad weather range of these regions. However, one of the main challenges on using taurine genetics in these areas is the high susceptibility of these animals to tick-borne diseases. Consequently, the present study evaluated from 10 November 2021–19 April 2022, the over 13 assessments, the Babesia bovis and Babesia bigemina DNA loads and the IgG anti-B. bovis and anti-B. bigemina levels in Angus (n = 17, 100% Taurine) and Ultrablack (n = 14, ∼82% taurine and 18% Zebu) calves. Data were analyzed using a multivariate mixed model with repeated measures of the same animal including the fixed effects of evaluation, genetic group, sex, Babesia spp., and their interactions. The repeatability values were estimated from the (co)variances matrix and expressed for each species. The correlations between the DNA loads (CNlog) and IgG titers (S/P) values for the two species were also estimated using the same model. Regarding the specific IgG antibody titers for both Babesia spp., no significant differences were observed between the two genetic groups. However, for B. bovis and B. bigemina DNA loads, Ultrablack calves presented significantly higher values than Angus calves. Under the conditions evaluated in this study, our findings suggest that the low percentage of Zebu genetic in the Ultrablack breed was insufficient to improve resistance against babesiosis. Further studies must demonstrate if the low percentages of Zebu genetics in Taurine breeds can modify the susceptibility to babesiosis infections.

The Differences in the Binding of IgM and IgG Antibodies with Erythrocytes and Epithelial Cells

Background:: The studies of agglutinating and adsorbing abilities of IgM and IgG antibodies towards the red blood cells (RBCs) and tissue cells are scarce. background: The studies of agglutinating and adsorbing abilities of IgM and IgG antibodies towards the red blood cells (RBCs) and tissue cells are scarce. Objectives:: The study aimed to estimate the differences in the avidity of blood group-specific IgM and IgG antibodies to RBCs and epithelial cells. Methods:: The reaction of hemagglutination, adsorption, mixed agglutination reaction and saliva inhibition test were used. Anti-B 2-54 monoclonal antibody, polyclonal citrated plasma and the heated plasma were used for investigation of IgM and IgG antibodies. Results:: IgM antibodies showed high adsorbing ability to RBCs and epithelial cells in an alkaline medium. On the contrary, the heated plasma containing IgG antibodies showed high adsorbing ability to RBCs and epithelial cells in the acid medium as compared to the alkaline medium. Complete adsorption of IgG antibodies was observed by epithelial cells as compared to erythrocytes. A mixed agglutination reaction confirmed the strong binding of anti-B IgG antibodies with group B epithelial cells in an acid medium. Conclusion:: The binding of polyclonal IgM and IgG group-specific antibodies with red blood cells and epithelial cells depends on the opposite values of pH of the medium. IgG antibodies completely adsorb on epithelial cells contrary to IgM antibodies. Highlights:: Blood group-specific IgG antibodies showed high avidity to epithelial cells as compared to red blood cells. IgG antibodies demonstrated high agglutinating ability in alkaline medium and strong adsorbing ability in acid medium contrary to IgM antibodies, demonstrating high adsorption properties in alkaline medium.

Parvovirus B19 and Parvovirus 4 infections among healthy blood donors; A prevalence report from Iran

BackgroundParvoviruses, characterized by their tropism for blood cells, can manifest as asymptomatic infections. With their ability to persist in blood, assessing the prevalence of Parvovirus B19 (B19V) and Parvovirus 4 (PARV4) among healthy blood donors is essential for evaluating the potential transmission risks through blood transfusions, emphasizing the need for comprehensive screening protocols. MethodsFour hundred blood donors participated in the study, with their blood specimens subjected to Real-Time PCR analysis for B19V and PARV4 nucleic acids after obtaining informed consent. Additionally, Complete Blood Count (CBC) assessments and determination of anti-B19 V-IgM and anti-B19 V-IgG antibody titers were performed using Enzyme-Linked Immunosorbent Assay (ELISA) for all collected samples. ResultsThe results reveal that 12 out of 400 individuals (3 %) exhibited positive results for B19V DNA, while 6 out of 400 individuals (1.5 %) tested positive for PARV4 DNA. Additionally, 8 out of 400 individuals (2 %) displayed positive results for anti-B19V IgM, and 306 out of 400 individuals (76.5 %) exhibited positive results for anti-B19 IgG. Notably, one donation from a donor presenting anti-IgM antibodies was subsequently confirmed as B19V DNA-positive through Real-Time PCR. In the analysis of CBC, a significant disparity in platelet levels was observed between B19V-positive donors, PARV4-positive donors, and B19V-negative donors. ConclusionsThe study suggests that individuals at high risk, lacking detectable B19V antibodies, should undergo systematic screening and exclusion. This precaution is intended to minimize potential contamination risks within the studied cohort, despite the undefined pathogenesis and clinical implications of PARV4.

An in vitro comparison of intra-operative isohemagglutinin and human leukocyte antigen removal techniques in pediatric heart transplantation

Background: Highly sensitized pediatric patients awaiting heart transplantation experience longer wait times and thus higher waitlist mortality. Similarly, children less than 2 years of age have increased waitlist times and mortality when compared to their older peers. To improve the likelihood of successful transplantation in these patients, various strategies have been utilized, including peri-operative plasmapheresis. However, limited data exists comparing plasmapheresis techniques for antibody reduction. This study’s aim was to compare the in vitro magnitude of isohemagglutinin titers (IT) and human leukocyte antigen (HLA) antibody removal and the time required between membrane-based plasmapheresis (MP) and centrifuge-based plasmapheresis (CP) incorporated into the extracorporeal (EC) circuit. Methods: Two MP (Prismaflex) and two CP (Spectra Optia, Terumo BCT) circuits were incorporated into four separate EC circuits primed with high titer, highly sensitized type O donor whole blood. Assays were performed to determine baseline IT and anti-HLA antibodies and then at 30-minute increments until completion of the run (two plasma volume exchanges) at two hours. Results: There was a decrease in anti-A and anti-B IgM and IgG titers with both MP and CP. Mean anti-A and anti-B titer reduction was by 4.625 titers (93.7% change) and 4.375 titers (93.8% change) using MP and CP, respectively. At 2 h of apheresis, CP reduced 62.5% of all ITs to ≤ 1:4, while MP reduced 50% of ITs to ≤ 1:4. Additionally, reduction of anti-HLA class II antibody to mean fluorescence intensity (MFI) <3000 was achieved with both MP and CP. At 2 h of apheresis, CP reduced MFI by 2–3.5 fold and MP reduced MFI by 1.7–2.5 fold. Both demonstrated similar hemolytic and thrombotic profiles. Conclusions: In this in vitro plasmapheresis model of IT and anti-HLA antibody reduction, both MP and CP incorporated into the EC circuit can be used quickly and effectively to reduce circulating antibodies. While CP may have some greater efficiency, further study is necessary to verify this in vivo.

An optimized microplate-based method to evaluate complement-dependent hemolysis mediated by intravenous immunoglobulins (IVIG)

Hemolytic reactions can cause serious complications after administration of Intravenous Immunoglobulin (IVIG), due to passive transfer of anti-A and anti-B IgG antibodies (isoagglutinins). A maximum allowable amount of isoagglutinins is established in the US and EU for licensed IVIG, as measured by a specified direct hemagglutination test (DHAT). Despite this limit, reports of hemolysis have increased over time, raising the question of how well the DHAT predicts clinically significant hemolysis. This study was undertaken to develop a microplate-based complement-dependent hemolysis assay (CDHA) that reproducibly measures functional hemolytic activity of IVIG, for assessment of IVIG products. An IVIG working reference reagent (NIBSC 14/160) was qualified as an assay control and for quantitation purposes. Hemolytic activities of 36 IVIG product lots encompassing seven brands and including 6 clinically hemolytic lots were measured. Hemolytic activity varied among IVIG product brands, and to a lesser extent, from lot-to-lot for individual brands. Correlation between the CDHA and DHAT was not robust which may reflect imprecision of the DHAT method or additional variables that influence complement-dependent hemolysis after opsonization. In conclusion, the CDHA provides a simple, specific, and sensitive tool for IVIG product characterization and investigation of hemolytic events by manufacturers, researchers, and regulatory authorities.

High titers of anti-A1 and anti-B antibodies among Peruvian group O platelet donors

Critical antibody titers have been described as factors associated with hemolysis in ABO plasma-incompatible platelet (PLT) transfusions. This study was carried out to describe the frequency of high-titers anti-A and antiB IgM and IgG antibodies in group O apheresis platelet donors, and to explore differences according to the donor characteristics. A cross-sectional study was carried out at the Blood Bank of a National Hospital in Peru from January to March 2019. IgM and IgG antibodies against A1 and B antigens were quantified in 339 platelet donors using the direct hemagglutination technique and the solid-phase adherence technique, respectively. For analysis purposes, two cut-off points; ≥128 and ≥64, were used to define a critical titer for IgM due to a lack of consensus. An IgG titer of ≥256 was also defined as critical. Of the donors, 22.1 % had critical IgM titers when the cut-off point was defined as ≥128. However, when the IgM cut-off was ≥64, the frequency of platelet donors with critical titers increased to 54.0 %. The frequency of donors with critical IgG titers was 23.5 %. Higher IgG titers were associated with female donors while higher IgM titers were negative associated with age. One in two or three platelet donors, depending on the cutoff point used to define a critical IgM titer, had at least one critical titer of anti-A or anti-B antibodies. Early identification of platelet donors with critical antibody titers could prevent passive transfusion of ABO antibodies to non-isogroup recipients.

From A to AB: A Caucasian Mother with High Anti-B Titer Causing Hemolytic Disease of the Newborn.

We report on a term infant with clinically significant hemolysis and hyperbilirubinemia. Testing revealed ABO incompatibility between maternal type A and infant type AB. The maternal alloantibody screen was negative. The infant's direct antiglobulin test was positive, and anti-B IgG was eluted off the infant's red blood cells (RBCs). Testing of the mother's plasma revealed a high anti-B titer. The infant was successfully treated with phototherapy and intravenous immunoglobulin. The bilirubin and hematocrit stabilized, and the infant was discharged home. This case was unusual because of its severity and unusual ABO constellation. Furthermore, this report is an exemplary educational case study on how effective collaboration between the clinical team and the blood bank laboratory is critical in reaching the correct diagnosis. In summary, the differential diagnosis of more unusual and atypical ABO-incompatible constellations must be considered when an infant presents with unexplained hemolysis.

Development of a Complement-Mediated Hemolysis Assay for Immune Globulin Intravenous Products and Characterization of Lots Associated with Clinical Hemolysis

Introduction: Immune Globulin Intravenous (Human) (IGIV), as known as Intravenous Gamma-Globulin (IVIg), are therapeutic polyclonal immunoglobulin preparations manufactured from pooled human plasma. Initially licensed for replacement therapy of primary immune deficiencies, IGIV indications have expanded to include various autoimmune and inflammatory conditions. Hemolytic reactions associated with IGIV infusion were historically considered as uncommon, but seem to be more frequent over the past 5-6 years, coinciding with widespread manufacturing changes to increase IgG yield, and use of higher dosages. Anti-A and anti-B IgG isoagglutinins in the IGIV preparations are believed to mediate these events. In 2014, FDA co-organized a workshop to discuss strategies to address hemolytic complications of IGIV infusions (Scott 2015). Although US products all comply with an anti-A and anti-B specification of ≤1:64, using the European Pharmacopoeia direct hemagglutination test (DHAT) (Thorp 2010), hemolytic events have persisted. The DHAT is semi-quantitative, imprecise, and does not measure hemolytic function. However, hemolysin tests have been difficult to standardize, especially for highly concentrated IGIV products (Scott 2015).This study reports development of a complement-dependent hemolysis assay (CDHA) suitable for testing IGIV products. We also tested a positive control preparation that is available from NIBSC (code 14/160), consisting of an IGIV lot that caused clinical hemolysis. Finally, we demonstrated differences among IGIV brands with respect to hemolytic activity.Methods: To provide consistent reagents, serum from whole blood donations were frozen as a large volume source of complement and a droplet-freezing method was used to store Red Blood Cells (Schmid 2011; Figure 1). Critical assay conditions were studied, including enzymatic treatment of RBC, removal of unbound IgG, complement source and concentration, divalent cation concentrations, and pH. Hemolytic activities of IGIV products and a positive control IGIV preparation were compared to the hemagglutination results tested using DHAT method. The CDHA was also used to evaluate hemolytic activity of IgG subclasses.Results: The most critical parameters for reproducible CDHA were complement source and preparation method, and assay pH. A pH of 6.5 (0.1M Tri-BIS buffer) was the most suitable condition for all of the tested IGIV analytes. Intra-laboratory variation was minimized by use of frozen RBCs and a consistent highly active complement preparation. The DHAT and CDHA results correlated with each other for most IGIV product lots (Figure 2). Hemolytic activity in IGIV lots varied among, in some cases within, brands. Enrichment of IgG subclass fractions from NIBSC 14/160 showed the highest hemolytic activity was present in IgG2 (Figure 3). In contrast, studies of recombinant anti-A monoclonal antibodies expressing the same anti-A F(ab'), but subclass-specific Fc receptors showed greatest activity of IgG3, consistent with evidence that IgG3 is the most potent complement-activating subclass. The role of IgG2 in hemolysis is not entirely surprising, as saccharide antigens often elicit IgG2 responses, and IgG2 can mediate complement activation (Diebolder 2014). Further subclass studies are planned.Conclusion: Hemolytic activity of IGIV products mediated by anti-A isoagglutinins can be reproducibly measured in IGIV products (Figure 4). Compared to the DHAT method, CDHA provides a continuous readout, is less variable, amenable to use of a standard curve, and more precise. Historical challenges to reproducibility are addressed through biological reagent standardization and attention to critical conditions. Studies comparing the CDHA and DHAT methods across a wider range of products and hemolytic lots will discern whether the CDHA provides a better measure of product safety than the current DHAT lot release assay. The CDHA can be helpful for characterization of new manufacturing methods and products, and investigation of hemolytic events. Studies of hemolysis-implicated lots may provide a basis for improvements in product safety. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Human Parvovirus B19 Infection Among Blood Donors in North-East of Iran

Background: Human parvovirus B19 is a single-stranded DNA and non-enveloped virus which belongs to the Parvoviridae family. The virus spreads through respiratory secretions and perinatal blood. B19 may cause serious complications in patients with cellular immunodeficiency that have no detectable antibodies to B19, such as aplastic crises in solid organs. Objectives: The present study aimed to investigate the prevalence of parvovirus B19 and to evaluate the presence of B19 DNA among blood donors in Golestan Province. Methods: Sera of 400 blood donors in Golestan Province who were negative for HIV, HbsAg, and HCV were tested for both IgG and IgM anti-B19 virus antibodies using the ELISA method, following which all sera were tested for the presence of B19 DNA by semi-nested PCR. Results: 242 (out of 400) subjects (60.5%; 95% CI) were positive for anti-B19 IgG, and 158 were negative for IgG antibody (39.5%; 95% CI). Moreover, all asymptomatic blood donors were negative for anti-B19 IgM. No viral genome was found in serum samples of donors. Conclusions: Since no viral genome was found in the donors' serum samples and the relatively high prevalence of anti HPV- B19 IgG in blood donors, it can be concluded that the possibility of B19 transmission through blood transfusion is low. However, special caution should be taken when transfusing blood to the high-risk groups such as pregnant women, immune-deficient patients, and those with hematological problems, particularly regarding IgG anti- HPV- B19 antibodies (HPV- B19-safe).

Analytical and clinical evaluation of new automated chemiluminescent immunoassays for the detection of IgG and IgM anti-Bartonella henselae antibodies

Serological diagnosis of Bartonella henselae infection mainly rely on microscopic immunofluorescence assays (IFA), which are however time-consuming and poorly standardized. The aim of the study was to assess the use of the new fully automated VirClia® chemiluminescent immunoassays for the detection of IgG and IgM anti-B. henselae antibodies. Eighty-one patients with a well-defined B. henselae infection as well as 80 patients with an alternative disease were included. The VirClia® IgG antibody assay showed a sensitivity of 79.0% and a specificity of 93.8% for the diagnosis of B. henselae infection. For the VirClia® IgM assay, results were more conflicting with a sensitivity of 42.0% and a specificity of 98.2% to predict IFA IgM results. In 11 additional patients with uninterpretable IFA due to autoimmune antibodies, VirClia® assays were able to deliver valuable quantitative results. The VirClia® IgG assay shows good analytical and clinical performances and could be easily integrated in the diagnostic workflow of B. henselae infection.

Prevalence and Quantity of Parvovirus B19 DNA Among Blood Donors from a Regional Blood Center in Turkey

ObjectiveParvovirus B19 causes a range of diseases and morbidity in humans and is transmissible by transfusion of blood, blood components and plasma derivatives. The objective of the study was to investigate the prevalence and quantity of B19 DNA among blood donors. Method: Totally 1053 samples were collected from March to July 2016 at a blood bank for detection of Parvovirus B19 DNA and serological status of blood donors. Testing of the presence of viral DNA was performed by a quantitative real-time PCR with a 101 copies/ml detection limit. All DNA positive and randomly selected 267 samples were tested for the presence of anti-B19 IgM and IgG by ELISA. ResultsAge distribution of donors was between 18-64; mean age was 27 and median was 23. Among the 1053 samples, 5 (0.47%) had PB19 DNA. All PB19 DNA positive donations had both B19 IgM and IgG antibodies. The DNA level for positive donations were between 0.9 × 102 to 3.1 × 104 copies/ml. IgG and IgM were present in 59.9% (160/267) and 0,74% (2/267) respectively among the healthy donors without PB19 DNA. ConclusionDetected DNA concentration was less than 105 copies/ml. The presence of IgM in low level PB19 DNA positive donors may indicate that there might be a risk in transmission of PB19 to particularly immunosuppressed recipients. The clinical follow-up of blood donation with low level of PB19DNA should be considered to answer the questions about blood safety.

A Comparison of Intra-Operative Isohemagglutinin Removal Techniques in Pediatric Heart Transplantation

ABO-incompatible (ABO-I) heart transplantation (HT) is used in children <2 years of age to reduce waitlist times and mortality with outcomes comparable to ABO-compatible HT. This strategy is made safer by reducing isohemagglutinin titers (IT). Limited data exists comparing techniques for IT reduction. This study's aim was to compare the in vitro magnitude of IT removal and time required between membrane-based plasmapheresis (MP) and centrifuge-based plasmapheresis (CP) incorporated into the cardiopulmonary bypass (CPB) circuit. Two MP (Prismaflex) and two CP (Spectra Optia, Terumo BCT) circuits were incorporated into four separate CBP circuits primed with high titer type O donor whole blood. Assays were performed to determine baseline isohemagglutinin titers and then at 30 minute increments until completion of the run (two plasma volume exchanges) at two hours. There was a decrease in anti-A and anti-B IgM and IgG titers with both MP and CP. Mean anti-A and anti-B titer reduction was 4.625 titers (93.7% change) and 4.375 titers (93.8% change) using MP and CP, respectively. At 2 hours of apheresis, CP reduced 62.5% of all ITs to ≤ 1:4, while MP reduced 50% of ITs to ≤1:4. Both demonstrated similar hemolytic and thrombotic profiles. In this in vitro plasmapheresis model of IT reduction, both MP and CP incorporated into the CPB circuit can be used quickly and effectively to reduce circulating IT. While CP may have some greater efficiency, further study is necessary to verify this in vivo.

Evaluation of anti-A and anti-B IgM and anti-AB IgG antibody titers in Group O blood Iraqi donors

BACKGROUND: Blood group O is transfused as a universal blood group in emergencies. The red cells of blood Group O possess no major ABO antigens, while the blood Group O plasma contains naturally occurring IgM anti-A and anti-B and cross-reacting IgG anti-AB antibodies. Thus, the transfusion of blood Group O units may cause marked destruction of the recipients' A or B red cells manifested as severe acute hemolytic transfusion reactions owing to potent ABO hemolytic antibodies.OBJECTIVES: This study was done to estimate the titers of anti-ABO antibodies in Group O blood donors and to suggest a possible scheme to introduce as a routine testing for high ABO antibodies' titers in blood Group O donors in Iraqi blood transfusion centers.SUBJECTS AND METHODS: Two hundred blood Group O Iraqi healthy blood donors (192 males and 8 females, age range: 18–59) were chosen to evaluate the titer of ABO antibodies (IgM and IgG) in their serums using tube test.RESULTS: The prevalence of high-titer anti-A and anti-B IgM antibody at 1:50 dilution in this study was 98.5% and 97.5%, respectively (P < 0.05). The prevalence of high-titer anti-AB IgG measured by dithiothreitol and 2-mercaptoethanol method of at least 50 was 63% and 74%, respectively (P < 0.05).CONCLUSIONS: There is a high prevalence of high-titer anti-A and anti-B IgM and IgG antibodies among blood Group O Iraqi donors. The detection of IgG antibodies is more practical by using dithiothreitol method as it needs much shorter incubation time; however, 2-ME is more sensitive. Detection of high-titer anti-A IgM antibody in dangerous universal donors, by immediate spin at 1:50 dilution, can be used alone in practice, replacing the need to use all portfolio of tests of anti-ABO antibodies.

Prevalence of high titered anti-A and anti-B antibodies among O blood group individuals and their associated factors

Background and Objectives: Severe hemolytic transfusion reactions due to minor incompatibility are usually associated with the so-called “High titred” antibodies in the plasma of donors especially the O group as it is generally regarded as universal donors. Critical titers of 1:64 or higher was considered high titered antibodies for IgM anti-A and anti-B. Critical titers of 1:256 or higher was considered high titered antibodies for IgG anti-A and anti-B. The objective of this study was to see the prevalence of such high titered antibodies among our donor population and to observe for an association if any with the age, gender, and diet type and probiotic intake of the donors. Methodology: This was a cross-sectional analytical study conducted over 20 months from September 2015 to May 2017. About 560 donors were chosen randomly after screening and counseling and titration of anti-A and anti-B antibodies was done by the double dilution tube agglutination technique. The information on age, gender, diet type, and probiotic intake was gathered from the donor history questionnaire and an association between the factors described and the distribution of critical titers of anti-A and anti-B was elicited. Results: In the present study, the prevalence of high titer anti-A and anti-B IgM antibodies was 36% and 32%, respectively. The prevalence of high titer anti-A and anti-B IgG antibodies was 9.6% and 5.5%, respectively. The median IgM antibody titers reduced with the increasing age of the donors starting 18 years. There was a statistically significant association between a vegetarian diet and high titers. Conclusion: There are a considerable proportion of O group donors with high titers of antibodies and hence a routine pretransfusion screening for such antibodies can prevent the development of hemolytic reactions by segregating units into “high titer” and “low titer” antibodies and issuing only low titer products for out of group transfusions.

Next Generation Sequencing-Based Fetal ABO Blood Group Prediction by Analysis of Cell-Free DNA from Maternal Plasma

Introduction: ABO blood group incompatibility between a pregnant woman and her fetus as a cause of morbidity or mortality of the fetus or newborn remains an important, albeit rare, risk. When a pregnant woman has a high level of anti-A or anti-B IgG antibodies, the child may be at risk for hemolytic disease of the fetus and newborn (HDFN). Performing a direct prenatal determination of the fetal ABO blood group can provide valuable clinical information. Objective: Here, we report a next generation sequencing (NGS)-based assay for predicting the prenatal ABO blood group. Materials and Methods: A total of 26 plasma samples from 26 pregnant women were tested from gestational weeks 12 to 35. Of these samples, 20 were clinical samples and 6 were test samples. Extracted cell-free DNA was PCR-amplified using 2 primer sets followed by NGS. NGS data were analyzed by 2 different methods, FASTQ analysis and a grep search, to ensure robust results. The fetal ABO prediction was compared with the known serological infant ABO type, which was available for 19 samples. Results: There was concordance for 19 of 19 predictable samples where the phenotype information was available and when the analysis was done by the 2 methods. For immunized pregnant women (n = 20), the risk of HDFN was predicted for 12 fetuses, and no risk was predicted for 7 fetuses; one result of the clinical samples was indeterminable. Cloning and sequencing revealed a novel variant harboring the same single nucleotide variations as ABO*O.01.24 with an additional c.220C>T substitution. An additional indeterminable result was found among the 6 test samples and was caused by maternal heterozygosity. The 2 indeterminable samples demonstrated limitations to the assay due to hybrid ABO genes or maternal heterozygosity. Conclusions: We pioneered an NGS-based fetal ABO prediction assay based on a cell-free DNA analysis from maternal plasma and demonstrated its application in a small number of samples. Based on the calculations of variant frequencies and ABO*O.01/ABO*O.02 heterozygote frequency, we estimate that we can assign a reliable fetal ABO type in approximately 95% of the forthcoming clinical samples of type O pregnant women. Despite the vast genetic variations underlying the ABO blood groups, many variants are rare, and prenatal ABO prediction is possible and adds valuable early information for the prevention of ABO HDFN.

False-Positive Flow-Cytometric T Cell Crossmatch caused by Blood Type Antigen Expressed on Lymphocytes in ABO Incompatible Kidney Transplantation

Background Flow-cytometric T cell crossmatch (FTXM) uses live donor lymphocytes to detect anti-donor-human leukocyte antigen (HLA) IgG antibodies. Therefore, the results can be affected by individual cell surface molecular expression. Herein we report three cases with FTXM false-positive living-related kidney transplants whose positivity was influenced by the expression of incompatible ABO blood-type antigens. Patients and Methods The kidney recipients were three men with blood type O who received from their wives with blood type B. Their anti-B IgG titers were 1:32, 1:128, and 1:256. Lymphocyte cytotoxic assays were negative in all cases. Median of mean channel shift (MCS) in FTXM was 78.7% (range, 52.8%–85.7%). There were no anti-HLA (either donor-specific or not) class I IgG antibodies in all 3 cases detected by a solid-phase bead assay. We hypothesized that non-HLA antigens expressed on T lymphocytes affect false-positive FTXM results. To examine this, we incubated the patients’ sera with donor-type red blood cells (RBCs) to adsorb anti-ABO antibodies prior to FTXM. Results When the incubated sera were tested in FTXM with donor T lymphocytes, the results were negative in all cases with median MCS 3.75% (0.0%–7.5%). To clarify the expression of ABO antigens on the donor T lymphocytes, we incubated donor T lymphocytes with recipients’ sera and then incubated the cells with acid solution to detach the bound antibodies from the T lymphocytes. The eluates showed agglutination in indirect Coombs test with blood type B RBCs but no agglutination with O blood type RBCs. This indicates that the donor T lymphocytes expressed blood type B antigen and anti-blood type IgG antibodies reacted with the B antigen. These results show that anti-blood type IgG antibodies can bind to ABO-blood type antigens on the surface of T lymphocytes, resulting in positive FTXM. The false-positive FTXM can be excluded by short-term incubation of recipients’ sera with donor-type RBCs. Conclusion In ABO-incompatible kidney transplantation, anti-blood type IgG antibodies can be detected as anti-donor IgG antibodies in FTXM.

Acrodermatitis chronica atrophicans: various faces of the late form of Lyme borreliosis.

IntroductionAcrodermatitis chronica atrophicans (ACA) is probably the most common late and chronic manifestation of the Lyme borreliosis seen in European patients.AimTo analyze epidemiological data, and to investigate the effects of treatment of patients with ACA.Material and methodsNine patients were included in the study. All patients had serological examinations (ELISA and Western blot) and histopathological examination of the skin lesions performed. Eight patients had PCR in the skin biopsy performed.ResultsThe duration of symptoms ranged from 2 months to 2 years. In 7 patients, skin lesions were located on lower limbs, in 2 patients – in a non-typical body area – abdomen. In 1 patient, scleroderma and in 3 patients, diabetes mellitus was diagnosed. Borrelia burgdorferi DNA was detected in 25% of the skin biopsy specimens. IgG anti-B. burgdorferi specific antibodies were present in serum of all patients (confirmed by Western blot). In all cases, the diagnosis was confirmed by histopathological examination. The response to ceftriaxone therapy varied. In 5 cases, the lesions resolved completely, in others they faded.ConclusionsDespite raising awareness of Lyme borreliosis, late forms of the disease such as ACA are still observed. Acrodermatitis chronica atrophicans skin lesions may be located in non-characteristic areas, e.g. abdominal skin. Symptoms are not irritating or painful, therefore patients do not seek medical help. The effect of antibiotic treatment varies.

Minimal variation in anti-A and -B titers among healthy volunteers over time: Implications for the use of out-of-group blood components.

Using potentially out-of-group blood components, like low titer A plasma and O whole blood, in the resuscitation of trauma patients is becoming increasingly popular. However, very little is known whether the donors' anti-A and/or anti-B titers change over time and whether repeated titer measurements on the same donor are required to ensure that each donation produces a low titer product. The anti-A and/or anti-B titers were measured on 56 healthy adult volunteers (47 blood donors; nine blood center personnel) every 3 months for 12 consecutive months using an automated solid phase analyzer. The results were expressed as log2 titer steps (e.g., titer 32 = 5 titer steps). Minor variations in the average anti-A and/or anti-B titers were seen over time; the maximum individual SD in each group was 1.50 (IgG anti-A) or 1.00 (IgM anti-A, IgM, and IgG anti-B). When the SDs for the four titer measurements from all 56 volunteers were combined as appropriate, the highest overall combined SD was 0.47 titer steps for IgG anti-A. This value corresponds to a 95% confidence interval for intraindividual variation in this antibody's titer over 12 months of 0.96 titer steps. Thus, based on one measurement, an IgG anti-A with a titer step of, for example, 6 would be expected to be in the range of titer step 5 to titer step 7 over the course of 1 year with 95% probability. The titers of anti-A and/or anti-B among healthy adults are stable over at least 1 year. This suggests that repeated titer measurements within a year on the same donor are not necessary if donations are made at 3 months or longer intervals. Diagnostic study, level V.