Evaluation of a quantitative PCR-based method for chimerism analysis of Japanese donor/recipient pairs.

Chimerism analysis is a surrogate indicator of graft rejection or relapse after allogeneic hematopoietic stem cell transplantation (HSCT). Although short tandem repeat PCR (STR-PCR) is the usual method, limited sensitivity and technical variability are matters of concern. Quantitative PCR-based methods to detect single nucleotide polymorphisms (SNP-qPCR) are more sensitive, but their informativity and quantitative accuracy are highly variable. For accurate and sensitive chimerism analysis, a set of KMR kits (GenDx, Utrecht, Netherlands), based on detection of insertions/deletions (indels) by qPCR, have been developed. Here, we investigated informativity and validated the accuracy of KMR kits in Japanese donor/recipient pairs and virtual samples of DNA mixtures representative of Japanese genetic diversity. We found that at least one recipient-specific marker among 39 KMR-kit markers was informative in all of 65 Japanese donor/recipient pairs. Moreover, the percentage of recipient chimerism estimated by KMRtrack correlated well with ratios of mixed DNA in virtual samples and with the percentage of chimerism in HSCT recipients estimated by STR-PCR/in-house SNP-qPCR. Moreover, KMRtrack showed better sensitivity with high specificity when compared to STR-PCR to detect recipient chimerism. Chimerism analysis with KMR kits can be a standardized, sensitive, and highly informative method to evaluate the graft status of HSCT recipients.

A newly devised flow cytometric antibody binding assay helps evaluation of dithiothreitol treatment for the inactivation of CD38 on red blood cells.

BACKGROUND AND OBJECTIVES: Anti-CD38 monoclonal antibodies, including daratumumab and isatuximab, often interfere with pretransfusion testing. Dithiothreitol (DTT) treatment of red blood cells (RBCs) negates this interference. However, the optimum DTT concentration and treatment time have not been well defined. Here, we quantified CD38 on RBCs before and after DTT treatment using a flow cytometric antibody binding assay (FABA) to specify the optimum conditions for CD38 inactivation. MATERIALS AND METHODS: For FABA, untreated or DTT-treated RBCs were incubated with fluorescein isothiocyanate-labelled anti-CD38 antibody, in the presence or absence of 100-fold or more excess of unlabelled anti-CD38 antibody, and then analysed by flow cytometry (FCM). Dissociation of CD38-positive and control histograms was determined from the D-value using the Kolmogorov-Smirnov test. The results from FABA were compared with those from conventional FCM, indirect antiglobulin test (IAT) and Western blotting. RESULTS: The results from FABA were more consistent than those from conventional FCM. The D-value was found to be reliable in the analysis of difference between CD38 before and after DTT treatment. Our data showed that 0·0075 mol/l DTT for 30 min is sufficient to inactivate CD38 on RBCs. These results were stable and consistent with the findings from IAT. CONCLUSION: Flow cytometric antibody binding assay is an objective way of evaluating the efficacy of DTT treatment for CD38 on RBCs. This approach allows the detection of a small number of cell surface antigens and will be useful for assessing the various chemical treatments to denature RBC antigens.

Erythrocyte Alloimmunity and Genetic Variance: Results from the Collaborative Study of Alloimmunity to Antigen Diversity in Asian Populations (All ADP).

As an East-Asian international study, we evaluated erythrocyte alloimmunity by gender and history of transfusion or pregnancy. In total, data from more than 1,826,000 patients were analyzed, from whom 26,170 irregular erythrocyte antibodies were detected in 22,653 cases. Antibody frequencies in these cases were as follows: anti-E, 26.8%; anti-Lea, 20.0%; anti-P1, 7.1%; anti-M, 6.4%; anti-Mia, 5.6%; anti-c + E, 5.6%; anti-Leb, 4.6%; anti-D, 2.8%; anti-Fyb, 2.6%; anti-Lea+Leb, 2.5%; anti-Dia, 2.0%; and others. For pregnant patients, anti-D (12.7%) was statistically more frequent. For transfused patients, anti-E (37.3%), anti-c + E (9.5%), anti-C + e (3.3%) and anti-Jka (3.1%) were significantly more frequent.

A Japanese multi-institutional collaborative study of antigen-positive red blood cell (RBC) transfusions in patients with corresponding RBC antibodies.

BACKGROUND AND OBJECTIVES: It is sometimes difficult to obtain antigen-negative red blood cells (RBCs) for patients with antibodies against RBCs. However, the frequency and severity of the adverse reactions have not been well elucidated. Here, we conducted a multi-institutional collaborative study to clarify the background, frequency and clinical significance of antigen-positive RBC transfusions to patients with the respective antibodies. MATERIALS AND METHODS: The survey included the background of patients, antigens on RBCs transfused, total amount of antigen-positive RBCs transfused, results from antibody screen and direct antiglobulin tests, specificity of antibodies, adverse reactions and efficacies. All antibodies were surveyed regardless of their clinical significance. RESULTS: In all, 826 cases containing 878 antibodies were registered from 45 institutions. The main reasons for antigen-positive RBC transfusions included 'negative by indirect antiglobulin test' (39%) and 'detection of warm autoantibodies' (25%). In 23 cases (3% of total), some adverse reactions were observed after antigen-positive RBC transfusion, and 25 antibodies (9 of 119 clinically significant and 16 of 646 insignificant antibodies) were detected. Non-specific warm autoantibodies were detected in 9 cases, anti-E in 5 cases, 2 cases each of anti-Lea , anti-Jra or cold alloantibodies, and 1 case each of anti-Dib , anti-Leb or anti-P1. Other antibodies were detected in 2 further cases. Five (22%) of these 23 cases, who had anti-E (3 cases) or anti-Jra (2 cases), experienced clinically apparent haemolysis. CONCLUSIONS: Adverse reactions, especially haemolysis, were more frequently observed in cases with clinically significant antibodies than those with clinically insignificant antibodies (P < 0·001).

Integrative genome analysis identified the KANNO blood group antigen as prion protein.

BACKGROUND: Anti-KANNO, a broadly reactive RBC alloantibody, is found among some Japanese pregnant women, but the genetic basis of the corresponding antigen remains unclear. STUDY DESIGN AND METHODS: We integrated a statistical approach to identify the coding gene for KANNO antigen by conducting a genome-wide association study (GWAS) on four KANNO-negative individuals and 415 healthy Japanese. We also applied whole-exome sequencing to them and performed a replication study to confirm the identified genome variation using independent 14 KANNO-negative individuals. A monoclonal antibody-specific immobilization of erythrocyte antigens (MAIEA) assay was used to locate KANNO antigen on RBC-specific membrane protein. In vivo and in vitro binding assays of anti-KANNO were further applied to the cells expressing a candidate protein. RESULTS: The GWAS revealed a genome-wide significant association of chromosome 20p13 locus (p = 2.76E-08; odds ratio > 1000 [95% confidence interval = 48-23,674]). The identified single-nucleotide polymorphism located in an intronic region of the prion protein (PRNP) gene. Whole-exome sequencing revealed a missense variant in the PRNP gene (rs1800014, E219K), which is in linkage disequilibrium with the single-nucleotide polymorphism identified in the GWAS. All 18 KANNO-negative individuals possessed the homozygous genotype of the missense variant. The MAIEA assay using anti-KANNO and mouse antihuman prion protein showed a clear difference between KANNO-positive and KANNO-negative RBCs. Anti-KANNO showed direct binding to CHO-K1 cells expressing wild-type PRNP but not to those expressing E219K PRNP. CONCLUSION: We first identified the coding gene of the high-frequency antigen KANNO located in PRNP and the missense variation (E219K) that affects the seropositivity of the KANNO antigen, which were confirmed by PRNP overexpressed cells.

Efficacy of D- red blood cell transfusion and rituximab therapy in autoimmune hemolytic anemia with anti-D and panreactive autoantibodies arising after hematopoietic stem cell transplant.

BACKGROUND: Autoimmune hemolytic anemia (AIHA) is caused by autoantibodies to red blood cells (RBCs), which can be panreactive and/or specific to Rh/other blood group antigens. We report a severe case of AIHA after bone marrow transplantation (BMT) due to autoanti-D triggered by reactivation of Epstein-Barr virus (EBV) infection. A combined strategy of D- RBC transfusion and administration of anti-CD20 monoclonal antibody (MoAb) resolved the hemolysis. CASE REPORT: A 33-year-old male underwent allogeneic BMT from an ABO-identical and HLA-matched unrelated male donor. Five months later, while having mild chronic graft-versus-host disease, he manifested AIHA, with a hemoglobin (Hb) level of 5.1 g/dL on AIHA Day 2 (Posttransplant Day 156) and was refractory to D+ RBCs, with a Hb level of 2.4 g/dL on AIHA Day 6. Anti-D-like autoantibodies (titer 1280, subclass immunoglobulin G1 , monocyte monolayer assay 28.7%) and panreactive (titer 40) were identified. Changing the RBC transfusion strategy to D- increased his Hb level to 6.7 g/dL on Day 10. Administration of anti-CD20 MoAb mitigated EBV-related B-cell proliferation and reduced anti-D autoantibody titer to 320 by Day 16 with normalized Hb concentration after 6 months. CONCLUSION: In severe AIHA, when standard treatment and regular RBC transfusions are ineffective, transfusion of RBCs lacking the target antigen(s) of autoantibodies and administration of anti-CD20 MoAb should be considered.

DOWN'S SYNDROME WITH NEONATAL ALLOIMMUNE THROMBOCYTOPENIA DUE TO HLA-A2 ANTIBODY.

Anti-HLA antibodies reportedly exist in the one third of pregnant women. But few occurrences of neonatal alloimmune thrombocytopenia (NAIT) caused by anti-HLA antibodies have been reported. Here a male baby, who was admitted for low birth weight with Down syndrome (DS), was suffered from thrombocytopenia without transient myeloproliferative disorder (TMD). Positive reactions of HLA-specific antibodies were detected in maternal serum. Cross-matching tests between maternal serum and paternal platelets and lymphocytes were strongly positive. It is most conceivable that the previous pregnancy of the mother induced the production of anti-HLA-A2 antibody, which crossed the placenta and subsequently caused an NAIT in the case presented. This is the first case of DS with NAIT due to anti-HLA antibodies.

Severe immune thrombocytopenia possibly elicited by the anti-influenza viral agent peramivir.

A 44-year-old man whose platelet count had been at the lower limit of the normal range for years visited the urgent care department of our hospital for treatment of a high fever and severe fatigue. The influenza A virus was detected, and the patient therefore received the intravenous antiviral agent, peramivir. One week later, he developed systemic petechial rashes. A peripheral blood examination showed a markedly decreased platelet count (3.0×10(9) cells/L), and the bone marrow findings were compatible with a diagnosis of immune thrombocytopenia (ITP). Furthermore, a drug-induced lymphocyte-stimulating test was positive for peramivir. The thrombocytopenia slowly responded to treatment with oral prednisolone. This case suggests that neuraminidase inhibitors, including peramivir, can elicit or worsen ITP.

Anti-KANNO: a novel alloantibody against a red cell antigen of high frequency.

We encountered a broadly reactive red cell alloantibody in 1991, reacting unlike any other known antibody, and named it anti-KANNO after the first patient. A total of 28 cases of anti-KANNO in the Japanese literature were reviewed. To distinguish KANNO from other antibodies against high-frequency antigens, including anti-JMH, anti-Ch/Rg, and anti-Jr(a), we conducted serologic studies with proteolytic enzyme and chemical treatments, complement sensitization against red cells, and serum neutralization techniques. Reactivity of anti-KANNO against red cells lacking high-frequency antigens and antisera to high-frequency antigens against KANNO cells were tested. Among the 28 patients, 26 were female, of whom 25 had a history of pregnancy. Red cells from patient KANNO were reactive with antisera against antigens of high frequency. Anti-KANNO reacted weakly with all cells known to lack high-frequency antigens. It reacted with 2-aminoethylisothiouronium bromide, so it can be distinguished from anti-JMH. Differences among anti-KANNO, anti-Ch/Rg, and anti-Jr(a) emerged with enzyme-treated cells, complement-sensitized cells, and the addition of normal serum. As yet, there are no reports of hemolytic transfusion reaction or hemolytic disease of the fetus and newborn attributable to anti-KANNO. It appears that anti-KANNO is a newly characterized antibody more likely stimulated by pregnancy than by transfusion and with little or no clinical significance. Further surveillance and investigation of anti-KANNO, its antigen biochemistry, and its genetics are warranted.

Hemolytic disease of the fetus and newborn with late-onset anemia due to anti-M: a case report and review of the Japanese literature.

Hemolytic disease of the fetus and newborn (HDFN) attributed to M/N-incompatibility varies from asymptomatic to lethally hydropic. Case reports are rare, and the clinical significance of anti-M is not completely understood. A challenging case of HDFN due to anti-M prompted an investigation of the Japanese literature, in order to characterize the clinical spectrum of M/N-incompatibility pregnancies in Japan and report results to English-language readers. Japanese reports of HDFN attributed to M/N incompatibility were compiled. Abstracted data include maternal antibody titers at delivery, fetal direct antiglobulin test, hemoglobin, total bilirubin, reticulocyte count at birth, and therapeutic interventions. We investigated characteristics of HDFN due to M/N-incompatible pregnancies in Japan after encountering a case of severe HDFN along with late-onset anemia in an infant born to a woman carrying IgG anti-M with a titer of 1. In total, thirty-three babies with HDFN due to anti-M and one due to anti-N have been reported in Japan since 1975. The median maternal antibody titer was 64 at delivery and was 16 or less in 10 of 34 women (29%). Five of 34 babies (15%) were stillborn or died as neonates. Twenty-one of 29 survivors (72%) had severe hemolytic anemia and/or hydrops fetalis. The reticulocyte count of neonates with anemia stayed below the reference interval. Sixteen (55%) developed late-onset anemia and 14 (48%) were transfused with M-negative RBCs. Significant positive correlation (P < .05) between the hemoglobin value and the reticulocyte count within 4 days of birth was obtained in 16 babies with anti-M HDFN. In the Japanese population, 21 of 34 cases of M/N-incompatible HDFN (72%) have manifested as severe hemolytic anemia and/or hydrops fetalis. Low reticulocyte count in neonates with late-onset anemia is consistent with suppressed erythropoiesis due to anti-M.

CD34+ cell enumeration by flow cytometry: a comparison of systems and methodologies.

CONTEXT: An increasing number of medical centers can collect bone marrow, peripheral blood, or umbilical cord stem cells. Pathology laboratories should accommodate this trend, but investment in additional equipment may be impractical. OBJECTIVES: To compare CD34(+) cell counting results by using 2 widely available flow cytometry systems, with and without the use of a separate hematology analyzer (ie, single-platform versus dual-platform methodologies). DESIGN: Whole blood and peripheral blood stem cell (PBSC) samples were analyzed from 13 healthy allogeneic PBSC donors and 46 autologous PBSC donors with various malignancies. The Cytomics FC500 (Beckman Coulter, Fullerton, California) was compared with the FACSCalibur (BD Biosciences, San Jose, California). Dual-platform CD34(+) cell counting incorporated data from a KX-21 hematology analyzer (Sysmex, Kobe, Japan). RESULTS: Subtle differences in CD34(+) cell counting between 2 systems and 2 methods did not achieve statistical significance. CONCLUSION: Different systems and methods for CD34(+) cell enumeration, properly validated, can support care for patients undergoing transplants and provide meaningful data for multicenter studies or meta-analyses.

Increased detection of clinically significant antibodies and decreased incidence of delayed haemolytic transfusion reaction with the indirect antiglobulin test potentiated by polyethylene glycol compared to albumin: a Japanese study.

BACKGROUND: The indirect antiglobulin test (IAT) can be potentiated by agents such as polyethylene glycol (PEG-IAT) and albumin (Alb-IAT). PEG-IAT is generally regarded as superior to Alb-IAT for the detection of clinically significant red blood cell (RBC) antibodies. However, supporting data come from Caucasian-dominant populations. Non-Caucasian populations should be investigated as well. MATERIAL AND METHODS: In this single-centre, retrospective, sequential study, Alb-IAT was used from 1989 to 1996 (8 years) and PEG-IAT from 1997 to 2008 (12 years). Pre-transfusion RBC alloantibody detection rates and specificity, post-transfusion alloantibody production, and the incidence of delayed haemolytic transfusion reaction were assessed and compared for the two periods. RESULTS: Although overall RBC alloantibody detection rates were comparable, PEG-IAT more frequently detected clinically significant antibodies such as anti-E, anti-Fy(b), and anti-Jk(a), and less frequently detected insignificant antibodies such as anti-Le(b) and anti-P(1). New alloantibodies emerged comparably during the two periods. Delayed haemolytic transfusion reaction was less frequent during the PEG-IAT period (0.30% versus 0.12%, p<0.05). CONCLUSION: PEG-IAT was superior in the detection of clinically significant antibodies, reduced the detection of insignificant antibodies, and prevented delayed haemolytic transfusion reaction better than Alb-IAT among Japanese transfusion recipients in this retrospective survey of limited power.

A noninvasive near infrared system for detection of platelet components contaminated with bacteria.

BACKGROUND: Platelet (PLT) transfusion-associated bacterial sepsis has remained a substantial patient risk, primarily due to lacking effective and point-of-issue measures to detect bacterial contamination. This study describes near infrared (NIR) spectroscopy to examine inoculated PLTs without sampling within a few seconds. STUDY DESIGN AND METHODS: This study evaluated apheresis PLTs inoculated with low concentrations of Bacillus cereus and Staphylococcus epidermidis, comparing with sterile bags. Short-wavelength NIR spectra over the range from 700 to 1100 nm in the transmittance mode were obtained using research (NIRS6500, Foss NIRSystems) and portable (NIRGun, Shizuoka Shibuya Seiki) instruments at 6-hour intervals from 0 to 72 hours after inoculation (HAI). RESULTS: The sensitivity of the NIRS6500 was 100% (43/43) and 98% (50/51) after incubating PLTs inoculated with B. cereus for 42 HAI or more and with S. epidermidis for 54 HAI or more, respectively. Difference spectra calculated by subtracting the NIR spectra of stored PLTs with that of the same PLTs measured at 0 HAI improved the discrimination results compared with conventional second derivative spectra. CONCLUSION: The NIRS6500 system can provide a PLT monitoring system based on difference spectra. The chemical components of PLTs that were influenced by bacterial metabolism seemed to play an important role in the calibration structure. Further studies should examine samples spiked with various species of prevalent bacteria.

Culture-based bacterial detection systems for platelets: the effect of time prior to sampling and duration of incubation required for detection with aerobic culture.

BACKGROUND: Bacterial contamination of platelet (PLT) products occurs at low concentrations requiring a period of incubation for growth to minimize sampling error. Culture-based detection methods also need sufficient incubation time; together these periods may limit the useful life of PLTs. This study characterizes the impact of sampling and detection times with two commercially available bacteria detection products. STUDY DESIGN AND METHODS: Apheresis PLTs inoculated with nine bacterial species at low concentrations were sampled immediately and 24 hours after inoculation. Test results were analyzed after incubation at 16, 20, and 24 hours after sampling with two bacterial detection systems. RESULTS: When sampled immediately after inoculation, two commercially available bacterial detection systems (BacT/ALERT, bioMérieux; and eBDS, Pall Corp.) failed to detect some PLTs inoculated with Staphylococcus epidermidis, Serratia liquefaciens, or Pseudomonas aeruginosa and S. epidermidis, S. liquefaciens, Bacillus cereus, or P. aeruginosa, respectively. The BacT/ALERT was better at 20 hours (p<0.02), but not at 16 or 24 hours for Time 0 sampling. When sampling occurred 24 hours after inoculation, there were no difference between the two systems. CONCLUSION: Results suggest that for either bacteria detection system, holding PLTs for 24 hours before sampling improves the detection sensitivity for PLTs contaminated with low concentrations of bacteria, and longer incubation periods improve detection.