Kupffer cells play a crucial role in monocrotaline-induced liver injury by producing TNF-α.

Monocrotaline (MCT), an unsaturated pyrrolizidine alkaloid (PA) in plants, is mainly toxic to the lung and liver of mammals. As a commonly used tool for liver injury model, the mechanism of MCT hepatoxicity has still not been fully clarified. Kupffer cells (KCs) are the liver-resident macrophages and have various responses to different toxicants and liver damage. However, the role of KCs in MCT-induced liver injury remains controversial. In current work, we investigated the effects of KCs on MCT-induced liver injury, especially on MCT-induced hepatocyte death. KCs were depleted in Balb/c mice by liposome-entrapped clodronate (Lip/Clo) (0.2 mL/mouse, i.p.) or GdCl3 (0.7 mg/kg, i.p.) before MCT administration (300 mg/kg, i.p.), we found that the Lip/Clo group showed higher efficiency in KCs depletion and stronger hepatoprotective effects against MCT. We also found TNF-α was remarkably decreased after KCs depletion, the experiment of administering anti-TNF-α antibody (20 µg/mouse, i.p.) to MCT-treated animals generated the similar results. To further elaborate the function of KCs, we compared the ALT levels released from co-culturing murine hepatic parenchymal cells (HPCs) and RAW264.7 cells with that from HPCs alone. After the treatment of MCT, the released ALT levels in co-culture system were shown to be dependent on the number of RAW264.7 cells, while the anti-TNF-α antibody could suppress it. Finally, we discovered RIPK3/MLKL pathway might be activated by TNF-α released from KCs, and subsequently induced hepatocyte necrosis. Noteworthy, the known mechanisms including ER stress and NF-κB pathways were also found to be involved in the activation of KCs. In conclusion, our study reveals a further mechanism to MCT-induced hepatoxicity mediated directly by KCs via producing TNF-α.

No increase in anti-A isohemagglutinin titer after SARS-CoV-2 infection: A retrospective cohort analysis of group O apheresis platelet donors.

The risk of a hemolytic reaction during the transfusion of ABO non-identical PC is determined by the presence of natural anti-A IgM antibodies, the titer of which may increase after infections. The aim of the study was to evaluate the titer of anti-A isohemagglutinins in platelet concentrate (PC) obtained by apheresis from group O donors who experienced SARS-CoV-2 infection, and to compare the titer before and after infection. A retrospective single-center analysis of 21 PC donors with a previous COVID-19 history was performed. The results showed neither a statistically important increase in the anti-A IgM antibody titers nor a significant correlation between the anti-A IgM antibody level and anti-SARS-CoV-2S1 antibody titer in the donors with an asymptomatic or mild COVID-19. Further population-based studies on anti-A titers are necessary for a comprehensive assessment of this phenomenon.

Pre-transplant donor-type red cell transfusion is a safe and effective strategy to reduce isohemagglutinin titers and prevent donor marrow infusion reactions in major ABO-mismatched transplants.

ABO incompatibility is not a barrier to allogeneic stem cell transplant but may result in acute hemolytic reactions. As stem cell product manipulation is cumbersome, we are reporting the effectiveness and safety of donor-type red cell infusion as a method of reducing acute hemolytic reaction while using marrow as stem cell source. In major ABO-mismatched bone marrow transplants, manipulation of marrow product requires expertise and expensive equipment, which may not be readily available to transplant centers in low- and middle-income regions. The aim behind our study is to report a safe and effective strategy to reduce isohemagglutinin titers and prevent donor marrow infusion reactions in major ABO-mismatched transplants. We retrospectively analyzed 303 consecutive allogeneic bone marrow transplants (BMTs) for beta thalassemia major, between August 2015 and March 2020, with either major (n = 41) or bidirectional (n = 14) mismatches. When isohemagglutinin titers were 1:32 or higher, donor-type packed red blood cell was divided into 4 aliquots, irradiated and administered over 4 days at incremental volumes. Patients were observed for hemolytic reaction, and if no reaction, bone marrow was infused without manipulation. Out of 55 patients, 20 received donor-type blood infusion. Twelve patients showed evidence of mild hemolysis. None developed severe hemolytic or anaphylactic reaction. Titers were rechecked in 14 patients and all had reduction in titers, except for one. Our experience demonstrated that donor-type PRBC infusion is safe and effective in preventing acute hemolysis in major ABO-mismatched stem cell transplants even with bone marrow as graft source.

Daratumumab for delayed RBC engraftment following major ABO mismatched haploidentical bone marrow transplantation.

BACKGROUND: Recent case reports have described the efficacy of daratumumab to treat refractory pure red cell aplasia (PRCA) following major ABO mismatched allogeneic hematopoietic stem cell transplantation (HSCT). In this report, we describe the use of daratumumab as a first-line agent for treatment of delayed red blood cell (RBC) engraftment following a major ABO mismatched pediatric HSCT and provide a review of the literature. STUDY DESIGN AND MATERIALS: We report on a 14-year-old with DOCK8 deficiency who underwent a myeloablative, haploidentical bone marrow transplant from her major ABO mismatched sister (recipient O+, donor A+) for treatment of her primary immunodeficiency. Despite achieving full donor chimerism, she had delayed RBC engraftment requiring ongoing transfusions. Due to iron deposition, symptomatic anemia, and persistence of anti-A iso-hemagglutinins despite discontinuation of immunosuppression, treatment for delayed RBC engraftment with the CD38-targeted monoclonal antibody daratumumab was selected as a less immunosuppressive agent that could more selectively target iso-hemagglutinin producing plasma cells without causing broad B-cell aplasia. RESULTS: Clinical effect with daratumumab was demonstrated by reduced iso-hemagglutinin titer, increased reticulocytosis, normalization of her hemoglobin, and transfusion independence. In the 11-month follow-up period to date, no additional transfusions or immunosuppression have been necessary, despite persistence of low-level anti-A iso-hemagglutinin. CONCLUSION: Our experience suggests that daratumumab was an effective first-line therapy for delayed RBC engraftment and that earlier consideration for daratumumab in treatment of delayed RBC engraftment may be warranted.

A prospective observational study of the incidence, natural history, and risk factors for intravenous immunoglobulin-mediated hemolysis.

BACKGROUND: Intravenous Immune Globulin (IVIG) is used to treat numerous immune-mediated and inflammatory conditions. There is growing awareness of hemolysis, occasionally severe, as a side-effect of this therapy. While most cases are associated with anti-A and/or anti-B isoagglutinins, the frequency and mechanism of hemolysis remain poorly characterized. STUDY DESIGN AND METHODS: A prospective observational study was conducted to determine incidence, natural history and risk factors for IVIG-mediated hemolysis. A total of 99 infusions of high-dose IVIG (2 g/kg or higher) administered to 78 non-group O patients were monitored and graded according to Canadian IVIG Hemolysis Pharmacovigilance Group. Serum ferritin and C3/C4 levels were monitored as indicators of macrophage activation and complement consumption, respectively. Supplementary investigations included assessment for ABO zygosity, Secretor status, FcR polymorphisms, eluate IgG subclass, monocyte monolayer assay, and a panel of cytokines. RESULTS: Hemolysis was observed in 32 of 99 (32%) of infusions, with 19 of 99 (19%) grade 2 or higher. Hemolysis was only apparent 5-10 days after a completed IVIG infusion in 84% of cases and was associated with increases in serum ferritin without complement-consumption. In univariate analysis, increased risk was observed in group AB patients, first-time IVIG recipients, those not taking immuosuppressive medications, or patients treated with a specific IVIG brand; however, in multivariate analysis, product association was no longer observed. No other patient- or practice-related risk factors were identified. CONCLUSION: IVIG-mediated hemolysis is common and frequently severe. Monitoring for 5-10 days following an infusion should be considered in non-O patients receiving high-dose IVIG with known risk factors.

Isoagglutinin removal by plasma centrifugation or filtration (single or double): A prospective study in a single center.

INTRODUCTION: ABO-incompatible (ABOi) kidney transplantation, a well-established procedure, has good long-term results provided pretransplant desensitization that includes immunosuppression and apheresis. OBJECTIVE: To compare, within the first pretransplant apheresis session given to 29 ABOi kidney-transplant candidates, the effect on isoagglutinin titers (both IgG and IgM isotypes) of three modalities: centrifugation therapeutic plasmapheresis (cTP; n = 10), filtration TP (fTP; n = 9), and double-filtration plasmapheresis (DFPP; n = 10). RESULTS: The three groups were comparable according to baseline demographics. Treated plasma volumes were similar across the three groups, that is, 4111 ± 403 mL (cTP), 3861 ± 282 mL (fTP), and 3699 ± 820 mL (DFPP): that is, 54 ± 7, 53 ± 7, and 53 ± 10 mL/kg respectively. One session of centrifugation or filtration TP reduced IgG anti-A/anti-B isoagglutinin titer by ~4, whereas one DFPP session reduced it by ~2. One session of cTP reduced IgM anti-A isoagglutinin titer by a little less than 4, whereas fTP and DFPP sessions reduced it by ~3. There were no statistical differences across the three groups regarding isoagglutinin rebound (IgG and IgM). However, isoagglutinin IgG rebound was >4 dilutions for anti-B titers compared with ~2 dilutions for anti-A titers. The median decreases in IgG level were -3.9 g/L (DFPP), -5.9 g/L (cTP), and - 6.06 g/L (fTP) (p = ns). Median fibrinogen depletions were ~ 60% (fTP), 64% (DFPP), and 76% (cTP). CONCLUSIONS: Isoagglutinin depletions within the first apheresis session were similar across cTP, fTP, and DFPP: this was numerically lower for DFPP.

Identifying correlations between donor demographics and isohemagglutinin titers as a potential method to screen for low-titer group O whole blood.

BACKGROUND: With more hospitals using low-titer group O whole blood in trauma resuscitation, having an efficient screening method for low-titer donors is critical. Our blood center uses an automated screen for high-titer isohemagglutinins in our platelet donations while collecting detailed donor demographic information. Using this data, we can identify key demographics often associated with titer status, thereby helping develop a donor-triaging method for titering. STUDY DESIGN AND METHODS: Titer results were read with an automated microplate system as either high or low, based on agglutination, with a cutoff equivalent to 1:256 (both anti-A and anti-B). Donor demographic data analyzed included date of donation, blood group, age, gender, and ethnicity. RESULTS: 57,508 donations were collected from 2073 unique donors between 2014 and 2018. We found the following demographics to be correlated with titer status: gender, ABO blood group, age, and ethnicity. Variability in titer status was identified in 215 individuals. This represented around 10 % of the total unique donors and was split equally amongst gender. We also found that donors between the ages of 41-60 ha d the highest likelihood of having variability in titer status, peaking at 13 %, and this proportion declined past age 60. CONCLUSION: Titer status is associated with the following donor demographics: gender, ABO type, age, and ethnicity. We also discovered that variability in titer status is correlated with age. In blood centers that do not have automated and routine titer screening procedure, these findings could be used as a method to efficiently identify low-titer donors a-priori.

Establishment of Ph. Eur. Bordetella pertussis mouse antiserum Biological Reference Preparation batches 2, 3 and 4.

A project aimed at establishing replacement batches for the European Pharmacopoeia (Ph. Eur.) Biological Reference Preparation (BRP) Bordetella (B.) pertussis mouse antiserum was started in 2013 under the aegis of the Biological Standardisation Programme (BSP) of the European Directorate for the Quality of Medicines & HealthCare (EDQM). This BRP is used for the immunogenicity assay in mice to assess the potency of acellular pertussis (aP) vaccines as described in Ph. Eur. general method 2.7.16. Assay of pertussis vaccine (acellular). In a preliminary phase of the project (referred to herein as BSP129 phase 1) a hyper-immune serum pool was produced in mice using a combined aP vaccine as immunogen. This pool was used to generate 3 freeze-dried candidate (c) B. pertussis anti-mouse serum BRP batches (cBRP2, cBRP3 and cBRP4). After the pre-qualification that showed their suitability as candidate batches, an international collaborative study (BSP129 phase 2) was carried out in order to standardise these 3 batches against the current BRP1 in terms of anti-PT, -FHA, -PRN and -FIM2/3 antibody contents. For the sake of continuity with the standardisation of BRP1, the corresponding WHO standard (1RR 97/642) was introduced as a second reference for the calibration of the 3 candidate BRPs. Eleven laboratories took part in phase 2. Ten of them performed the ELISA method they use routinely for aP vaccine batch release and one laboratory performed the Multiplex Immunoassay (MIA) as an alternative test. Four participants titrated the antibodies against all 5 pertussis antigens, 5 participants determined the antibody content against 3 antigens (PT, FHA, PRN), one participant titrated the antibodies against PT and FHA antigens and one laboratory determined the antibody content for the PT antigen only. Details of all ELISA methods used were analysed to evaluate their impact on the calibration of the cBRPs. The variability of the results in relation to the nature and methodology of the tests appeared rather limited. Discrepant titres of cBRPs were measured depending on the reference used: the use of the 1RR induced an overestimation (in 8 out of 11 laboratories) and a large inter-laboratory variation in the calculated titres. Regardless of the reference used, equivalency between the calculated titres of cBRP2 and cBRP3 was observed, whilst cBRP4 had systematically lower titres for all antibodies against the 5 acellular pertussis vaccine components. Based on these observations, it was decided to establish the candidate BRP batches against BRP1 and to assign the following potencies based on the mean values determined through centrally calculated results of the calibration assays performed by ELISA in BSP129 phase 2: For cBRP2 and cBRP3 Anti-pertussis toxin: 37 ELISA Units (ELU) per vial Anti-filamentous haemagglutinin: 114 ELU per vial Anti-pertactin: 44 ELU per vial Anti-fimbrial agglutinogens (FIM2/3): 25 ELU per vial For cBRP4 Anti-pertussis toxin: 32 ELU per vial Anti-filamentous haemagglutinin: 98 ELU per vial Anti-pertactin 38 ELU per vial Anti-fimbrial agglutinogens (FIM2/3):23 ELU per vial In February 2018, BRP2, BRP3 and BRP4 were adopted by correspondence by the Ph. Eur. Commission.

Incidentally discovered cold hemagglutinin disease with massive blood clots in the cardioplegia line and coronary artery, during coronary artery bypass graft.

BACKGROUND: Cold hemagglutinin disease (CHAD) is a rare autoimmune disease, in which patients manifest symptoms when the body temperature decreases. It causes critical problems with blood clotting and hemolysis during hypothermia in cardiac surgery. Although various methods are recommended, the CHAD discovered incidentally during cardiac surgery is still a clinical challenge. CASE PRESENTATION: A 76-year-old male visited our hospital for chest pain. Angiography revealed unstable angina, left-main and three-vessel disease. We performed coronary artery bypass graft (CABG) with cardiopulmonary bypass after heparin injection. Shortly after aorta cross-clamping (ACC) and infusion of cold blood cardioplegia, we found massive blood clots in the cardioplegia line. Upon suspicion of CHAD, we raised the temperature and infused warm blood cardioplegia in a retrograde manner. After performing cardiac arrest, we opened the coronary artery and found blood clots in the coronary artery. We eliminated the clots and washed with warm crystalloid cardioplegia simultaneously in an antegrade and retrograde manner. During the ACC, warm cardioplegia was infused every 15 min, via retrograde and antegrade techniques simultaneously. After distal anastomosis of the saphenous venous graft (SVG) to the coronary artery, we performed a direct SVG warm cardioplegia infusion. Finally, before the proximal SVG anastomosis to the aorta, we used warm cardioplegia to eliminate the remaining microemboli. The cold reactive protein test showed a positive result. The patient was discharged without any complications. CONCLUSION: In this rare case, we incidentally discovered CHAD associated with massive blood clots in the cardioplegia line and the coronary artery, during CABG. However, we performed CABG without any complications using a reasonable and appropriate cardioplegia infusion technique, including direct SVG warm cardioplegia infusion.

Analysis of the prevalence of systemic de novo thrombotic microangiopathy after ABO-incompatible kidney transplantation and the associated risk factors.

OBJECTIVES: To analyze the prevalence of systemic de novo thrombotic microangiopathy in ABO-incompatible kidney transplantation and risk factors associated with this condition. METHODS: A total of 201 patients who received living-donor kidney transplantation (114 patients with ABO-identical kidney transplantation and 87 patients with ABO-incompatible kidney transplantation) were retrospectively analyzed. Systemic de novo thrombotic microangiopathy was diagnosed clinically according to the presence of thrombocytopenia with microangiopathic hemolytic anemia and pathological findings of thrombotic microangiopathy. Anti-A and anti-B antibodies were purified from human plasma, and these antibodies' bindings to human kidney were investigated in vitro. RESULTS: ABO-incompatible kidney transplantation was a significant risk factor of systemic de novo thrombotic microangiopathy (odds ratio 55.9, 95% CI 1.8-8.9, P < 0.001) after transplantation. Multivariate logistic regression analysis showed that non-use of mycophenolate mofetil, pretreatment immunoglobulin G antibody titer ≥64-fold and pretransplant immunoglobulin M antibody titer ≥16-fold were significant risk factors for systemic de novo thrombotic microangiopathy in ABO-incompatible kidney transplantation. Microvascular inflammation of 1-h post-transplant biopsy could be observed more frequently in thrombotic microangiopathy patients than in non-thrombotic microangiopathy patients. Anti-A and anti-B antibodies purified from human plasma showed a strong in vitro reaction against human kidney when the antibody titer was ≥16-fold. CONCLUSIONS: Antibody titer should be decreased to ≤16-fold until the day of ABO-incompatible kidney transplantation by desensitization therapy including mycophenolate mofetil. The 1-h biopsy results might help to diagnose systemic de novo thrombotic microangiopathy.

Evaluating safety and cost-effectiveness of platelets stored in additive solution (PAS-F) as a hemolysis risk mitigation strategy.

BACKGROUND: Platelet inventory constraints can result in minor ABO incompatibility and possible hemolysis. The aims of this study were to determine the reduction of isoagglutinin in titers of platelets stored in additive solution (PAS) and compare its safety, efficiency, and cost-effectiveness with full-volume and plasma-reduced platelets. STUDY DESIGN AND METHODS: Isoagglutinin titers were performed in paired whole blood donor samples and apheresis platelets collected in PAS (PAS-PLT) aliquot samples by the tube method. RESULTS: A total of 149 pairs of donor/platelet samples were tested: 75 group O, 59 group A, and 15 group B. For group O donor samples, the median anti-A IgG and IgM were 64 and 16, respectively, and the median anti-B IgG and IgM were 64 and 16, respectively. For group O PAS-PLT samples the mean anti-A IgG and IgM, and anti-B IgG and IgM were 32 and 8, and 16 and 8, respectively. For group A donor samples, the mean anti-B IgG and IgM was 8 in both cases; and both titers decreased to 2 in PAS-PLT. For group B donor samples, mean anti-A IgG and IgM was 16 in both cases; and both titers decreased to 4 in PAS-PLT. PAS-PLT demonstrated a net reduction in cost and improved efficiency when compared to plasma reduction. The use of PAS-PLT resulted in a 40% reduction of allergic transfusion reactions. CONCLUSION: The use of PAS decreases plasma isoagglutinin titers, transfusion reactions, and is cost-effective when compared to routine plasma reduction as a strategy to mitigate hemolysis risk from minor incompatible platelet transfusion.

PAS-C platelets contain less plasma protein, lower anti-A and anti-B titers, and decreased HLA antibody specificities compared to plasma platelets.

BACKGROUND: Platelets (PLTs) collected and stored in PLT additive solution Intersol (PAS-C) are presumed to reduce recipient exposure to donor plasma components; however, the effects of PAS-C on PLT supernatant composition are poorly defined. Therefore, we compared the total protein concentration, isohemagglutinin titers, and HLA antibodies in supernatants of PAS-C PLTs to plasma PLTs. STUDY DESIGN AND METHODS: Apheresis PLTs from group O blood donors were collected into either 100% donor plasma (n = 50) or a mixture of 65% PAS-C/35% donor plasma (n = 50). Within 12 hours of collection, samples of the PLT supernatant were frozen and stored. PLT supernatants were assayed for total protein concentration and anti-A and anti-B titers and screened for HLA antibodies. Samples positive for HLA antibodies were further tested using single-antigen bead assays to determine antibody strength and specificity. RESULTS: Supernatants of PAS-C PLTs had significantly lower total protein concentration and anti-A and anti-B titers compared to plasma PLTs. There was no significant difference in the number of HLA antibody screen-positive PAS-C (3/50) compared to plasma PLT supernatants (2/50); however, the HLA antibody screen-positive supernatants of PAS-C PLTs had fewer HLA specificities (2) compared to those of the plasma PLTs (18). CONCLUSION: Decreased plasma proteins likely underlie lower rates of allergic and febrile, nonhemolytic transfusion reactions from the infusion of PAS-C PLTs. Decreased anti-A and anti-B titers may prevent hemolysis from minor ABO mismatch. Lower HLA antibody specificities may mitigate transfusion-related acute lung injury.

Induction of H7N9-Cross-Reactive Antibody-Dependent Cellular Cytotoxicity Antibodies by Human Seasonal Influenza A Viruses that are Directed Toward the Nucleoprotein.

Antibodies that mediate antibody-dependent cellular cytotoxicity (ADCC) against avian influenza virus subtypes, including H7N9 and H5N1, have been detected in human sera. Using NK cell activation and NK cytotoxicity assays, we compared ADCC-mediating antibodies (ADCC-Abs) in sera collected from healthy infants, children and adults against H7N9 virus-infected cells and recombinant hemagglutinin (HA), neuraminidase (NA), and nucleoprotein (NP) proteins. High titers of ADCC-Abs against H7N9 virus-infected cells were detected in sera from adults and children but not infants. ADCC-Abs titers directed against H7N9 HA or NA proteins. Further analysis showed that ADCC-Abs titers were significantly higher toward H7N9 NP, as compared with H7N9 HA or NA proteins, and correlated strongly with ADCC-Abs titers against H7N9 virus-infected cells. Indeed, ADCC-Abs to NPs of seasonal H1N1 and H3N2 viruses correlated strongly with ADCC-Abs to H7N9 NP, suggesting that seasonal influenza infections and vaccinations may induce these cross-reactive antibodies. Targeting ADCC-Abs to internal proteins may be a potential mechanism of universal vaccine design.

Reduction of Isoagglutinin in Intravenous Immunoglobulin (IVIG) Using Blood Group A- and B-Specific Immunoaffinity Chromatography: Industry-Scale Assessment.

BACKGROUND: Hemolysis, a rare but potentially serious complication of intravenous immunoglobulin (IVIG) therapy, is associated with the presence of antibodies to blood groups A and B (isoagglutinins) in the IVIG product. An immunoaffinity chromatography (IAC) step in the production process could decrease isoagglutinin levels in IVIG. OBJECTIVES: Our objectives were to compare isoagglutinin levels in a large number of IVIG (Privigen®) batches produced with or without IAC and to assess the feasibility of the production process with an IAC step on an industrial scale. METHODS: The IAC column comprised a blend of anti-A and anti-B resins formed by coupling synthetic blood group antigens (A/B-trisaccharides) to a base bead matrix, and was introduced towards the end of the industrial-scale IVIG manufacturing process. Isoagglutinin levels in IVIG were determined by anti-A and anti-B hemagglutinin direct and indirect methods according to the European Pharmacopoeia (Ph. Eur.) and an isoagglutinin flow cytometry assay. IVIG product quality was assessed with respect to the retention of immunoglobulin G (IgG) subclasses, specific antibodies, and removal of IgM using standardized procedures. RESULTS: The IAC step reduced isoagglutinins in IVIG by two to three titer steps compared with lots produced without IAC. The median anti-A and anti-B titers with IAC were 1:8 and 1:4, respectively, when measured by the Ph. Eur. direct method, and 1:2 and <1, respectively, when measured by the Ph. Eur. indirect method. The isoagglutinin flow cytometry assay showed an 87-90 % reduction in isoagglutinins in post-IAC versus pre-IAC fractions. IAC alone reduced anti-A and anti-B of the IgMs isotype by 92.5-97.8 % and 95.4-99.2 %, respectively. Other product quality characteristics were similar with and without IAC. CONCLUSIONS: IAC is an effective method for reducing isoagglutinin levels in IVIG, and it is feasible on an industrial scale.

Use of an Intravascular Warming Catheter during Off-Pump Coronary Artery Bypass Surgery in a Patient with Severe Cold Hemagglutinin Disease.

Cold hemagglutinin disease with broad thermal amplitude and high titers presents challenges in treating cardiac-surgery patients. Careful planning is needed to prevent the activation of cold agglutinins and the agglutination of red blood cells as the patient's temperature drops during surgery. We describe our approach to mitigating cold agglutinin formation in a 77-year-old man with severe cold hemagglutinin disease who underwent off-pump coronary artery bypass surgery without the use of preoperative plasmapheresis. This experience shows that the use of an intravascular warming catheter can maintain normothermia and prevent the activation and subsequent formation of cold agglutinins. To our knowledge, this is the first reported use of this technique in a patient with cold hemagglutinin disease. The chief feature in this approach is the use of optimal thermal maintenance-rather than the more usual decrease in cold-agglutinin content by means of therapeutic plasma exchange.

Donor screening reduces the isoagglutinin titer in immunoglobulin products.

BACKGROUND: Hemolysis reaction is a rare class effect of therapy with intravenously administered human normal immunoglobulin (IVIG). Anti-A/B isoagglutinins (isohemagglutinins) originating from donor plasma are considered a probable risk factor for hemolysis. We hypothesized that screening and exclusion of plasma obtained from donors with high isoagglutinin titers from the manufacturing process would produce a meaningful reduction of anti-A/B isoagglutinin titers of the final IVIG product. STUDY DESIGN AND METHODS: A donor screening method for anti-A isoagglutinins using an automated indirect agglutination test (IAT) in gel cards was developed. Industry-scale donor plasma pools and final IVIG product were prepared according to the manufacturing process of Privigen (human 10% liquid IVIG). Anti-A/B isoagglutinin levels were measured by IAT, direct agglutination test, and a flow cytometry-based assay. RESULTS: Screening of plasma from 705 randomly selected donors identified 6.8% donors with high anti-A isoagglutinin titers in plasma. Exclusion of plasma from these donors resulted in a one-titer-step reduction of anti-A isoagglutinin in laboratory-scale pooled plasma. The same donor screening method applied to industry-scale production resulted in exclusion of 5.1% of donors and produced a one-titer-step reduction of both anti-A and anti-B isoagglutinin titers in the final IVIG product. CONCLUSION: Anti-A/B isoagglutinin titers in IVIG products can be reduced on an industrial scale through implementation of anti-A donor screening, which may lower the risk of hemolysis after IVIG therapy.

ABO-incompatible cardiac transplantation in pediatric patients with high isohemagglutinin titers.

BACKGROUND: ABO-incompatible (ABOi) cardiac transplantation is now used widely in infants with isohemagglutinin titers <1:4, but there is increasing evidence that ABOi transplantation can also be used in children with significantly higher titers. We reviewed our high-titer ABOi transplants and report our results here. METHODS: Patients who underwent ABOi cardiac transplantation from 2000 to 2013 with pre-existing isohemagglutinin titers of ≥1:16 were identified from departmental databases. Outcomes were reviewed using medical and laboratory records. RESULTS: Thirty patients underwent ABOi cardiac transplantation between 2000 and 2013. Twelve (40%) had pre-transplant isohemagglutinin titers of ≥1:16 and were included for further study. Median age was 14.9 (range 9.8 to 107.3) months and median weight was 9.6 (range 7.6 to 25) kg. Five (42%) were male. Pre-transplant diagnosis was cardiomyopathy in 8 of 12 (67%) and congenital heart disease in 4 of 12 (33%). Highest pre-transplant isohemagglutinin titer was 1:256 in 2 patients. Four patients (33%) had early antibody-mediated rejection (AMR), all within 15 days post-transplant. Management included use of rituximab, bortezomib, immunoadsorption and eculizumab. Three patients died but no deaths were associated with high isohemagglutinin titers. CONCLUSIONS: ABOi cardiac transplantation in patients with isohemagglutinin titers ≥1:16 is possible. AMR may occur early and immunoadsorption has proven effective at decreasing antibody titers.

Anti-A and anti-B titers in donor plasma, plasma pools, and immunoglobulin final products.

BACKGROUND: High-dose intravenous immunoglobulin (IVIG) treatments are implicated in hemolytic events in some patients receiving treatment. The passive transfer of IgG anti-A and anti-B agglutinin is thought to play a role in the development of these events. The purpose of this study was to determine the prevalence of high-titer IgG anti-A and anti-B in plasma donors and investigate if there is any advantage of excluding these donors from the donor pool to limit anti-A and anti-B content in IVIG product. STUDY DESIGN AND METHODS: IgG anti-A and anti-B levels were assessed from group O donor plasma, manufacturing IgG plasma pools, and finished IVIG product (Gammagard Liquid). Antibody level in group O donors was also assessed by sex and age for their relative contribution of antibody to the plasma pool. RESULTS: The majority of group O donors (80%) had antibody titers of less than 1000. Of those with titers of at least 1000, theoretical estimates provide further evidence that the effects of high-titer donors are minimal. Antibody levels in plasma pools both during the manufacturing process and from the final IVIG product also support that anti-A and anti-B levels are low. In general, there were more females than males with higher antibody titer levels, with significantly more females than males with anti-A. CONCLUSION: Excluding donors with high anti-A and anti-B titers has minimal impact on the finished IVIG product titers due to ABO antibody neutralization and the dilution factor in the manufacturing pool.