Clinical consequences of the extremely rare anti-PP1Pk isoantibodies in pregnancy: a case series and review of the literature.

BACKGROUND: The absence of the red cell antigens P, P1 and Pk , known as 'p', represents an extremely rare red cell phenotype. Individuals with this phenotype spontaneously form anti-PP1Pk isoantibodies, associated with severe haemolytic transfusion reactions, recurrent spontaneous abortion and haemolytic disease of the fetus and newborn (HDFN). METHODS: We report a series of four successful pregnancies in three women with anti-PP1Pk isoantibodies, one complicated by HDFN, another by intrauterine growth restriction, all managed supportively. We also review the literature regarding the management of pregnancy involving anti-PP1Pk isoimmunization. RESULTS: The literature surrounding anti-PP1Pk in pregnancy is limited to a very small number of case reports. The majority report management with therapeutic plasma exchange (TPE) with or without intravenous immunoglobulin. The relationship between titre and risk of pregnancy loss remains unclear, though a history of recurrent pregnancy loss appears important. Although a positive cord blood direct antiglobulin test is frequently noted, clinically significant HDFN appears uncommon, though possible. CONCLUSION: Early initiation of TPE in high risk patients should be strongly considered. If possible, pregnancies should be managed in a high-risk obstetric or maternal fetal medicine service. The fetus should be monitored closely with interval fetal ultrasound and middle cerebral artery peak systolic volume Doppler to screen for fetal anaemia. Timely sourcing of compatible blood products is likely to be highly challenging, and both directed and autologous donation should be contemplated where appropriate. The International Red Cell Donor Panel may also provide access to compatible products.

Role of complement in alloimmunization and hyperhemolysis.

PURPOSE OF REVIEW: The purpose of this review is to summarize the role of complement in regulating the removal of a target alloantigen following an incompatible red blood cell (RBC) transfusion, the formation of alloantibodies following RBC alloantigen exposure, and the development of hyperhemolysis in patients with sickle cell disease (SCD). RECENT FINDINGS: Recent studies demonstrate that complement can accelerate alloantibody-mediated removal of target alloantigens from the RBC surface following incompatible transfusion. Complement also influences alloantigen availability during developing alloimmune responses and serves as a unique mediator of CD4 T-cell-independent alloantibody formation following RBC alloantigen exposure. Finally, alternative complement pathway activation appears to play a key role in the development of acute hemolytic episodes in patients with SCD, providing a potential druggable target to prevent acute complications in patients with this disease. SUMMARY: Recent studies suggest that complement can regulate a wide variety of processes germane to hematology, from transfusion complications to baseline hemolysis in patients with SCD. As the role of complement in various disease processes becomes more fully understood, the ability to leverage recently developed complement modulating drugs will only continue to enhance providers' ability to favorably intervene in many hematological diseases.

Biological mechanisms implicated in adverse outcomes of sex mismatched transfusions.

Transfusion of red cell concentrates is an essential lifesaving treatment for patients with massive bleeding or red blood cell disorders. However, correlations between blood transfusion from female donors, especially in sex mismatched transfusions, and a risk of mortality has been reported. A systematic understanding of how sex-mismatched transfusion can contribute to negative outcomes is still lacking. Here, we propose that variations in stored red blood cell products from female and male blood donors may be related to different characteristics of subpopulations of RBCs in units. As very little attention has been paid to this topic, the aim of this review is to investigate biological mechanisms implicated in negative outcomes of sex-mismatched transfusion. This review discusses basic hematology differences in the blood from female and male donors. Also, observational studies that linked donor sex with adverse transfusion outcome are reviewed. We present three physiological mechanisms (oxygen delivery, coagulation and microvesiculation) that could be impacted by sex-mismatched transfusions.

Evaluation of Automatic Blood Analyzer as Screening Method in Fetomaternal Hemorrhage.

Screening of fetomaternal hemorrhage (FMH) is essential in management of fetomaternal antigen incompatibilities of blood. The objective in this study was to evaluate the ability of automatic blood analyzer (ABA) to screen FMH, also comparing this method with flow cytometry (FCM). The contents of fetal red blood cells and fetal hemoglobin were evaluated by FCM and ABA, respectively, using both blood samples of male adults laced with umbilical cord blood diluted at 1/10, 1/100, 1/1,000, and 1/10,000, or blood from puerperal women collected within 48 hours following delivery. FCM had better performance (area under curve, AUC = 0.8723) than ABA (AUC = 0.6569) in detecting fetal blood laced with blood from male adults. At a critical level of 0.5%, ABA indicated that 27.5% of puerperal women would have FMH while FCM did not detect FMH. Our results showed that ABA overestimates FMH and disagrees with FCM on indicating puerperal women with FMH. ABA is inadequate for being used to screen for or to measure FMH.

Molecular Patterns Discriminate Accommodation and Subclinical Antibody-mediated Rejection in Kidney Transplantation.

BACKGROUND: Accommodation in ABO-incompatible (ABOi) transplantation and subclinical antibody-mediated rejection in HLA-incompatible (HLAi) transplantation share several morphological similarities. Because the clinical long-term outcomes differ, we hypothesized different molecular processes involved in ABOi transplantation and subclinical antibody-mediated rejection. METHODS: Using Illumina Human HT-12 v4 Expression BeadChips, the whole transcriptome was evaluated based on 3-month protocol C4d+ biopsies in otherwise stable ABOi and HLAi kidney grafts, as well as in C4d-negative HLA-compatible grafts exhibiting normal histological findings. Top differently regulated genes were further validated using real-time quantitative polymerase chain reaction in another patient cohort and complement regulatory proteins by immunohistochemistry. RESULTS: In the case of genes involved in immune response-related biological processes, ABOi and HLAi cohorts had similar transcriptomic profiles to C4d-negative controls. The majority of deregulated genes in the ABOi and HLAi groups consisted of metallothioneins and epithelial transporter genes. Increased expression of epithelial transporters (SLC4A1, SLC4A9, SLC17A3, SLC12A3, and SLC30A2) and class 1 metallothioneins (MT1F, MT1G, and MT1X) in HLAi transplantation was validated by real-time quantitative polymerase chain reaction. In comparison to controls, both incompatible cohorts were characterized by the upregulation of intrarenal complement regulatory genes. CD46 and CD59 transcripts were increased in the ABOi cohort, whereas CD46 solely in HLAi group, and CD59 protein expression was similar in both incompatible groups. CONCLUSIONS: Several epithelial transporters and metallothioneins discriminate subclinical antibody-mediated rejection in HLAi transplantation from accommodation in ABOi transplantation, which suggest different involved downstream mechanisms and increased risk of injury in HLAi settings. Metallothioneins with their antioxidative properties may help to attenuate the inflammation response induced by donor-specific anti-HLA antibody binding.

Multiplex blood group typing by cellular surface plasmon resonance imaging.

BACKGROUND: Blood-group typing of donors and patients is essential to avoid incompatible transfusions. Transfusion of incompatible RBCs may result in alloimmunization complicating future transfusions or in the presence of antibodies in adverse reactions. With more than 300 blood group antigens identified, it is difficult to provide fully compatible blood. Currently, standard practice is to match for the most immunogenic antigens. While the current agglutination-based RBC-typing methods are reliable for testing a selected number of antigens, they are not easily adaptable for high-throughput multiplex blood typing beyond the current standard. STUDY DESIGN AND METHODS: Surface plasmon resonance (SPR) is a label-free method to follow molecular-and, very recently, also cellular-interactions in real time. Demonstration of binding of RBCs to blood group antigen-specific antibodies by SPR has already been achieved. Here, we demonstrate the generation of an SPR array equipped with clinically relevant blood group antibodies (A, B, and Rh blood groups). To validate this method, we blindly compared typing of 946 blood donors with results of current diagnostic agglutination-based methods. RESULTS: RBC typing was achieved by monitoring RBC binding to blood group-specific antibodies on the sensor simultaneously within 5 minutes per sample. Regeneration of the chip was robust, allowing for typing of at least 100 samples. The typing results gave a 100% match with classical serology with all antibodies tested besides anti-E/e monoclonals, which gave inconsistent results due to low antibody specificity. CONCLUSION: This study demonstrates that SPR-based RBC typing for multiple antigens can be realized simultaneously with high-quality antibodies, enabling reduced hands-on time and possibly improving cost efficiency.

Underlying mechanism and specific prevention of hemolysis-induced platelet activation.

Thromboembolic complications significantly impair the outcome of hemolytic disorders. We hypothesized that red cell adenosine diphosphate (ADP) release results in significant platelet activation in hemolysis and that this prothrombotic state can be prevented by inhibition of the ADP P2Y12 receptor. In the current study, we therefore sought to investigate the mechanism and inhibition of hemolysis-induced platelet activation. The expression of activated integrin αIIbß3 was determined by flow cytometry, and platelet aggregation was assessed by multiple electrode platelet aggregometry. We demonstrate platelet activation and increased platelet aggregation by adding hemolytic blood (lysates) to whole blood, similarly to that achieved by the platelet agonist ADP. Enhanced platelet activation and reactivity in the presence of hemolytic blood were significantly abolished by apyrase, which catalyzes ADP degradation, and inhibited by blockade of the platelet ADP P2Y12 receptor with cangrelor. Platelets from patients treated with the ADP P2Y12 receptor antagonist clopidogrel showed a reduced response to lysates compared to platelets from healthy controls without antiplatelet treatment. Further, in vitro blood group ABO incompatibility induced hemolysis and led to increased platelet activation. Finally, "spontaneous" platelet aggregation seen in patients with cold agglutinin disease was completely abolished by cangrelor. In conclusion, hemolysis is associated with increased platelet activation and aggregation due to red cell derived ADP, which can be prevented by ADP receptor blockade.

Passenger lymphocyte syndrome in ABO and Rhesus D minor mismatched liver and kidney transplantation: A prospective analysis.

The increasing demand for solid organs has necessitated the use of ABO and Rhesus (Rh) D minor mismatched transplants. The passenger lymphocyte syndrome (PLS) occurs when donor lymphocytes produce antibodies that react with host red blood cell (RBC) antigens and result in hemolysis. Our aim was to evaluate prospectively the role of PLS in post transplant anemia and hemolysis in ABO and RhD minor mismatched recipients of liver and kidney grafts and to study the association of PLS with donor lymphocyte microchimerism. We examined 11 liver and 10 kidney recipients at Day +15 for anemia, markers of hemolysis, direct antiglobulin test and eluates, and serum RBC antibodies. Microchimerism was determined in peripheral blood lymphocytes by genotyping of simple sequence length polymorphisms encoding short tandem repeats. Immune hemolytic anemia and anti-recipient RBC antibodies were observed in 2 out of 11 liver (18.2%) and 2 out of 10 kidney (20%) transplants. RBC antibody specificity reflected the donor to recipient transplant, with anti-blood group B antibodies identified in 2 cases of O to B and 1 case of A to AB transplants while anti-D antibodies were detected in 1 case of RhD-negative to RhD-positive transplant. Donor microchimerism was found in only 1 patient. In conclusion, passenger lymphocyte mediated hemolysis is frequent in minor mismatched liver and kidney transplantation. Recognizing PLS as a potential cause of post transplant anemia may allow for early diagnosis and management to decrease the morbidity and mortality in some patients.

Pathobiology of transfusion reactions.

Antibody-induced hemolytic transfusion reactions were first described over 300 years ago. Indeed, during its early evolution, transfusion medicine focused almost exclusively on issues in immunohematology to prevent such events. However, despite the best of efforts to avoid them, incompatible transfusions still occur, through both error and an inability to obtain compatible red blood cells for patients who are alloimmunized against multiple antigens. Because transfusing units of incompatible blood is potentially lethal, studies on human volunteers are not ethical. Thus, understanding of hemolytic transfusion reactions is generated through clinical cases, animal models, inference from related human pathologies, or studies using small volumes of transfused red blood cells. Over the past several decades, substantial new knowledge has been accumulated regarding the mechanisms of hemolysis, the metabolism of products of hemolysis, and the effects of both on recipient biology. Using these data sources, this article traces the historical generation of this knowledge and describes recent advances.

Plasma cell-rich rejection accompanied by acute antibody-mediated rejection in a patient with ABO-incompatible kidney transplantation.

We report a case of plasma cell-rich rejection accompanied by acute antibody-mediated rejection in a patient with ABO-incompatible kidney transplantation. A 33-year-old man was admitted for an episode biopsy; he had a serum creatinine (S-Cr) level of 5.7 mg/dL 1 year following primary kidney transplantation. Histological features included two distinct entities: (1) a focal, aggressive tubulointerstitial inflammatory cell (predominantly plasma cells) infiltration with moderate tubulitis; and (2) inflammatory cell infiltration (including neutrophils) in peritubular capillaries. Substantial laboratory examination showed that the patient had donor-specific antibodies for DQ4 and DQ6. Considering both the histological and laboratory findings, we diagnosed him with plasma cell-rich rejection accompanied by acute antibody-mediated rejection. We started 3 days of consecutive steroid pulse therapy three times every 2 weeks for the former and plasma exchange with intravenous immunoglobulin (IVIG) for the latter histological feature. One month after treatment, a second allograft biopsy showed excellent responses to treatment for plasma cell-rich rejection, but moderate, acute antibody-mediated rejection remained. Therefore, we added plasma exchange with IVIG again. After treatment, allograft function was stable, with an S-Cr level of 2.8 mg/dL. This case report demonstrates the difficulty of the diagnosis of, and treatment for, plasma cell-rich rejection accompanied by acute antibody-mediated rejection in a patient with ABO-incompatible kidney transplantation. We also include a review of the related literature.

Pathology consultation on electronic crossmatch.

OBJECTIVES: The full crossmatch is traditionally the final step in compatibility testing, acting as a serologic double check for ABO compatibility and unexpected RBC antibodies. In this review, we discuss the development of electronic crossmatch (EXM), an approach for determining when EXM can be used, and its strengths and weaknesses. METHODS: Because EXM relies on highly sensitive screening assays, antibodies are frequently encountered whose clinical significance must be investigated and interpreted. Our approach is to obtain further history, perform enhanced tube testing, and consider tests of immune reactivity or RBC survival. RESULTS: For those without clinically significant antibodies, we found two alternatives: immediate-spin crossmatch (IS XM) and EXM. IS XM is prone to error related to serologic interference, whereas EXM depends on the accuracy of the sample label, accurate data entry, and informatics to avoid errors. CONCLUSION: EXM is an alternative to the serologic test in patients who have no clinically significant antibodies.

A novel role for C3 in antibody-induced red blood cell clearance and antigen modulation.

Hemolytic transfusion reactions (HTRs) due to incompatible red blood cell (RBC) transfusions are a leading cause of transfusion associated death. Although many transfused incompatible RBCs are cleared, some remain in circulation despite the presence of RBC-specific antibodies, potentially due to "antigen modulation." With a goal of better understanding incompatible RBC clearance, we generated a murine model with RBC-specific expression of a clinically significant human antigen (KEL2) known to be involved in antigen modulation as well as in HTRs. Wild-type (WT) recipients transfused with transgenic KEL2 RBCs generated anti-KEL glycoprotein alloantibodies, which fixed complement, led to intravascular hemolysis, and resulted in decreased levels of KEL2 antigen detectable on cells remaining in circulation. Antigen modulation did not appear to solely reflect removal of RBCs with higher antigen expression, because cells continued to display antigen modulation in the absence of significant clearance. Recipients genetically lacking complement exhibited lesser degrees of incompatible RBC clearance and antigen modulation in comparison with WT or FcγR knock-out (KO) animals, suggesting a role for complement in RBC clearance. In summary, this HTR model may serve as a platform to test strategies to downmodulate antigen and inhibit incompatible RBC clearance, thus potentially mitigating transfusion dangers.

A review of haptoglobin typing methods for disease association study and preventing anaphylactic transfusion reaction.

Haptoglobin, the product of the Hp gene, is a glycoprotein involved in the scavenging of free hemoglobin. Haptoglobin levels increase or decrease in response to various acquired conditions, and they are also influenced by genetic predisposition. There were 2 major alleles, Hp (1) and Hp (2), and 1 minor allele, Hp (del) . Many researchers have attempted to study the haptoglobin types and their association with disease; however, no definitive conclusions have been reached yet. It is reported that patients who are genetically deficient in haptoglobin are at risk of anaphylaxis against blood components containing haptoglobin. Haptoglobin genotypes also affect the reference intervals of haptoglobin levels. Many studies have attempted to establish simple and accurate typing methods. In this paper, we have broadly reviewed several methods for haptoglobin typing-phenotyping, Southern blotting, conventional PCR, real-time PCR, and loop-mediated isothermal amplification. We discuss their characteristics, clinical applications, and limitations. The phenotyping methods are time consuming and labor intensive and not designed to detect patients harboring Hp (del) . The rapid and robust haptoglobin genotyping may help in preventing fatal anaphylactic reactions and in establishing the relationships between the haptoglobin phenotypes and diseases.

Biphasic clearance of incompatible red blood cells through a novel mechanism requiring neither complement nor Fcγ receptors in a murine model.

BACKGROUND: Antibody binding to red blood cells (RBCs) can induce potentially fatal outcomes, including hemolytic transfusion reactions (HTRs), hemolytic disease of the fetus and newborn, and autoimmune hemolytic anemia. The mechanism(s) of RBC destruction following antibody binding is typically thought to require complement activation and/or the involvement of Fcγ receptors (FcγRs). In the current report, we analyzed mechanisms of HTRs during incompatible transfusions of murine RBCs expressing human glycophorin A (hGPA) into mice with anti-hGPA. STUDY DESIGN AND METHODS: C3 and Fcγ receptor knockout, splenectomized, Fcγ receptor blocking antibody-treated, and clodronate-treated mice were passively immunized with anti-hGPA (10F7 or 6A7) and transfused with RBCs expressing the hGPA antigen. Posttransfusion blood and serum were collected and analyzed via flow cytometry and confocal microscopy. RESULTS: This HTR model results in both rapid clearance and cytokine storm. Neither complement nor FcγRs were required for RBC clearance; in contrast, FcγRs were required for cytokine storm. Circulating aggregates of hGPA RBCs were visible during the HTR. Splenectomy and phagocyte depletion by clodronate had no effect on acute RBC clearance; however, incompatible RBCs reentered over 24 hours in clodronate-treated mice. CONCLUSION: These data demonstrate a biphasic HTR, the first phase involving sequestration of incompatible hGPA RBCs and the second phase involving phagocytosis of sequestered RBCs. However, the mechanism(s) of phagocytosis in the second phase required neither C3 nor FcγRs. These findings demonstrate novel mechanistic biology of HTRs.

Toward a prophylaxis against fetal and neonatal alloimmune thrombocytopenia: induction of antibody-mediated immune suppression and prevention of severe clinical complications in a murine model.

BACKGROUND: Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a severe bleeding disorder caused by maternal antibody-mediated destruction of fetal or neonatal platelets (PLTs). Results from our recent large screening study suggest that the pathophysiology of FNAIT is more similar to hemolytic disease of the fetus and newborn (HDFN) than previously thought. Immunization against HPA-1a might therefore be preventable by a prophylactic regimen of inducing antibody-mediated immune suppression (AMIS), which has been documented to be a useful prophylaxis against HDFN. This preclinical proof-of-concept study investigated whether passive administration of anti-ß3 integrin could induce AMIS and thereby prevent clinical complications of FNAIT. STUDY DESIGN AND METHODS: A murine model of FNAIT using ß3 integrin (GPIIIa)-deficient (ß3-/-) mice was employed for this study. AMIS in ß3-/- mice was induced by intravenous administration of human anti-HPA-1a immunoglobulin G or murine anti-ß3 antisera given as prophylaxis after transfusion of HPA-1a-positive human PLTs or murine wild-type PLTs, respectively. RESULTS: AMIS against both human and murine PLT antigens was induced using this prophylactic approach, reducing the amount of maternal PLT antibodies by up to 90%. Neonatal PLT counts were significantly increased and pregnancy outcome was improved in a dose-dependent manner. The incidence of intracranial hemorrhage, miscarriage, and dead-born pups in mice receiving high-dose prophylaxis was reduced to that of normal controls. We also observed that the severity of thrombocytopenia inversely correlated with birth weight. CONCLUSION: This work conceptually proves that prophylactic administration of PLT antibodies induces AMIS and prevents poor pregnancy outcome in FNAIT.

Maternal-fetal blood incompatibility and neuromorphologic anomalies in schizophrenia: Preliminary findings.

Prior research has shown that maternal-fetal Rhesus (Rh) and ABO blood incompatibility increase the risk for schizophrenia. In the present study, the relationship between blood incompatibility and volumes of brain structures previously implicated in schizophrenia was assessed in schizophrenia cases and controls from a large birth cohort. Rh/ABO incompatible cases had significantly reduced cortical gray matter volume compared to compatible cases, a finding which appears to be driven by significant volume reductions in the dorsolateral prefrontal cortex and inferior frontal cortex. Larger hippocampal and putamen volumes were also observed in exposed controls compared to unexposed controls. Although the sample size is small and replications are required, these data suggest that maternal-fetal blood incompatibility may increase the risk for altered brain morphology in both schizophrenia and in controls. The findings also suggest that the larger hippocampal volume in exposed controls may indicate a mechanism of adaptive resilience which diminishes the risk that controls will develop schizophrenia.

Can C4d immunostaining on endomyocardial biopsies be considered a prognostic biomarker in heart transplant recipients?

BACKGROUND: The aim of this study was to assess the significance of positive C4d capillary immunostaining of endomyocardial biopsies and its correlation to clinical outcome in adult heart transplant recipients. METHODS: Nine hundred eighty-five endomyocardial biopsies from 107 heart transplant recipients were evaluated. Immunostaining for detection of intragraft C4d capillary deposition was performed on paraffin-embedded tissue using anti-human C4d polyclonal antibody. RESULTS: Positive staining of C4d was present in 36 patients (34%) and antibody-mediated rejection in eight patients (7%). The patients were subdivided into four groups on the basis of their C4d, circulating antidonor antibodies (donor-specific antibodies [DSAs]), and graft function: group 1=C4d positive, DSA negative, and no graft dysfunction; group 2=C4d positive, DSA positive, and no graft dysfunction; group 3=C4d positive, DSA positive, and signs of graft dysfunction, and group 0 (control)=all negative. An higher mortality risk was found in C4d-positive patients, when compared with negative ones (unadjusted hazard ratios: group 1: 18, group 2: 61, and group 3: 32-fold risk; P<0.0001). CONCLUSIONS: Antibody-mediated rejection is a complex and ongoing phenomenon with different phenotypic features. C4d positive predicts worse prognosis. C4d positive [corrected] and DSA can be used as early mortality predictors in patients without signs of graft dysfunction.

Early humoral-mediated graft injuries in ABO-incompatible kidney transplantation in human beings.

INTRODUCTION: Acute humoral rejection is the most important risk factor for early graft loss in ABO-incompatible (ABO-i) renal transplantation (RTx) and is present from the early period after RTx. However, the characteristics of early humoral-mediated graft injury are pathologically uncertain. OBJECTIVE: To analyze tissue from 10 protocol graft biopsies performed in 10 patients within 30 days post-RTx to clarify the pathologic features of early humoral-mediated graft injuries in ABO-i RTx. METHODS: Pathologic findings were examined using light and electron microscopy and immunofluorescence studies for C4d. Protocol biopsies were performed within 30 days after RTx in the absence of an episode of dysfunction (creatinine concentration 1.21-1.81 mg/dL). RESULTS: The immunofluorescence study demonstrated C4d deposition in peritubular and glomerular capillaries. Acute glomerulitis with infiltration of mononuclear cells and neutrophils was observed in 3 patients. Furthermore, glomerulitis was accompanied by endothelial cell injuries, widening of subendothelial spaces with a double-contoured glomerular basement membrane, and mesangiolysis. CONCLUSION: In ABO-i RTx, early humoral-mediated graft injuries were observed in approximately 30% of patients despite normal graft function. They were characterized by C4d deposition and glomerular capillary injury. These findings suggest that renal glomeruli are the first site of graft injury by anti-A or anti-B blood type antibody with complement activation in ABO-i RTx.