Development of anti-Jk3 associated with silenced Kidd antigen expression and a novel single nucleotide variant of the JK gene.

Anti-Jk3 is a rare alloantibody to a high-prevalence antigen primarily seen in individuals of Polynesian descent and is associated with a handful of well-established variants of the SLC14A1 gene. We report a case of the Jknull phenotype, associated with formation of anti-Jk3, in a patient of non-Polynesian descent. This patient, a 51-year-old woman self-described as of Jamaican and Scottish ancestry, presented to our hospital for oncologic care. The patient's blood sample typed as blood group A, D+. All screening and panel reagent red blood cells showed reactivity, ranging from 2 to 4+; autocontrol and direct antiglobulin test were both negative. Antigen phenotyping revealed Jk(a-b-), leading to suspicion for anti-Jk3, which was subsequently confirmed by our immunohematology reference laboratory. Given her reported familial background, testing of the SLC14A1 gene was performed, revealing that the patient was heterozygous for the single nucleotide variant (SNV) at c.838G>A in exon 8 and therefore carries both JK*01 and JK*02 alleles that encode Jka and Jkb, respectively. However, the patient was found to be heterozygous for several additional SNVs: c.28G>A in exon 3; c.191G>A, c.226G>A, and c.303G>A in exon 4; and c.757T>C in exon 7. The patient's Jk(b-) phenotype can be explained by coinheritance of c.838A with c.191G>A, which defines null allele JK*02N.09. Coinheritance of SNVs c.28G>A and c.838G with rare SNV c.757C that is predicted to cause a non-conservative amino acid change (p.S253P) likely accounts for the complete serologic absence of Jka and the ability to form anti-Jk3 in this case. This finding would represent a new JK*01 null allele. This evaluation illustrates the importance of genetic analysis in identifying the factors preventing a high-prevalence antigen from being expressed, particularly when discovered outside of an expected racial or ethnic group.Anti-Jk3 is a rare alloantibody to a high-prevalence antigen primarily seen in individuals of Polynesian descent and is associated with a handful of well-established variants of the SLC14A1 gene. We report a case of the Jknull phenotype, associated with formation of anti-Jk3, in a patient of non-Polynesian descent. This patient, a 51-year-old woman self-described as of Jamaican and Scottish ancestry, presented to our hospital for oncologic care. The patient's blood sample typed as blood group A, D+. All screening and panel reagent red blood cells showed reactivity, ranging from 2 to 4+; autocontrol and direct antiglobulin test were both negative. Antigen phenotyping revealed Jk(a­b­), leading to suspicion for anti-Jk3, which was subsequently confirmed by our immunohematology reference laboratory. Given her reported familial background, testing of the SLC14A1 gene was performed, revealing that the patient was heterozygous for the single nucleotide variant (SNV) at c.838G>A in exon 8 and therefore carries both JK*01 and JK*02 alleles that encode Jka and Jkb, respectively. However, the patient was found to be heterozygous for several additional SNVs: c.28G>A in exon 3; c.191G>A, c.226G>A, and c.303G>A in exon 4; and c.757T>C in exon 7. The patient's Jk(b­) phenotype can be explained by coinheritance of c.838A with c.191G>A, which defines null allele JK*02N.09. Coinheritance of SNVs c.28G>A and c.838G with rare SNV c.757C that is predicted to cause a non-conservative amino acid change (p.S253P) likely accounts for the complete serologic absence of Jka and the ability to form anti-Jk3 in this case. This finding would represent a new JK*01 null allele. This evaluation illustrates the importance of genetic analysis in identifying the factors preventing a high-prevalence antigen from being expressed, particularly when discovered outside of an expected racial or ethnic group.

Red blood cell alloimmunization and delayed hemolytic transfusion reactions in patients with sickle cell disease.

Red blood cell (RBC) alloimmunization is more common in patients with sickle cell disease (SCD) than in any other studied patient population. The high prevalence of RBC alloimmunization is multi-factorial, likely involving the chronic hemolysis and inflammatory status of SCD itself, the transfusion burden of patients, and the RH genetic diversity of patients and blood donors, among other reasons. Antibody evanescence, or the decrease of RBC alloantibodies below levels detectable by blood bank testing, occurs frequently with fewer than 30% of alloantibodies estimated to be detected by current screening practices. Evanescence increases the likelihood that a patient with SCD will have a delayed hemolytic transfusion reaction upon future RBC exposure, with previously undetected alloantibodies coming roaring back in an anamnestic manner after exposure to the cognate RBC antigen. A subset of patients having delayed hemolytic transfusion reactions go on to experience hyperhemolysis; some but not all cases of hyperhemolysis are associated with previously evanescent RBC alloantibodies. There is an increasing appreciation of the association between RBC alloantibodies and RBC autoantibodies, as well as involvement of the alternative complement pathway in some instances of hyperhemolysis. A case report in this manuscript describes a highly alloimmunized patient with SCD who experiences a delayed hemolytic transfusion reaction with bystander hemolysis due to a previously evanescent, complement binding anti-M RBC alloantibody. Additional studies, including those involving multiple centers and countries, are needed to further understand RBC alloimmunization in patients with SCD and to develop strategies to prevent or mitigate potentially life-threatening hemolytic transfusion reactions.

A multicentre study investigating vital sign changes occurring in complicated and uncomplicated transfusions.

BACKGROUND AND OBJECTIVES: Many hospitals require transfusions to be discontinued when vital signs stray from predetermined ranges, regardless of clinical symptoms. Variations in vital signs may be unrelated to transfusion, however, and needlessly stopping a transfusion may delay medical care while increasing donor exposures and healthcare costs. We hypothesized that a detailed study of vital sign changes associated with transfusion of blood product by component, including those associated with potential reactions (complicated) and those deemed to be uncomplicated, would establish a useful framework of reference for treating clinicians and transfusion services alike. MATERIALS AND METHODS: A retrospective electronic record review of transfusion service and transfusion recipient data was completed on 3852 inpatient transfusion episodes over a 6-month period at four academic tertiary care hospitals across the United States. Vital signs pre- and post-transfusion were recorded by trained clinical research nurses. Serious reactions were adjudicated by a panel of transfusion medicine experts. RESULTS: In both uncomplicated transfusions (n = 3765) and those including an adverse reaction (n = 87), vital sign fluctuations were generally modest. Compared to uncomplicated transfusions, transfusions complicated by febrile reactions were associated with higher pretransfusion temperature and higher pretransfusion pulse rates. Episodes of transfusion circulatory overload were associated with higher pretransfusion respiration rates compared to uncomplicated transfusions. CONCLUSION: Most transfusions are associated with only modest changes in vital signs. Pretransfusion vital signs may be an important yet previously understudied predictor of vital sign changes during transfusion. The optimal role of vital sign assessment during blood transfusion deserves further study.

A patient with oxaliplatin immune-induced syndrome (OIIS) who also developed leucovorin and palonosetron-associated thrombocytopenia.

OBJECTIVES: We report a case of an 83 year old man who developed oxaliplatin immune-induced syndrome (OIIS) after his 19th cycle of FOLFOX (5FU, leucovorin, oxaliplatin). When oxaliplatin was omitted from his next cycle of chemotherapy he continues to show signs of drug-induced immune thrombocytopenia (DITP) and was found to have drug-dependent, platelet-reactive antibodies (DDPA) to leucovorin and palonosetron as well as oxaliplatin. METHODS: The patient was admitted for monitoring but required no transfusions and thrombocytopenia resolved without treatment during his first admission. Drug-dependent antibody testing was performed on his blood by the Blood Center of Wisconsin (Diagnostic Laboratories; Milwaukee, WI). RESULTS: No RBC or platelet IgG or IgM antibodies were detected in the absence of any drugs, but upon addition of palonosetron, leucovorin, or oxaliplatin, the tests became strongly positive for anti-RBC IgG and anti-platelet IgG antibodies. DISCUSSION: Repeated administration of oxaliplatin can result in drug-induced immune thrombocytopenia (DITP) or autoimmune hemolytic anemia (AIHA). This phenomenon has recently been termed OIIS and may additionally include Evan's syndrome or thrombotic microangiopathy (TMA). Here we describe a patient who developed OIIS with drug-dependent, platelet-reactive antibodies (DDPA) to leucovorin and palonosetron. To our knowledge, these two drugs have never been described in the literature as a cause of DDPA. We suggest that OIIS in addition to oxaliplatin-induced thrombocytopenia may be associated with the development of DDPAs to other drugs causing clinically significant thrombocytopenia which is important to recognize and manage with discontinuation of provoking agents.

The impact of vaccination on RBC alloimmunization in a murine model.

Emerging data in animal models and humans suggest that pathogen-associated and damage-associated molecular patterns variably impact RBC alloantibody formation. In this study, we tested the hypothesis that vaccinations may enhance immune responses to transfused RBCs. The Pneumovax23 vaccine decreased the magnitude of anti-KEL alloimmunization in a murine model, whereas the hepB vaccine did not impact the response; RBC transfusion did not alter immune responses to either vaccine. These data highlight the complexities of the intersection of innate and adaptive immunity and suggest that future studies investigating the pathways through which inflammation impacts alloimmunization are warranted.

A novel network analysis tool to identify relationships between disease states and risks for red blood cell alloimmunization.

We hypothesized that diagnoses may be associated with alloantibody 'responder' status and examined associations between disease states and alloimmunization. Patients with ≥1 alloantibody and non-alloimmunized controls were analysed. Pearson's coefficients were calculated to determine associations between alloimmunization and diseases; significant correlations were selected to construct a network. Inflammatory disorders and diseases requiring chronic transfusion support were associated with responder status. Mitigation steps may be considered in patients with these disorders.

Incidence and clinical characteristics of transfusion-associated circulatory overload using an active surveillance algorithm.

BACKGROUND: The concordance of haemovigilance criteria developed for surveillance of transfusion-associated circulatory overload (TACO) with its clinical diagnosis has not been assessed. In a pilot study to evaluate an electronic screening algorithm, we sought to examine TACO incidence and application of haemovigilance criteria in patients with post-transfusion pulmonary oedema. STUDY DESIGN AND METHODS: From June to September 2014, all transfused adult inpatients at four academic hospitals were screened with an algorithm identifying chest radiographs ordered within 12 h of blood component release. Patients with post-transfusion pulmonary oedema underwent case adjudication by an expert panel. TACO incidence was calculated, and clinical characteristics were compared with other causes of post-transfusion pulmonary oedema. RESULTS: Among 4932 transfused patients, there were 3412 algorithm alerts, 50 cases of TACO and 47 other causes of pulmonary oedema. TACO incidence was 1 case per 100 patients transfused. TACO classification based on two sets of haemovigilance criteria (National Healthcare Safety Network and proposed revised International Society for Blood Transfusion) was concordant with expert panel diagnosis in 57% and 54% of reviewed cases, respectively. Although the majority of clinical parameters did not differentiate expert panel adjudicated TACO from other cases, improved oxygenation within 24 h of transfusion did (P = 0·01). CONCLUSIONS: The incidence of TACO was similar to that observed in prior studies utilizing active surveillance. Case classification by haemovigilance criteria was frequently discordant with clinical diagnoses of TACO in patients with post-transfusion pulmonary oedema. Improvements in oxygenation within 24 h of transfusion merit further evaluation in the diagnosis of TACO.

Clinically significant anti-KEL RBC alloantibodies are transferred by breast milk in a murine model.

BACKGROUND AND OBJECTIVES: Fetuses affected by maternal RBC alloantibodies may have prolonged anaemia after birth, leading one to question whether maternal alloantibody transfer may occur outside the placenta. In response to a recent publication describing breast milk transfer of clinically significant amounts of maternal antiplatelet IgA antibodies from mother to nursing infant, we hypothesized that maternal RBC alloantibodies may also be capable of being transferred in breast milk. MATERIALS AND METHODS: The presence and clinical significance of breast milk alloantibody transfer were tested through a series of pregnancy, fostering and transfusion experiments, using a murine model in which transgenic RBCs express the human KEL glycoprotein. RESULTS: Maternal anti-KEL immunoglobulins, induced through transfusion or pregnancy, were detected in the aqueous phase of breast milk. Further, efficient transfer of maternal anti-KEL IgG and IgA to nursing pups was observed in fostering experiments. The breast milk-acquired alloantibodies were clinically significant in wild-type pups in a transfusion setting, binding to 'incompatible' KEL RBCs and leading to premature clearance from the circulation. Although breast milk-acquired alloantibodies also bound to the RBCs of transgenic KEL-positive fostered pups, no anaemia resulted. CONCLUSIONS: Taking these murine data in combination with recently published human data of maternal antiplatelet IgA antibodies in breast milk leading to sequelae in some infants, it is theoretically possible that maternal anti-RBC IgA alloantibodies may also be transferred in human breast milk and may lead to sequelae in some infants under some circumstances.

Measuring the influence of blood component infusion rate on recipient vital signs.

BACKGROUND AND OBJECTIVES: One of the challenges surrounding blood component administration is the determination of an appropriate rate of infusion. There are very few evidence-based guidelines available to guide healthcare providers looking for a 'standard' infusion rate for red blood cells (RBCs), plasma or platelets (PLTs). Our objective was to determine the extent to which blood component infusion rates were associated with changes in transfusion recipient vital signs. MATERIALS AND METHODS: We retrospectively examined records of 3496 component infusions (RBCs, n = 2359; PLTs, n = 478; plasma, n = 659) over a 1-year period at a 362-bed multispecialty hospital. The following data were collected for each transfusion: blood product volume and infusion time, recipient pre- and post-transfusion temperature, blood pressure and pulse rate, and hospital ward where transfusion occurred. RESULTS: Plasma (median 10.4 ml/min) was infused faster than PLTs (median 7.2 ml/min, P < 0.0001) or RBCs (median 2.3 ml/min, P < 0.0001). For all blood components, infusion rates varied based on the hospital unit performing the infusion. No association was found between relatively fast RBC, plasma or PLT infusion rates (>20 ml/min) and clinically significant reported changes in vital signs. CONCLUSIONS: There does not appear to be a strong correlation between infusion rate and significant changes in recipient temperature, blood pressure or pulse rate. Based on these data, a reasonable rate for routine transfusion is 2-3 ml/min for RBCs and 7-10 ml/min for plasma and PLTs. Faster infusion rates (>20 ml/min) likely can be applied with close patient monitoring if there is a more urgent need for transfusion.

Effects of genetic, epigenetic, and environmental factors on alloimmunization to transfused antigens: Current paradigms and future considerations.

Transfused red blood cells, platelets, or coagulation factors have the capacity to induce alloantibodies, which once formed, can be a clinical barrier to future transfusion therapy and/or transplantation. Large observational studies over the last 50 years have characterized some of the general properties of transfusion induced alloimmunization, which appear to vary to a considerable extent from what is generally observed for human responses to other immunogens, such as microbial pathogens and vaccines. Transfused cells and factor only induce immune responses in the minority of recipients. There are data to suggest that differences in the unit may play a role. However, there are clearly differences in recipient biology, as once a recipient makes one antibody they are much more likely to make additional antibodies; indeed, recipients have been categorized as "responder" and "non-responder" by the field. Recent mechanistic studies have begun to define potential causes for such differences in alloimmunization from patient to patient, but much progress needs to be made to understand how, why, and in whom alloimmunization occurs. This review gives a general background on immunology in the context of transfusion, summarizes recent progress in the field, and discusses future directions for exploration. Particular attention is paid to the general concept that the human immune system is melded by the wide range of antigens encountered in our environment, and that the effects of such on the immune system may have a profound effect upon response to transfused cells.

MHC II on transfused murine blood is not required for alloimmunization against MHC I.

BACKGROUND AND OBJECTIVES: Transfusion of allogeneic platelet products can result in antibodies against donor major histocompatibility complex (MHC) I antigens, leading to a refractory state to subsequent platelet transfusions. However, there is disagreement in the field regarding the molecular mechanisms of humoral alloimmunization. One hypothesis states that donor MHC II is a requirement for alloimmunization. However, other studies have suggested that donor MHC I is alone sufficient and MHC II is not required. MATERIALS AND METHODS: We utilized a mouse model of anti-MHC I alloimmunization to transfused blood, which employed donors with a complete deletion of all MHC II genes. BALB/c (H-2(d)) recipients were transfused with blood from either C57BL/6 (H-2(b)) or MHC II null donors on a C57BL/6 background. Anti-MHC I alloimmunization was monitored by indirect immunofluorescence. RESULTS: Recipients of either wild type or MHC II null blood produced equivalent humoral responses against donor MHC I antigens. However, there was variation in the relative amounts of IgG subclasses. CONCLUSION: These data reject the hypothesis that donor MHC II expression is required for alloimmunization to MHC I antigens.

Use of mouse models to study the mechanisms and consequences of RBC clearance.

Mice provide tractable animal models for studying the pathophysiology of various human disorders. This review discusses the use of mouse models for understanding red-blood-cell (RBC) clearance. These models provide important insights into the pathophysiology of various clinically relevant entities, such as autoimmune haemolytic anaemia, haemolytic transfusion reactions, other complications of RBC transfusions and immunomodulation by Rh immune globulin therapy. Mouse models of both antibody- and non-antibody-mediated RBC clearance are reviewed. Approaches for exploring unanswered questions in transfusion medicine using these models are also discussed.

Unexpected familial recurrence in Angelman syndrome.

We report on two instances of familial recurrence of Angelman syndrome which, from pedigree analysis, appear incompatible with currently known mechanisms of inheritance of this disorder. In these two families, deletion-positive Angelman syndrome has recurred in cousins. Several established mechanisms for deletion-positive familial recurrence have been ruled out. In each family, molecular cytogenetic studies show typical chromosome 15 deletions, and DNA methylation analysis verifies the maternal origin of the deleted chromosomes in all four individuals. Since the mothers of the affected individuals in each family are not known to be related, these recurrences appear to be secondary to coincidental, de novo events. This conclusion is consistent with direct and indirect estimates of the population frequency of Angelman syndrome.

Cis and trans interactions between the iab regulatory regions and abdominal-A and abdominal-B in Drosophila melanogaster.

The infra-abdominal (iab) elements in the bithorax complex of Drosophila melanogaster regulate the transcription of the homeotic genes abdominal-A (abd-A) and Abdominal-B (Abd-B) in cis. Here we describe two unusual aspects of regulation by the iab elements, revealed by an analysis of an unexpected complementation between mutations in the Abd-B transcription unit and these regulatory regions. First, we find that iab-6 and iab-7 can regulate Abd-B in trans. This iab trans regulation is insensitive to chromosomal rearrangements that disrupt transvection effects at the nearby Ubx locus. In addition, we show that a transposed Abd-B transcription unit and promoter on the Y chromosome can be activated by iab elements located on the third chromosome. These results suggest that the iab regions can regulate their target promoter located at a distant site in the genome in a manner that is much less dependent on homologue pairing than other transvection effects. The iab regulatory regions may have a very strong affinity for the target promoter, allowing them to interact with each other despite the inhibitory effects of chromosomal rearrangements. Second, by generating abd-A mutations on rearrangement chromosomes that break in the iab-7 region, we show that these breaks induce the iab elements to switch their target promoter from Abd-B to abd-A. These two unusual aspects of iab regulation are related by the iab-7 breakpoint chromosomes that prevent iab elements from acting on Abd-B and allow them to act on abd-A. We propose that the iab-7 breaks prevent both iab trans regulation and target specificity by disrupting a mechanism that targets the iab regions to the Abd-B promoter.

Angelman syndrome in a daughter with del(15) (q11q13) associated with brachycephaly, hearing loss, enlarged foramen magnum, and ataxia in the mother.

We report on a 4-year-old girl with Angelman syndrome who has an apparent de-novo del(15) (q11q13) originating from a maternally derived chromosome. Her mother had severe brachycephaly, sensorineural hearing loss, speech impediment, and mild ataxia. CT brain scans showed an enlarged foramen magnum in the mother and daughter but magnetic resonance imaging (MRI) showed no brainstem abnormality in either. This family demonstrates that some Angelman syndrome cases may be dominantly transmitted with variable expression and associated with abnormal or cytogenetically apparently normal chromosome 15.

Growth of normal human keratinocytes and fibroblasts in serum-free medium is stimulated by acidic and basic fibroblast growth factor.

Keratinocytes and fibroblasts isolated from human neonatal foreskin can be plated and grown through multiple rounds of division in vitro under defined serum-free conditions. We utilized these growth conditions to examine the mitogenic potential of acidic and basic fibroblast growth factor (aFGF and bFGF) on these cells. Our results demonstrate that both aFGF and bFGF can stimulate the proliferation of keratinocytes and fibroblasts. aFGF is a more potent mitogen than bFGF for keratinocytes. In contrast, bFGF appears to be more potent than aFGF in stimulating the growth of fibroblast cultures. Heparin sulfate (10 micrograms/ml) dramatically inhibited the ability of bFGF to stimulate the proliferation of keratinocytes. In comparison, heparin slightly inhibited the stimulatory effect of aFGF and had no effect on epidermal growth factor (EGF) stimulation in keratinocyte cultures. In fibroblast cultures the addition of heparin enhanced the mitogenic effect of aFGF, had a minimal stimulatory effect on the mitogenic activity of bFGF, and had no effect on EGF-stimulated growth. Our results demonstrate that the proliferation in vitro of two normal cell types found in the skin can be influenced by aFGF and bFGF and demonstrate cell-type specific differences in the responsiveness of fibroblasts and keratinocytes to these growth factors and heparin.