Emily Cooley was a highly-regarded medical technologist and morphologist. The “Emily Cooley Lectureship and Award” was established to honor her, in particular, and medical technologists, in general. This article reviews some basic concepts about the “life of a red blood cell” and uses these to discuss the actual and potential consequences that occur in patients following clearance of transfused refrigerator storage-damaged red blood cells by extravascular hemolysis.
red blood cells; transfusion; macrophages; cytokines; non-transferrin bound iron
Background and Purpose
Interleukin-6 (IL6) is a pro-inflammatory cytokine with known auto-regulatory feedback mechanisms. We hypothesized elevated high-sensitivity C-reactive protein (hsCRP) relative to IL6 confers an increased risk of ischemic stroke (IS), and low hsCRP relative to IL6 a decreased risk, for individuals in the prospective, multi-ethnic, population-based Northern Manhattan Study (NOMAS).
Serum hs-CRP and IL6 were measured in NOMAS participants at baseline. We created a trichotomized predictor based on the dominant biomarker in terms of quartiles: hsCRP-dominant; IL6-dominant; and co-dominant groups. Cox proportional hazards models were used to calculate hazard ratios and 95% confidence intervals (HR, 95%CI) for the association between inflammatory biomarker group status and risk of incident IS.
Of 3298 participants, both hsCRP and IL6 were available in 1656 participants (mean follow-up 7.8 years, 113 incident IS). The hsCRP-dominant group had increased risk of IS (adjusted HR 2.62, 95%CI 1.56–4.41) and the IL6-dominant group had decreased risk (adjusted HR 0.38, 95%CI 0.18–0.82), compared to the referent group, after adjusting for potential confounders. Model fit was improved using the inflammation dominant construct, over either biomarker alone.
In this multi-ethnic cohort, when hsCRP quartile was higher than IL6 quartile, IS risk was increased, and conversely when IL6 quartiles were elevated relative to hsCRP, IS risk was decreased. Construct validity requires confirmation in other cohorts.
RBC transfusion is a life-saving therapy, the logistical implementation of which requires RBC storage. However, stored RBCs exhibit substantial donor variability in multiple characteristics, including hemolysis in vitro and RBC recovery in vivo. The basis of donor variability is poorly understood.
Study Design and Methods
We applied a murine model of RBC storage and transfusion to test the hypothesis that genetically distinct inbred strains of mice would demonstrate strain-specific differences in RBC storage. In vivo recoveries were determined by monitoring transfused RBCs over 24 hours. Timed aliquots of stored RBCs were subjected to tandem chromatography/mass spectrometry analysis to elucidate metabolic changes in the RBCs during storage.
Using independent inbred mouse strains as donors, we found substantial strain-specific differences in post-transfusion RBC recovery in vivo following standardized refrigerated storage in vitro. Poor post-transfusion RBC recovery correlated with reproducible metabolic variations in the stored RBC units, including increased lipid peroxidation, decreased levels of multiple natural antioxidants, and accumulation of cytidine. Strain-dependent differences were also observed in eicosanoid generation (i.e. prostaglandins and leukotrienes).
These findings provide the first evidence of strain-specific metabolomic differences following refrigerated storage of murine RBCs. They also provide the first definitive biochemical evidence for strain specific variation of eicosanoid generation during RBC storage. The molecules described that correlate with RBC storage quality, and their associated biochemical pathways, suggest multiple causal hypotheses that can be tested regarding predicting the quality of RBC units prior to transfusion and developing methods of improved RBC storage.
The hallmark of glucose-6-phosphate dehydrogenase (G6PD) deficiency is red blood cell (RBC) destruction in response to oxidative stress. Patients requiring RBC transfusions may simultaneously receive oxidative medications or have concurrent infections, both of which can induce hemolysis in G6PD-deficient RBCs. Although it is not routine practice to screen healthy blood donors for G6PD deficiency, case reports identified transfusion of G6PD-deficient RBCs as causing hemolysis and other adverse events. In addition, some patient populations may be more at risk for complications associated with transfusions of G6PD-deficient RBCs because they receive RBCs from donors who are more likely to have G6PD deficiency. This review discusses G6PD deficiency, its importance in transfusion medicine, changes in the RBC antioxidant system (of which G6PD is essential) during refrigerated storage, and mechanisms of hemolysis. In addition, as yet unanswered questions that could be addressed by translational and clinical studies are identified and discussed.
glucose-6-phosphate dehydrogenase deficiency; transfusion; oxidative stress; sickle cell disease; hemolysis
Although mucin-type O-glycans are critical for Cryptosporidium infection, the enzymes catalyzing their synthesis have not been studied. Here, we report four UDP N-acetyl-α-d-galactosamine:polypeptide N-acetylgalactosaminyl transferases (ppGalNAc-Ts) from the genomes of C. parvum, C. hominis and C. muris. All are Type II membrane proteins which include a cytoplasmic tail, a transmembrane domain, a stem region, a glycosyltransferase family 2 domain and a C-terminal ricin B lectin domain. All are expressed during C. parvum infection in vitro, with Cp-ppGalNAc-T1 and -T4 expressed at 24 h and Cp-ppGalNAc-T2 and -T3 at 48 and 72 h post-infection, suggesting that their expression may be developmentally regulated. C. parvum sporozoite lysates display ppGalNAc-T enzymatic activity against non-glycosylated and pre-glycosylated peptides suggesting that they contain enzymes capable of glycosylating both types of substrates. The importance of mucin-type O-glycans in Cryptosporidium–host cell interactions raises the possibility that Cp-ppGalNAc-Ts may serve as targets for intervention in cryptosporidiosis.
O-glycosylation; Mucin; Mucin-like glycoprotein; Cryptosporidium; UDP GalNAc:polypeptide; N-acetylgalactosaminyl transferase
A subgroup of the cholesterol-dependent cytolysin (CDC) family of pore-forming toxins (PFTs) has an unusually narrow host range due to a requirement for binding to human CD59 (hCD59), a glycosylphosphatidylinositol (GPI)-linked complement regulatory molecule. hCD59-specific CDCs are produced by several organisms that inhabit human mucosal surfaces and can act as pathogens, including Gardnerella vaginalis and Streptococcus intermedius. The consequences and potential selective advantages of such PFT host limitation have remained unknown. Here, we demonstrate that, in addition to species restriction, PFT ligation of hCD59 triggers a previously unrecognized pathway for programmed necrosis in primary erythrocytes (red blood cells [RBCs]) from humans and transgenic mice expressing hCD59. Because they lack nuclei and mitochondria, RBCs have typically been thought to possess limited capacity to undergo programmed cell death. RBC programmed necrosis shares key molecular factors with nucleated cell necroptosis, including dependence on Fas/FasL signaling and RIP1 phosphorylation, necrosome assembly, and restriction by caspase-8. Death due to programmed necrosis in RBCs is executed by acid sphingomyelinase-dependent ceramide formation, NADPH oxidase- and iron-dependent reactive oxygen species formation, and glycolytic formation of advanced glycation end products. Bacterial PFTs that are hCD59 independent do not induce RBC programmed necrosis. RBC programmed necrosis is biochemically distinct from eryptosis, the only other known programmed cell death pathway in mature RBCs. Importantly, RBC programmed necrosis enhances the growth of PFT-producing pathogens during exposure to primary RBCs, consistent with a role for such signaling in microbial growth and pathogenesis.
In this work, we provide the first description of a new form of programmed cell death in erythrocytes (RBCs) that occurs as a consequence of cellular attack by human-specific bacterial toxins. By defining a new RBC death pathway that shares important components with necroptosis, a programmed necrosis module that occurs in nucleated cells, these findings expand our understanding of RBC biology and RBC-pathogen interactions. In addition, our work provides a link between cholesterol-dependent cytolysin (CDC) host restriction and promotion of bacterial growth in the presence of RBCs, which may provide a selective advantage to human-associated bacterial strains that elaborate such toxins and a potential explanation for the narrowing of host range observed in this toxin family.
Objective: to study the association between soluble tumour necrosis factor receptor 1 (sTNFR1) levels and mortality in the population-based Northern Manhattan Study (NOMAS).
Methods: NOMAS is a multi-ethnic, community-based cohort study with mean 8.4 years of follow-up. sTNFR1 was measured using ELISA. Cox proportional hazards models were used to calculate hazard ratios and 95% confidence intervals (HR, 95% CI) for the association of sTNFR1 with risk of all-cause mortality after adjusting for relevant confounders.
Results: sTNFR1 measurements were available in 1,862 participants (mean age 69.2 ± 10.2 years) with 512 all-cause deaths. Median sTNFR1 was 2.28 ng/ml. Those with sTNFR1 levels in the highest quartile (Q4), compared with those with sTNFR1 in the lowest quartile (Q1), were at an increased risk of all-cause mortality (adjusted HR: 1.8, 95% CI: 1.4–2.4) and non-vascular mortality (adjusted HR: 2.5, 95% CI: 1.5–3.6), but not vascular mortality (adjusted HR: 1.3, 95% CI: 0.9–1.9). There were interactions between sTNFR1 quartiles and medical insurance-status [likelihood ratio test (LRT) with 3 degrees of freedom, Pinteraction = 0.02] and alcohol consumption (LRT with 3 degrees of freedom, Pinteraction < 0.01) for all-cause mortality. In participants with no insurance or Medicaid, those with sTNFR1 in the top quartile had nearly a threefold increased risk of total mortality than the lowest quartile (adjusted HR: 2.9, 95% CI: 1.9–4.4).
Conclusion: in this multi-ethnic cohort, sTNFR1 was associated with all-cause and non-vascular mortality, particularly among those of a lower socioeconomic status.
inflammation; insurance status; mortality risk; alcohol use; older people
Glucose-6-phosphate dehydrogenase (G6PD) deficiency is characterized by red blood cell (RBC) destruction in response to oxidative stress. Although blood donors are not routinely screened for G6PD deficiency, the transfusion of stored G6PD-deficient RBCs may have serious adverse outcomes. By measuring G6PD enzyme activity of RBC units from a large metropolitan hospital transfusion service we sought to determine 1) the prevalence of G6PD-deficient RBC units, 2) if G6PD activity changes during storage, and 3) if G6PD activity in segments correlates with its activity in the bags.
Study Design and Methods
Quantitative G6PD activity was measured in 301 randomly selected packed RBC (pRBC) units and 73 D+C-E- (i.e. R0r or R0R0) pRBC units, all stored in additive solutions. G6PD deficiency was defined as activity <60% of the normal mean.
The frequency of G6PD-deficient units in the general inventory was 0.3% (1/301) [95% CI <0.01%–2.1%]. In contrast, its frequency in D+C-E- pRBC units was 12.3% (9/73) [95% CI 6.4%–22.0%]. G6PD activity did not significantly change during the 42 day storage period, and G6PD activity measured in pRBC storage bags and attached segments correlated well (r=0.7–0.9, p≤0.001, Spearman rank correlation).
Although the frequency of G6PD-deficient pRBC units in the transfusion service general inventory was relatively low, it was significantly higher among a subset of R0r or R0R0 units. The latter are preferentially allocated for transfusion to patients with sickle cell disease to decrease the risk of RBC alloimmunization, possibly allowing more of these units to be inadvertently targeted to these patients.
glucose-6-phosphate dehydrogenase deficiency; hemolysis; oxidative stress; sickle cell disease
Red blood cell (RBC) alloimmunization can be a serious complication of blood transfusion, but factors influencing the development of alloantibodies are only partially understood. Within FDA-approved time limits, RBCs are generally transfused without regard to length of storage. However, recent studies have raised concerns that RBCs stored for more than 14 days have altered biologic properties that may affect medical outcomes. To test the hypothesis that storage time alters RBC immunogenicity, we utilized a murine model of RBC storage and alloimmunization.
STUDY DESIGN AND METHODS
Blood from transgenic HOD donor mice, which express a model antigen (hen egg lysozyme [HEL]) specifically on RBCs, was filter leukoreduced and stored for 14 days under conditions similar to those used for human RBCs. Fresh or 14-day-stored RBCs were transfused into wild-type recipients. The stability of the HOD antigen and post-transfusion RBC survival were analyzed by flow cytometry. RBC alloimmunization was monitored by measuring circulating anti-HEL immunoglobulin levels.
Transfusion of 14-day-stored, leukoreduced HOD RBCs resulted in 10- to 100-fold higher levels of anti-HEL alloantibodies as detected by enzyme-linked immunosorbent assay than transfusion of freshly collected, leukoreduced RBCs. RBC expression of the HOD antigen was stable during storage.
These findings demonstrate that HOD murine RBCs become more immunogenic with storage and generate the rationale for clinical trials to test if the same phenomenon is observed in humans. Length of storage of RBCs may represent a previously unappreciated variable in whether or not a transfusion recipient becomes alloimmunized.
Increased rates of RBC alloimmunization in patients with sickle cell disease may be due to transfusion frequency, genetic predisposition, or immune dysregulation. To test the hypothesis that sickle cell pathophysiology influences RBC alloimmunization, we utilized two transgenic mouse models of sickle cell disease.
Study Design and Methods
Transgenic sickle mice, which express human α and βS globin, were transfused with fresh or 14-day stored RBCs containing the HOD (hen egg lysozyme, ovalbumin, and human Duffyb) antigen; some recipients were inflamed with poly (I:C) prior to transfusion. Anti-HOD alloantibody responses were subsequently measured by ELISA and flow crossmatch; a cohort of recipients had post-transfusion serum cytokines measured by bead array.
Both Berkeley and Townes homozygous (SS) and heterozygous (AS) mice had similar rates and magnitude of anti-HOD RBC alloimmunization following fresh HOD RBC transfusion compared with control animals; under no tested condition did homozygous SS recipients make higher levels of alloantibodies than control animals. Unexpectedly, homozygous SS recipients had blunted cytokine responses and lower levels of anti-HOD alloantibodies following transfusion of 14-day stored RBCs, compared with control animals.
In sum, homozygous βS expression and the ensuing disease state are not alone sufficient to enhance RBC alloimmunization to transfused HOD RBCs in 2 distinct humanized murine models of sickle cell disease under the conditions examined. These data suggest other factors may contribute to the high rates of RBC alloimmunization observed in humans with sickle cell disease.
red blood cells (sickle cell disease); alloimmunization; transfusion medicine
Although a subset of recent studies have suggested red blood cell (RBC) storage length is associated with adverse patient outcomes, others have shown no such relationship. Adults may be transfused with RBC units of different storage lengths, and existing studies do not take into consideration that fresh RBCs may alter responses to concurrently transfused stored RBCs. To test this possibility, we utilized a murine model and investigated transfusion outcomes of fresh, stored, or fresh plus stored RBCs.
Study Design and Methods
Fresh, 14-day stored, or fresh plus 14-day stored leukoreduced RBCs from HOD transgenic donors (with RBC specific expression of hen egg lysozyme, ovalbumin, and human Duffyb) were transfused into naïve C57BL/6 recipients. Serum cytokines and anti-HOD alloimmunization were evaluated following transfusion.
In 6 of 6 experiments (n=90 mice total), a pro-inflammatory serum cytokine storm of interleukin-6, keratinocyte-derived chemokine/CXCL1, and monocyte chemoattractant protein-1 was observed in transfusion recipients of stored but not fresh RBCs, along with high degrees of anti-HOD alloimmunization. However, concurrent transfusion of fresh HOD RBCs along with stored HOD RBCs significantly decreased these adverse outcomes (p<0.05).
These results are consistent with fresh murine HOD RBCs losing protective properties during storage, and introduce a previously unrecognized variable in RBC storage studies. If translatable to humans, uniform “old blood” groups may be needed in future clinical studies to most accurately investigate the biological effects of older RBC units.
14-day stored RBCs containing an RBC specific transgenic antigen (HOD) induce a recipient pro-inflammatory cytokine storm and are significantly more immunogenic compared to fresh RBCs. Given that recipient mice clear transfused stored RBCs more rapidly than fresh RBCs, we hypothesized that rapid RBC clearance was associated with adverse transfusion outcomes.
Study Design and Methods
HOD RBCs were treated by two distinct methodologies known to lead to rapid post-transfusion RBC clearance: phenylhydrazine or heat. HOD antigen expression was analyzed on the treated cells prior to transfusion, and RBC recovery, recipient cytokine response, and recipient anti-HOD alloimmunization response were measured post-transfusion.
Phenylhydrazine and heat treatment each led to near complete RBC clearance in recipients by 24 hours post-transfusion, without significantly altering HOD antigen expression on the transfused RBCs. Recipients of phenylhydrazine or heat-treated RBCs had elevated circulating levels of KC/CXCL-1, MCP-1, and IL-6 following transfusion. Furthermore, phenylhydrazine or heat treated RBCs were significantly more immunogenic than control RBCs, with a mean 25.1-fold enhancement and 10.3 fold enhancement, respectively, of anti-HOD alloimmunization magnitude by flow cytometric crossmatch.
Three separate insults to RBCs (storage, phenylhydrazine, or heat treatment) result in rapid post-transfusion clearance, with a recipient pro-inflammatory cytokine storm and enhanced alloimmunogenicity. These data are consistent with the hypothesis that rapid clearance of RBCs is causally involved in these outcomes, and suggest that human donor RBCs with favorable post-transfusion clearance profiles may be less immunogenic.
post-transfusion clearance; alloimmunogenicity; RBCs
To assess the accuracy of novel and traditional biomarkers in patients with suspected appendicitis as a function of duration of symptoms.
This was a prospective cohort study, conducted in a tertiary care emergency department (ED). The authors enrolled children 3 to 18 years old with acute abdominal pain of less than 96 hours, and measured serum levels of Interleukin-6 (IL-6), Interleukin-8 (IL-8), C - reactive protein (CRP), white blood cell count (WBC), and absolute neutrophil count (ANC). Final diagnosis was determined by histopathology or telephone follow-up. Trends in biomarker levels were examined based on duration of abdominal pain. The accuracy of biomarkers was assessed with receiver operating characteristic (ROC) curves. Optimal cut-points and test performance characteristics were calculated for each biomarker.
Of 280 patients enrolled, the median age was 11.3 years (IQR 8.6 to 14.8), 57% were male, and 33% had appendicitis. Median IL-6, median CRP, mean WBC, and mean ANC differed significantly (p < 0.001) between patients with non-perforated appendicitis and those without appendicitis; median IL-8 levels did not differ between groups. In non-perforated appendicitis, median IL-6, WBC, and ANC levels were maximal at less than 24 hrs of pain, while CRP peaked between 24 and 48 hours. In perforated appendicitis, median IL-8 levels were highest by 24 hours, WBC and IL-6 by 24 to 48 hours, and CRP after 48 hours of pain. The WBC appeared to be the most useful marker to predict appendicitis in those with fewer than 24 or more than 48 hours of pain, while CRP was the most useful in those with 24 to 48 hours of pain.
In this population, the serum levels and accuracy of novel and traditional biomarkers varies in relation to duration of abdominal pain. IL-6 shows promise as a novel biomarker to identify children with appendicitis.
Hemolytic transfusion reactions (HTRs) can produce serious and potentially life-threatening complications in sickle cell disease (SCD) patients; however, the mechanisms underlying these complications remain undetermined. We established a model of alloimmune, IgG-mediated HTRs in a well-characterized humanized murine model of SCD. HTRs induced acute vaso-occlusive crisis (VOC), resulting in shortened survival of SCD mice. Acute VOC was associated with elevated circulating inflammatory chemokine levels, including striking elevation of the levels of the neutrophil chemoattractant CXCL1. Recombinant CXCL1 administration was sufficient to induce acute VOC in SCD mice, characterized by leukocyte recruitment in venules, capture of circulating red blood cells, reduction of venular flow, and shortened survival. In contrast, blockade of the CXCL1 receptor, CXCR2, prevented HTR-elicited acute VOC and prolonged survival in SCD mice. These results indicate that CXCL1 is a key inflammatory mediator of acute VOC in SCD mice. Targeted inhibition of CXCL1 and/or CXCR2 may therefore represent a new therapeutic approach for acute VOC in SCD patients.
Storage of RBCs is necessary for an adequate blood supply. However, reports have identified potential negative sequelae of transfusing stored RBCs. An animal model would be useful to investigate the pathophysiology of transfusing stored RBCs. However, it has been reported that storage of rat RBCs in CPDA-1 resulted in an unexpected sudden decline in post-transfusion survival. We developed a mouse model of RBC storage and transfusion to assess survival kinetics of mouse RBCs.
Study Design and Methods
RBCs expressing green fluorescent protein were collected in CPDA-1, filter leukoreduced, adjusted to a 75% hematocrit, and stored at 4°C. At weekly intervals, stored RBCs were transfused into C57BL/6 recipients. RBC survival was measured by flow cytometry and 51Chromium labeling. Phosphatidylserine externalization and CD47 expression was also evaluated.
Mean 24-hour survival of transfused RBCs was 99%, 91%, 64%, 54%, 30%, and 18% following 0, 7, 14, 21, 28, and 35 days of storage, respectively. Stored RBCs showed an initial rapid clearance with subsequent extended survival. Increased surface phosphatidylserine and decreased CD47 expression was also observed.
Mouse RBCs showed a progressive decline in survival, as a function of storage time, unlike the precipitous loss of viability previously reported for rat RBCs. Moreover, changes in the measured surface markers were analogous to trends reported for human RBCs. Together, these findings provide an initial characterization of a novel mouse model of RBC storage with the potential to serve as an experimental platform for studying the pathophysiological consequences of transfusing stored RBCs.
The overall burden of prior infections may contribute to atherosclerosis and stroke risk. We hypothesized that serological evidence of common infections would be associated with carotid plaque thickness in a multi-ethnic cohort.
Antibody titers to five common infectious microorganisms (i.e. Chlamydia pneumoniae, Helicobacter pylori, cytomegalovirus, and herpesvirus 1 and 2) were measured among stroke-free community participants, and a weighted index of infectious burden (IB) was calculated based on Cox models previously derived from for the association of each infection with stroke risk. High-resolution carotid duplex Doppler studies were used to assess maximum carotid plaque thickness (MCPT). Weighted least squares regression was used to measure the association between IB and MCPT after adjusting for other risk factors.
Serological results for all five infectious organisms were available in 861 participants with MCPT measurements available (mean age 67.2+/−9.6 yrs). Each individual infection was associated with stroke risk after adjusting for other risk factors. The IB index (n=861) had a mean of 1.00 ± standard deviation 0.35, median 1.08. Plaque was present in 52% of participants (mean 0.90+/−1.04 mm). IB was associated with MCPT (adjusted increase in MCPT 0.09 mm, 95% confidence interval 0.03–0.15 mm, per standard deviation increase of IB).
A quantitative weighted index of infectious burden, derived from the magnitude of association of individual infections with stroke, was associated with carotid plaque thickness in this multi-ethnic cohort. These results lend support to the notion that past or chronic exposure to common infections, perhaps by exacerbating inflammation, contributes to atherosclerosis. Future studies are needed to confirm this hypothesis and to define optimal measures of infectious burden as a vascular risk factor.
Purpose of review
Hemolytic transfusion reactions (HTRs) are potentially fatal complications of blood transfusions. Many studies, primarily performed in vitro, have provided a great deal of insight into the initiating events of HTRs; however, it is not clear how they are modulated and how they combine to lead to one or more of the final common pathways. Recently developed mouse HTR models now make it possible to enhance our understanding of the pathogenesis of HTRs; this will allow for the rational design of specific therapies to prevent or ameliorate this serious complication in transfusion medicine.
Mouse models support the hypothesis that “cytokine storm” plays an important role in the pathogenesis of HTRs. Nitric oxide and endothelial cell dysfunction are also implicated in the pathophysiology of these reactions. In addition, the intriguing phenomenon of “antigen loss,” where antigen crosslinking by alloantibody leads to antigen removal rather than RBC clearance, has been modeled and explored. Finally, these mouse models were used to evaluate new therapeutic targets employing complement receptor 1 peptide homologues and the anti-macrophage agent, liposomal clodronate.
Models of HTRs are valuable for gaining a better understanding of the pathophysiology of these potentially fatal complications of blood transfusion. The participation of various inflammatory mediators was shown to play a role in these reactions in vivo. This knowledge will lead to novel treatment options.
Hemolysis; Cytokines; Antigen loss; Hemolytic transfusion reaction
Classical anaphylaxis is the most severe, and potentially fatal, type of allergic reaction, manifested by hypotension, bronchoconstriction, and vascular permeability. Similarly, a hemolytic transfusion reaction (HTR) is the most feared consequence of blood transfusion. Evidence for the existence of an alternative, IgG-mediated pathway of anaphylaxis may be relevant for explaining the pathophysiology of IgG-mediated-HTRs. The purpose of this review is to summarize the evidence for this alternative pathway of anaphylaxis and to present the hypothesis that an IgG-mediated HTR is one example of this type of anaphylaxis.
hemolytic transfusion reaction; anaphylaxis; platelet activating factor