Ralph R. Vassallo

A critical comparison of platelet preparation methods.

Purpose of reviewPlatelet concentrates may be prepared from whole blood or by plateletpheresis. Currently, the non-evidence-based preponderance of apheresis units in the United States and the 50: 50 ratio in Europe may not optimize the gifts of whole-blood donors or minimize healthcare costs. Post-storage pooled, whole-blood-derived platelets, on the other hand, do not provide the convenience of or an equivalent level of safety as apheresis platelets. Recent findingsSome data suggest that different methods of manufacture of whole-blood-derived platelets (platelet-rich plasma or buffy coat intermediate steps) result in differing degrees of platelet activation, which may impact on the quality of stored concentrates. Recent studies have observed superior radiolabel recovery and post-transfusion increments for platelets derived from apheresis compared with platelet-rich plasma whole-blood-derived platelets. A pre-storage pooling system for whole-blood-derived platelets has just been licensed in the USA, and may eventually combine the benefits of apheresis-derived and whole-blood-derived platelets. The advantages of the European method of manufacture of buffy coat whole-blood-derived platelet concentrate have convinced the Canadian Blood Services to abandon platelet-rich-plasma-derived concentrates. SummaryWe present a literature-based review of the relative merits of apheresis-derived and whole-blood-derived platelets. Additional studies are needed in order to define the optimal proportion of the platelet supply from apheresis collections and the choice of whole-blood-derived production method for US blood providers.

It's time to phase in RHD genotyping for patients with a serologic weak D phenotype

In 2014, the College of American Pathologists (CAP) Transfusion Medicine Resource Committee (TMRC) reported the results of a survey of more than 3100 laboratories concerning their policies and procedures for testing serological weak D phenotypes and administration of Rh immune globulin (RhIG).1 Among the findings of this survey is the observation that there is a lack of standard practice in the United States for interpreting the RhD type when a serological weak D phenotype is detected. In some laboratories, an individual with a serological weak D phenotype, especially if a blood donor, is interpreted to be RhD-positive. In the same or other laboratories, especially if a serological weak D phenotype is detected in a female of child-bearing potential, the individual is likely to be managed as RhD-negative for transfusions and, if pregnant, considered a candidate for RhIG. Also, the performance characteristics of serological typing methods for RhD vary. For patients, including pregnant women, the majority of laboratories have policies and procedures that do not use the indirect antiglobulin (weak D) test, thereby avoiding detection of a serological weak D phenotype so that the RhD type will be interpreted to be RhD-negative. Other laboratories typically perform a weak D test for the same category of patients. For blood donors and newborns, it is standard practice for laboratories to have policies and procedures for RhD typing to ensure that serological weak D phenotypes are detected and interpreted as RhD-positive.1 n nThe goal of these RhD typing practices is to protect RhD-negative persons from inadvertent alloimmunization to the D antigen by exposure to RhD-positive RBCs, including RBCs expressing a serological weak D phenotype. Although there has not been a recent prospective study in the United States, it is estimated that current RhD typing practice, together with contemporary obstetrical practices for administration of antepartum and postpartum RhIG, is 98.4 to 99 percent successful in preventing RhD alloimmunization and RhD hemolytic disease of the fetus/newborn.2 However, there are unwarranted consequences associated with the practice of not determining the RHD genotype of persons with a serological weak D phenotype, including unnecessary injections of RhIG and transfusion of RhD-negative RBCs -- always in short supply -- when RhD-positive RBCs could be transfused safely. n nCAP’s TMRC reviewed the current status of RHD genotyping and proposed that selective integration of RHD genotyping in laboratory practices could improve the accuracy of RhD typing results, reduce unnecessary administration of RhIG in women with a serological weak D phenotype, and decrease unnecessary transfusion of RhD-negative RBCs to recipients with a serological weak D phenotype.1 In response to the findings of the CAP TMRC survey, AABB and CAP convened a Work Group on RHD Genotyping and charged it with developing recommendations to clarify clinical issues related to RhD typing in persons with a serological weak D phenotype. As an initial step for formulating recommendations, the Work Group reviewed the current state of molecular science of RHD, including more than 140 publications covering background;1-12 D variants with anti-D;13-29 molecular basis of serological weak D phenotypes;30-92 reviews, editorials and commentaries;93-129 technical resources;130-142 and standards and guidelines.143-149 This Commentary summarizes the proceedings and recommendations of the Work Group.

Guidance on platelet transfusion for patients with hypoproliferative thrombocytopenia.

Patients with hypoproliferative thrombocytopenia are at an increased risk for hemorrhage and alloimmunization to platelets. Updated guidance for optimizing platelet transfusion therapy is needed as data from recent pivotal trials have the potential to change practice. This guideline, developed by a large international panel using a systematic search strategy and standardized methods to develop recommendations, incorporates recent trials not available when previous guidelines were developed. We found that prophylactic platelet transfusion for platelet counts less than or equal to 10 × 10(9)/L is the optimal approach to decrease the risk of hemorrhage for patients requiring chemotherapy or undergoing allogeneic or autologous transplantation. A low dose of platelets (1.41 × 10(11)/m2) is hemostatically as effective as higher dose of platelets but requires more frequent platelet transfusions suggesting that low-dose platelets may be used in hospitalized patients. For outpatients, a median dose (2.4 × 10(11)/m2) may be more cost-effective to prevent clinic visits only to receive a transfusion. In terms of platelet products, whole blood-derived platelet concentrates can be used interchangeably with apheresis platelets, and ABO-compatible platelet should be given to improve platelet increments and decrease the rate of refractoriness to platelet transfusion. For RhD-negative female children or women of child-bearing potential who have received RhD-positive platelets, Rh immunoglobulin should probably be given to prevent immunization to the RhD antigen. Providing platelet support for the alloimmunized refractory patients with ABO-matched and HLA-selected or crossmatched products is of some benefit, yet the degree of benefit needs to be assessed in the era of leukoreduction.

Financial implications of RHD genotyping of pregnant women with a serologic weak D phenotype

Hemolytic disease of the fetus and newborn, classically caused by maternal–fetal incompatibility of the Rh blood group D antigen, can be prevented by RhIG prophylaxis. While prophylactic practices for pregnant women with serologic weak D phenotypes vary widely, RHD genotyping could provide clear guidance for management. This analysis evaluated the financial implications of using RHD genotyping to guide RhIG prophylaxis among pregnant females.

New paradigms in the management of alloimmune refractoriness to platelet transfusions.

Purpose of reviewFollowing transfusion or pregnancy, a significant number of patients develop antibodies to class I human leukocyte antigen. Some will exhibit platelet transfusion refractoriness, defined as inappropriately low platelet count increments after two or more consecutive transfusions. Unfortunately, failure of at least two products is required before an immunologic work-up is undertaken. Among those diagnosed with immune refractoriness, there is no standard method for identifying platelet products likely to be effective. Recent findingsRecent advances in detection and identification of human leukocyte antigen antibody may permit pretransfusion screening of selected patients and provide guidance in choosing the optimal product. An approach more like that for red cell alloimmunized patients, in which one provides products guided solely by the antibody profile, is preferable to selection based on educated guesswork when human leukocyte antigen identical units are unavailable, and offers some advantages over platelet crossmatching. SummaryThis review presents a literature-based algorithm with which to approach the management of platelet refractory individuals, focusing on newer technology to maximize the post-transfusion yield of matched units. Strategies are presented that allow selection of more effective products for difficult, broadly alloimmunized individuals, including patients who have developed antibodies to human platelet antigens.

Utility of cross-matched platelet transfusions in patients with hypoproliferative thrombocytopenia: a systematic review.

Multiply transfused hypoproliferative thrombocytopenic (HT) patients with alloimmune transfusion refractoriness require specially selected platelets (PLTs). Cross‐matching apheresis PLTs is a popular support option, avoiding requirements for large panels of typed donors for HLA‐based selection. We undertook a systematic review of the utility of various cross‐matching techniques on mortality reduction, prevention of hemorrhage, alloimmunization and refractoriness, and improvement in PLT utilization or count increments.

Lipemic plasma: a renaissance.

I n this issue of TRANSFUSION, Peffer and colleagues address the issue of lipemic donations with a case-control study of 272 donors of milky, turbid plasma. They outline many well-known risks for postprandial hypertriglyceridemia and correlate visual and spectrophotometric measures of turbidity. In the Netherlands, recovered plasma is sent to fractionation, where standards for fractionation require that units exhibit no more than slight turbidity. In the United States, where no such rule exists, the exclusion of turbid plasma is driven by the interference of suspended lipid particles with infectious disease testing. In either case, donation loss may be minimized by an understanding of donor and dietary factors, which increase the likelihood of plasma and serum sample turbidity. Turbidity has been observed since physicians first began analyzing blood samples. It is described as smoky, opalescent, or lactescent, akin to the appearance of milk, whose suspended lipids cause this characteristic appearance. There may be a separated layer of fat as well as evident particulates in lipemic blood. Studies in the 1950s identified triglycerides (TGs) as the lipid associated with lactescent samples. Hydrophobic TGs are transported in the blood within two types of lipoprotein particles, hepatically derived very-low-density lipoproteins (VLDLs) and their remnants and intestinally derived chylomicrons and their remnants. VLDLs are small particles (25-200 nm), while the much larger chylomicrons range in size from 70 to 1000 nm. Both have a hydrophobic core predominantly composed of TGs with some esterified cholesterol, surrounded by a phospholipid shell containing various regulatory apolipoproteins. Hepatic VLDL secretion peaks as insulin levels decrease during fasting, providing fuel in the form of fatty acids to muscle and other tissues. After a meal, VLDL levels decline while enterocyte-packaged chylomicrons, rich in dietary-derived long-chain TGs, traverse the intestinal lymphatics and enter the bloodstream via the thoracic duct. Both chylomicrons and VLDLs provide their fatty acids to adipose (after meals) and other tissues through the action of endothelial lipoprotein lipase. Over time, smaller remnant particles are cleared from the circulation by hepatic uptake and, in the case of VLDLs, by hepatic metabolism to low-density lipoprotein (LDL) particles which mediate cholesterol transport. Chylomicrons circulate for 6 to 8 hours, progressively accumulating throughout the day after successive meals. Hypertriglyceridemia, defined by the National Cholesterol Education Program’s (NCEP) Adult Treatment Panel III as fasting levels greater than 199 mg/dL (2.3 mmol/L; mmol/L = mg/dL 88.57), is considered very high when levels reach 500 mg/dL for which TG-targeted pharmacologic intervention is recommended as first-line therapy. Fewer than 1% of the population has extreme fasting hypertriglyceridemia ( 1000 mg/dL), which results in frequent attacks of acute pancreatitis (approx. 20% of individuals). It is fasting lipids which define cardiovascular risk, but healthy western individuals spend most of their waking hours in a fed, rather than fasting state. Successive meals result in a gradual rise in TG levels. In healthy people, blood TGs are commonly twice their fasting level by bedtime and may only return to baseline 10 to 12 hours after the evening meal. Because of the dynamic remodeling of VLDLs and chylomicrons and the intermittent postprandial entry of chylomicrons into the circulation, particle concentration and diameter show significant temporal variance. These two characteristics determine the light-scattering properties of lipemic serum or plasma. The very largest particles, chylomicrons, scatter light more efficiently than smaller particles (VLDLs and remnants) while higher particle concentrations similarly increase light scatter. Because the TG content of particles can change independently of particle concentration and to a smaller degree, diameter, subjective visual estimates of turbidity correlate weakly with measured TG concentrations. As visual and automated spectrophotometric assessments of turbidity show moderate concurrence, TG concentration and spectrophotometric turbidity are also less well correlated. Lipoprotein particles scatter light, raising the absorbance of the blank and reducing the operating scale of colorimetric and chemiluminescence assays, possibly resulting in false-negative results in borderline-reactive samples. Manufacturers of US FDA-approved assays for transfusion-transmitted infectious antigens and relevant antibodies now validate their assays to TG concentrations generally up to 3000 mg/dL. US blood centers often perform an initial visual check of samples for lipemia against a comparator chart and only those exceeding predetermined eye-read levels require a TG concentration determination. The majority of US donor-screening facilities use Abbott’s PRISM assays and/or the ORTHO Summit Processor (OSP) to screen for infectious markers for hepatitis B and C viruses (HBV and HCV, respectively), human immunodeficiency virus (HIV), human T-cell TRANSFUSION 2011;51:1136-1139.

Changing paradigms in matched platelet support.

A s conversion to universally leukoreduced red cell (RBC) and platelet (PLT) inventories has progressed, the face of alloimmune refractoriness to PLT transfusion is changing. PLT matching services have observed declining numbers of patients requiring support, anecdotally accompanied by an increased breadth of human leukocyte antigen (HLA) alloimmunization. Increasingly aggressive transfusion support for many diseases may reverse that trend to some degree, however. In the late 1960s, Yankee and coworkers identified HLA-A and -B antibodies as the principal cause of alloimmune transfusion refractoriness and demonstrated the efficacy of HLA-identical PLT support. The development of apheresis technology to collect large numbers of PLTs from an HLA-identical donor enabled ongoing support of refractory patients. Development of increasingly efficient plateletpheresis instruments, the necessity to recover fixed costs associated with apheresis technology, the perceived superiority of apheresis PLTs, and their convenience have all driven the explosive growth of plateletpheresis programs. Almost 80 percent of PLTs transfused in the United States in 2004 were apheresis-derived. Theoretically, through their blood suppliers, hospitals have a wide variety of single-donor products from which to choose when treating PLT-refractory patients. Even with a panel of 2500 or more HLA-typed apheresis donors and 50 or more apheresis collections per day, few blood centers can provide more than 5 to 20 percent identical matches directly off their shelves. In response to difficulties in fulfilling immediate patient needs, Duquesnoy and coworkers developed a grading scheme in the 1970s to identify “best mismatches.” Several cross-reactive groups (CREGs) of HLA-A and -B antigens are identifiable serologically. These have been shown to be related to “public” epitopes shared by groups of HLA antigens. Patients cannot develop antibodies against public epitopes on their own Class I HLA molecules. The original Duquesnoy system depends on the fact that 70 to 90 percent of HLA antibodies are directed against “foreign” public epitopes. There are data demonstrating that one to two in-CREG mismatches or one out-of-CREG mismatch (of a patient’s two to four HLA-A and -B antigens) are superior to random selections. Unfortunately, patients can also produce antibodies to private (single antigen) epitopes within CREGs in which their own HLA-A and -B antigens are grouped. CREG matching therefore will inevitably lead to the immune-based failure of a significant fraction of the mostly nonidentical products provided for transfusion. Another validated strategy, designated “antibody specificity prediction” or the “antigen-negative approach,” attempts to provide matched PLTs containing none of the antigens to which refractory patients have been alloimmunized. This selection method relies on precise detection of HLA antibody specificity, similar to the approaches currently used in pretransfusion testing for RBC mismatch. Until recently, techniques were not sufficiently robust to determine the specificity of all HLA antibodies present in PLT-refractory patient sera. High-definition ELISA and flow cytometry–based techniques with transfected or recombinant antigens promise to change the current paradigm which emphasizes CREG-based selection. Because most PLT-refractory patients quickly establish a stable breadth of HLA alloimmunization after only a few antigenic exposures, the risk for forming additional antibodies from ongoing nonidentical transfusion appears to be relatively low. The most important study to date in assessing the equivalency of PLT-matching techniques has compared this antigen-negative approach (employing less reliable detection techniques) with both classical CREG-based selection and PLT crossmatching. Success rates with transfusions selected to be antigennegative were apparently equivalent to crossmatched PLTs and higher (though not statistically so) than CREGselected products. This third approach to matched PLT support involves crossmatching of segments from apheresis units in inventory with serum from alloimmunized recipients. One would never similarly screen RBCs for compatibility without identifying patients’ antibodies for fear of missing mismatched cells due to dosage effects. Unfortunately, PLT crossmatching also fails to detect some PLT antibodies, although the consequences of failure are not as dire. Petz reported that using a relatively insensitive technique for antibody identification, significant HLA antibodies in 17 percent of serum samples were missed by crossmatching. In one large study employing the most common method for crossmatching, only 17 percent of crossmatched units were compatible, demonstrating another important limitation of this approach. Finally, in most laboratories, crossmatching is not available on nights or weekends and requires 4 to 6 hours for completion, leading to significant delays in providing matched products. On the positive side, PLT crossmatching may TRANSFUSION 2008;48:204-206.

Anaphylactic transfusion reactions

A llergic transfusion reactions are common, occurring with an estimated incidence of 1 to 3 per 100 transfusions. However, anaphylactic transfusion reactions are uncommon events, occurring with an estimated incidence of 1.7 to 4.3 per 100,000 red blood cell (RBC) and plasma transfusions and 62.6 per 100,000 platelet (PLT) pools. Based on our experiences at the American Red Cross National Reference Laboratory, the number of anaphylactic transfusion reactions would be even smaller if reported cases were limited to those fully satisfying standard diagnostic criteria for anaphylaxis. Fatal anaphylactic transfusion reactions are rare. In fiscal year 2008, three fatal anaphylactic transfusion reactions were reported to the US Food and Drug Administration, although a total of 23,669,000 units of blood components were transfused during that year. As a consequence of the infrequency and sporadic occurrence of anaphylactic transfusion reactions, our understanding of the pathophysiology has been based almost exclusively on case reports using a variety of diagnostic standards and laboratory test methods. An illustrative example of the difficulty encountered in identifying the etiology of an anaphylactic transfusion reaction is the entity of immunoglobulin A antibody (antiIgA)-related anaphylaxis, first described in 1968. Subsequently, more than 40 case reports of anti-IgA–related anaphylactic or anaphylactoid reactions have been published in medical journals, almost all employing variations of the originally described hemagglutination assay using IgA myeloma-coated reagent RBCs. Even when performed under optimal laboratory conditions, hemagglutination assays for anti-IgA are nonspecific. When the American Red Cross National Reference Laboratory tested 32,376 serum samples from healthy blood donors using the same assays that were routinely employed to establish a diagnosis of anti-IgA–related anaphylactic transfusion reaction, 1 per 1200 samples were totally IgA deficient (<0.05 mg/dL) and contained hemagglutinating anti-IgA. If these laboratory test results were specific and truly predictive of an anti-IgA–related anaphylactic transfusion reaction, we would observe more than 60 anti-IgA–related anaphylactic reactions every day in the United States from the approximately 64,800 blood components transfused daily. Further, of 359 serum samples from patients with suspected IgA-related anaphylaxis that were referred to the Red Cross National Reference Laboratory, only 18.1% were IgA deficient with anti-IgA. More recent results from this laboratory from an additional 223 samples revealed a similarly low rate of anti-IgA detection (17.9%; American Red Cross National Reference Laboratory, 1996-2011 data). These findings indicate that anti-IgA is only one of many causes of anaphylactic transfusion reactions. The shortcoming of current anti-IgA assays for predicting anaphylactic transfusion reactions is highlighted by the lack of observations of anaphylaxis after exposure to IgA in most individuals with anti-IgA. Conversely, in the 1994 Red Cross study, 4.7% of patients had total IgA deficiency and no demonstrable anti-IgA, yet had experienced an anaphylactic transfusion reaction. Clearly, there is a need for improved diagnostic criteria, more predictive analytes, and more accurate test methods to better sort out the various causes of transfusion-related anaphylaxis. Among the many other causes of severe allergic reactions associated with transfusions are infusion of allergens (donor-ingested foods and medications) and polymorphic forms of serum proteins other than IgA (e.g., haptoglobin, C3/4, transferrin, albumin, and others) to which the patient has been presensitized, passive transfer of IgE antibodies to common environmental allergens, and anaphylatoxins or PLT biologic response mediators generated during storage. In this issue of TRANSFUSION, Abe and colleagues identify yet another trigger for anaphylaxis, describing two recipients of PLT concentrates whose posttransfusion clinical presentations and increased tryptase concentrations satisfied standard diagnostic criteria for anaphylaxis. Both PLT concentrates were traced to a common donor whose plasma contained mast cell degranulation activity, as well as an increased concentration of total immunoglobulin (Ig)E (approx. 150,000 ng/mL). Gel filtration chromatography, cation-exchange chromatography, anion-exchange chromatography and mass spectrometry determined that the donor’s plasma mast cell degranulation activity was associated with abnormal high-molecular-weight IgE oligomers. The investigators tested serum from 10 atopic individuals with abnormally high total IgE concentrations (18,000-1,180,000 ng/mL), but none contained mast cell degranulation activity. They concluded that their findings strongly suggest that the mast cell degranulation activity occurred because of the donor’s uniquely abnormal IgE oligomers. They referred the donor for medical evaluation and he was diagnosed as having early stage IgE k-type multiple myeloma. How does this case report contribute to our understanding of anaphylactic transfusion reactions? The TRANSFUSION 2011;51:2265-2266.