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In recent years, polymerase chain reaction–based genotyping platforms, which provide a predicted phenotype, have increased in both patient and high-throughput donor testing, especially in situations where serologic methods or reagents are limited. This study looks at the concordance rate between two platforms commercially available in the United States when used for testing samples from patients with sickle cell disease (SCD), a group particularly vulnerable to alloimmunization. DNA extracted from samples from 138 patients with SCD was tested by human erythrocyte antigen (HEA) BeadChip (Immucor, Norcross, GA) and by ID CORE XT (Progenika-Grifols, Barcelona, Spain). Predicted phenotype results were compared, and a concordance rate was calculated. Discrepancies were resolved by Sanger sequencing. All testing was done under an institutional review board–approved protocol. A concordance rate of 99.9 percent was obtained. Sanger sequencing was performed on four samples with discrepancies in the Rh blood group system. Three samples had a similar allelic variant detected by ID CORE XT. Two of the three discrepant samples were correctly identified as V+w, VS− by ID CORE XT but not by HEA BeadChip. The third sample, predicted to have a phenotype of V+, VS+ by sequencing, was called correctly by HEA BeadChip but not by ID CORE XT, which had predicted V+w, VS−. The fourth discrepancy was identified in a sample that ID CORE XT accurately identified as RHCE*ce[712G] and predicted a partial c phenotype. This result was confirmed by Sanger sequencing, whereas HEA BeadChip found no variants and predicted a c+ phenotype. The high concordance rate of the two methods, along with the known limitations of serology, warrant further discussion regarding the practice of serologic confirmation of extended phenotypes. Clinical significance of the identified discrepancies remains to be determined.

In recent years, polymerase chain reaction-based genotyping platforms, which provide a predicted phenotype, have increased in both patient and high-throughput donor testing, especially in situations where serologic methods or reagents are limited. This study looks at the concordance rate between two platforms commercially available in the United States when used for testing samples from patients with sickle cell disease (SCD), a group particularly vulnerable to alloimmunization. DNA extracted from samples from 138 patients with SCD was tested by human erythrocyte antigen (HEA) BeadChip (Immucor, Norcross, GA) and by ID CORE XT (Progenika-Grifols, Barcelona, Spain). Predicted phenotype results were compared, and a concordance rate was calculated. Discrepancies were resolved by Sanger sequencing. All testing was done under an institutional review board-approved protocol. A concordance rate of 99.9 percent was obtained. Sanger sequencing was performed on four samples with discrepancies in the Rh blood group system. Three samples had a similar allelic variant detected by ID CORE XT. Two of the three discrepant samples were correctly identified as V+w, VS- by ID CORE XT but not by HEA BeadChip. The third sample, predicted to have a phenotype of V+, VS+ by sequencing, was called correctly by HEA BeadChip but not by ID CORE XT, which had predicted V+w, VS-. The fourth discrepancy was identified in a sample that ID CORE XT accurately identified as RHCE*ce[712G] and predicted a partial c phenotype. This result was confirmed by Sanger sequencing, whereas HEA BeadChip found no variants and predicted a c+ phenotype. The high concordance rate of the two methods, along with the known limitations of serology, warrant further discussion regarding the practice of serologic confirmation of extended phenotypes. Clinical significance of the identified discrepancies remains to be determined.

Objectives Next-generation sequencing (NGS) for the determination of rare blood group genotypes was tested in 72 individuals from different ethnicities. Background Traditional serological-based antigen detection methods, as well as genotyping based on specific single nucleotide polymorphisms (SNPs) or single nucleotide variants (SNVs), are limited to detecting only a limited number of known antigens or alleles. NGS methods do not have this limitation. Methods NGS using Ion torrent Personal Genome Machine (PGM) was performed with a customised Ampliseq panel targeting 15 different blood group systems on 72 blood donors of various ethnicities (Caucasian, Hispanic, Asian, Middle Eastern and Black). Results Blood group genotypes for 70 of 72 samples could be obtained for 15 blood group systems in one step using the NGS assay and, for common SNPs, are consistent with previously determined genotypes using commercial SNP assays. However, particularly for the Kidd, Duffy and Lutheran blood group systems, several SNVs were detected by the NGS assay that revealed additional coding information compared to other methods. Furthermore, the NGS assay allowed for the detection of genotypes related to VEL, Knops, Gerbich, Globoside, P1PK and Landsteiner-Wiener blood group systems. Conclusions The NGS assay enables a comprehensive genotype analysis of many blood group systems and is capable of detecting common and rare alleles, including alleles not currently detected by commercial assays.

Molecular genotyping platforms provide a quick, high-throughput method for identifying red blood cell units for patients on extended phenotype-matching protocols, such as those with sickle cell disease or thalassemia. Most of the antigen prevalence data reported are for non-Hispanic populations. Therefore, this study sought to determine the phenotype prevalence in a single blood center's Hispanic population and to compare those results with previously reported rates in non-Hispanic donor populations. We performed a retrospective review of all serologic and molecular typing from donors who self-reported as Hispanic. The phenotype prevalence was reported and compared with rates from other racial/ethnic groups. A total of 1127 donors who selfidentified as Hispanic were screened by serologic methods for Rh and Kell antigens, and 326 were subsequently selected for molecular typing. The most prevalent probable Rh phenotypes were R1r (26.6%), R1R2 (21.5%), and R1R1 (20.7%); rr was found in 7.8 percent of donors tested. The percentage of K+ donors in this population was 2.8 percent. The most prevalent Duffy phenotypes were Fy(a+b+) (35.9%), Fy(a+b-) (35.6%), and Fy(a-b+) (27%). Of the donors studied, 15.3 percent had an FY GATA mutation. Only 1.5 percent of the donors were Fy(a-b-). The Jk(a+b+) phenotype was found in nearly half of the population. M+N+S+s+ was the most prevalent MNS phenotype from that group, constituting 22.4 percent. A total of 95.7 percent of the donors were Lu(a-b+), and Di(a-b+) was observed in 94.4 percent. The most prevalent Dombrock phenotype was Do(a+b+), constituting 46.9 percent, followed closely by Do(a-b+) at 40.5 percent. Hispanic donor antigen prevalence is distinctly different from other racial/ethnic groups and should be considered when attempting to find extended matched units for these patients.

BACKGROUND: Platelet (PLT) storage adversely affects PLT structure and function in vitro and is associated with decreased PLT recovery and function in vivo. In pediatric transfusion medicine, it is not uncommon for small residual volumes to remain in parent units after aliquot preparation of leukoreduced apheresis-derived PLTs (LR-ADP). However, limited data exist regarding the impact of storage on residual small-volume LR-ADP. STUDY DESIGN AND METHODS: Standard metabolic testing was performed on residual volumes of LR-ADP after aliquot removal and PLT aggregometry using a dual agonist of ADP and collagen was performed on stored, small-volume aliquots (10-80 mL) created from an in vitro model of PLT storage. RESULTS: Seventy-seven LR-ADP underwent metabolic (n = 67) or metabolic and aggregation (n = 10) studies. All products maintained a pH value of more than 6.89 throughout storage. Lactate and pCO2 increased proportionally with longer storage time. Regardless of acceptable metabolism during storage, aggregation in 10- to 20-mL aliquots was impaired by Day 4 and aliquots less than 40 mL demonstrated the most dramatic decrease in aggregation from baseline. CONCLUSIONS: Despite maintenance of acceptable metabolic conditions, residual volumes of LR-ADP develop impaired aggregation in vitro that may adversely affect PLT survival and function in vivo. At volumes below 40 mL, LR-ADP revealed reduced aggregation. As a result, it is recommended to monitor and record volumes of LR-ADP used for pediatric transfusion. Moreover, once LR-ADP attain a volume of 50 mL or less on Day 4 or Day 5 of storage, consider discarding these products until their in vivo efficacy can be studied.

The purpose of our study was to improve the quality of care in an emergency department (ED) as measured by length of stay (LOS), total turnaround time (TAT) for laboratory result reporting, and the blood culture contamination rate. Data were included for patients who had at least 1 of 5 laboratory tests performed as part of their care. The study was conducted in 2 phases. First, phlebotomy was performed by a dedicated phlebotomist or nonlaboratory personnel. The second phase added a dedicated laboratory technologist. There was a significant reduction in total TAT for all tests (at least 46 and 75 minutes in the respective interventions), and blood culture contamination rates dropped from 5.0% to 1.1%, although the overall LOS did not change. Estimated cost avoidance is more than $400,000 annually. Quality of care in an ED is improved when samples are collected by a dedicated phlebotomist, but overall LOS does not change.

Thrombin is a key hemostatic enzyme, which propagates its own generation by activating factors V, VIII, and XI. Sustained thrombin generation also activates thrombin-activatable fibrinolysis inhibitor (TAFI), which stabilizes fibrin clot against fibrinolysis. Recombinant activated factor VII (rFVIIa) is considered a novel hemostatic intervention for refractory bleeding, but rebleeding episodes related to fibrinolysis still occur. The present study aimed to investigate the antifibrinolytic effects of rFVIIa in relation to thrombin generation. Using thrombelastography, the effects of rFVIIa on thrombin-activated fibrin formation and on fibrinolysis induced by tissue plasminogen activator were evaluated in various factor-deficient plasma samples. A Thrombinoscope was used to quantitate thrombin generation. Thrombin increased antifibrinolytic activity in a concentration-dependent manner as demonstrated by a longer clot lysis time. In plasma deficient in factors V, VIII, IX, X, or XI, clot lysis occurred early (20 min), and rFVIIa addition had minimal effect, except for improved antifibrinolytic effect in factor-XI-deficient plasma. A normal clot lysis time was observed in factor-XIII-deficient or dual antithrombin/factor-VIII-deficient plasma. Inhibition of TAFI increased the rate of fibrinolysis. Thrombin generation was delayed or decreased in single factor-deficient plasma except for factor XIII deficiency. After rFVIIa addition, the peak thrombin generation reached over 100 nmol/l in factor-XI-deficient plasma, but not in plasma deficient in factors V, VIII, IX, or X. Thrombin generation and subsequent activation of TAFI were important for clot stability. We conclude that rFVIIa therapy does not compensate for increased susceptibility to fibrinolysis due to lack of factor(s) necessary for the formation of tenase and prothrombinase.

Many patients with nonmalignant hematological disorders rely on blood transfusion at some point in the course of their treatment. Additionally, these patients may require modifications to blood products to prevent alloimmunization or complications from iatrogenic immunosuppression. Occasionally, these patients may become transfusion dependent for a period of time, if not indefinitely, and thus are at increased risk for experiencing complications from this therapy. The case scenarios in this chapter will help the reader develop a better understanding on how to select platelet (PLT) products, manage transfusions in patients with sickle cell disease, and discuss some of the newest literature from randomized controlled trials on the red blood cell (RBC) storage lesion (Table 1).

Heparin, an anticoagulant used for the treatment and prophylaxis of thrombosis, is most commonly monitored using the activated partial thromboplastin time (APTT). Despite its widespread use, the APTT has limitations which stem from the coagulation factors it measures and the technical variability of the

With the success of reducing the risk of transfusion-transmitted infectious diseases, noninfectious serious hazards of transfusion have come to the forefront with respect to transfusion safety. Transfusion-related acute lung injury has emerged as a dominant noninfectious serious hazard of transfusion. Improved understanding of its pathophysiology is needed to improve clinical strategies to deal with the risk. Such understanding, in turn, will depend on the continued progress in development of good model systems, in vitro and in vivo, for experimental studies. As the pathologic mechanisms are elucidated, a universal definition and strategies for the prevention and/or mitigation may become more tangible. This article reviews the clinical manifestations, evolving definition, incidence, pathophysiology, animal modeling, and donor screening and deferral algorithms as they relate to transfusion-related acute lung injury.

Publisher Summary This chapter discusses the testing of donated blood products for syphilis, human t-cell lymphotropic virus (HTLV) and Chagas' disease. The testing of blood donors for these diseases is either mandated or had become a de facto community standard in the US. Syphilis is stated to be the most commonly recognized disease transmitted by transfusion pre-World War II and since then the disappearance of transfusion-associated syphilis infection have been attributed to multiple factors including decline in and control of syphilis infection, no longer performing direct donor-to-patient transfusions and the rarity in blood donors of spirochetemia. The testing methods of syphilis include non-treponemal antibody tests, treponemal antibody test and particle aggregation tests. Testing for HTLV uses enzyme immunoassay (EIAs) which can detect both HTLV-1 and HTLV-2 antibodies. For the detection of Chagas' disease, a FDA approved EIA method is used to screen blood donors for antibodies to Trypanosoma cruzi and a confirmatory test for Chagas' disease is done by repeat EIA test, radioimmunoprecipitation assay (RIPA) among others.

BACKGROUND: Thromboembolic complications can occur with recombinant activated factor VII (rFVIIa) treatment in trauma and surgical patients but they are infrequent in hemophiliacs. Bleeding diathesis in these conditions is often attributed to reduced thrombin generation, which may be improved with rFVIIa. Normally, thrombin that diffuses from local vascular injury sites is quickly inactivated by antithrombin (AT). Evaluating the influence of AT levels on thrombin generation in hypocoagulable FVIII-deficient plasma would be a simple approach to better understand how procoagulant stimuli, such as rFVIIa, might result in postoperative thrombotic complications. We hypothesize that reduced AT concentrations would increase the procoagulant effects of rFVIIa in FVIII-deficient plasma. METHODS: Thrombin generation was evaluated in vitro in FVIII-deficient and AT/FVIII-deficient plasma using thrombelastography and a thrombin generation assay (Thrombinoscope™). The effect of added rFVIIa on these variables was evaluated. RESULTS: Delayed thrombus formation based on thrombelastography in FVIII-deficient plasma was predictably reversed by rFVIIa. Improved thrombus formation and responses to rFVIIa were observed when AT levels were 20%–50% of normal. Thrombin generation in FVIII-deficient plasma increased in an inverse relationship to AT levels. Supplemental rFVIIa decreased the lag time of thrombin generation but not the amount of thrombin generated. CONCLUSIONS: Using FVIII-deficient plasma as a model of reduced thrombin generation, we demonstrate that low AT levels enhance in vitro hemostatic responses to rFVIIa. Reduced AT levels in trauma and surgical patients with normal or increased FVIII levels may be considered potentially prothrombotic. Monitoring of AT levels during rFVIIa therapy may thus reduce thrombotic complications in nonhemophiliacs.

Red blood cell (RBC) transfusions were requested for a 60-year-old woman with hypoplastic myelodysplastic syndrome (MDS) treated with cyclosporine to reverse progressive pancytopenia. During routine forward grouping, the patient typed as group A, but with mixed-field reactivity in the anti-A column of the gel card (Left panel, red box). Reverse typing with Al and B cells in the right-most two gel columns was consistent with blood group A. Common causes of mixed-field agglutination were excluded, including transfusion within the last 3 months and the presence of A-sub-groups. To further investigate the mixed-field result, we utilized an automated fluorescence cytometric blood typing method previously described by our group1'2 (Right panel. The density of antigen expression [PE fluorescence] increases from left to right; isotype control [iso] is purified polyclonal IgM). These studies showed that the patient's RBCs were group A, but could be separated into two distinguishable subpopulations that differed in their levels of A antigen expression (red line). Note that the population on the left (dashed arrow), with lower PE fluorescence, nonetheless does express residual A antigen as it is shifted to the right of the isotype control reaction (blue line). The population on the right (solid arrow) shows more usual levels of A antigen expression. In agreement with the typing results on the gel card, the patient's RBCs were negative for B antigen (black line). Thus, the mixed-field reaction seen on the gel card was due to the loss of A-antigen expression on a subpopulation of the patient's RBCs-a phenomenon that has been described in MDS patients. In addition to routine blood typing 1,2 , fluorescence cytometry is a powerful method for resolving complicated immunohematologic problems.

We read with interest the Technical Brief by Smogorzewska et al. (1) describing an artificially increased total bilirubin in a patient with a monoclonal IgM paraprotein. Monoclonal paraproteins have been shown to artifactually influence several automated assays of different methodologies, including nephelometry, turbidometry, and immunologic assays, by forming precipitates during the assay procedure (2)(3)(4)(5)(6)(7). The total bilirubin assay on the Hitachi 917 automatic chemistry analyzer (Roche Diagnostics) has been reported to yield falsely increased bilirubin values as a result of paraprotein interference (1)(8). Smogorzewska et al. (1) and Pantanowitz et al. (8) described this artifact as rare, but we have identified 6 patients at 2 hospitals with documented paraproteins who had falsely increased serum total bilirubin. Notably, patients with artifactually high total …