DNA Reference Reagents for Genotyping RH Variants.

Patients who carry Rhesus (RH) blood group variants may develop Rh alloantibodies requiring matched red blood cell transfusions. Serologic reagents for Rh variants often fail to specifically identify variant Rh antigens and are in limited supply. Therefore, red blood cell genotyping assays are essential for managing transfusions in patients with clinically relevant Rh variants. Well-characterized DNA reference reagents are needed to ensure quality and accuracy of the molecular tests. Eight lyophilized DNA reference reagents, representing 21 polymorphisms in RHD and RHCE, were produced from an existing repository of immortalized B-lymphoblastoid cell lines at the Center for Biologics Evaluation and Research/US Food and Drug Administration. The material was validated through an international collaborative study involving 17 laboratories that evaluated each DNA candidate using molecular assays to characterize RHD and RHCE alleles, including commercial platforms and laboratory-developed testing, such as Sanger sequencing, next-generation sequencing, and third-generation sequencing. The genotyping results showed 99.4% agreement with the expected results for the target RH polymorphisms and 87.9% for RH allele agreement. Most of the discordant RH alleles results were explained by a limited polymorphism coverage in some genotyping methods. Results of stability and accelerated degradation studies support the suitability of these reagents for use as reference standards. The collaborative study results demonstrate the qualification of these eight DNA reagents for use as reference standards for RH blood group genotyping assay development and analytical validation.

Posttransfusion Sepsis Attributable to Bacterial Contamination in Platelet Collection Set Manufacturing Facility, United States.

During May 2018‒December 2022, we reviewed transfusion-transmitted sepsis cases in the United States attributable to polymicrobial contaminated apheresis platelet components, including Acinetobacter calcoaceticus‒baumannii complex or Staphylococcus saprophyticus isolated from patients and components. Transfused platelet components underwent bacterial risk control strategies (primary culture, pathogen reduction or primary culture, and secondary rapid test) before transfusion. Environmental samples were collected from a platelet collection set manufacturing facility. Seven sepsis cases from 6 platelet donations from 6 different donors were identified in patients from 6 states; 3 patients died. Cultures identified Acinetobacter calcoaceticus‒baumannii complex in 6 patients and 6 transfused platelets, S. saprophyticus in 4 patients and 4 transfused platelets. Whole-genome sequencing showed environmental isolates from the manufacturer were closely related genetically to patient and platelet isolates, indicating the manufacturer was the most probable source of recurrent polymicrobial contamination. Clinicians should maintain awareness of possible transfusion-transmitted sepsis even when using bacterial risk control strategies.

Validated Reference Panel from Renewable Source of Genomic DNA Available for Standardization of Blood Group Genotyping.

Extended blood group genotyping is an invaluable tool used for prevention of alloimmunization. Genotyping is particularly suitable when antigens are weak, specific antisera are unavailable, or accurate phenotyping is problematic because of a disease state or recent transfusions. In addition, genotyping facilitates establishment of mass-scale patient-matched donor databases. However, standardization of genotyping technologies has been hindered by the lack of reference panels. A well-characterized renewable reference panel for standardization of blood group genotyping was developed. The panel consists of genomic DNA lyophilized and stored in glass vials. Genomic DNA was extracted in bulk from immortalized lymphoblastoid cell lines, generated by Epstein-Barr virus transformation of peripheral blood lymphocytes harvested from volunteer blood donors. The panel was validated by an international collaborative study involving 28 laboratories that tested each DNA panel member for 41 polymorphisms associated with 17 blood group systems. Overall, analysis of genotyping results showed >98% agreement with the expected outcomes, demonstrating suitability of the material for use as reference. Highest levels of discordance were observed for the genes CR1, CD55, BSG, and RHD. Although limited, observed inconsistencies and procedural limitations reinforce the importance of reference reagents to standardize and harmonize results. Results of stability and accelerated degradation studies support the suitability of this panel for use as reference reagent for blood group genotyping assay development and standardization.

Extended blood group molecular typing and next-generation sequencing.

Several high-throughput multiplex blood group molecular typing platforms have been developed to predict blood group antigen phenotypes. These molecular systems support extended donor/patient matching by detecting commonly encountered blood group polymorphisms as well as rare alleles that determine the expression of blood group antigens. Extended molecular typing of a large number of blood donors by high-throughput platforms can increase the likelihood of identifying donor red blood cells that match those of recipients. This is especially important in the management of multiply-transfused patients who may have developed several alloantibodies. Nevertheless, current molecular techniques have limitations. For example, they detect only predefined genetic variants. In contrast, target enrichment next-generation sequencing (NGS) is an emerging technology that provides comprehensive sequence information, focusing on specified genomic regions. Target enrichment NGS is able to assess genetic variations that cannot be achieved by traditional Sanger sequencing or other genotyping platforms. Target enrichment NGS has been used to detect both known and de novo genetic polymorphisms, including single-nucleotide polymorphisms, indels (insertions/deletions), and structural variations. This review discusses the methodology, advantages, and limitations of the current blood group genotyping techniques and describes various target enrichment NGS approaches that can be used to develop an extended blood group genotyping assay system.

Red blood cell transfusion: a clinical practice guideline from the AABB*.

DESCRIPTION: Although approximately 85 million units of red blood cells (RBCs) are transfused annually worldwide, transfusion practices vary widely. The AABB (formerly, the American Association of Blood Banks) developed this guideline to provide clinical recommendations about hemoglobin concentration thresholds and other clinical variables that trigger RBC transfusions in hemodynamically stable adults and children. METHODS: These guidelines are based on a systematic review of randomized clinical trials evaluating transfusion thresholds. We performed a literature search from 1950 to February 2011 with no language restrictions. We examined the proportion of patients who received any RBC transfusion and the number of RBC units transfused to describe the effect of restrictive transfusion strategies on RBC use. To determine the clinical consequences of restrictive transfusion strategies, we examined overall mortality, nonfatal myocardial infarction, cardiac events, pulmonary edema, stroke, thromboembolism, renal failure, infection, hemorrhage, mental confusion, functional recovery, and length of hospital stay. RECOMMENDATION 1: The AABB recommends adhering to a restrictive transfusion strategy (7 to 8 g/dL) in hospitalized, stable patients (Grade: strong recommendation; high-quality evidence). RECOMMENDATION 2: The AABB suggests adhering to a restrictive strategy in hospitalized patients with preexisting cardiovascular disease and considering transfusion for patients with symptoms or a hemoglobin level of 8 g/dL or less (Grade: weak recommendation; moderate-quality evidence). RECOMMENDATION 3: The AABB cannot recommend for or against a liberal or restrictive transfusion threshold for hospitalized, hemodynamically stable patients with the acute coronary syndrome (Grade: uncertain recommendation; very low-quality evidence). RECOMMENDATION 4: The AABB suggests that transfusion decisions be influenced by symptoms as well as hemoglobin concentration (Grade: weak recommendation; low-quality evidence).

Thrombolytic-associated coagulopathy and management dilemmas: a review of two cases.

Thrombolytic therapy improves the overall outcome of many patients with acute ischemic stroke, but it is associated with complications such as symptomatic intracranial hemorrhage. Several factors predict the risk of hemorrhage. Dramatic changes in the coagulation profile following thrombolytic therapy have not been well studied. However, it is unknown if commonly used laboratory tests for coagulation are of predictive value. Yet these tests are commonly requested to predict or treat symptomatic intracranial hemorrhage. When such tests are abnormal, they may present a management dilemma. In this report, we present two cases of coagulopathy following thrombolytic therapy without symptomatic intracranial hemorrhage that were managed differently. Our report suggests that dramatic changes occur in the coagulation profile of patients who receive thrombolytic therapy, but may not clearly predict symptomatic intracranial hemorrhage. Therefore, other factors should be considered when managing these patients.

Infection as a cause of early relapse in patients recovering from thrombotic thrombocytopenic purpura.

Thrombotic thrombocytopenic purpura (TTP) is a rare but severe disorder characterized by hemolytic anemia, thrombocytopenia, fever, renal failure, and neurologic manifestations. Plasma exchange is the most effective treatment for this condition reducing mortality from 90% in untreated patients to 10%. However, infections acquired during the course of therapy could lead to early relapse of TTP. In this case report, we report three patients with TTP who initially responded well to plasma exchange treatments but suffered early relapses following bacterial infections. All these patients achieved remission once appropriate antibiotic therapy was instituted although one patient eventually received four courses of rituximab. This report emphasizes the need to be vigilant for new infections especially urinary tract infections in TTP patients undergoing plasma exchange. Instituting appropriate antibiotic therapy once an infection is suspected may reduce the need for prolonged plasma exchange procedures and extended hospital stay.

Mucosal tolerance to E-selectin and response to systemic inflammation.

Mucosal tolerance to E-selectin has been shown to prevent stroke and reduce brain infarcts in experimental stroke models. However, the effective E-selectin dose range required to achieve mucosal tolerance and the precise mechanisms of neuroprotection remain unclear. We sought to examine the mechanisms of cytoprotection using gene expression profiling of tissues in the setting of mucosal tolerance and inflammatory challenge. Using spontaneously hypertensive rats (SHRs), we achieved immune tolerance with 0.1 to 5 microg E-selectin per nasal instillation and observed a dose-related anti-E-selectin immunoglobulin G antibody production. We also show the distinct patterns of gene expression changes in the brain and spleen with the different tolerizing doses and lipopolysaccharide (LPS) exposure. Prominent differences were seen with such genes as insulin-like growth factors in the brain and downregulation of those encoding the major histocompatibility complex class I molecules in the spleen. In all, mucosal tolerance to E-selectin and subsequent exposure to LPS resulted in significant tissue changes. These changes, while giving an insight to the underlying mechanisms, serve as possible targets for future studies to facilitate translation to human clinical trials.

Antibiotics for vascular diseases: a meta-analysis of randomized controlled trials.

BACKGROUND: Trials of antibiotic treatment of vascular diseases, in attempts to eradicate possible microbial initiators, have had mixed results. We sought to evaluate the efficacy of antibiotics in treating patients with atherosclerotic vascular diseases, using a meta-analysis. METHODS: We searched MEDLINE, EMBASE, and Cochrane Central Register of Controlled Trials and also used cross-references. Randomized controlled trials of antibiotic treatment of vascular diseases were included. Two independent raters assessed the trials for quality. We performed summary estimates, subgroup analyses and tests for homogeneity. RESULTS: Twelve trials, with a total of 12,236 patients, were included. Antibiotic treatment resulted in a non-significant reduction in the risk of new vascular events or death (odds ratio (OR), 0.84; 95% confidence interval (CI), 0.67-1.05). There was significant heterogeneity between the sub-groups in type of vascular disease (coronary heart disease, CHD versus non-CHD (p=0.01)). Among the 72 non-CHD patients, a trend appears for treatment benefit in reducing recurrent events or death (OR, 0.22; 95% CI, 0.07-0.66). CONCLUSIONS: Overall, antibiotic treatment did not significantly reduce occurrence of new vascular events or death. However, further trials are needed to confirm the benefit demonstrated in non-CHD patients.

Current applications of flow cytometry in the diagnosis of primary immunodeficiency diseases.

CONTEXT: To review the applications of flow cytometry in the diagnosis and management of primary immunodeficiency disease. DATA SOURCES: Articles describing the use of flow cytometry in the diagnosis of several primary immunodeficiency diseases were obtained through the National Library of Medicine database. STUDY SELECTION: Publications that described novel and known applications of flow cytometry in primary immunodeficiency disease were selected. Review articles were included. Articles describing the different immunodeficiency diseases and methods of diagnosis were also selected. DATA EXTRACTION: Approximately 100 data sources were analyzed, and those with the most relevant information were selected. DATA SYNTHESIS: The diagnosis of many primary immunodeficiency diseases requires the use of several laboratory tests. Flow cytometry has become an important part of the workup of individuals suspected to have such a disorder. Knowledge of the pathogenesis of many of these diseases continues to increase, hence we acquire a better understanding of the laboratory tests that may be helpful in diagnosis. CONCLUSIONS: Flow cytometry is applicable in the initial workup and subsequent management of several primary immunodeficiency diseases. As our understanding of the pathogenesis and management of these diseases increases, the use of many of these assays may become routine in hospitals.