CD59 gene: 143 haplotypes of 22,718 nucleotides length by computational phasing in 113 individuals from different ethnicities.

BACKGROUND: CD59 deficiency due to rare germline variants in the CD59 gene causes disabilities, ischemic strokes, neuropathy, and hemolysis. CD59 deficiency due to common somatic variants in the PIG-A gene in hematopoietic stem cells causes paroxysmal nocturnal hemoglobinuria. The ISBT database lists one nonsense and three missense germline variants that are associated with the CD59-null phenotype. To analyze the genetic diversity of the CD59 gene, we determined long-range CD59 haplotypes among individuals from different ethnicities. METHODS: We determined a 22.7 kb genomic fragment of the CD59 gene in 113 individuals using next-generation sequencing (NGS), which covered the whole NM_203330.2 mRNA transcript of 7796 base pairs. Samples came from an FDA reference repository and our Ethiopia study cohorts. The raw genotype data were computationally phased into individual haplotype sequences. RESULTS: Nucleotide sequencing of the CD59 gene of 226 chromosomes identified 216 positions with single nucleotide variants. Only three haplotypes were observed in homozygous form, which allowed us to assign them unambiguously as experimentally verified CD59 haplotypes. They were also the most frequent haplotypes among both cohorts. An additional 140 haplotypes were imputed computationally. DISCUSSION: We provided a large set of haplotypes and proposed three verified long-range CD59 reference sequences, based on a population approach, using a generalizable rationale for our choice. Correct long-range haplotypes are useful as template sequences for allele calling in high-throughput NGS and precision medicine approaches, thus enhancing the reliability of clinical diagnostics. Long-range haplotypes can also be used to evaluate the influence of genetic variation on the risk of transfusion reactions or diseases.

IgA plasma cell myeloma presenting as cold agglutinin-induced haemolytic transfusion reaction.

Cold agglutinins produced in the setting of B cell neoplasms, such as lymphoplasmacytic lymphoma and plasma cell myeloma, can mediate autoimmune haemolytic anemia. Transfusion of these patients can exacerbate cold agglutinin-mediated haemolysis. Moreover, the workup for these reactions represents a diagnostic challenge due in part to false negative direct antiglobulin tests (DATs). Here, we report an anaemic patient who after a red blood cell (RBC) transfusion performed without blood warming, experienced a DAT-negative haemolytic transfusion reaction, and was later diagnosed with IgA-multiple myeloma, which showed an uncommon granular pattern by CD138 immunohistochemistry. Extensive workup excluded other diagnostic possibilities, including the presence of Donath-Landsteiner antibodies and cryoglobulins. Successful treatment with CyBorD (cyclophosphamide, bortezomib and dexamethasone) achieved complete remission, and additional RBC transfusions using warmers were completed uneventfully.

Long-range haplotype analysis of the malaria parasite receptor gene ACKR1 in an East-African population.

The human ACKR1 gene encodes a glycoprotein expressing the Duffy blood group antigens (Fy). The Duffy protein acts as a receptor for distinct pro-inflammatory cytokines and malaria parasites. We determined the haplotypes of the ACKR1 gene in a population inhabiting a malaria-endemic area. We collected blood samples from 60 healthy volunteers in Ethiopia's southwestern low-altitude tropical region. An assay was devised to amplify the ACKR1 gene as a single amplicon and determine its genomic sequence. All haplotypes were resolved at 5178 nucleotides each, covering the coding sequence (CDS) of the ACKR1 gene and including the 5'- and 3'-untranslated regions (UTR), intron 1, and the 5'- and 3'-flanking regions. When necessary, allele-specific PCR with nucleotide sequencing or length polymorphism analysis was applied. Among the 120 chromosomes analyzed, 18 ACKR1 alleles were confirmed without ambiguity. We found 18 single-nucleotide polymorphisms (SNPs); only one SNP was novel. The non-coding sequences harbored 14 SNPs. No SNP, other than c.-67T>C, indicative of a non-functional allele, was detected. We described haplotypes of the ACKR1 gene in an autochthonous East-African population and found 18 distinct ACKR1 alleles. These long-range alleles are useful as templates to phase and analyze next-generation sequencing data, thus enhancing the reliability of clinical diagnostics.

Obstetric hemorrhage and safe blood for transfusion in Ethiopia: the challenges of bridging the gap.

BACKGROUND: Obstetric hemorrhage is a leading cause of maternal death in sub-Saharan Africa, and the shortage of blood for transfusion is a contributory factor. In Ethiopia, the National Blood Bank Service continues to be confronted with challenges in its efforts to ensure the availability of blood for health care facilities. This paper reviews the available data on the contribution of obstetric hemorrhage to maternal mortality and examines the current status of the blood supply in Ethiopia. STUDY DESIGN AND METHODS: We reviewed the published literature and data from the Ethiopian Federal Ministry of Health. To assess the status of the current blood supply, we applied the five cornerstones of a safe and effective blood donor service advocated by the World Health Organization. RESULTS: Our review indicates that there are insufficient national data on the prevalence of obstetric hemorrhage and the contribution of blood supply shortage to maternal death. Also, transfusion safety may be compromised by inadequate testing of donated blood and ineffective hospital transfusion policies. CONCLUSION: To overcome the shortage of blood to treat obstetric hemorrhage, the first step is to evaluate the demand and supply gap by acquiring comprehensive data on the current status of the blood supply and the prevalence of obstetric hemorrhage in Ethiopia. Subsequent steps would include the implementation of transfusion policies, the optimization of whole blood collection, ensuring quality-assured testing of donated blood, and the implementation of transfusion guidelines for the appropriate use of blood products. Strategies for long-term, viable solutions to maintain an adequate blood supply should be simultaneously developed.

Beta radiation for renal nerve denervation: initial feasibility and safety.

AIMS: In small clinical trials, sympathetic renal denervation using radiofrequency (RF) energy shows promise in treating resistant hypertension. However, the RF procedure is lengthy and is associated with pain during ablation. Vascular brachytherapy, a proven treatment for in-stent restenosis, has the potential to cause nerve fibrosis. The purpose of the present study was to assess the safety and feasibility of renal artery brachytherapy for sympathetic renal denervation. METHODS AND RESULTS: A total of 10 normotensive domestic swine underwent vascular brachytherapy to left and right renal arteries using the Beta-Cath 3.5 Fr delivery system at doses of 25 Gy (n=8) and 50 Gy (n=8) at 2 mm from the source centre. These groups were compared to untreated arteries that served as control (n=4). Follow-up obtained at one or two months included angiogram, intravascular ultrasound, and histopathology analysis. The vascular brachytherapy procedure was safe and no apparent angiographic or ultrasound injuries to the vessel were seen. Histology showed a varying degree of thermal injury more pronounced in the 50 Gy group. The majority of examined nerves showed some degree of injury; there was a dose-related effect on nerve injury severity. There were varying degrees of arteriolar changes in the examined sections, with most showing a 2-20% degree of endothelial cell loss. CONCLUSIONS: This initial feasibility and safety study of renal nerve denervation, mediated by low and intermediate ß-radiation dosages, indicates that this approach can cause nerve fibrosis while avoiding significant damage to the renal artery.

The clinical relevance of persistent recombinant immunoblot assay-indeterminate reactions: insights into the natural history of hepatitis C virus infection and implications for donor counseling.

BACKGROUND: Recombinant immunoblot assay (RIBA) is used to determine the specificity of antibody to hepatitis C virus (anti-HCV). The RIBA result is recorded as positive, negative, or indeterminate. The interpretation and significance of RIBA-indeterminate reactions are unclear. We addressed the clinical relevance of these reactions in the context of the natural history of HCV infection in a prospectively followed cohort of anti-HCV-positive blood donors. STUDY DESIGN AND METHODS: Donor demographics, exposure history, and humoral and cell-mediated immunity (CMI) were compared in 15 RIBA-indeterminate subjects, nine chronic HCV carriers, and eight spontaneously recovered subjects. Serum samples were tested for anti-HCV by a quantitative, liquid luciferase immunoprecipitation system (LIPS). CMI was assessed by interferon-γ enzyme-linked immunosorbent spot assay. RESULTS: In the LIPS assay, the sum of antibody responses to six HCV antigens showed significant (p < 0.001) stepwise diminution progressing from chronic carriers to spontaneously recovered to RIBA-indeterminate subjects. CMI responses in RIBA-indeterminate subjects were similar to spontaneously recovered subjects and greater than chronic carriers and controls (p < 0.008). A parenteral risk factor was identified in only 13% of RIBA-indeterminate subjects compared to 89% of chronic carriers and 87% of spontaneously recovered subjects. RIBA-indeterminate donors were older than the other groups. CONCLUSION: The CMI and LIPS results suggest that persistent RIBA-indeterminate reactions represent waning anti-HCV responses in persons who have recovered from a remote HCV infection. In such cases, detectable antibody may ultimately disappear leaving no residual serologic evidence of prior HCV infection, as reported in a minority of long-term HCV-recovered subjects.

Atypical teratoid rhabdoid tumor (AT/RT) in adults: review of four cases.

Atypical teratoid/rhabdoid (AT/RT) tumor is a rare, highly malignant tumor of the central nervous system (CNS) most commonly found in children less than 5 years of age. Although the vast majority of cases are diagnosed in young children, there have been isolated case reports in adults. Since its histological appearance can be confused with other tumors, especially in adults, separating AT/RT from other neoplasms may be difficult. In many instances, a reliable diagnosis is not possible without demonstrating the lack of nuclear INI1 protein expression by immunohistochemical methods. The patients (three males and one female) ranged in age from 23 to 42 years (mean age, 32 years). Radiographically, two tumors were localized in the right fronto-parietal region, one was frontal and the other was found in the left temporal lobe. Varying degrees of hydrocephalus and heterogeneous enhancement were present on MRI. In all cases, diagnosis during intraoperative consultation and preliminary diagnosis was different from the final diagnosis after immunohistochemical analysis. Immunohistochemical staining showed that the tumor cells were positive for vimentin and reacted variably for keratin, epithelial membrane antigen (EMA), synaptophysin, neurofilament protein, CD34, and smooth muscle actin (SMA). All were negative for GFAP, S-100, desmin and CD99. Three of the four cases lacked nuclear expression of INI1. One patient is alive with no evidence of disease 17 years after the diagnosis. In adult examples of AT/RT, the diagnosis requires a high index of suspicion, with early tissue diagnosis and a low threshold for investigation with INI1 immunohistochemistry to differentiate this entity from other morphologically similar tumors. Although the prognosis is dismal in pediatric population, long term survival is possible in adult AT/RT cases after surgery and adjuvant radiotherapy and chemotherapy.

Oligodendroglioma with neurocytic differentiation versus atypical extraventricular neurocytoma: a case report of unusual pathologic findings of a spinal cord tumor.

Differentiating oligodendroglioma from extraventricular neurocytoma by conventional light microscopy alone can present a diagnostic challenge. We report pathologic findings of an unusual spinal cord tumor from a 33-year-old male patient which showed hybrid features of oligodendroglioma and extraventricular neurocytoma. Magnetic resonance imaging (MRI) showed an enhancing intramedullary mass in the cervicothoracic region (C7 through T6). Histologic examination revealed a clear cell neoplasm containing ganglion-like cells and calcifications, prompting the differential diagnosis of oligodendroglioma and extraventricular neurocytoma. The immunohistochemical analysis disclosed neural differentiation of the neoplastic cells with strong synaptophysin and neurofilament staining consistent with extraventricular neurocytoma, as well as strong S-100 and glial fibrillary acidic protein (GFAP) expression. Molecular studies with fluorescent in situ hybridization (FISH) revealed chromosome 1p/(partial) 19q deletions, a finding commonly observed in oligodendroglioma. The proliferation index (using antibody MIB1) of the tumor was approximately 30%. The morphologic findings and these results strengthen the hypothesis that these tumors may share a common progenitor cell, which has also been observed by others. Because there are differences in patient management and long-term prognosis, it is important to attempt to distinguish between oligodendroglioma and neurocytoma. This unusual case and similar rare reported cases support the need to reclassify tumors showing pathologic features common to both neurocytoma and oligodendroglioma as a unique entity, while the effort continues to identify the cell of origin.

Lipoprotein-associated phospholipase A2 protein expression in the natural progression of human coronary atherosclerosis.

OBJECTIVE: Although lipoprotein-associated phospholipase A2 (Lp-PLA2) has received recent attention as a biomarker of inflammation and risk for acute coronary events, its relative expression in coronary plaque phenotypes, including unstable lesions, has not been established. METHODS AND RESULTS: Coronary segments (n=30) were prospectively collected from 25 sudden coronary death patients for immunolocalization of Lp-PLA2. Lesion morphologies were classified as pathologic intimal thickening, fibroatheromas, thin-cap fibroatheromas (fibrous cap thicknesses <65 microm), and rupture. The expression of Lp-PLA2 was detected using a specific monoclonal antibody. Apoptosis was identified by DNA end-labeling using terminal deoxynucleotidyl transferase (TdT). Lp-PLA2 staining in early plaques was absent or minimally detected. In contrast, thin-cap fibroatheromas and ruptured plaques showed intense Lp-PLA2 expression within necrotic cores and surrounding macrophages including those in the fibrous cap. The degree of macrophage apoptosis was greater in thin-cap fibroatheroma and ruptures compared with less advanced plaques with additional double labeling studies showing Lp-PLA2 present in apoptotic cells in regions of high macrophage density. CONCLUSIONS: Lp-PLA2 is strongly expressed within the necrotic core and surrounding macrophages of vulnerable and ruptured plaques, with relatively weak staining in less advanced lesions. These findings together with the association of Lp-PLA2 in apoptotic macrophages suggest a potential role in promoting plaque instability.

Temperature-sensitive labels for containers of RBCs.

Temperature-sensitive labels are adhesive tags that display color changes at preset temperatures. There have been no studies of the suitability of this technology for measuring the temperature of blood components during transportation and storage. We used a digital thermometer to measure temperature in different locations inside containers of RBC as they were allowed to warm to ambient temperatures following removal from refrigeration. We compared these temperature readings with those of 3 temperature-sensitive labels. These labels are marketed to alert transfusion services if the temperature of blood bags exceeds 10 degrees C, which is the maximum permissible by Food and Drug Administration and American Association of Blood Banks requirements for transporting RBCs. The contents of refrigerated RBC units changed from one homogeneous temperature to a range of temperatures when containers were allowed to warm (undisturbed) to ambient temperatures. Color changes of all 3 temperature-sensitive labels correlated more with core compared with surface temperatures of RBCs units. These devices add an additional dimension of safety to the conventional 30-minute rule, which limits storage of blood components at ambient temperature to 30 minutes.