A hemolytic anti-LKE associated with a rare LKE-negative, "weak P" red blood cell phenotype: alloanti-LKE and alloanti-P recognize galactosylgloboside and monosialogalactosylgloboside (LKE) antigens.

BACKGROUND: "Weak P" is a rare red blood cell (RBC) phenotype, characterized by a global decrease in P(k) and P antigens. We now describe a second weak P individual who also typed LKE-negative (LKE-N) and possessed a clinically significant anti-LKE. STUDY DESIGN AND METHODS: Patient RBCs and plasma were examined by standard serology and flow cytometry. Glycosphingolipids (GSLs) from patient, P(k) , and LKE-strong (LKE-S) RBCs were isolated and analyzed by high-performance thin-layer chromatography (HPTLC). To confirm antibody specificity, patient serum and 30 human polyclonal controls, including alloanti-P and anti-PP1 P(k) , were tested against a panel of GSLs by HPTLC immunostaining. RESULTS: The patient typed P1 +, P+, and LKE-N and possessed a "P-like" panagglutinin. In a two-stage indirect antiglobulin test, the patient's plasma caused hemolysis of LKE-S cells but not p, P(k) , or LKE-N cells. Clinically, transfusion of P+ RBCs compatible by a prewarmed technique had shortened RBC survival with laboratory evidence of hemolysis. Analysis of the patient's isolated RBC GSLs showed a 30% relative decrease in Gb3 (P(k) ) and Gb4 (P) and a 90% decrease in monosialogalactosylgloboside (MSGG, LKE), accompanied by increased lactosylceramide (CDH), paragloboside, and GM3. On HPTLC immunostaining, the patient's plasma strongly bound MSSG with weak binding to galactosylgloboside (Gb5). Binding to MSGG, Gb5, and Gb4 was also observed with some examples of alloanti-P from P(k) individuals, but not anti-PP1 P(k) , autoanti-P, or normal controls. CONCLUSIONS: We describe the first example of a clinically significant anti-LKE in the setting of a rare weak P background. Human alloanti-LKE and some alloanti-P recognized Gb5 and MSGG.

Infusion of P-Capt prion-filtered red blood cell products demonstrate acceptable in vivo viability and no evidence of neoantigen formation.

BACKGROUND: Transmission of variant Creutzfeldt-Jacob disease (vCJD) is a major concern in blood transfusion. The P-Capt filter has been shown to remove around 4 log ID(50) prion infectivity from prion-spiked human red blood cells (RBCs). STUDY DESIGN AND METHODS: Two independent, single-center, randomized, open-label studies were designed to analyze the safety of P-Capt-filtered RBCs. RBCs prepared from leukoreduced whole blood from 43 eligible subjects were randomly assigned to P-Capt filtration and/or storage in plasma or SAGM and stored for 28 or 42 days. Stored RBCs were analyzed for in vivo 24-hour recovery, hemolysis, metabolic variables, blood group antigen expression, neoantigen formation, and safety after autologous infusion. RESULTS: Mean P-Capt filtration times for leukoreduced RBCs were 41 (SAGM) to 51 (plasma) minutes. Thirteen of 14 subjects receiving P-Capt-filtered RBCs had 24-hour RBC recoveries of 75% or more after 42-day storage, with a mean hemolysis of less than 0.6%. No loss of RBC antigen expression or formation of neoantigens was observed. In both studies, RBCs had white blood cell counts of less than 1 × 10(6)/unit after leukofiltration. P-Capt prion filtration provided an additional greater than 0.8 log leukoreduction. No serious or unexpected adverse events were observed after infusion of P-Capt-filtered full-volume RBC units. CONCLUSIONS: P-Capt-filtered, stored RBCs demonstrated acceptable viability and no detectable neoantigen expression, immunogenic responses. or safety issues after infusion of a complete unit. The additional filtration time and modest reduction in RBC content are within acceptable levels for implementation in countries with transfusion transmission of vCJD.

Rh discrepancies caused by variable reactivity of partial and weak D types with different serologic techniques.

BACKGROUND: RhD discrepancies between current and historical results are problematic to resolve. The investigation of 10 discrepancies is reported here. STUDY DESIGN: Samples identified were those that reacted by automated gel technology and were negative with an FDA-approved reagent. Reactivity with a commercially available panel of monoclonal anti-D was performed. Genomic DNA was evaluated for RHD alleles with multiplex RHD exon polymerase chain reaction (PCR), weak D PCR-restriction fragment length polymorphism, and RHD exon 5 and 7 sequence analyses. RESULTS: The monoclonal anti-D panel identified two samples as DVa, yet possessed the DAR allele. Two weak D Type 1 samples had a similar monoclonal anti-D profile, but only one reacted directly with one of two FDA-approved anti-D. Only two of four weak D Type 2 samples reacted directly with one FDA-approved anti-D, and their D epitope profile differed. CONCLUSIONS: The monoclonal anti-D reagents did not distinguish between partial and weak D Types 1 and 2. Weak D Types 1 and 2 do not show consistent reactivity with FDA-approved reagents and technology. To limit anti-D alloimmunization, it is recommended that samples yielding an immediate-spin tube test cutoff score of not more than 5 (i.e., < or =1+ agglutination) or a score of not more than 8 (i.e., < or =2+ hemagglutination) by gel technology be considered D- for transfusion and Rh immune globulin prophylaxis. That tube test anti-D reagents react poorly with some Weak D Types 1 and 2 red cells is problematic, inasmuch as they should be considered D+ for transfusion and prenatal care. Molecular tests that distinguish common partial and Weak D types provide the solution to resolving D antigen discrepancies.

Case report and literature review: transient Inab phenotype and an agglutinating anti-IFC in a patient with a gastrointestinal problem.

BACKGROUND: The Inab phenotype is a rare deficiency of all Cromer antigens. These antigens are carried on the decay-accelerating factor (DAF, CD55) molecule that is attached to the red blood cell (RBC) membrane by a glycosylphosphatidylinositol (GPI) anchor. Although typically inherited, an acquired and transient form of the Inab phenotype also exists. A patient with the triad of transient Inab phenotype, a direct-agglutinating anti-IFC, and gastrointestinal (GI) abnormalities is reported. CASE REPORT: An 18-month-old boy with gastroesophageal reflux disease requiring a feeding tube, milk and soy intolerance, and severe growth retardation, as well as vision and hearing deficits from cytomegalovirus infection, was identified when pretransfusion testing revealed a potent panagglutinin (titer > 2000 at 4 degrees C). This antibody did not react with Dr(a-) and IFC RBCs, and the autocontrol was negative. The patient's RBCs lacked CD55 by flow cytometric techniques but had normal levels of CD59 and antigens such as Yt(a) and Emm, carried on GPI-linked proteins, thus excluding paroxysmal nocturnal hemoglobinuria. Several months after initial detection, the anti-IFC was virtually undetectable and his cells reacted weakly with anti-IFC, anti-Dr(a), and anti-CD55. RBCs from the propositus' parents and brother demonstrated normal CD55 and CD59 expression. CONCLUSION: This is the first example of a direct-agglutinating anti-IFC. The cause of the transient depression in CD55 protein (and thus Cromer system antigens) and appearance of anti-IFC remains unknown, as does the relationship between the patient's GI system abnormalities and these serologic findings.

Noninvasive prenatal RHD genotyping by real-time polymerase chain reaction using plasma from D-negative pregnant women.

OBJECTIVE: Prenatal noninvasive determination of fetal Rh status is an important aid to the management of hemolytic disease of the fetus and newborn. We performed real-time polymerase chain reaction on fetal DNA derived from maternal plasma to determine fetal Rh status. STUDY DESIGN: Cell-free plasma DNA from 98 D-negative pregnant women was tested for the presence of exons 4, 5, and 10 of RHD. The presence of fetal DNA was confirmed by detection of SRY or biallelic insertion/deletion polymorphisms in the maternal plasma and buffy coat. RESULTS: Seventy-two D-positive infants and 26 D-negative infants were determined by serologic studies. All 3 RHD exon sequences were detected in 68 of 72 mothers of D-positive infants. The presence of fetal DNA in mothers of D-negative infants was confirmed in all 10 boys and in 14 of 16 girls. CONCLUSION: Fetal RHD genotyping in this study correctly predicted fetal Rh status in 92 of 98 (94%) cases.

Persistence of cefotetan on red blood cells.

BACKGROUND: Cefotetan can cause severe immune hemolytic anemia that may persist long after the drug is discontinued. To study the binding of cefotetan to RBCs, patients who received cefotetan were followed and tested for the presence of antibody to cefotetan. STUDY DESIGN AND METHODS: Patients receiving cefotetan were identified from pharmacy and nursing records. Blood samples obtained for routine hematology tests were analyzed. Cefotetan binding to patients' RBCs was tested using a previously characterized high-titer anticefotetan serum by gel technique. To determine the minimum amount of drug necessary for binding to occur, RBCs were incubated with serial dilutions of cefotetan at pH 7.4. RESULTS: Sixty patients receiving 1 to 25 g i.v. (median, 2 g) of cefotetan were followed for 1 to 123 days (median, 18 days). All were initially positive, for cefotetan on RBCs. Positivity persisted for up to 98 days after the last dose of drug. Fifteen patients became negative during follow-up. The first negative sample occurred at Day 30 to 123. Using the midpoint between the last positive and first negative to estimate of the duration of positivity, we estimate that cefotetan remains RBC-bound for 16.5 to 92 days (median, 67.5 days). During the follow-up period, five patients developed anticefotetan detectable in the serum. Twenty patients receiving other cephalosporin antibiotics showed no specific reactivity of their RBCs with anticefotetan. In vitro studies showed a minimum necessary drug concentration of 1 micromol/L at physiologic pH, which was not significantly altered by RBC pretreatment with ficin, sialydase, or DTT. CONCLUSIONS: Cefotetan is tightly bound to RBCs after intravenous administration and remains detectable for weeks after the last dose. Antibodies to cefotetan may occur in about 8 percent of patients receiving the drug. The minimum necessary concentration for RBC binding is low compared to an estimated plasma concentration of 240 micromol/L from a single i.v. dose of 1 g.

Immobilized Marasmius oreades agglutinin: use for binding and isolation of glycoproteins containing the xenotransplantation or human type B epitopes.

The type B-specific lectin from the mushroom Marasmius oreades was immobilized onto Sepharose 4B. The immobilized lectin bound murine laminin and bovine thyroglobulin, glycoproteins that contain the Galalpha1,3Galbeta1,4GlcNAc epitope. This epitope is responsible for hyperacute rejection of xenotransplants from lower mammals to humans, Old World monkeys, or apes. The immobilized lectin also bound a fraction of serum proteins from type B human serum but little or none from type A or O(H) serum. The major protein bound from human B serum was a portion of the alpha2-macroglobulin. Treatment of this fraction with N-glycosidase F resulted in decreased molecular weight of bands associated with alpha2-macroglobulin and loss of their M. oreades lectin reactivity, whereas on treatment with coffee bean alpha-galactosidase, this bound fraction also lost reactivity with M. oreades lectin but became reactive with Ulex europaeus I lectin, suggesting the presence of L-fucosyl-alpha1,2-terminated structures. The presence of blood group epitopes on alpha2-macroglobulin has been detected previously by immunological methods, but this is the first isolation and characterization of the specifically glycosylated fraction of this serum protein. The immobilized lectin also bound a number of proteins from pig, rabbit, and rat serum that were distinct in electrophoretic mobility from the human B-serum components and presumably contain the xenotransplantation epitope among their glycan structures. This report further demonstrates the utility of immobilized lectins in isolating and characterizing glycan structures of naturally occurring glycoproteins.