Comparison of CD34+ cell collection efficiency on the COBE Spectra and Fenwal CS-3000 Plus.

Optimal collections of mobilized CD34+ cells are important in terms of both patient toxicity and cost. The factors that determine CD34+ collection efficiency (CD34eff) of cell separators have not been well studied. In addition, because several cell separators are available, the type of collection device may also be a significant variable. Previous studies comparing the Baxter-Fenwal CS3000 and the COBE Spectra have not yielded consistent conclusions. Therefore, we retrospectively analyzed the collection outcomes of 163 consecutive donors with a peripheral CD34+ cell concentration (pCD34) of > or =5 cells/microl on the first collection that had been harvested on one or the other device. The CS3000 was found to yield a significantly higher CD34eff (50% vs. 39%, P = 0.006). However, donors were not balanced for several prognostic factors, which may contribute to CD34eff including mobilization with G-CSF vs. chemotherapy+G-CSF, average flow rate, and total volume of peripheral blood processed. When appropriate variables were included in a stepwise multiple variable analysis, cell separator type emerged as a significant independent predictive factor for CD34eff (P = 0.018). Our data indicates that the CS3000 will, on average, show a higher absolute CDeff of 8%. Furthermore, since the two devices differ in mechanism, prognostic factors may also differ. Comparisons suggest that peripheral blood WBC and hematocrit may be more important predictors for the CS3000.

Anti-G in a pregnant patient.

BACKGROUND: Anti-G is a red cell (RBC) antibody of the Rh system. It has been described in pregnant women only in association with anti-D or anti-C; therefore, the ability of this antibody alone to cause hemolytic disease of the newborn is uncertain. One case in which this antibody caused no clinical sequelae is reported. CASE REPORT: The patient was a 35-year-old primigravida with type O, D-, C-, E-, c+ RBCs who was given 4 units of type O, D- allogeneic RBCs and 2 units of autologous RBCs 2 years antepartum. She was found to have anti-D and anti-C by an outside laboratory as part of a routine prenatal work-up. Further evaluation by our laboratory revealed the presence of anti-G and possible anti-C without anti-D. Titers at 22 weeks' gestation were 64 against r'r RBCs and 16 against R2R2 RBCs; these remained unchanged throughout the pregnancy. Amniocentesis performed at Weeks 28 and 32 showed no evidence of hemolytic disease of the newborn. A healthy 3.3-kg infant was delivered at 36 weeks' gestation. Prophylactic Rh immune globulin was administered antepartum and postpartum. The infant's RBCs were type O, D+, c+ C-, E-, and the direct antiglobulin test was positive. An acid eluate prepared from the baby's RBCs revealed anti-G. The total bilirubin was 5.5 mg per dL at birth, and the hematocrit was 66 percent. Total bilirubin peaked on Day 5 at 11.9 mg per dL, and no therapeutic intervention was required. CONCLUSIONS: Anti-G alone caused little if any fetal or neonatal hemolysis in this case. Although further study is needed, invasive fetal monitoring may be unnecessary if anti-G is the sole cause of fetomaternal RBC incompatibility.

DNA from urine sediment or buccal cells can be used for blood group molecular genotyping.

Accurate blood group antigen typing of red blood cells with a positive direct antiglobulin test or from a recently transfused patient has been a long-standing problem. To overcome this problem, we evaluated the feasibility of using somatic cells as a source of DNA for molecular genotyping. Two sources of cells that could be obtained by noninvasive procedures were chosen for analysis: urine samples, which were already available in the clinical laboratory, and buccal epithelial cells collected with cotton wool swabs. DNA, prepared using a commercial kit, was subjected to polymerase chain reaction amplification and followed by digestion with the appropriate restriction enzyme. Genotyping was performed for three alleles encoded by polymorphic genes on three different chromosomes, namely KEL1/KEL2, JKA/JKB, and FYA/FYB. Genotyping results were compared to the results of typing performed on red blood cells using standard hemagglutination techniques. Results given by samples freshly collected from volunteer donors were concordant. Although results obtained with samples collected from hospital patients were initially not in agreement with the phenotyping results, adjustments to the test protocol resulted in concordance. DNA from blood, urine sediment, or buccal cells can be used for blood group molecular genotyping.

Difficulties in identifying antibodies in the Dombrock blood group system in multiply alloimmunized patients.

BACKGROUND: Although red cell (RBC) antibodies of the Dombrock blood group system have been reported to cause acute and delayed hemolytic transfusion reactions, the difficulty in identifying these antibodies in patients with multiple RBC alloantibodies has not previously been discussed. The cases of four sickle cell disease patients who developed Dombrock system antibodies after transfusion, three of which were discovered in association with hemolytic transfusion reactions, are reported. CASE REPORTS: Patient 1 was a 36-year-old woman with multiple RBC alloantibodies. Because of the lack of an increment in hematocrit after transfusion, an investigation was performed; it revealed anti-Do(b) in the serum. Patient 2 was a 30-year-old woman with known anti-C, -E, -K, -S, -Fya, and -Bga. She had received a transfusion 10 days previously. Before further transfusion was begun, antibody identification revealed weak nonspecific reactions, which were thought to be HLA antibodies. She developed acute hemolysis during RBC exchange for acute chest syndrome; anti-Doa was identified in both the serum and eluate. She received 2 units of Do(a-) RBCs without complication. Patient 3 was a 35-year-old woman with known anti-C, -E, -K, -Fya, and -N and a warm autoantibody. Two weeks after transfusion, she had a delayed hemolytic transfusion reaction coincident with the identification of anti-Doa. Patient 4 was a 33-year-old woman with known anti-C, -V, -K, -Fya, -Fy3, Jkb, -S, -N, and -Ytb, who developed anti-Doa 8 weeks after transfusion. CONCLUSION: An association of Dombrock blood group system antibodies with hemolytic reactions is demonstrated in alloimmunized sickle cell disease patients. In all four cases, identification of Dombrock system antibodies was delayed because high-titer low-avidity antibodies, HLA antibodies, or autoantibodies were thought to explain the serologic findings. The presence of Dombrock system antibodies should be considered when unexplained serologic reactivity occurs during antibody identification in this population.

The first reported case of anti-Dob causing an acute hemolytic transfusion reaction.

The antibodies of the Dombrock blood group system have only rarely been encountered in transfusion practice, and anti-Dob has not previously been implicated in an acute hemolytic transfusion reaction. We have encountered the first such case involving a chronically transfused black female with hemoglobin SS disease and multiple antibodies in her serum. During a previous admission for sickle cell crisis, the patient received 3 units of compatible blood with no untoward effects. Serum obtained 21 days later contained, in addition to the known antibodies, anti-S plus an unidentified antibody showing characteristics of HTLA. Blood lacking the E, K1, Fy(a), Jk(b) and S antigens was obtained, and 2 least incompatible units were transfused. While administering the second unit, the patient complained of fever and low back pain, and hemoglobinemia was detected. Anti-Dob was identified in the post-reaction samples by absorption-elution tests, and the patient was confirmed to be Do(a+b-). The first unit transfused during this hemolytic episode tested Do (b+). This case, and a similar case involving anti-Doa reported in 1986, strengthens the belief that Dombrock antibodies are clinically significant and illustrates the need for their differentiation, prior to transfusion from less clinically significant HTLA antibodies.

Identification of cyclophilin as a proinflammatory secretory product of lipopolysaccharide-activated macrophages.

We have isolated an 18-kDa peptide (designated sp18, for 18-kDa secreted protein) from the conditioned medium of lipopolysaccharide-stimulated RAW 264.7 murine macrophages. Purified sp18 had in vivo inflammatory activity and in vitro chemotactic activity for human peripheral blood polymorphonuclear leukocytes and monocytes. Surprisingly, N-terminal sequencing and tryptic mapping studies revealed that sp18 and cyclophilin, an intracellular protein that binds the immunosuppressive drug cyclosporin A, are highly homologous. The in vitro chemotactic activity of sp18 on monocytes was blocked by cyclosporin A but not by cyclosporin H, a structural analog of cyclosporin A that does not bind cyclophilin. Like purified porcine cyclophilin, mouse sp18 exhibited peptidyl-prolyl cis-trans isomerase activity. Medium conditioned by lipopolysaccharide-stimulated resident peritoneal exudate macrophages isolated from C57BL/6 mice contained substantially higher levels of sp18/cyclophilin than medium conditioned by nonstimulated macrophages. The observation that sp18/cyclophilin exhibits proinflammatory activity and is secreted by macrophages in response to endotoxin suggests that this protein may function as a cytokine, and invites the hypothesis that the immunosuppressive action of cyclosporin A results in part from interaction with an extracellular form of cyclophilin released as a mediator of immune and inflammatory functions.