Barry Wenz

Detection of bacteria in WBC-reduced PLT concentrates using percent oxygen as a marker for bacteria growth

BACKGROUND:  The risk of receiving a PLT concentrate (PC) contaminated with bacteria may be 1000‐fold greater than that of pathogenic viral transmission, yet surveillance for this risk is not generally practiced. A novel bacteria detection system (BDS) that overcomes the limitations of current systems is described. The BDS monitors percent oxygen (%O2) in air above aliquots of PCs that have been filtered to remove the confounding effect of respiring PLTs and residual WBCs.

Preparation of Granulocyte‐Poor Red Blood Cells by Microaggregate Filtration

Abstract. A simple, effective method for removing granulocytes from stored blood is described. Microaggregate filtration removes approximately 95% of the granulocytes from blood which has been stored for 2 weeks, centrifuged and filtered. The mean number of remaining leukocytes is 8 ± 3.7 × 108/unit. The residual white cell population, which is composed almost entirely of lymphocytes, is substantially less than the average number of cells previously associated with febrile reactions. 45 patients were selected for the study. All had significant febrile transfusion reaction histories, and averaged one reaction for every 3.6 U of conventional red cell product transfused. Administration of 212 units of microaggregate filtered granulocyte poor red cells caused a 95% reduction in the incidence of fibrile reactions. The technique is inexpensive, easily incorporated into the routine of the clinical blood bank, and does not require ‘open‐system’ processing. These considerations make microaggregate filtration a logical first choice method for the preparation of granulocyte‐poor red blood cells.

Enhancement of a culture-based bacterial detection system (eBDS) for platelet products based on measurement of oxygen consumption

BACKGROUND: An enhanced bacterial detection system (Pall eBDS) was developed that distinguishes itself from its predecessor (Pall BDS) by removal of the platelet (PLT)‐retaining filter allowing for optimal bacterial transfer, modification of the culture tablet to reduce the confounding effects of respiring PLTs while enhancing bacterial growth, and facilitation of nutrients and gas exchange by agitating the sample pouch during incubation at 35°C. The objective was to evaluate the performance of the new eBDS.

Prevention of growth of Yersinia enterocolitica in blood by polyester fiber filtration

The ability of polyester white cell‐reduction blood filters to prevent the growth of Yersinia enterocolitica in units of donated blood was studied. Sixteen units of freshly drawn blood were inoculated with 10, 50, 100, or 150 colony‐forming units (CFU) per mL of a clinical isolate of Y. enterocolitica (serotype O:3). The units were subsequently fractionated into red cell concentrate and resuspended in AS‐1 or AS‐3 solution. One‐half of the red cell concentrates in each solution were filtered within 15 hours of phlebotomy and stored for 42 days. The remaining units served as unfiltered controls. Bacterial growth was monitored by weekly cultures and, on the last storage day, by the presence of endotoxin and the formation of methemoglobin. One hundred twelve primary cultures (560 plates) were performed. Units collected in AS‐1 and filtered remained sterile when initially inoculated with 50 CFU or less. Filtered units spiked with 100 CFU or less and collected in AS‐3 remained sterile throughout their shelf life. All unfiltered units supported bacterial growth and the formation of endotoxin and methemoglobin. The filtration of freshly donated blood proves to limit the growth of Y. enterocolitica in red cell components.

False positive immunometric assays caused by anti-immunoglobulin antibodies: a case report

A serological phenomenon causing aberrant results with monoclonal immunoenzymetric assays (IEMAs) is reported. Two different commercial IEMA kits detected low levels of choriogonadotropin (hCG) in the serum of a non-pregnant woman. These assays detected between 32 and 55 IU/l of serum hCG over a 3-wk period; however, an RIA for beta-subunit and two monoclonal immunoradiometric assays (IRMAs) detected no hCG (less than 5 IU/l). An IEMA measurement of creatine kinase MB isozyme was also elevated. Antisera to either human immunoglobulin or specifically to human IgM, added to the serum prior to assay, substantially decreased these IEMA reactions. Addition of either mouse serum or purified mouse IgG totally abolished them. It is concluded that these spurious reactions were most likely caused by an IgM antibody which binds to native and enzyme-labelled mouse IgG, but not to iodinated IgG.

Leukocyte Reduction's Role in the Attenuation of Infection Risks among Transfusion Recipients

Despite advances in the screening of donated blood for infectious agents, the risk of transmitting viral, bacterial, and protozoal infections, as well as newly emerging diseases, via transfusion persists. A complementary approach is leukocyte reduction (LR), the removal of leukocytes from donated blood by filtration. Published evidence, establishing the benefit of LR in reducing the risk of febrile nonhemolytic reactions, cytomegalovirus transmission, and human leukocyte antigen alloimmunization has led to its use for some time for the care of immunosuppressed and other individuals considered to be at high risk for such complications. Recent literature suggests that LR may be effective in reducing the risk of transmission of a number of additional transfusion-transmitted infectious agents, including herpesviruses, retroviruses, bacteria, protozoa, and prions. There is also evidence that LR may reduce the risk of transfusion-related immunomodulation, further contributing to protection against infections that would complicate treatment. With the mounting evidence of potential benefit, a number of countries, as well as many hospitals and blood centers in the United States, have adopted a policy of performing LR for all donated blood. Physicians who care for immunosuppressed patients and those who are responsible for institutional infection-control practices should remain informed of the growing body of literature on LR.

Studies on the mechanism of decreased NMR-measured free magnesium in stored erythrocytes

31P-NMR spectra have been recorded on erythrocytes stored at 4 degrees C in various preservation media. Storage was always associated with an upfield shift of the inorganic phosphate (Pi) resonance and a pronounced upfield shift of the ATP beta resonance, indicating decreased intracellular pH (pHi) and decreased intracellular free magnesium ([Mg2+]i). The decreased [Mg2+]i occurred in preservation media not containing citrate and even in media supplemented with Mg2+. It could not be attributed to the changes in pHi, Na+, K+, lactate, Pi or 2,3-diphosphoglycerate, that occur with storage. The decrease in [Mg2+]i was largely reversed when stored cells were incubated for 1 h at 37 degrees C in fresh plasma. Stored cells were found to contain significant amounts of inorganic pyrophosphate, up to about 200 mumol per liter cell water. Being a tight binder of Mg2+, pyrophosphate could account for some of the observed decrease in [Mg2+]i. Additional mechanisms may involve precipitation of some other Mg2+ complex during cold storage or enhancement of Mg2+ binding to membrane components.

Development of direct antiglobulin reaction accompanying alloimmunization in a patient with Rhd (D, category III) phenotype.

Abstract. New examples of the Rhd (D, category III) red cell phenotype are described in three siblings. One of these individuals who had previously been transfused, received three units of Rho(D) positive blood several years later and developed a high titer anti‐RhD antibody. This anamnestic response was associated with the development of an autoantibody which persisted for a period of at least 8 months. This observation represents another example of autoimmunization which has rarely been observed to accompany alloimmunization.

A mimicking red blood cell autoantibody accompanying transfusion and alloimmunization

A patient with sickle cell disease who concomitantly developed red cell autoimmunity and alloimmunization is reported. The implied but ‘wrong’ specificity of the autoantibody mimicked one of the alloantibodies in the patients serum. Although the patients red blood cells phenotyped at Ro4, anti‐rh” was eluted from them on several occasions. Absorption and secondary elution from selected cells proved the cell bound antibody had a unique and independent specificity from the anti‐rh” in his serum. Standard antibody identification procedures did not distinguish these differences.

Multicenter Evaluation of the 3% Paraformaldehyde Method for White Cell Counting in Leukocyte‐Reduced Red Blood Cells

Background/Aim: The 3% paraformaldehyde (PFA) method is a simple technique for counting residual white blood cells (WBC) in leukocyte‐depleted red blood cells (RBC). Preliminary data suggested that its sensitivity is at least equal to PCR and flow cytometry. We report the results of a multicenter study conducted by the BEST Working Party to determine precision and accuracy of the 3% PFA method. Study Design: In the 7 participating laboratories, 5 sets of samples containing nominal concentrations of 200, 100, 50, and 10 WBC/ml were prepared by diluting whole blood into ‘WBC‐free’ RBC. Ten milliliters of each sample were processed using the 3% PFA method, which is based on erythrocyte lysis and WBC concentration into 5% of the original sample volume; a Nageotte chamber is used to count concentrated WBC. Results: The precision of the technique varied according to the nominal concentration, ranging from a CV of 12% at 200 WBC/ml to 57% at 10 WBC/ml. The technique measured fewer than the nominal WBC concentrations (mean of all laboratories, ‐12.4%); underestimation was probably due to cell loss during sample manipulation. Overall accuracy was however acceptable, because statistical considerations establish that the actual WBC concentration would unlikely exceed 2 times the estimated count. Conclusions: The 3% PFA method is suitable for the enumeration of residual WBC at concentrations ≥50/ml. It represents a useful tool for evaluation of high performance filters by reference laboratories.