Diversion of initial blood flow to prevent whole-blood contamination by skin surface bacteria: an in vitro model.

BACKGROUND: Sepsis arising from the transfusion of bacterially contaminated platelet components continues to be an infrequent, yet serious transfusion complication. Skin organisms are implicated in a number of these septic episodes. A model system was used to investigate if a skin organism's bioburden in blood components could be reduced by diverting the first few mL of whole blood away from the primary container. STUDY DESIGN AND METHODS: A sterile medication site was inserted into a bag containing sterile saline or whole blood; the site was deliberately contaminated with Staphylococcus aureus and allowed to dry. After needle puncture of the contaminated medication site, bacteria levels were measured 1) in successive 7-mL tubes of blood or saline drawn through a diversion arm, 2) in 40 mL of a connected transfer pack, and 3) in blood or saline from a needle puncture of the original container via another sterile medication port. RESULTS: Diverting the first 21 to 42 mL of saline or whole blood reduces the downstream bioburden of deliberately introduced surface S. aureus by approximately 1 log. CONCLUSION: Development of a diversion system for collection of whole blood in sample tubes before filling the primary container may reduce the bioburden of subsequently prepared components and thereby the frequency of sepsis due to skin organisms.

Viability and in vitro properties of AS-1 red cells after gamma irradiation.

BACKGROUND: Irradiation has been shown to adversely affect both in vivo 24-hour recovery (recovery [%]) and in vitro properties of stored red cells (RBCs). There is uncertainty as to how these changes are related to the day of irradiation and the length of storage after irradiation. STUDY DESIGN AND METHODS: Four protocols used day of irradiation and storage time after irradiation as the independent variables. At the conclusion of the storage period, viability was measured with radiolabeled RBCs as the recovery and the long-term survival time for RBCs that were circulating beyond 24 hours. In addition, in vitro values including RBC ATP, hemolysis level, and supernatant potassium were measured. Each subject donated 2 units of whole blood (CPD) and received autologous irradiated and untreated control RBCs (AS-1) on two separate occasions. RESULTS: Reduced recovery in irradiated units was noted when compared to that in control units, and the reduction was most apparent with long periods of storage after irradiation, irrespective of the day of irradiation. With irradiation on Day 1 of storage and a total storage period of 28 days, mean +/- SD recovery (single label) was 84.2 +/- 5.1 percent for control RBCs and 78.6 +/- 5.9 percent for irradiated RBCs (n = 16; p<0.01). With irradiation on Day 14 and storage through Day 42, the recoveries were 76.3 +/- 7.0 percent for control RBCs and 69.5 +/- 8.6 percent for irradiated RBCs (n = 16; p<0.01). Less reduction in recovery was observed with shortening of the postirradiation storage time. When the total storage period was reduced to 28 days after Day 14 irradiation, the recoveries were not significantly different. With an additional 2-day storage period after irradiation on Day 26, the recoveries were also comparable. Long-term survival times for control and irradiated RBCs were not significantly different in any of the four protocols. RBC ATP levels and hemolysis were minimally, but significantly influenced by irradiation. Supernatant potassium levels, however, were substantially increased after irradiation in each of the four protocols. CONCLUSION: Irradiation has only a small effect on the properties of RBCs treated and stored according to the utilized protocols. Longer storage times after irradiation resulted in progressively reduced recovery while long-term survival remained unaffected.

Assessment of blood donor privacy during health history interviews.

BACKGROUND: Given the personal nature of health history interviews, it is important to provide donors with both visual and auditory privacy. Privacy is affected by variables such as background noise, the use of visual screens, and the loudness of the donor's voice. STUDY DESIGN AND METHODS: In Phase I of this study, an interview station and waiting area were simulated. To measure auditory privacy, a speech intelligibility test was given to subjects with and without the use of a free-standing privacy screen and masking noise device. Phase II was a field trial designed to evaluate screens and masking noise. Background noise was measured during each blood collection operation, and donors completed a survey. RESULTS: In Phase I, speech intelligibility test scores ranged from 78 to 5.1 percent, depending on the type of visual screen and the number of masking noise devices used. In Phase II, with the use of screens, 94 percent of donors rated visual privacy as "good to excellent," compared with 74 percent who did so when no screens were used. At many blood drives, the background noise level exceeded the level of the masking noise. CONCLUSION: The use of visual screens increases donors' perception of visual privacy. The use of masking noise is effective only when the health history interview is conducted under conditions of low background noise levels.

Development and evaluation of a shipping system for platelet components.

BACKGROUND: Platelet concentrates and apheresis platelets must be maintained at a temperature as close as possible to 20 to 24 degrees C during transport. To improve temperature control, ensure component quality, and meet handling and freight carrier needs, a new insulated shipping container system was developed and evaluated. STUDY DESIGN AND METHODS: Molded polyurethane-insulated shipping containers were loaded with different payloads of simulated platelet components, with or without gel-based temperature stabilizing packs (TSPs). The containers were subjected to constant ambient temperature of 37, 4 or -10 degrees C. Payload temperatures were continuously monitored, in situ, for 24 hours. RESULTS: Temperature data are reported as the mean number of hours needed for components to warm or cool by 1 degree C. The temperature of payloads exposed to a constant 37 degrees C ambient temperature increased by 1 degree C in 2.5 to 3.8 hours when no TSPs were included in the shipment and in 6.1 to 6.9 hours when TSPs were used. Exposure to a constant 4 degrees C ambient temperature resulted in a 1 degree C temperature decrease in 1.8 to 3.4 hours without TSPs and in 4.6 to 5.6 hours with TSPs. At a -10 degrees C ambient temperature, there was a 1 degree C drop within 1.0 to 1.6 hours without TSPs and within 2.7 to 2.9 hours with TSPs. CONCLUSION: The container and packing methods described moderate the rate of change in the temperature of platelet components during their exposure to challenging ambient conditions. The use of TSPs substantially improves the performance of the system. In addition, the system meets freight carrier requirements and is easy to use, environmentally friendly, and durable.

Comparison of bacteria growth in single and pooled platelet concentrates after deliberate inoculation and storage.

BACKGROUND: The ability to store pools of platelet concentrates (PCs) for extended periods would provide logistical flexibility. However, reports of severe adverse reactions due to the transfusion of contaminated PCs led to an examination of whether the total bacteria levels after storage of pools containing a deliberately inoculated platelet unit would be significantly different than the levels in paired unpooled concentrates. STUDY DESIGN AND METHODS: A single PC was deliberately inoculated on Day 0 with one of three bacterial species (0.1-8.0 colony-forming units/mL). On Day 1, the deliberately inoculated PC was divided into three equal parts and either 1) pooled with 5 half-volume, ABO- and Rh-identical PCs; 2) similarly pooled and white cell reduced; or 3) kept as a control. Sterile connections were used during pooling; modified storage containers were used to ensure the correct surface-to-volume ratio of the single unit. RESULTS: Between Day 2 and Day 5 of storage, in 26 of 36 paired samples, nonfiltered pools containing Escherichia coli had greater total numbers of bacteria than did the paired single PCs. Day 2 pools had total bacteria levels approximately five times higher (colony-forming units/mL x container volume) than those in single units (p < 0.05). There was rapid growth of Staphylococcus aureus by Day 2 in pooled and unpooled PCs; by Day 3, total bacteria levels were approximately five times higher in pools than in single units (p < 0.05). Between Days 3 and 5 of storage, in 23 of 27 paired samples, nonfiltered pools containing S. aureus had greater total bacteria levels than the single PCs. By Day 5, 15 of 16 non-white-cell reduced pools had total levels of Staphylococcus epidermidis bacteria approximately five times those in the paired single PCs. Greater total bacteria levels in pooled units than in single units generally occurred when bacteria in pools reached the stationary phase of growth (when bacteria concentration became constant), and they were well correlated with the sixfold volume of pooled units. White cell reduction did not substantially affect the time required to attain stationary phase. CONCLUSION: The potential during storage for greater total bacteria levels in pools than in single PCs is a consequence of the greater volume of the pool.

Reducing the infectivity of blood components--what we have learned.

The safety of the nation's blood supply has improved over the last several years as a result of more intensive donor screening and viral testing. Concurrently, there has been more judicious use of blood components. Although the risk is small, transmission of blood borne viruses, bacteria and parasites can occur. Investigators have studied a myriad of processes for pathogen depletion and/or inactivation, including the use of chemicals, extended storage, filtration, heating, irradiation, photochemicals and washing. Pasteurization, methylene blue and solvent-detergent processes have been introduced in parts of Europe for improving the safety of plasma used for transfusion. The FDA is reviewing a license application for the solvent-detergent process. For red cells, use of highly efficient leukodepletion filters is believed to be equivalent to antibody testing for the prevention of CMV disease transmission. Otherwise, no successful treatments have yet been identified for red cells or platelets. Several photochemicals, which may be useful for treating these components, are being studied. However, there appear to be trade-offs between the extent of pathogen inactivation, platelet or red cell damage, and genotoxicity. These as well as other biological parameters and operational issues will need to be further evaluated before implementation can be considered.

Effects of new brochures on blood donor recruitment and retention.

BACKGROUND: Currently, donors may arrive at blood collection sites without prior knowledge of eligibility and deferral criteria. STUDY DESIGN AND METHODS: The effects of distributing newly developed recruitment brochures 2 weeks in advance of blood drives and the provision of brochures on temporary deferral at the taking of health history were examined in four southeastern regional blood collection centers. Twenty-four similar pairs of worksites, with employee-only recruitment, were randomly assigned to a control (C) or experimental (E) group. Information about sponsor recruitment strategies, worksite factors, and first-time, repeat, and temporarily deferred donors was obtained at three collection drives per site over a 1-year period. Drive 1 was used as a baseline. Two weeks before Drives 2 and 3, the recruitment brochures were distributed to all Group E employees, with temporary deferral brochures provided as needed when the health history was taken. RESULTS: No significant differences between groups or drives were found in the total percentage of employees recruited or returning as a result of recruitment or deferral brochure distribution (Wilcoxon's signed rank test and t test). Substantiating previous observations by donor recruiters, the study results showed decreased donations during vacation periods and busier times at the workplace. Loudspeaker announcements led to decreased donations; increased donations followed special appeals in relation to a specific patient, an accident, or a natural disaster. CONCLUSION: The brochures may have encouraged previous donors to return, but their use did not significantly increase the recruitment of new donors or the return of temporarily deferred donors.

Transfusion-associated bacterial sepsis.

The incidence of sepsis caused by transfusion of bacterially contaminated blood components is similar to or less than that of transfusion-transmitted hepatitis C virus infection, yet significantly exceeds those currently estimated for transfusion-associated human immunodeficiency and hepatitis B viruses. Outcomes are serious and may be fatal. In addition, transfusion of sterile allogenic blood can have generalized immunosuppressive effects on recipients, resulting in increased susceptibility to postoperative infection. This review examines the frequency of occurrence of transfusion-associated sepsis, the organisms implicated, and potential sources of bacteria. Approaches to minimize the frequency of sepsis are discussed, including the benefits and disadvantages of altering the storage conditions for blood. In addition, the impact of high levels of bacteria on the gross characteristics of erythrocyte and platelet concentrates is described. The potentials and limitations of current tests for detecting bacteria in blood are also discussed.

Loss of red blood cell viability associated with limited thermal inactivation of extracellular HIV-1.

The effects of incubation at mildly elevated temperatures on HIV-1 inactivation and in vitro red blood cell properties were investigated. Red cells (55% Hct) were leukodepleted (3 log10) by filtration, maintained at 45 or 47 degrees C for 4 or 8 h, and then stored at 4 degrees C. Hemolysis was twice that of controls after 42-day storage for samples treated for 4 h at 45 degrees C, and five times larger for samples heated at 47 degrees C. There was also a significant increase in the rate of potassium loss, an early decrease in ATP levels, and an initial drop in pH for samples treated at either temperature. Larger differences were observed for samples exposed to these elevated temperatures for 8 h. Osmotic deformability curves obtained by ektacytometry showed dramatic decreases in red cell deformability at both temperatures and for both time periods. HIV-1 inactivation in red cells treated at 45 degrees C (approximately 0.25 log10/h) was considerably less than that obtained in tissue culture medium (1-2 log10/h). Since the decrease in red cell deformability is likely to indicate reduced red cell function and survival, and the rate of HIV-1 inactivation is low, mild heat treatment is not an adequate process for viral inactivation of red cell products.

Red cell alterations associated with virucidal methylene blue phototreatment.

Several red cell storage properties were evaluated following phototreatment with methylene blue (MB) under conditions that inactivated > or = 6 log10 of added vesicular stomatitis virus. Red cell 2,3 DPG levels were similar to untreated controls throughout conventional 42-day storage at 4 degrees C. Plasma hemoglobin levels were elevated approximately twofold in MB-phototreated samples, and morphology scores were 5 percent lower after 42-day storage. ATP levels declined 30 percent in phototreated samples and in a control sample containing MB and not exposed to light. Lipid peroxidation was not observed in treated or control cells, nor were differences observed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis of ghost membranes derived from phototreated and control samples. Phototreated cells exhibited enhanced ion permeability; sodium and potassium levels approached equilibrium with the suspending medium within 4 to 7 days after treatment. Direct agglutination tests using rabbit anti-human IgG or rabbit anti-human serum albumin on MB-phototreated cells indicated that serum proteins had absorbed to the surface of treated red cells. Plasma depletion by washing red cells prior to phototreatment did not prevent protein binding upon subsequent addition of untreated autologous or group AB plasma. To a much smaller extent, phototreatment of plasma resulted in IgG association with untreated red cells. The addition of glutathione to red cell suspensions prevented IgG binding to phototreated red cells but did not prevent enhanced ion permeability. Taken together, these data suggest that the red cell surface is altered by virucidal MB phototreatment of vesicular stomatitis virus.

Viral inactivation and reduction in cellular blood products.

Even though the risks associated with the transfusion of blood products are lower than ever before, considerable efforts are being employed to improve the safety of the blood supply. Based upon available data, a six log (99.9999%) reduction in virus level from screened and tested blood components should significantly reduce or eliminate the risk of post-transfusion infection. The objective has been to identify "generic" methods, that is, one that would be applicable to all virus. For red cells, physical and chemical approaches have been studied; for platelets, the approaches have been limited to chemical. The physical methods include depletion of leukocytes by filtration, removal of plasma by washing, and viral inactivation by heat. Among the chemicals investigated to inactivate or help displace virus are ozone, detergents, and hypochlorous acid. Several photochemicals have also received intensive investigation: merocyanine 540, a benzoporphyrin derivative, aluminum phthalocyanine, and methylene blue. For platelets, photochemical inactivation methods using merocyanine 540, and two psoralen derivatives, 8-methoxypsoralen (8-MOP) and aminomethyl trimethyl psoralen (AMT), have also been studied. Approaches which include washing are not suitable. For the most part, either viral removal or inactivation has been insufficient, or red cell or platelet damage unacceptable. However, there are a few indications that at least inactivation of a specific virus, such as HIV, may be possible without major cell damage. These studies are in their early stages and significant work remains. If feasibility is clearly shown in vitro, it is likely that in vivo primate studies to demonstrate safety and efficacy will be required.

Effects of oral donor questioning about high-risk behaviors for human immunodeficiency virus infection.

The purposes of this study were 1) to compare blood donor deferrals resulting from additional, oral questions about human immunodeficiency virus risk behaviors with deferrals resulting from currently used, written screening questions; 2) to examine differences in donor deferral resulting from use of an indirect (IQ) versus direct (DQ) additional oral question format; and 3) to evaluate written survey responses of donors and staff members to the additional questions. The IQ group (n = 3050) were asked if they understood the seven ineligible-donor risk behaviors, and the DQ group (n = 4753) were asked if they had engaged in any of these behaviors. Owing to positive answers or refusal to answer the additional questions, there was an increase in donor deferrals, over the level seen with customary screening. Only 1 percent of donors indicated they would not return if the questions were asked in the future. Embarrassment was indicated by 3 percent of the IQ group and 7 percent of the DQ group; 14 to 15 percent preferred to write their answers rather than give them orally. The staff members generally felt training was adequate (IQ = 92%, DQ = 83%) and were comfortable asking the questions (IQ = 82%, DQ = 78%). Mean screening times were 5.7 minutes before the addition of the oral questions, 7.5 minutes with IQ, and 7.6 minutes with DQ. This study confirms the value of IQ and DQ formats in identifying potentially infectious donors and suggests that the DQ format may be slightly more effective.

Experimental 6 log10 white cell-reduction filters for red cells.

White cell (WBC) reduction of blood components has been receiving increased attention as a way of reducing transfusion-related complications such as WBC-associated HLA alloimmunization and transmission of cell-associated viral diseases. Currently available filters are limited to removing approximately 3 log10 (99.9%) of WBCs from red cells (RBCs). The performance of two experimental filters that were designed to remove 6 log10 WBCs from fresh RBCs during component preparation was evaluated. Both filters were able to meet this objective in less than 40 minutes with RBC losses of less than 15 percent under nonoptimized conditions. Filtered RBCs showed storage parameters within the normal range over a 42-day period. The use of these filters, if combined with a sterile docking device or if incorporated into a collection set, should provide the means to supply highly WBC-reduced RBCs with a normal shelf life.

Virucidal levels of ozone induce hemolysis and hemoglobin degradation.

The animal virus, vesicular stomatitis virus (VSV), and the bacterial virus, phi 6, were inactivated by greater than 4 log10 in response to incubation with 13 to 14 mL of 1.4 mmol per L (65 micrograms/mL) to 1.6 mmol per L (75 micrograms/mL) of overlaid ozone in virus-spiked, dilute, red cell suspensions. Virus inactivation was greatly inhibited when ozone was overlaid in the presence of high-hematocrit red cells or, to a lesser degree, high levels of plasma. At hematocrits at which 5 to 6 log10 of VSV were inactivated, ozone caused 30-percent hemolysis, as measured by the loss of total cellular hemoglobin. Unexpectedly, this level of hemolysis could not be observed in supernatants because of the ozone-induced destruction (bleaching) of extracellular hemoglobin. These results suggest that ozone may have little biological specificity for damaging viruses over red cells.

Methods for measuring a 6 log10 white cell depletion in red cells.

Methodology is presented for enumerating very low concentrations of white cells (WBCs) in red cells (RBCs) by two separate measurement techniques. Both techniques rely on the method of harvesting WBCs from a 300- to 350-mL unit of RBCs and concentrating them to a volume of approximately 0.5 to 1.0 mL, which is equivalent to a WBC concentration of approximately 550 to 1. The WBC separation and concentration steps require less than 3 hours to complete, and multiple RBC units can be processed in parallel. Cell counting is carried out in a fluorescence hemocytometer or by a modified cytospin technique. As few as 1000 WBCs in a unit of RBCs, which corresponds to a more than 6 log10 WBC depletion, can be measured without reaching the sensitivity limit of either technique (800 and 200 WBC/unit, respectively). The harvesting method and counting techniques are relatively simple and inexpensive.

The effect of flow on the interaction of isolated megakaryocytes with subendothelial extracellular matrix.

We have previously shown that human, guinea pig, or rat megakaryocytes, incubated under static conditions on an extracellular matrix (ECM) produced by endothelial cells, readily adhered to the matrix and underwent platelet-like shape change and thromboxane A2 secretion. We have now exposed megakaryocytes to ECM in a perfusion system similar to that used to study platelets circulated over aortic subendothelium. We used a continuous flow circuit incorporating a parallel plate perfusion chamber. Megakaryocytes were isolated to high purity from guinea pig marrow by centrifugal elutriation and velocity sedimentation. The cells were introduced into the flowing medium while the surface of an ECM-coated coverslip mounted in the chamber was observed continuously by phase-contrast video microscopy for up to 18 hours. Megakaryocytes from the flowing suspension started to adhere to the ECM within seconds. Significant adhesion occurred over a range of shear rates, from 10 to 190 seconds-1, did not appear above 300 seconds-1 and was greatest at a shear rate of 60 seconds-1. Adhesion to the ECM was specific, since there was no adherence to glass coverslips, glutaraldehyde-fixed ECM-coated coverslips, or to endothelial cells cultured on ECM-coated coverslips. At low shear rates large aggregates of megakaryocytes formed on the ECM surface; these could be detached and washed away by higher shear forces. Megakaryocytes thus acquire, even before platelet formation, an adhesive capacity similar to that of platelets. In addition, a significant fraction of the adherent megakaryocytes underwent elongation and pseudopod formation similar to that seen in marrow sinusoids.

Evaluation of technical and behavioral issues in predonation alanine aminotransferase testing.

To evaluate the technical and behavioral impact of predonation alanine aminotransferase (ALT) testing, one group of 2914 donors (Reflotron group) were tested with the Boehringer Mannheim Reflotron instrument; 3042 control group donors had no predonation test. Mobile blood collection sites in three regional blood centers were studied. More Reflotron group donors than control group donors thought that predonation testing did not add time and was a good idea. Both groups were equally satisfied with the donation process and indicated they would donate again even if testing added time. Comparison of the Reflotron and standard laboratory tests showed excellent correlation with low variability. The mean r value was 0.934, with a mean sensitivity of 94.4 percent and a specificity of 99.8 percent. The staff working with the Reflotron group found predonation testing easy to incorporate into donor screening. They observed that donors were pleased with testing and accepting of deferral counseling for elevated ALT. The control group staff was concerned that predonation testing would require more time and would have a negative effect on the donor return rate. This did not appear to be the case as judged by donor response and the 6-month return rates of 50 percent for Reflotron group and 54 percent for control group donors.