Validation of a microbial detection system for use with ACD-A platelets with PAS III platelet additive solution.

BACKGROUND: The BacT/ALERT microbial detection system (BTA) is used for testing leukoreduced apheresis platelets (LR-AP) in plasma. Platelet additive solutions (PASs) such as InterSol (PAS III) may be used to reduce the amount of plasma transfused in LR-AP. This study evaluated the performance of the two-bottle BTA testing scheme in the recovery of seeded microorganisms from LR-AP in InterSol-plasma compared to a reference plate culture method. STUDY DESIGN AND METHODS: Hyperconcentrated, double LR-AP were collected from healthy donors; InterSol was added (65% Intersol:35% plasma), equally divided into two containers, and then inoculated with an isolate of 1 of 10 clinically relevant index organisms at two levels. Aerobic (BPA) and anaerobic (BPN) BTA bottles were inoculated with 4 mL each of the inoculated LR-AP, and blood agar plates (BAPs) for aerobic and anaerobic culture (0.5 mL each). RESULTS: Zero false-positives from 103 bottle pairs were observed. All 400 two-bottle BTA tests were positive within 24 hours, except for Propionibacterium acnes (maximum time-to-detection of 86.4 hr) and 13 of 20 pairs of Streptococcus viridans (maximum time-to-detection of 31.7 hr). Thirteen of 400 BAP two-plate tests were negative for starting bacterial concentrations of 10 colony-forming units (CFUs)/mL or less. At 40 CFUs/mL or less, BTA was 100% positive while BAP was 94% positive. CONCLUSION: Seeded organism recovery was superior in the two-bottle BTA test system compared to the two-plate BAP system using InterSol platelets (PLTs). This performance is comparable to previously published results for PLTs in plasma. The use of InterSol does not appear to have a detrimental effect on the performance of the two-bottle BTA system.

In vitro and in vivo evaluation of apheresis platelets stored for 5 days in 65% platelet additive solution/35% plasma.

BACKGROUND: In the United States, apheresis platelets (PLTs) are suspended in autologous plasma. PLT additive solutions, long used in Europe, decrease recipient allergic reactions and may reduce the risk of transfusion-related acute lung injury. We evaluated Amicus-collected PLTs stored in platelet additive solution (PAS) III (InterSol) for 5 days. STUDY DESIGN AND METHODS: In Study 1, 71 subjects donated two products on a single day-one each stored in 100% plasma or 65% PAS III/35% plasma. Products underwent standard in vitro testing on Days 1 and 5. In Study 2, 43 additional subjects provided Amicus products stored for 5 days in 65% PAS III/35% plasma for in vivo radiolabeled recovery and survival determinations. The effect of approximately 2500cGy Day 1 gamma irradiation was evaluated in a subset of products. RESULTS: PAS III PLTs (n=70) had a median Day 5 pH(22°C) of 7.2 (lower 95%, 95% tolerance limit, 6.9). Mean Day 5 recovery and survival of radiolabeled PAS III PLTs (n=33) were, respectively, 80.5 and 72.1%, of fresh autologous PLTs. With 95% confidence, these values were at least 66% of fresh PLT recovery and 58% of survival. All in vitro variables remained within ranges seen in licensed products for irradiated and nonirradiated PAS III PLTs. CONCLUSION: Leukoreduced Amicus PLTs stored in 65% PAS III/35% plasma in PL-2410 containers maintained pH ≥6.9 throughout 5 days' storage. Radiolabeled PLT recovery and survival values met US Food and Drug Administration statistical criteria. Gamma-irradiated PAS III PLTs demonstrated no significant adverse effects due to irradiation in in vitro testing.

Prediction of red cell and blood volumes distribution by various nomograms: do current nomograms overestimate?

BACKGROUND: Currently used formulas for estimation of a person's red cell volume (RCV) by weight and height are decades old and were based on the use of 51Cr isotopes and on a sample population, which may not be reflective of today's population. In this study, the accuracy and precision of the use of 99mTc RCV measurements in volunteers more typical of today's population were evaluated. STUDY DESIGN AND METHODS: The subjects were volunteers who met the requirements for a standard blood donation. RESULTS: The mean +/- standard deviation (SD) 99mTc RCV for 127 males (mean weight, 83.2 kg; height, 180 cm) was 2062 +/- 339 mL, and for 101 females (mean weight, 69.5 kg; height, 166 cm) it was 1320 +/- 201 mL. These results were highly correlated with RCV results with the standard extrapolation 51Cr method with stored red blood cells (RBCs) and highly consistent (within +/-10%) by repeated measurements with the same 22 donors over a 3.5-year period. The RCV results correlated with estimates from the current formulas, but were on average 11 to 14 percent lower. CONCLUSION: The studies demonstrated that 99mTc is a reproducible and precise method for determination of a person's RCV and that current formulas may significantly overestimate the RCV of today's population. This is likely the result of a shift in population characteristics over the past four decades as reflected by an increased mean body mass index (from 25 to 28 kg/m2), which has not resulted in a proportionally increased RCV.

In vitro pH effects on in vivo recovery and survival of platelets: an analysis by the BEST Collaborative.

BACKGROUND: The pH environment of stored platelet (PLT) products is recognized as an important factor and is generally used as a key surrogate measure of PLT viability. It is the only in vitro measurement that has been translated into industry standards and regulatory rules or specifications for storage of PLT products. The objective of this study was to evaluate the effect of in vitro pH on the in vivo recovery and survival of autologous PLT products. STUDY DESIGN AND METHODS: Data from individual autologous radiolabeled PLT kinetic studies were solicited from independent laboratories. PLTs stored for at least 5 days in 100 percent autologous plasma with a pH(22 degrees C) of at least 6.2 were analyzed. Data were fit to a mixed-effects regression model with fixed effects of pH(22 degrees C), time of storage, and preparation method-storage bag combination. RESULTS: Eight research laboratories reported 476 individual recovery and survival results with associated pH before labeling from a variety of autologous, radiolabeled PLT kinetic studies from September 1999 to March 2005. These results are from 254 individual subjects who donated a total of 386 PLT units, with up to nine collections per subject reported. The effect of pH on either PLT recovery (p = 0.86) or survival (p = 0.55) was not significant. Time of storage and the method-bag combination both had significant effects on these outcomes (p < 0.0001). CONCLUSION: These data suggest that there is no relationship between in vitro pH at a pH(22 degrees C) of at least 6.2 and in vivo PLT viability as measured by radiolabeled recovery and survival of autologous PLTs.

Collection of two units of leukoreduced RBCs from a single donation with a portable multiple-component collection system.

BACKGROUND: A portable automated component collection system that produces double (2) units of leukoreduced RBCs (DRBCs) from a single donation was evaluated. This study analyzed quality of the collected and final products, the efficacy of automated leukoreduction, and donor safety. STUDY DESIGN AND METHODS: The system was used to collect 120 DRBCs. WBCs were removed from 90 products with machine-controlled filtration. DRBCs were collected in ACD-A and stored in AS-1 for 42 days at 1 to 6 degrees C. Pre- and postprocedure donor vital signs and hematologic parameters were measured. Procedure time, product characteristics, and adverse events were also recorded. In vitro studies were performed on all products on Day 0 and at end of storage. In vivo recoveries of 28 leukoreduced and 9 nonleukoreduced products were measured on Day 42. RESULTS: Day 0 mean percentage of hemolysis for leukoreduced and nonleukoreduced units was 0.05 percent. DRBCs had residual WBC counts of less than 1 x 106 cells per unit and mean RBC recovery after filtration of 91.9 +/- 2.7 percent. Mean 24-hour recovery after infusion for leukoreduced units at end of storage was 80.9 +/- 6.9 percent and nonleukoreduced units was 77.6 +/- 5.8 percent (p> 0.05). No clinically significant changes in donor vital signs or serious adverse events were observed. CONCLUSIONS: The quality of leukoreduced RBCs collected with this portable automated component collection system met or exceeded FDA requirements. This automated system is safe and effective for collection and processing of 2 units of RBCs suitable for transfusion.

Autologous transfusion recovery of WBC-reduced high-concentration platelet concentrates.

BACKGROUND: This study evaluates the recovery and survival of high-concentration platelets (HCPs) compared to standard apheresis platelets (APCs) in a double-label autologous human system. METHODS: Nine HCP units paired with APC units were stored, labeled with either 51Cr and 111In, and returned, and recovery and survival were determined. Standard in vitro platelet biochemical and functional parameters were monitored over the storage period and evaluated in a secondary analysis. RESULTS: Three each HCP units containing more than 2.2 x 10(11), 1.5 x 10(11) to 2.1 x 10(11), and 0.8 x 10(11) to 1.1 x 10(11) platelets in 59.4 +/- 2.5 mL were stored for 1, 2, or 5 days, respectively, and simultaneously with matched APC units (3.8 x 10(11) platelets, 282 mL). Recoveries were 72.3 +/- 8.6, 60.8 +/- 14.6, and 52.5 +/- 6.7 percent for HCPs, respectively; and 59.4 +/- 6.4 percent for APCs (p=0.37). HCP survivals were 202.0 +/- 14.9, 204.9 +/- 10.2, and 162.6 +/- 17.0 hours; APC survivals were 155.4 +/- 20.3 hours (p=0.001). Secondary analysis with P-selectin added as a predictor in the model resulted in significant difference in recoveries for Day 1 HCPs versus Day 5 APCs (p=0.024) with no difference shown for HCPs on Days 2 or 5 versus APCs. No significant difference was found in survival (p=0.16). CONCLUSION: HCPs may be stored 24 hours for high yield, 48 hours for intermediate yield, and up to 5 days for yields less than 1.6 x 10(11) platelets per bag with equivalent to superior recovery and survival of platelets in the autologous transfusion model compared to APCs.

In vivo and in vitro characteristics of double units of RBCs collected by apheresis with a single in-line WBC-reduction filter.

BACKGROUND: A novel apheresis procedure for a blood separator (MCS+, Haemonetics) enables the collection of 2 WBC-reduced RBC units in a single donation by using one disposable set with one in-line WBC-reduction filter (RC2H, Pall Corp.). The objective of this study was to evaluate the filtration performance in connection with different prefiltration RBC storage conditions and with the in vitro and in vivo storage quality of the filtered units. STUDY DESIGN AND METHODS: Sixty-six 2-unit RBC collection and gravity-filtration procedures were completed at three sites, resulting in 132 RBC units. Filtration of the double RBC units was performed at room temperature (RT) within 8 hours of collection (n = 36) and under refrigeration (1-6 degrees C) for up to 24 hours (n = 10) and 72 hours (n = 20) before filtration. RBC quality was compared to that of nonfiltered apheresis RBC units (n = 10). RESULTS: Median filtration time was 6.5 and 14 minutes for units stored at RT and under refrigeration, respectively. All 132 RBC units had residual WBC counts <0.4 x 10(6). The refrigerated units showed a greater mean log reduction in WBCs: 5.06 +/- 0.16 (24 hour) and 4.74 +/- 0.48 (72 hour), respectively, than did RT units: 4.47 +/- 0.28 (p<0.05). RBC loss was less than 12 percent in all cases (mean, 7.8 +/- 1.8%). Minimal differences in volume were observed between the paired RBC units. In vitro RBC storage characteristics of the filtered units were as expected and similar to those of the nonfiltered units. For RBC units held at RT (n = 24), the mean in vivo 24-hour recovery was 81.8 +/- 8.4 percent (double-label). CONCLUSION: Satisfactory filter performance in terms of WBC removal and RBC loss was observed with all 66 procedures, irrespective of storage conditions before filtration.

The effect of leukocyte-reduction method on the amount of human cytomegalovirus in blood products: a comparison of apheresis and filtration methods.

This study examined the effectiveness of 3 leukocyte-reduction (LR) methods in depleting the residual level of cytomegalovirus (CMV) in blood products measured by quantitative polymerase chain reaction (QA-PCR). At 2 locations over 3 allergy seasons, apheresis platelets and whole blood were collected from 52 healthy CMV seropositive subjects having an elevated titer of CMV DNA (median = 2400 genome equivalents [GE]/mL) resulting in 32 evaluable LR apheresis platelets, 31 filtered platelets from whole blood, and 31 filtered red blood cells (RBCs) from whole blood. Leukoreduction by apheresis and filtration resulted in substantial reduction of detectable CMV DNA levels with 99.9% of the LR products expected to have less than 500 GE/mL of CMV DNA. No difference was found between methods (P =.52). CMV genomic leukocyte subset localization was determined by QA-PCR of fluorescence-activated cell sorter (FACS)-sorted peripheral blood from 20 seropositive subjects (n = 10 > 100 GE/mL, n = 10 QA-PCR negative). CMV was detected in monocyte (13 of 20) and granulocyte (3 of 20) fractions. Presence of competent virus in QA-PCR positive (> 100 GE/mL) peripheral blood samples was verified with 4 of 19 subjects positive in shell vial assay, and 8 of 18 positive for CMV gene products (messenger RNA). We observed a seasonal DNAemia variation in seropositive subjects. CMV seropositive subjects (n = 45) entered into longitudinal monitoring in March/April 1999 were QA-PCR negative at baseline. Subjects converted to a positive QA-PCR coincident with increased seasonal allergen levels (Norfolk 15 of 18 evaluable in 43.4 +/- 9.48 days; Denver, 16 of 23 evaluable in 96 +/- 26.3 days). These data demonstrate effective reduction of CMV load by LR during periods of DNAemia in CMV seropositive subjects. (Blood. 2001;97:3640-3647)

Evaluation of an automated system for the collection of packed RBCs, platelets, and plasma.

BACKGROUND: This study evaluated the quality of WBC-reduced platelets, RBCs, and plasma collected on a new system (Trima, Gambro BCT) designed to automate the collection of all blood components. The study also evaluated donor safety and suitability of these components for transfusion. STUDY DESIGN AND METHODS: In Phase I, the quality of the components collected on the new system was evaluated by standard in vitro and in vivo testing methods. Results were compared to those from control components collected by currently approved standard methods. In Phase II, additional collections were performed to evaluate the acceptability of the new system and the safety of platelets collected. RESULTS: In vivo 24-hour RBC recovery was 76.8 +/- 3.1 percent for the test RBC units and 77.1 +/- 4.4 percent recovery for whole-blood (control) RBCs. The differences between test and control platelet results in the in vivo and in vitro assays were not clinically significant. Plasma clotting factors and fibrinogen levels met international standards. The system was well accepted by donors, and no major adverse donor reactions were reported for the 68 procedures performed. No problems were reported with transfusing the blood components collected. CONCLUSION: Blood components collected with the Trima are equivalent to currently available components, and they meet the applicable regulatory standards. This system provides consistent, standardized components with predictable yields. It provides the option of fully automating the collection of all blood components.

Quality and clinical response to transfusion of prestorage white cell-reduced apheresis platelets prepared by use of an in-line white cell-reduction system.

BACKGROUND: This study evaluated the quality and clinical effectiveness of white cell (WBC)-reduced apheresis platelets collected by the use of a new technology, fluidized particle-bed separation. STUDY DESIGN AND METHODS: In phase 1, six suitable donors underwent two separate plateletpheresis procedures on one occasion, each separated by less than 10 minutes. In random order, a control unit was collected with the COBE Spectra and a test unit with the Spectra Leukocyte-Reduction System (LRS). The quality of apheresis platelet components was assessed by an in vitro test panel, and residual WBCs were counted by Nageotte chamber and flow cytometric methods. For the in vivo studies, the test and control units were randomly labeled with either 51Cr or 111In at the end of storage and transfused simultaneously to the donor. Samples were taken for calculation of platelet survival and recovery. In phase II, 109 thrombocytopenic patients were given platelets collected by use of the Spectra LRS. RESULTS: Test platelets had significantly fewer residual WBCs (median 7.6 x 10(4)) than control platelets (median 3.9 x 10(5)), with equivalent in vitro function values. Test and control platelets had similar recovery and survival. Transfused platelets collected by use of the LRS achieved a mean 1-hour corrected-count increment of 19.3. CONCLUSION: The LRS collects platelet components with significantly lower WBC contamination without adverse effects on the function or in vivo survival of the platelets.

Evaluation of in vivo and in vitro quality of apheresis-collected RBC stored for 42 days.

BACKGROUND AND OBJECTIVES: New technological developments make it possible to collect red blood cells (RBCs) by apheresis, which allows for better product consistency and has the potential for improved RBC quality. The purpose of these studies was to evaluate the quality and consistency of units of RBCs collected by apheresis using the MCS+(R) machine (Haemonetics Corp., Braintree, Mass., USA). MATERIALS AND METHODS: Two studies were performed. In study 1 (n = 10), using containers and CP2D/AS-3 solutions from Medsep Corp. (Covina, Calif. USA), one-unit apheresis RBCs were compared to manually collected RBCs in a random crossover design. In study 2 (n = 12), 6 subjects had one unit collected, while the remaining 6 subjects had two units of RBCs collected with comparison to previously manually collected RBCs from the same donors. Haemonetics containers and solutions were used in study 2. RESULTS: Low RBC volume variability was found for the apheresis collections with a standard deviation of only 6 ml difference between actual and target volumes. Combining the data from the two studies (n = 21 pairs), at 42 days of storage, the apheresis units showed slightly lower hemolysis (0.44+/-0.26 vs. 0.61+/-0.50%), lower supernatant potassium levels (50+/-3 vs. 53+/-3 mEq/l), and improved tolerance to osmotic shock (47+/-3 vs. 49+/-3%) as compared to manual units (p < 0.05). There was no statistically significant difference in RBC ATP (3.0+/-0.6 vs. 2.9+/-0.5 micromol/g Hb) or in 24-hour percent recoveries (81+/-6 for apheresis vs. 81+/-4% for apheresis red cells). Apheresis RBC quality was not affected by the manufacturer (Haemonetics vs. Medsep) of solutions and containers. CONCLUSIONS: RBC units collected by apheresis demonstrated low variability in volume of RBC mass collected, and showed similar RBC properties as compared to manually collected RBCs after processing and after 42 days of storage.

In vitro interference of the red cell substitute pyridoxalated hemoglobin-polyoxyethylene with blood compatibility, coagulation, and clinical chemistry testing.

OBJECTIVES: Pyridoxalated hemoglobin-polyoxyethylene (PHP) is a prototypical red cell substitute approved for phase I studies. Peripheral blood smears of human blood mixed with PHP in 1 to 4 g/dL concentrations showed dose-dependent red cell aggregation and rouleaux. Whether this aggregation limits interpretation of blood compatibility testing and whether the intense coloration of serum or plasma containing PHP affects routine coagulation and clinical chemistry measurements was tested. DESIGN: In vitro studies. SETTING: University hospital laboratory. PARTICIPANTS: Four healthy volunteers, blood types A, B, AB, and O. All were Rh+. MEASUREMENTS AND MAIN RESULTS: ABO typing, Rh typing, and antibody screening and coagulation studies were performed on blood: PHP admixtures having final concentrations of 1, 2, and 4 g/dL. For clinical chemistry interference studies, known concentrations of analytes were added to a serum matrix containing PHP. ABO (forward) and Rh typing showed no interference in the three concentrations tested. Reverse ABO typing and antibody screening showed rouleaux at 4 g/dL, which corrected with routine saline replacement. Partial thromboplastin time (PTT), prothrombin time (PT), and fibrinogen showed no clinically significant differences from the controls. Results for electrolytes, renal function analytes, and markers of cardiac injury were acceptable by standard laboratory methods. However, results of liver function tests were unacceptable in PHP-containing specimens. CONCLUSIONS: PHP-induced aggregation was observed with high PHP concentration; however, compatibility testing was not affected because agglutination was corrected by saline replacement, which is standard practice. Although routine blood banking, coagulation, and most clinical chemistry analytes can be measured reliably, alternative methods and strategies are needed for assessing liver function in the presence of PHP.

The expression of p-selectin during collection, processing, and storage of platelet concentrates: relationship to loss of in vivo viability.

BACKGROUND: Recent studies suggested that platelet activation with surface expression of p-selectin on stored platelets may be related to a loss of viability. At present, there has been no thorough investigation of the extent or significance of p-selectin expression during the collection, processing, and storage of platelet concentrates (PCs) under various conditions. STUDY DESIGN AND METHODS: Platelet surface expression of p-selectin (CD62) was determined on fixed platelet samples using fluorescein-conjugated monoclonal antibodies. Platelet viability was assessed by autologous transfusion of platelets stored for 5 days and labeled with either 51Cr or 111in. RESULTS: Little (2-10%) platelet expression of p-selectin was found in whole blood and platelet-rich-plasma preparations, whereas PCs showed a substantial increase in p-selectin expression to levels of 20 to 30 percent. Both fresh PCs and those stored for 5 days, obtained with one cell separator (MCS, Haemonetics) showed substantially lower levels of p-selectin expression than PCs from two other cell separators (Spectra, COBE, and CS-3000 with TNX-6, Baxter Healthcare). Exposure of platelets to EDTA, cold, or a pH below 6.2, conditions that are known to result in the loss of viability upon transfusion, produced substantial and irreversible p-selectin expression. PCs with a pH of 6.2 to 6.8 (conditions in which no loss of viability has been demonstrated) also showed pronounced p-selectin expression, which returned to control values after incubation at 37 degrees C in plasma at pH 7.0 to 7.2. With storage under current conditions the in vivo studies (n = 61) demonstrated a rather poor correlation between p-selectin expression and the percentage of recovery (r = -0.25) but a somewhat better correlation with survival (r = -0.42). Better correlations were observed with the extent of shape change, lactate, and hypotonic shock response. CONCLUSION: These studies show that p-selectin expression on the platelet surface is a predictor of platelet viability, although the extent of shape change and the hypotonic shock response may be more sensitive.

Evaluation of a new prestorage leukoreduction filter for red blood cell units.

BACKGROUND AND OBJECTIVES: Prestorage leukoreduction offers a variety of potential benefits and is becoming more commonly practiced. The LeukoNet prestorage leukoreduction filtration system is intended for leukoreduction of red blood cells and uses a vent to allow automatic drainage of red cells from the filter. MATERIALS AND METHODS: We studied the functional characteristics and the in-vivo and in-vitro properties of leukoreduced AS-1 Red Blood Cells prepared with this new system. Units of AS-1 Red Blood Cells were filtered at 4 degrees C through the LeukoNet filter 24-48 h after collection and stored under usual conditions for 42 days. Residual leukocytes were enumerated using a Nageotte chamber or with a polymerase chain reaction (PCR) technique. In the clinical trial (phase one), 21 donors had units stored with and without leukoreduction for 42 days; biochemical assays were done before and after storage, and 51Cr/99mTc red cell recovery studies at the end of the storage period. RESULTS: Leukocyte content after filtration was 3.2 +/- 2.6 x 10(4)/unit (n = 21), and all units had < 1 x 10(5) leukocytes (median: 3.8 x 10(4)). In-vivo paired studies showed no difference in 24-hour recovery (control: 82.1 +/- 5.8%; test: 82.9 +/- 6.0%). Hemolysis was halved with leuko-reduction (0.59 +/- 0.30 vs. 0.29 +/- 0.11%; p < 0.05), and glucose consumption was reduced by 5% compared to control units (p = < 0.05). Other biochemical parameters showed no differences. In the practical trial (phase two), filtration time was 41 +/- 23 min. With a residual leukocyte content of 6.6 +/- 4.9 x 10(4)/unit and 14 +/- 3% red cell loss (n = 84). Six additional units underwent leukocyte enumeration by PCR and had 2.6 +/- 1.1 x 10(4) residual leukocytes. CONCLUSIONS: Under the conditions studied, the LeukoNet leukoreduction filtration system produces about 4-5 log10 leukocyte content reduction.