Safety of platelet transfusion: past, present and future.

Platelet components became routinely available to many institutions in the late 1960s and since then utilization has steadily increased. Platelets are produced by three principal methods and their manufacturing process is regulated by multiple agencies. As the field of platelet transfusion has evolved, a broad array of strategies to improve platelet safety has developed. This review will explore the evolution of modern platelet component therapy, highlight the various risks associated with platelet transfusion and describe risk reduction strategies that have been implemented to improve platelet transfusion safety. In closing, the reader will be briefly introduced to select investigational platelet and platelet-mimetic products that have the potential to enhance platelet transfusion safety in the near future.

Multi-institutional randomized control study of haemolysis in stored red cell units prepared manually or by an automated system.

BACKGROUND: The haemolysis level at the end of storage is a performance parameter for RBC preparations. In the evaluation of new devices or new processes for processing blood, it is relevant to evaluate whether the haemolysis is linked to (1) specific characteristics of the blood donor, or (2) the nature of the blood-processing methodologies. MATERIALS AND METHODS: As part of the validation of a new automated whole blood processing system compared to the current manual methods, randomized, paired crossover studies were conducted evaluating measures of blood component quality, including RBC haemolysis over 42 days of storage. RESULTS: The association between haemolysis and the individual subject was evaluated by modelling haemolysis with independent predictors of treatment (control and test processing) and leucocyte reduction as fixed factors with donor and laboratory as random effects in a mixed-effects ANOVA model. It was found that the day 42 haemolysis values were strongly dependent on the donor subject, with an intraclass correlation coefficient of 0.81. CONCLUSIONS: The data reported in this study suggest a link between the specific whole blood donor and the haemolysis levels observed in red-blood-cell units stored refrigerated for 42 days. Additional research to identify possible donor characteristics associated with haemolysis during storage is warranted.

Reducing the variation in performance of antibody titrations.

BACKGROUND: Antibody titration is difficult to standardize. We investigated whether a detailed, uniform procedure for antibody titration would reduce variation in both tube-based and gel card titres in an international study. METHODS: Laboratories (n = 35) tested proficiency testing material provided by the College of American Pathologists each according to (i) their routine method; (ii) a detailed, uniform method; and (iii) the uniform method titrating the serum sample against a red cell of specified phenotype (D+ C- c+ E+ e- for anti-D; A(1) for anti-A) instead of the red cell of the same phenotype provided in the proficiency testing kit. Uniform method results were reported with 1+ and w+ end-points. Paired statistical analyses of variance were conducted using the F-test. RESULTS: The variance between laboratories was not significantly reduced with the uniform method using a 1+ end-point. However, a statistically significant reduction in the variance of anti-D and anti-A titres by the tube-based uniform technique after 37 degrees C incubation and conversion to the antiglobulin (AHG) phase was seen when 19 laboratories reanalysed their results using a w+ end-point. Too few laboratories reported results with a w+ end-point in gel card testing to allow analysis. Titration against red cells of the specified phenotype provided by the participating laboratory did not appear to introduce additional variance. Overall, results reported based on the gel card technique at the AHG phase (1+ end-point) showed reduced variance compared to tube-based techniques. CONCLUSIONS: A detailed, uniform method for antibody titration at 37 degrees C and read at the AHG phase in a tube-based method with a w+ end-point reduced interlaboratory variability.

Reducing the variation in performance of antibody titrations.

BACKGROUND: Antibody titration is difficult to standardize. We investigated whether a detailed, uniform procedure for antibody titration would reduce variation in both tube-based and gel card titres in an international study. METHODS: Laboratories (n = 35) tested proficiency testing material provided by the College of American Pathologists each according to (i) their routine method; (ii) a detailed, uniform method; and (iii) the uniform method titrating the serum sample against a red cell of specified phenotype (D+ C- c+ E+ e- for anti-D; A(1) for anti-A) instead of the red cell of the same phenotype provided in the proficiency testing kit. Uniform method results were reported with 1+ and w+ end-points. Paired statistical analyses of variance were conducted using the F-test. RESULTS: The variance between laboratories was not significantly reduced with the uniform method using a 1+ end-point. However, a statistically significant reduction in the variance of anti-D and anti-A titres by the tube-based uniform technique after 37 degrees C incubation and conversion to the antiglobulin (AHG) phase was seen when 19 laboratories reanalyzed their results using a w+ end-point. Too few laboratories reported results with a w+ end-point in gel card testing to allow analysis. Titration against red cells of the specified phenotype provided by the participating laboratory did not appear to introduce additional variance. Overall, results reported based on the gel card technique at the AHG phase (1+ end-point) showed reduced variance compared to tube-based techniques. CONCLUSIONS: A detailed, uniform method for antibody titration at 37 degrees C and read at the AHG phase in a tube-based method with a w+ end-point reduced interlaboratory variability.

Sampling errors and the precision associated with counting very low numbers of white cells in blood components.

Attention to the accurate and precise measurement of the white cell (WBC) content of transfused products has risen in response to awareness of the potential benefits of WBC-depleted components and the development of technical capabilities to produce these components. The techniques thus far reported have focused on the reliability of detecting a WBC, provided it is present in the test system. The likelihood of selecting a WBC from the product of interest for counting in the analytical system--that is, the sampling error--must also be considered. The occurrence of a WBC in a WBC-depleted component is a rare event and may be modeled with the binomial or the Poisson distribution. Several assay techniques were analyzed by using these distribution models to determine the confidence intervals of the WBC content. The 95-percent confidence intervals spanned more than 2 logs10 for some methods at 3 x 10(5) WBCs per product. It is concluded that the reporting of WBC content for research provides not only the estimate of the mean but also a confidence interval for this estimate. Quality control procedures should be designed to verify that the WBC content is less than the targeted amount and should provide an associated statement of confidence.

Microdroplet fluorochromatic assay for the enumeration of white cells (WBCs) in WBC-reduced blood components: validation and application for evaluating newly developed WBC-reduction filters.

BACKGROUND: Sensitive and accurate counting methods are required to assess the residual white cells (WBCs) in WBC-reduced blood components. The Nageotte hemocytometer, widely used for this purpose, is cumbersome, and its efficacy is dependent upon the skill of the operator. The performance of a simple fluorochromatic assay using tissue-typing microdroplet trays is presented here. STUDY DESIGN AND METHODS: Undiluted samples of blood components were mixed with a fluorochromatic dye in trays. WBCs were counted under an epifluorescence microscope. The accuracy and sensitivity of this method were compared with those of the reference Nageotte hemocytometer method by using serial dilution of samples of platelets and red cells containing known concentrations of WBCs and by calculating the standard curves. The Nageotte hemocytometer and the microdroplet fluorochromatic assay (MFA) were also compared in terms of count correlation and reproducibility in 320 paired counts of plateletpheresis samples. MFA was used to evaluate a newly developed WBC-reduction red cell filter. RESULTS: The MFA for platelets and red cells was linear to 0.1 and 0.03 WBCs per microL, respectively. The linear regression line of log10 MFA versus log10 Nageotte method had a slope of 0.963, intercept of -0.04, and r2 of 0.968. The Nageotte method gave an estimation of WBC content 12 to 20 percent greater than that of the MFA. The MFA, with a larger neat sample volume, showed precision comparable to that of the Nageotte method. The filters demonstrated a median WBC reduction of 4.8 log10. CONCLUSION: The MFA is a sensitive and accurate method for quality control processes to assess the residual WBCs in WBC-reduced blood components.

Enhanced flow cytometric method for counting very low numbers of white cells in platelet products.

BACKGROUND: The increased demand for leukocyte-reduced platelet products has prompted actions both by blood suppliers and device manufacturers to develop effective quality assurance methods and improved devices for leukocyte-reduced platelet preparation. White cell (WBC) counting methods capable of counting WBCs substantially below 1,000 WBC/mL enhance these activities. The purpose of this study was to enhance and validate a method capable of counting 5-10 WBC/mL in platelets. METHODS: We added marker chicken red blood cells (cRBC) at a known concentration to an aliquot of platelet concentrate containing propidium iodide stain solution (PI). A modified BD FACScan was used to analyze this mixture. The ratio of cRBC and WBC events observed was used with the cRBC concentration to calculate the WBC concentration. Validation was performed by analyzing standard curves prepared with leukocyte-depleted platelet diluent. Methods of preparing leukocyte-reduced platelets by apheresis and filtration were evaluated using this method. RESULTS: The method was linear from 5,000 WBC/mL to 10 WBC/mL (r2 = 0.983). Residual WBCs in the platelet diluent hampered validation below 10 WBC/mL. Repeatability (CV) was 44% at 8 WBC/mL and 6.5% at 1,000 WBC/mL. The FACScan required frequent mixing of the sample and flushing with cleaning agents during acquisition. CONCLUSIONS: This method has good linearity and reproducibility from 10-5,000 WBC/mL for platelets. It is suited for research and development work, and may be a useful adjunct method for quality assurance in the blood center.

Making risk statements stick.

Estimates of risk associated with blood transfusion are reported from a variety of sources using different numerical constructs. These data must be judged for validity and generalizability to facilitate decisions for interventions and to estimate potential benefits of interventions. Risk estimates reported in consistent terms, such as occurrences per million units transfused, will assist in comparisons of risks and the expected effect observed at the practitioner level. Use of the estimated number needed to treat puts the effect of an intervention in perspective for the individual practitioner and for national health authorities. We re-evaluated data reported from several recent studies of transfusion risk to highlight this approach. In the USA, the number needed to treat estimated to prevent one HIV transmission is 4.3 million (mini-pool NAT); to prevent one death from bacterial sepsis is 21 thousand (conversion to single donor platelets), and 16 thousand (bacterial screening of platelet concentrates). As interventions are continuing to drive infectious disease transmission rates lower and lower, expressing residual risk as the number needed to treat demonstrates that further improvements in safety are unlikely to be recognized at the local level even though the overall impact at the national level is significant.

Clinical trial and local process evaluation of an apheresis system for preparation of white cell-reduced platelet components.

BACKGROUND: A new method for the consistent preparation of white cell (WBC)-reduced plateletpheresis components, the Spectra Leukoreduction System (LRS), was evaluated by clinical trial and local process validation. The centrifuge-based system was projected to decrease the WBC content of plateletpheresis components to a level below 1 x 10(6) per unit. Phase I and II clinical trials were performed. The manufacturer's claims were then tested at the local level with an ongoing quality assurance program. STUDY DESIGN AND METHODS: In Phase I, a cross-over analysis of five subjects compared LRS to standard plateletpheresis procedures in collection efficiency and component quality: a panel of in vitro measures was taken on Day 0 and Day 5. In Phase II, the LRS process was tested on a larger scale (n = 57; control = 58) with component transfusion. Finally, validation, determination of degree of conformance with standards, and ongoing quality control were performed locally on a newly installed instrument. RESULTS: Phase I and II trials revealed no significant differences between LRS and control units in donor or recipient safety and comfort, platelet function and yield, or component volume. WBC per-unit values were significantly different: the LRS median per unit was 3.2 x 10(4) WBCs, versus 81.4 x 10(4) for control units. Assessment of process capability gave an estimate of 99-percent confidence that 99.5 percent of LRS units would be WBC reduced to < 1 x 10(6) WBCs. Local process validation and quality control revealed 90-percent confidence that 99 percent of the units would be WBC reduced and 99.9-percent confidence that 75 percent would exceed platelet yield standards; the process was stable over time. CONCLUSIONS: The LRS is safe for apheresis and the component produced is safe for transfusion with platelet function and yield equivalent to controls and WBC reduction superior to controls. Local process evaluation confirmed that component quality meets the goals of the institution.

Practical guidelines for process validation and process control of white cell-reduced blood components: report of the Biomedical Excellence for Safer Transfusion (BEST) Working Party of the International Society of Blood Transfusion (ISBT).

BACKGROUND: The increased use of white (WBC)-reduced blood components has prompted many institutions to develop quality assurance programs directed to such component preparation processes. For consistent preparation of WBC-reduced blood components that meet clinical needs as well as national standards, a program of process validation and control should be instituted. This involves controlling key factors that affect WBC reduction as well as periodic monitoring of the residual cellular content of components. Practical guidelines for the implementation of such a program are provided. STUDY DESIGN AND METHODS: A program involving three phases of monitoring was developed by individuals belonging to an international working party of the International Society of Blood Transfusion. RESULTS: The first phase, process validation, evaluates a minimum of 20 consecutive units (a minimum of 60 units when nonparametric measurements are used) to document the successful local implementation of a new or substantially modified process. Ongoing process control employing Levey-Jennings type control charts is used to demonstrate that the process remains stable over time. Process capability assessment and conformance with standards are evaluated once residual WBCs are counted in a sufficient number of units. This enables a facility to claim with a specified degree of confidence that a stated proportion of WBC-reduced units will meet national standards. Two approaches to determine the number of units that should be selected for counting are presented. The first approach considers units as either acceptable or not acceptable and assumes that the distribution of failed (or nonconforming) units approximates the Poisson distribution. The second approach takes into consideration the observed WBC content of the tested units, with the assumption that the residual WBC content in WBC-reduced components follows a lognormal distribution. A method to assess the lognormal distribution of residual WBCs is presented. Specific tables based on each of these approaches are provided to guide the reader in the design of a program that will verify conformance with any national standard at specific confidence levels. The approach can be generalized to other process control applications. CONCLUSION: Guidelines are presented for process validation, process control, and assessment of conformance in the production of WBC-reduced blood components. Policy makers retain the responsibility to establish, on the basis of the expected use of WBC-reduced components, requirements for the frequency of testing and for the proportion of prepared units that are expected with a stated degree of confidence to meet the standards. Facilities preparing WBC-reduced components can monitor key factors that influence the preparation of WBC-reduced blood, can periodically assess their conformance with the standards, and can intervene to correct adverse changes in the process. This approach can be used to ensure the consistent quality of WBC-reduced blood components.

Detecting failed WBC-reduction processes: computer simulations of intermittent and continuous process failure.

BACKGROUND: By regulation, ongoing process control of WBC-reduced processes is performed on 1 percent of WBC-reduced components, typically four to five samples per month. However, prospective study of the power of this small sample has been difficult. Using computer-generated "residual WBC" distributions, sample size sensitivity to continuous or intermittent WBC-reduction failure was examined. STUDY DESIGN AND METHODS: Populations of log-normally distributed values (mean +/- SD, 4.5+/-0.5; n = 10(5)) were generated. Continuous failure (log-normality maintained) was simulated by incrementally increasing the population mean or its SD. Intermittent failure (bimodal distributions with discrete subpopulations of WBCs > the FDA cutoff) was simulated by admixing increasing percentages of secondary outlier populations. Sample sizes of 4 to 60 were examined (500 repetitions each) for their power to detect drift or failure by standard control criteria. RESULTS: Normally distributed low variance failure was easily detected by comparison of the mean of four samples to an upper control limit (95% confidence of detecting 2% failure). However, 40 samples were required to detect > 5 percent intermittent (bimodal) failure or high variance failure with 90-percent confidence, and only if individual WBC values were compared to cutoff. CONCLUSION: Sampling error limits the detection of high variance or bimodal distributions. While the mean of a small sample is highly sensitive to shifts in a low-variance normal distribution, the detection of a high-variance bimodal population requires a large number of individual values compared to cutoff. Therefore, the number of samples required for confident failure detection depends on both the nature of the underlying distribution and the interpretive criteria. Further research is necessary to determine the true distributions of WBC-reduction process failure, as well as clinically relevant quality limits.

Preparation and storage characteristics of white cell-reduced high-concentration platelet concentrates collected by anapheresis system for transfusions in utero.

BACKGROUND: Important concerns with regard to in utero platelet transfusions are avoidance of volume overload and the immunomodulatory effects of residual white cells (WBCs). This study evaluated a modification of a leukocyte-reduction system (LRS, Spectra, COBE BCT) for apheresis, which collects high-concentration WBC-reduced platelets (HCPs) for in utero transfusion. STUDY DESIGN AND METHODS: The LRS procedure was modified by running the platelet collection pump at specified low flow rates (Q(col)) for the first part of the procedure, collecting HCPs by gently purging them from the LRS chamber into a designated collection bag and then restoring the original LRS procedure settings to collect a second standard apheresis platelet concentrate (PC). Two centers carried out 32 procedures. Platelet yield, residual WBCs, and in vitro platelet function studies were evaluated. RESULTS: Platelet concentrations in 60 mL of HCPs were predictable according to Q(col) (r(2) = 0.735). HCP yields varied from 0.9 to 3.2 x 10(11), depending on the desired final platelet concentrations in 60 mL, with an overall average of 1. 92 x 10(11) (n = 32). Apheresis PCs had a mean platelet yield of 2.9 x 10(11) (1.3-4.4 x 10(11), n = 20) and 3.9 x 10(11) (2.2-5.8 x 10(11), n = 12) at concentrations of 1.3 x 10(12) per L for single-needle and dual- needle procedures, respectively. Median WBC counts were 5.6 x 10(3) for HCPs and 2.0 x 10(4) for apheresis PCs, with >99 percent expected to be less than 1 x 10(6). HCP in vitro characteristics were equivalent to those of apheresis PCs at 24 hours after collection. In vitro performance declined over storage as a function of HCP yield. HCP pH at 22(o)C was maintained at a level of >6.2 for more than 3 days for yields >1.6 x 10(11), less than 2 days for yields 1.6 to 2.2 x 10(11), and less than 24 hours for yields >2.2 x 10(11). HCPs showed good in vitro characteristics and could be stored for 1 to 3 days, depending on the total number of platelets collected. CONCLUSION: A standard apheresis PC and an HCP requiring no secondary processing can be collected with the Spectra LRS. The platelet concentration may be determined by clinical need. HCPs meet the requirements for components that are transfused in utero.

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)

Platelet storage solution affects on the accuracy of laboratory tests for platelet function: a multi-laboratory study.

BACKGROUND AND OBJECTIVES: Extent of shape change (ESC) and hypotonic shock response (HSR) have been widely used to characterize the in vitro function of platelets and have been shown to correlate with in vivo viability. These assays have been routinely performed using platelet-poor plasma (PPP) as the test sample diluent. Because of the increasing popularity of storing platelets in synthetic media, it is important to understand the effects of using these synthetic media as test diluents for ESC and HSR measurements. The objective of this study was to determine the effect of using platelet storage solutions vs. plasma for the in vitro testing of ESC and HSR. MATERIALS AND METHODS: Six laboratories participated in this study. Platelets were prepared by apheresis, the platelet-rich plasma (PRP) method, or derived from buffy-coats. Each platelet preparation was divided, half being stored in plasma and the other half in storage solution. ESC and HSR testing were performed in duplicate on days 1 and 5, using each of three diluents: autologous plasma; fresh-frozen plasma; or storage solution. RESULTS: For both ESC and HSR, dilutions made in each of the three diluents yielded significantly different results. Dilutions made in storage solutions were more than 30% lower for ESC and HSR than those made in autologous plasma (P < 0.0001). Dilutions made in thawed fresh-frozen plasma were more than 16% lower for ESC and HSR than those made in liquid autologous plasma (P < 0.0005). CONCLUSIONS: ESC and HSR test results are significantly affected by the test diluent. Platelets should be diluted in plasma (preferably autologous) for the in vitro testing of ESC and HSR, regardless of the media in which they are stored.