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.     

Validation of use of the Nageotte hemocytometer to count low levels of white cells in white cell-reduced platelet components

BACKGROUND: Determination of the white cell (WBC) count in WBC-reduced platelet components requires methods that have a detection limit in the range of approximately 5.0 × 10(2) to 5.0 × 10(4) per mL. STUDY DESIGN AND METHODS: With a 50-microL Nageotte hemocytometer and bright-field microscopy (200x magnification), studies were conducted to develop and validate a method that could be used routinely with filtered and apheresis-harvested platelets. A 1-in-5 dilution of sample with a commercially available blood-diluting fluid was used because, with a lower (1-in-2) dilution, the observed number of WBCs was substantially less than the number expected at relatively high platelet counts (> 1.9 × 10(9)/mL). RESULTS: The observed and expected WBC counts in WBC- reduced platelet samples correlated well at levels between approximately 5 and 1100 WBCs per counting area (5.0 × 10(2)-1.1 × 10(5)/mL). At levels of more than 300 to 400 WBCs per counting area, accurate counts were obtained when 10 of the 40 rectangles were counted. CONCLUSION: These studies provide data to confirm that the 50- microL Nageotte hemocytometer can be used to accurately count low levels of WBCs in platelet components.     

A multicenter study evaluating three methods for counting residual WBCs in WBC-reduced blood components: Nageotte hemocytometry, flow cytometry, and microfluorometry

BACKGROUND: A multicenter study was conducted to evaluate the performance characteristics of flow cytometry and microfluorimetry for counting low concentrations of WBCs and to compare the results with Nageotte hemocytometry. STUDY DESIGN AND METHODS: A two-phase study involving 10 centers located in the United States and in Europe was performed. Coded samples of RBCs and platelets were distributed by 24-hour (Phase 1) or 2-day (Phase 2) courier service to each test site for analysis. Samples were prepared to include concentrations of WBCs slightly above and below the concentration corresponding to the threshold standards for WBC-reduced RBCs and platelets. All centers tested samples by Nageotte hemocytometry plus one or both of two automated methods. RESULTS: Both flow cytometry and microfluorometry gave better results than Nageotte hemocytometry in testing freshly prepared samples. At WBC concentrations >5 per microL (RBCs) or >3 per microL (platelets), the intersite CV was <20 percent for the automated methods but >30 percent for the Nageotte hemocytometer method (p<0.001). Accuracy was greater for the automated methods than for the Nageotte hemocytometer method (p<0. 001). Nageotte hemocytometry showed a bias to underestimation relative to the results obtained with the automated methods. All methods had poorer performance in testing samples that required > or =2 days' shipment than in testing of those requiring overnight shipment. CONCLUSION: Automated methods for counting residual donor WBCs in WBC-reduced cellular components offer advantages of improved precision and greater accuracy than are seen with the Nageotte hemocytometer method. Automated methods are less labor-intensive but more costly than microscopic methods. Preparation and shipping methods will need further refinement for samples to be counted more than 24 hours after sample collection.     

Automatic volumetric capillary cytometry for counting white cells in white cell-reduced plateletpheresis components

BACKGROUND: As the benefits of white cell (WBC)-reduced blood components become increasingly apparent, the need has arisen for a simple, automated WBC-counting technique that is sensitive to low WBC concentrations. Automated volumetric capillary cytometry was evaluated for its ability to quantify residual WBCs in WBC-reduced plateletpheresis components. STUDY DESIGN AND METHODS: The volumetric capillary cytometry system evaluated uses a laser to excite fluorescent dye-labeled nucleated cells. The number of nucleated cells per microliter is reported. Four studies were performed: linearity, precision of results near the value of 5 × 10(6) WBCs per unit, the limit of detection, and correlation to the Nageotte manual counting method. RESULTS: Assay values correlated to expected values (range, 0- 125 WBC/microliter) with an r2 > 0.99. In the range of 5 × 10(5) WBCs per unit the CV was 8.5 percent, and concentration differences of 0.15 log10 were detectable. The limit of detection was 1.0 WBCs per microliter (95% upper confidence limit). The assay correlated to the Nageotte method with an r2 of 0.98, slope of 1.0, and y-intercept of 2.0 WBCs per microliter. Assay results were 10 to 15 percent higher than Nageotte results, in samples with values near 5 × 10(6) WBCs per unit. Technician time per sample was 2 to 3 minutes. CONCLUSION: Volumetric capillary cytometry is precise and sensitive to small differences in WBC concentration in the range of clinical interest. The device provides an efficient new method for quality assurance and control of WBC-reduced plateletpheresis products.     

A general method for concentrating blood samples in preparation for counting very low numbers of white cells

BACKGROUND: To count extremely low levels of white cells (WBCs) in WBC- reduced blood components, a larger volume of sample must be processed. The goal was to develop an all-purpose method for concentrating the samples obtained from WBC-reduced red cells or platelets. The method was designed to be compatible with a variety of counting techniques. STUDY DESIGN AND METHODS: Coded samples of red cell concentrates with an expected WBC concentration of 200, 100, 50, and 10 per mL and of the diluent (undetectable WBCs/mL) were sent to three sites on five occasions and counted by the use of the concentration method, crystal violet stain, and a Nageotte counting chamber. Additional samples were tested by flow cytometry, polymerase chain reaction, and volumetric capillary cytometry. RESULTS: The results from the three test sites showed good linearity, with an overall r2 = 0.9994. The lower limit of accurate detection of the assay was 10 WBCs per mL. The results were biased toward underestimation, particularly at one of the test sites (Site A). There were no significantly different results in Sites B and C. The intra-assay CV was acceptable. Precision (reproducibility) at the three test sites varied. CONCLUSION: This method allows reliable determination of WBC concentrations as low as 0.01 per microL in blood. Despite the use of technologists trained in Nageotte chamber counting, validation testing demonstrated that one test site's performance was significantly different from that of the other two sites, because of both underestimation bias and variation in count results. The sample concentration method, when used in conjunction with an automated assay for WBC identification, should permit larger volume analysis with a greater degree of precision and a lower limit of detection than is found in assays that do not concentrate the sample before counting.     

Multicenter evaluation of two flow cytometric methods for counting low levels of white blood cells

BACKGROUND: Flow cytometric methods can be used to count residual white blood cells (WBCs) in WBC-reduced blood products, which should contain fewer than 1 x 10(6) WBCs per unit (approximately 3.3 WBCs/ microL). In this study two flow cytometric methods for counting WBCs under routine conditions in nine laboratories were evaluated. STUDY DESIGN AND METHODS: Panels of red blood cells (RBCs), platelets (PLTs), and plasma were prepared containing 33.3, 10.0, 3.3, 1.0, and 0.3 WBCs per microL and counted with flow cytometric methods (either LeucoCOUNT, BD Biosciences, four laboratories; or LeukoSure, Beckman Coulter, five laboratories). Requirements were that at the level of 3.3 WBCs per microL, coefficient of variation was < or =20 percent and accuracy was > or =80 percent. Routine flow cytometric quality control (QC) data of WBC-reduced blood products from two laboratories were analyzed. RESULTS: At the level of 3.3 WBCs per microL, none of the laboratories met the requirements for all three blood products. The LeucoCOUNT method met requirements at more laboratories than the LeukoSure method for RBCs and PLTs, but the opposite was true for plasma. Routine QC data showed that >99 percent of the flow cytometric measurements for WBC-reduced products was below the 95 percent prediction interval at 3.3 WBCs per microL. CONCLUSION: None of the laboratories met the requirements for accuracy and precision for all three blood products. Nevertheless, routine results showed that in >99 percent of the products, WBC counts were below guideline limits. Therefore, both flow cytometric methods are suitable for QC with pass-fail criterion.     

Validation of a simple method to count very low white cell concentrations in filtered red cells or platelets

The increased performance of white cell (WBC) filters makes it difficult to count precisely the number of residual WBCs. Concentrations as low as 0.01 WBC per microL cannot be determined with electronic cell counters, conventional hemocytometers, or the flow cytometric techniques currently being used. This article describes a simple, manual method using a Nageotte hemocytometer with a large-volume chamber (50 microL) to count the number of WBCs contained in red cell (RBC) suspensions (preparations A, B, and C) and in platelet suspensions (preparation D) diluted 1 in 10 pure, or concentrated two fold. To validate the method, several reference ranges, prepared by successively adding mononuclear cells to a suspension of pure RBCs or platelets, were used. Among the different series, validation ranges varied from 0.2 to 12 to 0.01 to 0.5 WBCs per microL and correlation coefficients ranged from 0.929 to 0.996. To determine the limit of accurate detection, accuracy tests (n = 160) were carried out by two experienced operators on samples with WBC concentrations of about 5, 10, and 120 times the concentration at the theoretical limit of detection (1 WBC/chamber). No significant difference was observed in the various types of preparations (A, B, C, D) in the tests performed by the two operators. However, intra-assay coefficients of variation were 18, 9.5, and 2.2 percent, respectively, at WBC concentrations of 5, 10, and 120 times that at the theoretical limit of detection. These observations show that a limit of accurate detection (10%) seems to be reached when 10 cells are observed in a Nageotte hemocytometer.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Effects of white cell reduction on the resistance of blood components to bacterial multiplication

BACKGROUND: While prestorage white cell (WBC) reduction by filtration may improve platelet and red cell quality, it also may remove an important anti-bacterial defense mechanism, especially if blood is WBC- reduced shortly after collection. STUDY DESIGN AND METHODS: The question of whether WBC reduction of platelet concentrates and red cells altered bacterial proliferation kinetics in components prepared from deliberately contaminated, freshly collected blood was investigated. Two-unit pools of whole blood were inoculated, at a concentration of approximately one colony-forming unit per mL, with one of 17 bacterial species reported to have caused septicemia in transfusion recipients. Each pool was divided after inoculation, and components were prepared from the 2 units after a 7-hour room- temperature holding period. One unit of each AS-1 red cell or platelet pair was WBC-reduced, and the pairs were then stored for 42 days at 4 degrees C (red cells) or for 10 days at 22 degrees C (platelets). Quantitative bacterial cultures were performed at periodic intervals. RESULTS: In red cells, clinically significant bacterial proliferation occurred in only one instance (Serratia marcescens), and growth was less rapid in the WBC-reduced unit than in the control. Three patterns of growth were seen in platelet concentrates. In four cases, there was rapid proliferation in both test and control units, while on 13 occasions there was minimal replication in either pair. On six occasions, substantial growth was noted in control units, while few or no bacteria could be found in the WBC-reduced units. There was no evidence in either red cells or platelets that bacteria proliferated more rapidly in units that had been WBC-reduced before storage than they did in units in which WBCs were retained. CONCLUSION: Rather than increasing the risk of bacterial proliferation through removal of active phagocytic cells, WBC reduction by filtration before blood storage may act to reduce the likelihood of significant bacterial proliferation, possibly by removal of microorganisms along with WBCs.     

Flow cytometric determination of residual white blood cell levels in preserved samples from leukoreduced blood products

BACKGROUND: In preparation for a proposed consolidated testing service, Canadian Blood Services undertook the evaluation of a commercial test kit for the enumeration by flow cytometry of residual white blood cells (rWBCs) present in preserved samples recovered from leukoreduced (LR) blood and platelet products. STUDY DESIGN AND METHODS: The stability of preserved WBCs, the equivalency of WBCs used for spiking, test method precision, specificity, reliability, accuracy, and sensitivity were investigated. For comparative purposes, WBC counts were also determined by Nageotte as well as by flow cytometry. RESULTS: WBCs were stable up to 4 weeks at room temperature for all components by either method. Within methods, no differences were observed due to the source of WBC used for spiking purposes. By either method, test precision was acceptable (<20% coefficient of variation) and of similar reliability at a target value of 10 +/- 5 WBCs per microL. The flow cytometric method was shown to be more specific and accurate than the Nageotte method. Sensitivity by either method was 0.1 WBCs per microL. On average, Nageotte counts were lower than those observed by flow cytometry. CONCLUSIONS: These results demonstrate that WBCs in WBC stabilizing solution-treated samples from LR blood components were stabilized up to 4 weeks at room temperature and that rWBC determinations made with a WBC enumeration kit by flow cytometry have the required precision, specificity, reliability, and accuracy in the relevant test range. This validated WBC stabilization and flow cytometric counting method is considered acceptable as part of a quality control program for leukoreduced blood products.     

Process control of filtered red blood cells: which counting method?

Various counting methods have been described and reported for process control of leucodepleted blood components. The recent production of high-efficiency leucocyte removal filters intensifies the need for sensitivity in determining the ever lower residual concentration of white cells (WBCs) in filtered units.
In order to assess which method was the most efficient and feasible in the laboratory for the control of WBC-reduced packed red blood cells, we compared the sensitivity of four counting methods: Nageotte chamber analysis, flow cytometry, the fluorochrome method by Borzini and Nageotte chamber analysis as modified by Prati.
We observed a difference in the post-filtration WBC content depending on which method of counting was used and we feel it reasonable to ask what method should be employed in blood component process control.
The answer must naturally consider that the method is for use by a large number of laboratories, while the sensitivity of the method needs to be appropriate to the goal desired.     

A national quality assessment scheme for counting residual leucocytes in unfixed leucodepleted products: the effect of standardisation and 48 hour storage.

BACKGROUND: WBC counting, an essential part of quality monitoring of WBC-reduced blood components, is carried out logistically within 48-72 h of collection. The between-laboratory variability and effects of 24-48 h storage were investigated using three major counting technologies. STUDY DESIGN AND METHODS: Samples of RBC and platelets with WBC in the range 0-50/microl were transported by courier. WBC counting was performed on days 1 and 2, by IMAGN 2000, flow cytometry and Nageotte, initially using local protocols and then using a national flow protocol. Up to 15 laboratories participated in each exercise. RESULTS: For "real failed leucodepleted" red cell products, higher levels of variability were observed for flow and Nageotte, as compared to IMAGN. For spiked RBC samples at critical decision making point (3-20 WBC/microl), between-laboratory the coefficients of variation (CVs) were low for IMAGN and were the highest for Nageotte. Flow cytometry CVs were generally high but improved subsequent to standardisation of sampling and the gating strategy. A similar pattern in the variability of results was observed for platelet concentrates. Sign tests using all samples (carried out for each method in each exercise; 25 in total) demonstrated no overall tendency for larger WBC counts to be recorded on day 1 when compared to day 2, although this difference was significant (p < 0.001) in certain cases depending on the nature of the spiked product. CONCLUSIONS: We conclude that while a good performance is achieved using validated automated technologies for low residual leucocyte counting, the unification of reagents and standardisation of sampling and gating strategies are essential in obtaining interchangeable results. Unfixed RBC and platelet samples can generally be stored for 48 h before WBC counting.     

The effect of unmodified or prestorage white cell-reduced allogeneic red cell transfusions on the immune responsiveness in orthopedic surgery patients

BACKGROUND: The immunomodulatory effects of allogeneic blood transfusions have been attributed to the white cells (WBCs) present in the cellular blood components transfused to patients. STUDY DESIGN AND METHODS: The effect of the transfusion of allogeneic red cells (RBCs) or allogeneic prestorage WBC-reduced RBCs (WBC-reduced RBCs) on host immune responsiveness was evaluated by measuring the lymphocyte subsets and the in-vitro cytokine production in response to phytohemagglutinin stimulation of WBCs of orthopedic surgery patients. Forty-seven patients undergoing hip replacement surgery were randomly assigned to receive allogeneic RBCs (n = 17) or WBC-reduced RBCs (n = 14; 99.95% WBC removal). Sixteen patients were not transfused. Patient blood samples taken before surgery and on Days 1 and 4 after surgery were tested for complete blood count, lymphocyte subset analysis, and measurement of cytokine levels. RESULTS: After surgery, the lymphocyte count was significantly decreased in patients transfused with > or = 3 units of allogeneic RBCs (2.0 +/- 0.5 vs. 1.3 +/- 0.3 x 10(9)/L; p = 0.017), but not in patients transfused with > or = 3 units of WBC-reduced RBCs (2.0 +/- 0.9 vs. 1.7 +/- 0.8 x 10(9)/L). Compared with preoperative levels, on Day 4 after surgery, patients transfused with > or = 3 units of allogeneic RBCs also had a decrease in the number of natural killer cells (0.07 +/- 0.05 vs. 0.04 +/- 0.03 x 10(9)/L; p = 0.018). Postoperatively, interleukin-2 was decreased in one patient who received WBC-reduced RBCs compared with that in four patients transfused with allogeneic RBCs (p = 0.32), and eight untransfused patients (p = 0.01). On Day 4, about 70 percent of patients transfused with allogeneic RBCs showed a 20-percent decrease in the interferon gamma level. CONCLUSION: Taken together, these data support the hypothesis that transfusion of > or = 3 units of allogeneic RBCs is associated with early postoperative lymphopenia in otherwise healthy individuals undergoing surgery. These findings were not observed in those individuals transfused with RBCs that had undergone prestorage WBC reduction.     

Optimal conditions for white cell reduction in red cells by filtration at the patient's bedside

Background: A quality control program of white cell (WBC) reduction in red cells at the bedside was implemented, based on postfiltration counting in a Nageotte chamber of the residual WBCs from samples taken from a segment of the transfusion set, after 1-in-10 sample dilution with Turks's solution. During a 1-year quality control program, 5.1 percent of counted units had apparent filtration failures, that is, WBC counts exceeding 5 × 10⁶ per unit. The cause(s) for these apparent failures were investigated. Study Design and Methods: In Study 1, residual WBCs from 150 buffy coat-free red cells filtered through one type of filter at 4°C, 20 to 24°C, or 27°C in 5 to 10 minutes, 50 to 100 minutes, or 100 to 200 minutes were counted as described above. In Study 2, residual WBCs in samples collected from segments of the transfusion set and from the postfiltration bags were counted in parallel by a new, more sensitive counting method. In this method, 5 mL of filtered red cells was diluted with 20 mL of 3-percent paraformaldehyde and centrifuged, the pellet was resuspended to 500 μL with a lysis solution, and the WBCs were counted in a Nageotte chamber. In Study 3, residual WBCs were counted by the 3-percent paraformaldehyde method in samples from postfiltration bags of 1- to 2- day-old buffy coat-rich red cell units filtered through a second type of filter. Filtration was started within 30 minutes of the removal of the unit from the refrigerator, ambient temperature was 20 to 24°C, and the median filtration time was 90 minutes per unit. results: Study 1: Median WBC counts per unit increased progressively from 51,000 at 4°C to 934,000 at 27°C, with intermediate values at 20 to 24°C. In no unit did the WBC count exceed 5 × 10⁶ if filtration at 20 to 24°C was completed within 100 minutes, while counts in excess of 50 × 10⁶ were found at 20 to 24°C and at 27°C with filtration times of 100 to 200 minutes, and 50 to 100 minutes, respectively. Study 2: The relation between segment and postfiltration bag WBC counts obtained by the 3- percent paraformaldehyde method was poor, with the latter being almost always lower than the former. Study 3: None of the 120 units filtered through the second type of filter at 20 to 24°C in 50 to 100 minutes contained more than 3.2 × 10⁶ WBCs; the median value was 147,000 WBCs per unit. Conclusion: On the basis of the results with the 3-percent paraformaldehyde method, which showed the unreliability of segment counts, a new policy was adopted for quality control of bedside WBC reduction, based on controlling the time of and temperature at transfusion. Bedside WBC reduction in 1- to 2-day-old red cells performed with the second type of filter at 20 to 24°C in less than 100 minutes per unit allowed the preparation of units that meet the standard of fewer than 5 × 10⁶ WBCs in all tested cases. Bedside WBC reduction with the second type of filter and under the controlled conditions reported seems effective.     

The effect of whole-blood storage time on the number of white cells and platelets in whole blood and in white cell-reduced red cells

BACKGROUND: Whole blood (WB) can be stored for some time before it is processed into components. After introduction of universal white cell (WBC) reduction, it was observed that longer WB storage was associated with more residual WBCs in the WBC-reduced red cells (RBCs). Also, weak propidium iodide (PI)-positive events were observed in the flow cytometric WBC counting method, presumably WBC fragments. The effect of storage time on the composition of WB and subsequently prepared WBC-reduced RBCs was studied. STUDY DESIGN AND METHODS: WB was collected in bottom-and-top collection systems with inline filters, obtained from Baxter, Fresenius, or MacoPharma. Units were stored at room temperature and separated into components in 4-hour intervals between 4 and 24 hours after collection. RBCs were WBC-reduced by inline filtration (approx. 50/group). RESULTS: Platelet (PLT) counts were lower in WB stored for 4 to 8 hours compared to 20 to 24 hours (mean +/- SD): 79 +/- 31 versus 102 +/- 30 for Baxter (p < 0.01); 91 +/- 31 versus 101 +/- 35 for Fresenius (not significant); and 73 +/- 47 versus 97 +/- 31 (all x 10(9) per unit) for MacoPharma (p < 0.01), respectively. The median residual WBC counts in WBC-reduced RBCs for WB stored for 4 to 8 and 20 to 24 hours were 0.03 versus 0.17 for Baxter (p < 0.001), 0.00 versus 0.06 for Fresenius (p < 0.001), and 0.13 versus 0.26 (all x 10(6) per unit) for MacoPharma (not significant), respectively. All WBC-reduced RBCs contained fewer than 5 x 10(6) WBCs per unit. A longer storage time of WB was associated with more weak PI-positive events, irrespective of the filter. CONCLUSION: Longer storage of WB before processing results in counting higher numbers of PLTs in WB, higher numbers of WBCs in WBC-reduced RBCs, and more weak PI-positive events.     

Preparation of white cell-reduced platelet concentrates from whole blood during component preparation

This article introduces a new method of component preparation that is capable of producing white cell (WBC)-reduced platelet concentrates (PCs) from whole blood. Whole blood is separated into packed red cells (RBCs) and platelet-rich plasma (PRP) by centrifugation, and the PRP is expressed through a newly designed WBC removal filter into the platelet storage bag. The filtered PRP is then centrifuged and yields WBC-reduced PCs and plasma for freezing as fresh-frozen plasma (FFP). The method uses standard triple-pack blood bags and centrifugation protocols. Fifteen WBC-reduced PCs prepared with this technique had an average volume of 56.7 mL, an average Day 5 platelet content of 8.6 x 10(10) per unit, and an average Day 5 WBC content of 0.83 +/- 0.7 x 10(4) per unit (0.14 WBCs/microL). This represents WBC removal equal to at least 99.9 percent (3 log10) of the WBCs found in standard PCs prepared in our laboratory by an identical centrifugation protocol. Paired studies documented a 4.5-percent platelet loss by filtration. Filtration had no effect on the plasma prepared for FFP as measured by prothrombin time; activated partial thromboplastin time; factors I, V, VIII:C, and VIII:von Willebrand factor; antithrombin-III; albumin; globulin; or total protein. This method holds promise as a simple and highly effective technique for the production of WBC-reduced PCs by filtration during component preparation.     

Phenotypic characterization of white cells in white cell-reduced red cell concentrate using flow cytometry

The residual white cell (WBC) content of donated units of red cell concentrate rendered WBC-reduced by filtration through commercially available polyester filters was quantified and phenotypically analyzed. All studies were performed by flow cytometery. Quantification studies were performed with a DNA/RNA fluorophore, propidium iodide. WBC subset analyses were performed with fluorescence-labeled monoclonal antibodies directed against various cluster differentiation (CD) loci. The results indicate that the filter removes in excess of 3 log10 total WBCs from the red cell components and depletes granulocytes to or beyond the specific assay's sensitivity of 3 log10. Total T and B cells, T4 and T8 lymphocytes, and monocytes are reduced by approximately 4 log10. These analyses provide plausible explanations for the clinical success of the filter and suggest other potential applications.     

血小板单采样品中残留白细胞计数两种方法的评价

为评价流式细胞计数法和Nageotte血细胞计数法计数单采血小板中残留白细胞,应用系列的稀释实验研究其准确性;对已知白细胞浓度的同一样本,反复检测14次,研究其重复性;分别用流式细胞计数法和Nageotte血细胞计数法检测102份去白细胞的单采血小板样本,对其结果进行比较.结果发现,当白细胞浓度为0.8＜WBC/μl＜10时,两方法无显著差异（P＞0.05）.结论：两种方法均可用于少白细胞成分的质量控制.     

Cytokine generation in stored, white cell-reduced, and bacterially contaminated units of red cells

BACKGROUND: Proinflammatory cytokines were measured in the supernatant portion of stored, bacterially contaminated, and/or white cell (WBC)- reduced units of red cells (RBCs). Previous studies from this laboratory and others have shown that cytokines are generated in platelet concentrates during storage. This earlier work has been expanded to the study of stored RBCs. STUDY DESIGN AND METHODS: Units of AS-1 RBCs (n ? 10 non-WBC-reduced; n ? 10 WBC-reduced) were obtained from a regional blood center, and each was split on Day 3 of storage into three equal portions by sterile techniques. One portion was kept sterile (control), and the other two were inoculated with Yersinia enterocolitica and Staphylococcus aureus, respectively, at 1 to 3 colony-forming units per mL. The RBCs were stored at 1 to 6 degrees C for 42 days. Sequential samples were taken during storage and assayed for interleukin 8 (IL-8), interleukin 1 beta (IL-1 beta), interleukin 6, WBC count, and bacteria count. For the WBC-reduced group (n ? 10), WBC removal was done by filtration on Day 3 of storage, before bacterial inoculation. RESULTS: IL-8 was detected in the supernatant portion of all 42-day-old, non-WBC-reduced (mean WBCs ? 4760 ± 3870/μL) units of AS-1 RBCs at levels ranging from 63 to 1610 pg per mL. By contrast, at 2 to 3 days of storage, lower levels of IL-8 (range, 0-280 pg/mL) were detected in the same units. IL-8 levels increased progressively during storage in most (7/10) units. The highest mean levels of IL-8 were reached by outdate at Day 42. Y. enterocolitica-contaminated units had statistically higher levels of IL- 8, with a range of 170 to 2100 pg per mL, by 42 days of storage. S. aureus grew poorly in stored units of RBCs and failed to further stimulate cytokine production. No WBC-reduced unit (mean WBCs ? 0.5 ± 0.6/μL), even when contaminated with bacteria, had more than 260 pg per mL of IL-8. Although IL-1 beta was not detected in any unit of RBCs at 3 days of storage, it increased to low levels (5-13 pg/mL) in all units tested at 42 days. Interleukin 6 was not detected in any unit at any storage time. CONCLUSION: IL-8 and IL-1 beta accumulated in the supernatants of stored RBCs despite cold storage conditions. IL-8 reached levels > 1000 pg per mL in the supernatants of some RBC units. IL-1 beta increased to significant but low levels (< 13 pg/mL). WBC filtration early in storage prevented the accumulation of IL-8. The physiologic significance to transfusion recipients of IL-8 in RBC supernatants is currently unknown and deserves further investigation.