Manual and automated methods for the determination of leukocyte counts at extreme low levels: comparative evaluation of the Nageotte chamber and the Abbott Cell Dyn 3500 analyser

Leukodepleted or leukocyte-poor blood products (fresh-frozen plasma, packed red cell and platelet concentrates in particular) are widely used in current clinical practice. However, because the monitoring of leukodepletion efficiency is generally carried out (if at all) using the labour-intensive and relatively inaccurate manual Nageotte chamber technique, it is clear that any increased demand for leukodepletion monitoring would be difficult, if not impossible, to meet. As the need to identify an automated alternative to the Nageotte technique is important, this study was undertaken to evaluate such a possibility. White blood cells were enumerated in a representative series of filtered and non-filtered human blood components by both microscopic counting in the Nageotte chamber, and with the Abbott CD3500 automated haematology analyser. For the Nageotte estimate, a single analysis was made in accordance with standard procedures, whereas the automated analysis was achieved by making six replicate counts and determining the mean of four replicates after excluding the highest and lowest estimates. To determine linearity limits of the manual and automated procedures, freshly isolated leukocytes were admixed with cell-free plasma-pheresis plasma. Reasonable reproducibility (mean CV 10% for cell counts exceeding 100 cells/microL) and good linearity (r > 0.9) were observed for CD3500 determinations in four separate experiments. The manual and automated measurements also correlated well (r > 0.9) with no obvious inter-method bias for cell counts up to 40 cells/microL although there was some suggestion of lower absolute CD3500 counts in the range 40-130 cells/microL. For the comparative studies with filtered and non-filtered blood products, no significant method bias was seen with 70 individual red cell concentrates, but systematically higher CD3500 white blood cell counts were observed in the series of 68 platelet concentrates (probably due to the presence of platelet clumps). This study concludes that automation of white cell counts in blood products with the CD3500 analyser is feasible for quality control in the preparation of fresh-frozen plasma and red cell concentrates but is limited for the analysis of filtered platelet concentrates.     

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.     

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.     

Preparation of white cell-reduced red cells by filtration: comparison of a bedside filter and two blood bank filter systems

BACKGROUND: Concern has been raised about the quality of white cell (WBC)-reduced red cells (RBCs) obtained by bedside filtration. The bedside performance and workload of a routine bedside filter have been compared to the laboratory performance and workload of two blood bank filter systems. STUDY DESIGN AND METHODS: Buffy coat-depleted saline- adenine-glucose-mannitol (SAGM) RBCs (90 units) were prepared. Thirty units were filtered with each of the two blood bank filter systems, and 30 units were filtered (but not transfused) with the bedside filter in a clinical department after 8 to 24 days of storage. The RBCs lost and the postfiltration WBC content (Nageotte chamber) were determined for all filtered units, and the workload associated with filtration by each of the filter systems/filter was assessed. Units with a postfiltration content of > or = 2 × 10(6) WBCs were regarded as filtration failures. RESULTS: Four (13%) of the 30 units filtered at the bedside were filtration failures, compared to no failures with either of the blood bank filter systems. In addition, the median WBC content (0.14 × 10(6)) of the units filtered at the bedside (2 units/filter) was significantly higher than that of the units filtered in the blood bank (0.05 × 10(6)). The RBC loss was significantly higher with the filter systems than with the bedside filter, provided 2 units per filter were processed with the latter. The timed workload of the filter systems was 45 to 75 minutes per 12 units, which was similar to the time required for bedside filtration. CONCLUSION: Bedside filtration of 2 units of stored buffy coat-depleted SAGM RBCs per filter resulted in a higher incidence of filtration failure and higher postfiltration WBC content than did laboratory filtration of 1 unit of fresh buffy coat-depleted SAGM RBCs per filter with either of two blood bank filter systems.     

Quantitation of residual white cells in filtered blood components by polymerase chain reaction amplification of HLA DQ-A DNA

BACKGROUND: Over the past several years, blood filtration technology has improved dramatically, such that currently available experimental filters are capable of reducing white cells (WBCs) in blood components to less than 0.1 WBC per microL. These residual WBC concentrations are below the sensitivity of automated cell counters, as well as of large- volume (Nageotte) hemocytometers. STUDY DESIGN AND METHODS: A quantitative polymerase chain reaction (PCR) amplification assay directed at HLA DQ-A DNA sequences has been developed for the enumeration of WBCs in filtered blood. To ensure quantitative recovery of WBCs at very low residual cell concentrations, a direct red cell lysis and WBC concentration protocol using 0.5 mL of filtered blood was perfected. Amplified product is detected by oligomer hybridization using 32P-labeled probes, with quantitation by image analysis of autoradiographic signals relative to a standardized dilution series processed in parallel. RESULTS: Recovery of residual WBCs in filtrates was shown to be enhanced by the addition of xenogeneic WBCs or polystyrene beads, which served as "carrier" particles during red cell lysis and wash steps. A contribution of nuclear fragments in filtered blood to PCR signal in the range of 0.01 to 0.5 WBCs per microL was observed; a modified protocol was developed to minimize this effect. Parallel analysis of spiked dilution series and evaluations of 39 red cell components filtered through commercial filters indicated good correlation between PCR and standard Nageotte counts in the range of 0.1 to 10 WBCs per microL (r2 = 0.94); only PCR was able to detect residual WBCs in filtrates from prototype 6 log10 WBC-reduction filters. CONCLUSION: This assay should prove useful for the development and quality assurance of increasingly efficient WBC-reduction filters.     

Prestorage inline filtration of whole blood for obtaining white cell- reduced blood components

BACKGROUND: Preliminary studies have indicated that the inline filtration of whole blood is a feasible method of obtaining white cell (WBC)-reduced packaged red cells (RBCs) and WBC-reduced fresh-frozen plasma (FFP) while using only one filter. STUDY DESIGN AND METHODS: An inline WBC-reduction filter, specially designed for this purpose and integrated in a "top-top" system, was used in the preparation of 24 units of WBC-reduced RBCs (RBC-F) and FFP (FFP-F) in each of two transfusion centers (Vienna and Gottingen). Twelve conventionally prepared units of RBCs (RBC-C) and FFP (FFP-C) served as controls. WBC contamination was assessed in each unit with the Nageotte chamber. Several coagulation measures were evaluated by using standardized test systems. RESULTS: The median WBC contamination in RBC-F was 27,000 per unit in Vienna and 50,000 in Gottingen. In FFP-F, the median WBC contamination was 13,000 (Vienna) and 31,000 (Gottingen) per unit. Coagulation factors I, V, VIII, and XI in FFP-F were not different from those in FFP-C. In addition, markers for the activation of coagulation and fibrinolysis–that is, factor XIIa, prothrombin fragments, thrombin- antithrombin complexes and fibrinogen degradation products–were not greater in FFP-F. CONCLUSION: Blood components prepared from inline- filtered whole blood meet the standards for WBC-reduced RBCs and FFP. The protein profile of FFP-F is not altered, and markers for the activation of coagulation and fibrinolysis show no increase.     

A multicenter study on the efficiency of white cell reduction by filtration of red cells

To evaluate accurately the current performance of filtration, the French Produits Sanguins Labiles study group, composed of 21 transfusion teams, conducted a large-scale 6-month study involving over 1400 filtrations and 3000 controls. Some 745 standard red cell concentrates (RBC concentrates) and 690 concentrates previously white cell (WBC)-reduced by removal of buffy coat (BC-poor RBC concentrates) were filtered using six commercially available filters: at least 170 results were collected per filter, spread among a minimum of three teams. Prefiltration controls show that the removal (manual and automated) of the buffy coat results in an initial WBC reduction of approximately 63 percent, along with a hemoglobin loss of 4 g (7%). After filtration, residual WBCs were counted in the Nageotte manual counting chamber. The reliability of this counting method, which is simple and adapted to low WBC concentrations, was characterized in this study by a 25-percent coefficient of variation (CV) for a concentration of 2.5 WBCs per microL (i.e, 0.6 x 10(6) WBCs/filtered unit). The analysis of the results shows that, for five of six filters (1 filter was excluded), the postfiltration median value of residual WBCs was 1.1 x 10(6) in filtered RBC concentrates (n = 590), whereas it was 0.34 x 10(6) in filtered BC-poor RBC concentrates (n = 581). The difference is significant (p less than 10(-8), Wilcoxon test). Hemoglobin loss due to filtration varies according to the filter, from 5.7 +/- 2.2 to 17.3 +/- 2.5 g.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Increasing hemoglobin oxygen saturation levels in sickle trait donor whole blood prevents hemoglobin S polymerization and allows effective white blood cell reduction by filtration

BACKGROUND Red blood cell (RBC) components from donors with sickle cell trait (Hb AS) often occlude white blood cell (WBC) reduction filters. Techniques were investigated to successfully filter Hb AS donor blood by increasing the Hb oxygen saturation with storage bags and conditions suitable for transfusion products. STUDY DESIGN AND METHODS: Oxygenation kinetics were measured over 3 days in whole-blood units stored in standard-sized 600-mL polyvinylchloride (PVC) bags and whole-blood units divided into three equal parts and stored in standard-sized blood bags made from PVC, tri-2-(ethylhexyl)trimellitate (CLX) plastic, or Teflon. The filterability of Hb AS blood stored for 3 days was tested with whole-blood filters. RESULTS: Oxygen saturation levels did not increase in full whole-blood units from donors without sickle cell trait during 3 days of storage in 600-mL PVC bags. In divided Hb AS whole-blood units stored for 3 days, oxygen saturation levels increased from baseline levels of 45 to 56, 66, and 94 percent after storage in 600-mL PVC, CLX, and Teflon bags, respectively (n = 5, p < 0.02), and all components filtered completely. When full Hb AS whole-blood units from eight donors were stored for 3 days in 1.5-L CLX bags, all units filtered completely, but one had a high residual WBC count. CONCLUSION: Storage of Hb AS whole blood in large-capacity oxygen-permeable bags increases oxygen tension and allows more effective WBC reduction by filtration.     

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 flow cytometric method for counting very low levels of white cells in blood and blood components

Reduction of white cells (WBCs) in blood components may reduce the risk of virus transmission and HLA alloimmunization. Filtration provides a means by which to achieve high-efficiency WBC reduction. A method has been developed using flow cytometry to quantitate the number of WBCs in WBC-reduced packed red cells or platelet concentrates. This method uses a detergent and propidium iodide (PI) solution to label the WBC nuclei and incorporates a known amount of fluorescein isothiocyanate (FITC)- labeled chicken red cells (cRBCs) into the mixture as an indicator of the volume examined. The number of observed WBCs per mL is calculated as follows: Number of PI WBC nuclei events/Number of FITC cRBC events × Number of FITC cRBCs added to mixture/Volume of blood in mixture. The method may allow the detection of WBCs at a concentration as low as 0.01 per microliters (10/mL) in a blood sample. It is an efficient method of collecting data, as it requires less than 10 minutes per sample. This flow cytometric technique is suitable for research purposes and for quality control of WBC-reduced blood components, because it is precise and can be used to quantitate WBCs in large or small numbers in a sample.     

Quantitation of residual WBCs in filtered blood components by high-throughput, real-time kinetic PCR

BACKGROUND: The effort to eliminate transfusion complications associated with WBCs has led to the widespread use of filters able to reduce WBC concentrations to 相似文献   

流式细胞术在成分血残留白细胞检测中的应用

目的 探讨流式细胞术(FCM)绝对计数法在成分血残留白细胞检测中的应用价值。方法 采用FCM绝对计数法检测不同稀释度的浓缩血小板样本中自细胞残留量，并与Nageotte血细胞计数板法比较。结果 两种方法计数微量白细胞具有高度相关性(r=0．9964，P＜0．001)。低浓度白细胞计数时，FCM法变异系数小于Nageotte计数板法。两种方法检测结果相比，Nageotte计数板法计数值偏低。结论 流式细胞术绝对计数法具有快速、客观、精确和重复性好等优点，是一种稳定的检测成分血中微量残存白细胞数的方法。     

Quality control of red cell filtration at the patient's bedside

BACKGROUND: Concern has been raised about the quality of white cell (WBC) reduction in blood components when it is performed by filtration at the patient's bedside. Thus, the quality of red cell (RBC) filtration performed at the bedside through two new flatbed WBC- reduction filters was evaluated. STUDY DESIGN AND METHODS: In the laboratory, 25 and 10 RBC units suspended in additive solution were stored for 1 to 2 and 14 to 21 days, respectively, and filtered through each filter. At the end of filtration, an automated complete blood count and a manual WBC count (Nageotte chamber) were determined in two samples collected from 1) a segment clamped at 5 and 25 cm below the filter along the line connecting prefiltration and postfiltration bags and 2) the postfiltration bag. In addition, 10 of the 11 nurses of the hematology outpatient clinic administered to hematologic patients 25 RBC units stored for 1 to 2 days through each type of filter. At the end of transfusion, a segment was collected from the transfusion set and a WBC count was performed as described above. No filter priming or rinsing with saline was done. RESULTS: WBC counts obtained after laboratory filtration in the segments were similar to those obtained from the postfiltration bags and from the segments collected at the bedside in all cases, with the exception of 14- to 21-day-old RBCs filtered in the laboratory through one of the filters, which produced slightly higher WBC counts in the segments than were seen in the postfiltration bags. The difference was not significant. In no case was the count in the postfiltration bag higher than that in the segment. Nurse training was easy, and bedside filtration was associated with no untoward effects. CONCLUSION: The RBC filtration at the patient's bedside can be equal in quality to that performed in the laboratory, at least in such clinical settings as hematology and oncology departments in which blood transfusion is common practice, and if simple training is provided to the nursing staff. Under the conditions of this study, it seems that quality control of RBC bedside filtration is feasible and simple.     

Comparison of COBE white cell-reduction and standard plateletpheresis protocols in the same donors

BACKGROUND: It is necessary to protect patients from white cell (WBC)- caused side effects of platelet transfusion by reducing the WBC contamination in single-donor platelets. STUDY DESIGN AND METHODS: A new COBE Spectra WBC (leuko)-reduction system (LRS) was compared to the COBE standard plateletpheresis (standard) procedure. Each of 62 donors underwent plateletpheresis under the two protocols (LRS and standard). The collection efficiency and WBC contamination in the components collected using the techniques were compared. Platelets were counted in a cell counter and WBCs were counted using two full grids of a Nageotte chamber. RESULTS: The preseparation and postseparation numbers for red cells, WBCs, and platelets as well as the number of collected platelets were not different in the two techniques. Collection efficiency in the LRS procedures was 96.2 +/− 13.0 percent of that in the standard procedures. Median WBC contamination in the platelet components was 10,160 per LRS procedure and 56,500 per standard procedure. The purity of the LRS components was significantly improved (p = 0.001), as seen in a comparison of the WBC numbers in components per procedure after log10 transformation (LRS: 0.096 +/− 0.195 × 10(6); standard: 0.390 +/− 1.075 × 10(6)). CONCLUSION: These data suggest that the LRS procedure produces platelet concentrates with a collection efficiency that is comparable to that obtained with the standard technique and with a residual WBC content that satisfies even the most stringent criteria for filtered platelets. As this purity can be achieved without platelet loss or alteration, conventional fiber filtration no longer seems necessary or useful in this type of single-donor platelet component.     

White cell reduction during plateletpheresis: a comparison of three blood cell separators

BACKGROUND: White cell (WBC)-reduced single-donor platelet concentrates (SDPs) can be collected by the newest generation of blood cell separators. Three WBC-reduction techniques during plateletpheresis were investigated in the present study with respect to WBC content and platelet yield. STUDY DESIGN AND METHODS: The Amicus device used the elutriation principle for WBC reduction, and separations with periodically alternating interface position (PAIP) were employed in the AS.TEC 204. WBC reduction by in-line filtration was performed in the MCS+. Platelets were measured electronically and WBCs were determined manually (Nageotte chamber). RESULTS: In-line filtered SDPs showed significantly lower WBC content (0.088+/-0.178 x 10(6)) than SDPs that were WBC reduced by elutriation (0.31+/-0.48 x 106) or PAIP technique (0.89+/-1.57 x 10(6), p = 0.0001). Platelet yield (5.0+/-0.46 x 10(11)) was significantly higher in components obtained with the Amicus device (p = 0.0001). The AS.TEC 204 and MCS+ gave similar results for platelet yields: 3.15+/-0.63 and 3.28+/-0.71 x 10(11), respectively. CONCLUSIONS: The plateletpheresis systems studied allow the collection of WBC-reduced SDPs. In-line filtration resulted in the best WBC reduction. Some SDPs collected with the devices studied had a WBC content >1 x 10(6) per unit. Platelet yield was significantly higher in SDPs from the Amicus device.     

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)