Leukocyte counts in cerebrospinal fluid with the automated hematology analyzer CellDyn 3500 and the urine flow cytometer UF-100

BACKGROUND: The counting of leukocytes and erythrocytes in cerebrospinal fluid (CSF) is still performed microscopically, e.g., using a chamber in most laboratories. This requires sufficient practical experience, is time-consuming, and may constitute a problem in emergency diagnostics. Specific automated systems for CSF cell counting are not available at present. METHODS: We tested the hematology analyzer CellDyn 3500 (CD) and the urine flow cytometer UF-100 (UF), which are not designed for CSF analysis. We studied >104 samples with both analyzers, and the counts obtained were compared with the reference method (Fuchs-Rosenthal chamber). RESULTS: Good linearity in the medically relevant range of 15 x 10(6) to 1000 x 10(6) leukocytes/L and a high degree of within-run accuracy were seen for both analyzers. Cell counting on the UF was excellent, especially when low cell counts were encountered (CV, 4. 9% compared with 28% observed for the CD). Method comparison showed that identical results could be detected for a majority of the count pairs. For a few samples, there was a discrepancy between the results from the analyzers and the counting chamber. In most cases, these were CSF samples containing a high proportion of lymphocytes. For these samples, the CD result led to a false-positive high leukocyte count, and on the UF these cells were not allocated to the leukocyte population, thus leading to false-negative counts. CONCLUSIONS: Both analyzers should not be used for CSF cell counting in all cases at present. However, once the technical and software problems have been solved, routine use of the two analyzers for CSF analysis should be seriously contemplated.     

Determination of leukocyte counts in cerebrospinal fluid with a disposable plastic hemocytometer

Manual microscopic cell counting in a Fuchs-Rosenthal (FR) chamber has been the gold standard for quantification of leukocytes in cerebrospinal fluid (CSF). However, for accurate determination of the number and differentiation of cells by chamber counting, hemocytometers must be prepared carefully and kept clean. Improper fitting of the chamber and coverslip changes the volume of sample introduced into the chamber well. Moreover, because conventional hemocytometers are used repeatedly and are breakable, there is a risk of exposure to potentially infectious material. To address these issues, disposable plastic hemocytometers have been developed. However, the accuracy, precision, and clinical usefulness of disposable chambers for CSF cells counting have not been determined. In the present study, we evaluated use of a disposable plastic counting chamber (C-Chip DHC-F01) by comparing its performance with that of an FR chamber for counting of CSF specimens and cell suspensions. Within-run precision of C-Chip counting was comparable or superior to that of FR chamber counting, and excellent correlation between cell counts obtained with the C-Chip chamber and FR chamber was observed. However, C-Chip chambers that were kept at 4 degrees C yielded significantly low cell counts. The disposable hemocytometer will reduce the risk of exposure to potentially infectious material. However, use of C-Chip chambers should be avoided in cold environments.     

Measurement of total and differential white blood cell counts in synovial fluid by means of an automated hematology analyzer

We developed a rapid and accurate method for quantifying total and differential white blood cell (WBC) counts by pretreating synovial fluid with hyaluronidase and using an automated hematology analyzer. Forty-seven samples of synovial fluid that had been placed in blood-collection tubes containing ethylenediamine- N,N,N',N' -tetraacetic acid as an anticoagulant were treated with hyaluronidase at 37 degrees C for 10 minutes, and then the total and differential WBC counts were determined by means of the automated hematology analyzer. Results were compared with those achieved by traditional manual counting methods. For the automated method, the coefficient of variation values for within-run precision of the WBC count were 5.27%, 3.56%, and 3.01% at 0.54, 1.12, and 2.05 x 10(9) /L, respectively; the run-to-run coefficient of variation values was less than 10.0%. The total and differential WBC counts obtained by this automated method showed good correlation with those obtained by the hemocytometer method ( r = .998; P < .0001; regression formula, y = 0.986 x - 0.072). Bland-Altman plots indicated no significant discrepancy between the methods. Our evaluation supports the use of this automated hematology analyzer method to measure total and differential WBC counts, which should aid clinical diagnosis.     

Automated analysis of pleural fluid total and differential leukocyte counts with the Sysmex XE-2100.

BACKGROUND: Determination of leukocyte (WBC) counts in pleural fluid is routinely performed by microscopic examination. In this study, we evaluated the performance of automated (differential) WBC counting in comparison with manual counting. METHODS: Pleural fluid samples (n=45) were obtained from patients undergoing diagnostic thoracocentesis. The manual total WBC count was determined after Samson staining in a Fuchs-Rosenthal hemocytometer; microscopic differential counts were performed on May-Grünwald Giemsa-stained cytospin slides. The Sysmex XE-2100 hematology analyzer was used for automated (differential) WBC counting. The functional detection limit was determined by serial dilution of continuous ambulatory peritoneal dialysis (CAPD) fluid and replicate measurements of each dilution. RESULTS: The automated WBC count (x10(6)/L) was highly correlated with that of the microscopic reference method (r(2)=0.95; WBC-analyzer=0.97 x WBC-reference method+16; n=45). Good agreement was also observed for the absolute lymphocyte count (r(2)=0.92; WBC-analyzer=0.99 x WBC-reference method+32; n=36), neutrophil count (r(2)=0.94; WBC-analyzer=0.91 x WBC-reference method+6; n=35), and monocyte count (r(2)=0.73; WBC-analyzer=0.83 x WBC-reference method+6; n=38). The functional detection limit for WBCs was calculated at 50 x 10(6)/L (coefficient of variation 20%). CONCLUSIONS: With some limitations, total and differential WBC counts in pleural fluid can be reliably determined using the Sysmex XE-2100 instrument.     

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.     

Automated white blood cell counts in cerebrospinal fluid using the body fluid mode on the platform Sysmex XE-5000

Abstract

Background. The Sysmex XE-5000 offers automated quantification of red blood cells and white blood cells (WBCs) in body fluids, with differentiation of polymorphonuclear cells (PMNs) and mononuclear cells (MNCs). Methods. We evaluated automated WBC counting in cerebrospinal fluid (CSF) using the body fluid mode on the Sysmex XE-5000, comparing it with flow cytometry as the reference method, and also with manual counting by microscopy. Experimental analysis for linearity and limit of detection was performed by diluting isolated WBCs in cell-free CSF. To study the ability to discriminate between PMNs and MNCs, samples were spiked using MNCs separated from peripheral blood. Comparison of WBC counts between a counting chamber and the XE-5000 was performed for 198 CSF samples. Results. In the experimental set-up, within-run (CV 19%) and between-day imprecision (CV 15.3%) in quantitating total number of WBC on XE-5000 was acceptable for WBC counts ≥ 25 × 10⁶/L. Compared with expected cell counts, mean bias was + 2.6% for flow cytometry, + 5.5% for XE-5000 and ? 73.2% for manual counting. Differentiation between PMNs and MNCs was in concordance with flow cytometry. In comparisons of clinical CSF samples, overall agreement between the XE-5000 and manual counting was observed in 81% of the samples, but mean difference in WBC differentiation was higher for PMN (51.1 × 10⁶/L) than for MNC (7.95 × 10⁶/L). Conclusion. Despite limited precision at low WBC counts, XE-5000 could be a favourable alternative to the labour-intensive, time-consuming and less reliable manual counting and cuts turnaround times in routine CSF-based diagnosis.     

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.     

Quantitative PCR for counting residual white blood cells in blood products.

Leukocyte depleted blood components are frequently used to reduce alloimmunization and the risk of transfusion transmitted infection. Counting residual white blood cells in filtered blood products requires sensitive and reliable techniques. After separation of white blood cells from 500 microliters of 20 non-filtered and 54 filtered blood products we used polymerase chain reaction (PCR) and fluorimetric detection for the quantification of genomic DNA. The results were compared with results from Nageotte chamber counting. The accurate limit of detection of PCR was determined at 1 WBC/microliter (intra-assay coefficient of variation: 16.3%). PCR correlated well with Nageotte chamber counts (r = 0.77, p < 0.001, n = 74). Concordant results were obtained in 51 filtered and 20 non-filtered blood products. Discrepant results were obtained in 3 filtered whole blood units: In these blood products > 12 WBC/microliters were counted in Nageotte chamber and PCR gave a negative result. After component preparation fresh-frozen plasma and red cell concentrates of these units contained < 1 WBC/microliter using both methods. In conclusion we describe a quantitative PCR method which had about the same sensitivity and specificity as Nageotte chamber testing. However, PCR is more laborious than the standard method. As well, as reliable PCR testing requires expensive instruments and staff experienced in molecular biology, the standard method is more cost effective.     

White cell-reduced red cells prepared by filtration: a critical evaluation of current filters and methods for counting residual white cells

White cell (WBC) reduction, red cell (RBC) recovery, and filtration time were determined in 1-day-old standard and buffy coat-depleted RBCs filtered in the laboratory through six commercial filters for WBC reduction. Residual WBCs were counted with a Burker chamber (BC), with a Nageotte chamber (NC), and by flow cytometry (FC). Results show that BC counts were 0 in several cases in which WBCs were detected with NC and FC, which indicated that the traditional BC method is too insensitive in use with currently available filters. Calibration curves performed by FC and with NC with samples containing known concentrations of WBCs from 1000 to 1 per microL showed that both FC and NC detected, on average, 67 percent of WBCs present in the samples (efficiency). However, the efficiency of FC showed small variability (61-70%) at different WBC levels, whereas the variability with NC was large (39-91%). This greater variability prevented the correction of NC counts by using a single factor and indicated difficulty in NC standardization. Therefore, because our main aim was to compare different filters rather than to define absolute levels of WBC contamination, uncorrected FC and NC counts were chosen to be reported. True WBC counts per unit should not exceed values that can be obtained by dividing uncorrected counts by the lowest efficiencies (61% for FC and 39% for NC). Uncorrected NC and FC counts were below 2 × 10(6) per unit in all units processed through three of the filters and below 5 × 10(6) per unit in all units processed through the other three.(ABSTRACT TRUNCATED AT 250 WORDS)     

New generation IQ‐200 automated urine microscopy analyzer compared with KOVA cell chamber

Objective: The examination of the urine remains to be one of the most commonly performed tests in laboratory practice. Currently, laboratories also need to accredit their urine diagnostics by comparing their measurement methods to acceptable references. In this study we compared particle counts obtained by new generation automated technique, image capture analysis (IQ‐200) with those of a standardized chamber counts. Design and Methods: The same 258 urine samples from different departments of a hospital assayed by IQ‐200 were analyzed in parallel with the KOVA cell chamber system. Clinically significant discrepancy results (positive vs. negative) for red blood cell (RBC) and white blood cell (WBC) were also compared with those obtained by dipstick testing. Results: There was a good agreement between the automated system and sediment microscopy for RBCs, WBCs, and squamous epithelial cells (SCs) (r=0.90; r=0.80; r=0.72, respectively: P<0.001). The IQ‐200 was more sensitive for determining RBCs, WBCs, and SCs than other formed elements. Conclusions: IQ‐200 can perform accurate quantification of microscopic element in urine. However, automated techniques are not completely free of error. Therefore, by adopting an appropriate algorithm and combining the results with stript analysis and other laboratory tests allows further reduction of clinically important errors. J. Clin. Lab. Anal. 24:67–71, 2010. © 2010 Wiley‐Liss, Inc.     

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.     

Early total white blood cell recovery is a predictor of low number of apheresis and good CD34(+) cell yield

OBJECTIVE: We analysed peripheral blood progenitor cell (PBPC) mobilisation and collection in order to assess the main factors related to CD34(+) cell yields in patients affected by haematological malignancies. PATIENTS AND METHODS: The features of CD34(+) cell mobilisation of patients with haematological malignancies that underwent autologous bone marrow transplantation were examined. Mobilisation chemotherapy consisted mainly of cyclophosphamide (CY) 4 or 7 g/m(2) followed by growth factors. Leukapheresis was started when the WBC counts reached 1.0x10(9)/l with the aim to collect at least 5x10(6) CD34(+) cells/kg body weight. The aphereses were performed on continuous-flow blood cell separators. The analysed variables were: age, diagnosis, CT mobilisation regimen, type of growth factor, number of previous CT lines, prior radiotherapy, days for WBC recovery and number of aphereses procedures to achieve the target of CD34(+) cells. RESULTS: There were 41 consecutive patients (26 M/15 F): 21 non-Hodgkin's lymphoma (NHL), 15 Hodgkin's disease (HD), two chronic myeloid leukaemia (CML) and three multiple myeloma (MM). Eleven patients could not collect the proposed threshold of CD34(+) cells. CY 4 mobilised patients recovered WBC counts in less days (P=0.03). By ANOVA, the days to WBC recovery had a linear function of the predictors "number of aphereses" and "type of mobilisation CT" (coefficients: 0.86 and 0.95, respectively). For the number of aphereses and WBC recovery after CT mobilisation, we obtained a correlation coefficient of 0.36 (P=0.02). CONCLUSION: This study shows that it is feasible to mobilise and collect PBPC in patients previously treated with CT with or without RT. There was a linear correlation between the days for WBC recovery and the number of aphereses needed to collect the target number of CD34(+) cells. The study suggests that early WBC recovery, using mainly CY 4 mobilisation chemotherapy, is an important predictor of a low number of aphereses to achieve a good CD34(+) yield.     

Quality control of white cell-reduced red cells: white cell preservation and simplified counting

BACKGROUND: White cell (WBC) degradation restricts the interval between the filtration process and the assay for residual WBCs. Maintaining WBC integrity would permit extended sample storage for batching and/or shipment to centralized laboratories. The usual quality control assay for WBC-reduced red cell units requires determining the number of WBCs in the entire counting area of a Nageotte hemocytometer, which consists of 40 rows. Reducing the counting area would simplify the quality control procedure. STUDY DESIGN AND METHODS: Adsol red cell units were prepared either on the day of collection (Day 0) or on Day 1 and WBC reduced by filtration on the same day. By using prefiltration and postfiltration red cells, samples containing WBC concentrations of 15, 10, and 3 WBCs per microL were prepared by serial dilution. Identical samples were treated with glutaraldehyde and stored at either 20 to 24 degrees C or 1 to 6 degrees C. All samples were assayed on the day of component preparation and on Days 7 and 14. The numbers of WBCs corresponding to 10- and 40-row areas of the Nageotte hemocytometer were determined. RESULTS: For the conditions and WBC concentration range studied, no significant changes in WBC concentrations were observed through Day 14 for glutaraldehyde-treated samples stored at either temperature, although there were substantial decreases in untreated samples. A 10-row measurement was determined to be sufficient for identifying WBC-reduced red cell units passing the present limit of 5 × 10(6) residual WBCs. CONCLUSION: Glutaraldehyde treatment can preserve WBCs in red cell samples at least up to Day 14, which provides increased efficiency in quality control for laboratories. Current red cell WBC-reduction filters produce components that, when assayed, contain fewer than 10 WBCs per full counting area. The simplified procedure would allow reduction of the counting area by 75 percent.     

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.     

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)     

UF-1000i flow cytometry is an effective screening method for urine specimens

This study was undertaken to evaluate the UF-1000i™ (UF) flow cytometer to count urine constituents including bacteria. The objective was to screen urine samples and determine what white blood cell (WBC) and/or bacteria screening criteria would minimize the number of specimens cultured yet ensuring that all true positives were cultured. UF screening and culture on CHROMagar™ Orientation (CO) medium were performed on 2496 specimens. Various combinations of WBC/bacterial counts were assessed as screening criteria and correlated with significant growth on CO medium. A bacterial count of ≥20 from UF gave an overall screening sensitivity of 92.6%, allowing 35% of specimens to be screened out and not cultured. The sensitivity was 99.2% and 85.0% for Gram-negative and Gram-positive organisms, respectively, using the same bacterial count. Our study indicated that UF was a simple, rapid, and reliable method for urine screening when the bacterial count of ≥20 was used as the sole screening criterion.     

The predictive value of white cell or CD34+ cell count in the peripheral blood for timing apheresis and maximizing yield

BACKGROUND: The collection of peripheral blood stem and progenitor cells (PBPCs) for transplantation can be time-consuming and expensive. Thus, the utility of counting CD34+ cells and white cells (WBCs) in the peripheral blood was evaluated as a predictor of CD34+ cell yield in the apheresis component. STUDY DESIGN AND METHODS: The WBC and CD34+ cell counts in the peripheral blood and the apheresis components from 216 collections were assessed. Sixty-three patients underwent mobilization with chemotherapy plus filgrastim, and 17 patients and 14 allogeneic PBPC donors did so with filgrastim alone. The relationship between the number of WBC and CD34+ cells in the peripheral blood and in the apheresis component was analyzed by using rank correlation and linear regression analysis. RESULTS: The correlation coefficient for CD34+ cells per liter of peripheral blood with CD34+ cell yield (x 10(6)/kg) was 0.87 (n = 216 collections). This correlation existed for many patient and collection variables. However, patients with acute myeloid leukemia had fewer CD34+ cells in the apheresis component at any level of peripheral blood CD34+ cell count. Components collected from patients with CD34+ cell counts below 10 x 10(6) per L in the peripheral blood contained a median of 0.75 x 10(6) CD34+ cells per kg. When the WBC count in the blood was below 5.0 x 10(9) per L, the median number of CD34+ cells in the peripheral blood was 5.6 x 10(6) per L (range, 1.0-15.5 x 10(6)/L). A very poor correlation was found between the WBC count in the blood and the CD34+ cell yield (p = 0.12, n = 158 collections). CONCLUSION: The number of CD34+ cells, but not WBCs, in the peripheral blood can be used as a predictor for timing of apheresis and estimating PBPC yield. This is a robust relationship not affected by a variety of patient and collection factors except the diagnosis of acute myeloid leukemia. Patients who undergo mobilization with chemotherapy and filgrastim also should undergo monitoring of peripheral blood CD34+ cell counts, beginning when the WBC count in the blood exceeds 1.0 to 5.0 x 10(9) per L.     

Statistical process monitoring of WBC-reduced blood components assessed by two types of software

BACKGROUND: Statistical process control is required for monitoring of the WBC-reduction process. This study focused on some factors that may influence the outcomes of statistical process monitoring, such as WBC-reduction technologies, the anticoagulant used, and WBC-counting technologies, by using two types of software. STUDY DESIGN AND METHODS: Data were collected from January to September 1999, before the implementation of universal WBC reduction. The effects of three major factors were investigated: methods of preparation, the addition of EDTA to the sample, and the WBC-counting technologies used (microvolume fluorimetry, flow cytometry, and Nageotte chamber). The WBC-reduction process capability was assessed by two types of software, EZQC (Gambro BCT) and NWA (Northwest Analytical). In addition, the differences between various sets of results were compared by the t test or ANOVA. RESULTS: There was no statistical difference (at the 0.05 level of significance) in WBC content when the three types of platelets in citrate samples were compared with EDTA samples. In general, the Nageotte chamber appeared to count the lowest, and microvolume fluorimetry appeared to count lower than flow cytometry. There were minor but significant methodologic differences between the software packages. However, these differences had negligible effects on the percentage of conforming components at both <1 x 10(6) and <5 x 10(6) WBCs per unit. CONCLUSION: Only the counting technologies were sufficiently different to warrant consideration. This difference may make unacceptable the interchange of results obtained from various counting methods.     

BECKMAN-COULTER LH750血液分析仪“WBC Correct”功能评估

目的对BECKMAN-COULTER LH750血液分析仪(简称LH750)关于白细胞(WBC)计数的一项特殊功能——"WBC Correct"的准确性及可靠性进行统计及评估。方法以卫生部推荐的《红细胞和白细胞计数的参考方法》对WBC计数结果受到仪器自动修正的样本进行手工计数,并将计数结果与仪器结果进行统计学分析。结果 WBC Correct修正后的WBC计数结果与手工法计数结果具有非常好的相关性,比没有该功能的同系列机型的WBC计数结果更准确、可靠,但在420例被检的标本中有5例结果有明显误修正。结论 LH750修正功能有较高的实用价值,但也不排除误修正的可能性,因此在工作中应密切注意仪器的修正提示,必要时应进行手工复检。     

急性髓细胞白血病中婆罗双树样基因4的表达及其临床意义

目的 探讨婆罗双树样基因4(SALL4)在急性髓细胞白血病(AML)中的表达及其临床监测意义.方法 采用逆转录PCR、实时荧光定量PCR、细胞培养、流式细胞术、骨髓涂片及血细胞分析仪等技术检测了68例AML患者(急性期36例、缓解期32例)、30名健康对照者、AML细胞系Kasumi-1和THP-1中SALL4的表达水平,评价其与骨髓原始细胞数、外周血WBC、未染色大细胞(LUC)计数及骨髓白血病免疫表型CD34表达率的关系.动态观察5例AML患者化疗前后3个时间点(化疗前急性期、治疗中第2～3周、化疗后缓解期)SALL4的表达水平.结果 急性期AML患者SALLA表达水平[69.01(17.20～120.28)]是缓解期AML患者SALL4表达水平[2.64(1.35～5.41)]的26倍,是健康对照组SALL4表达水平[1.14(0.50～1.62)]的61倍(Z=-6.48、-6.83,P均<0.01);缓解期AML患者SALL4表达水平是健康对照组的2.3倍(Z=-3.61,P<0.01).动态观察5例AML患者,SALL4基因表达水平随着治疗和病情缓解呈下降趋势,化疗前急性期、化疗中第2～3周和化疗后缓解期SALLA基因表达水平分别为79.74(33.76～89.09)、7.19(5.97～20.21)和3.40(1.44～15.53).骨髓原始细胞数增高组、LUC计数增高组及CD34表达率增高组中SALL4表达水平分别为33.82(16.00～144.01)、30.70(23.75～72.50)、56.25(23.79～153.81),高于相应正常组的2.74(1.59～5.13)、5.71(2.52～22.40)、20.82(14.03～55.12),差异有统计学意义(Z=-4.64、-2.18、-3.66,P<0.01或<0.05);外周血WBC计数增高组SALL4表达水平为89.26(23.75～154.34),高于相应的WBC计数正常组的3.86(2.03～6.01)和WBC计数减低组的6.66(2.51～17.06),差异有统计学意义(Z=-4.91、-4.21,P均<0.01);SALL4表达率与骨髓原始细胞数以及外周血WBC计数呈正相关(r=0.45、0.40,P均<0.01).结论 成功建立了人SALL4基因表达水平的实时荧光定量PCR方法,SALL4表达有望成为监测AML病情和判断预后的新指标.