An evaluation of the CELL-DYN 1700® haematology analyser: automated cell counting and three-part leucocyte differentiation

The performance of the CELL-DYN 1700® (Abbott Diagnostics, Abbott Park, IL, USA) was evaluated in a tertiary care hospital laboratory using the guidelines proposed by the German Society of Clinical Chemistry. Precision, accuracy, linearity, background counts, and carry-over were satisfactory for all measured standard parameters including haemoglobin concentration, haematocrit, red blood cell count, mean corpuscular volume (MCV), red cell distribution width (RDW), white blood cell count and platelet count. With 259 selected normal and abnormal blood samples the results of the CELL-DYN 1700® (CD1700) compared very well (r > 0.96 for all parameters with exception of RDW) with those obtained with the Bayer Diagnostic H-1 and the Hoffmann-La Roche Cobas Argos systems. This study considered in particular the performance of the CD1700 three-part leucocyte differential. For those samples without instrument-generated suspect flags, the neutrophil and lymphocyte percentages were highly correlated with the results of the H-1 blood cell counter (r = 0.97 and 0.98, respectively) and with manual 400-cell differentials (r = 0.91 and 0.88, respectively). In contrast, the CD1700 mid-fraction which comprised the composite total of mono cytes, eosinophils, basophils and precursor white cells (when present) could not be directly compared to the differentials from the H-1 system or from manual microscopy. For those samples with CD1700 instrument suspect flags, the neutrophil and lymphocyte differential results also compared well with both the H-1 (r = 0.93 and 0.93, respectively) and manual estimates (r = 0.89 and 0.87, respectively). In conclusion, the CD1700 is an accurate haematology analyser for cellular blood counts and three-part leucocyte differentiation.     

Laboratory evaluation of the Sysmex SE‐9500 automated haematology analyser

The Sysmex SE‐9c500 is a new, fully automated haematology analyser, providing a complete blood count (CBC), including a five‐part differential count (DC), with flagging of morphological abnormalities. The SE‐9500 was evaluated according to guidelines published by the International Committee for Standardisation in Haematology (ICSH). The results demonstrated minimal carryover (< 0.01%) and excellent linearity for WBC, RBC, HGB and platelet (PLT) (r > 0.995). Samples were stable with regard to CBC parameters after storage for up to 48 h at room temperature (RT) and 4 °C. Imprecision was generally acceptable for all CBC parameters (CV < 5%). Correlation between the SE‐9500 and reference methods was excellent (r > 0.97) for all the major CBC parameters (WBC, RBC, HGB, PLT). There was minimal interference for WBC, RBC, HGB and PLT at high concentrations of bilirubin (BIL=224 μmol/l) or triglyceride (TG=7.78 mmol/l). SE‐9500 reference values for CBC parameters are presented. Our results indicate that the SE‐9500 is an excellent tool for routine haematological examination.     

Performance evaluation of the Celltac F haematology analyser

Introduction: Performance of the Celltac F haematology analyser (MEK‐8222) which provides 22 parameters, including a 5‐part differential, was compared with the Sysmex XE‐2100. Methods: 242 EDTA samples were investigated. Differential results from both instruments were compared with the reference microscopic count. Flagging performance was compared with cell morphology seen in the blood films. Results: Precision met or exceeded manufacturer’s specifications, carryover was minimal (≤1.37%) and linearity was excellent (R ≥ 0.99). Results were stable for at least 8 h at room temperature and for 24 h at 4 °C. Comparisons were excellent for white blood cells, red cell count, Hb, HCT and platelets (R ≥ 0.98). All other red cell and platelet parameters showed good correlation with the XE‐2100 (R ≥ 0.93) except for mean cell haemoglobin concentration. The differential was comparable to the XE‐2100 for neutrophils, lymphocytes and eosinophils and acceptable for monocytes. Correlation of automated differentials with manual reference counts and the efficiency of flagging of blasts, immature granulocytes and platelet clumps were similar for both instruments. Celltac F demonstrated better efficiency for atypical lymphocyte and platelet clumps. Conclusion: The Celltac F shows broadly comparable analytical performance to the XE‐2100 for the parameters assessed. The Celltac F is recommendable for medium‐sized laboratories or as a back‐up instrument in larger laboratories.     

Performance evaluation of the Sysmex XS-1000i automated haematology analyser

The Sysmex XS-1000i is a compact new, fully automated haematology analyser, designed to generate complete blood counts with five-part leucocyte differential. In our study, a Sysmex XS-1000i instrument was evaluated according to Clinical Laboratory Standards Institute (CLSI) and International Council for Standardization in Haematology (ICSH) guidelines. Precision, carry-over and linearity were determined. Using a total of 700 patient samples, results from the Sysmex XS-1000i were compared with those from a Sysmex XE-2100, an Abbott Cell Dyn 4000 and the manual reference leucocyte differential. Using quality control material, total and within-run imprecision was less than 3% except for platelets. The system demonstrated good linearity over the entire reporting range and no carry-over (<0.5%). The Sysmex XS-1000i showed good correlation with XE-2100, CD-4000 and the manual reference leucocyte differential. Overall flagging sensitivity and specificity were 91% and 48%, respectively. In conclusion, the Sysmex XS-1000i demonstrated good analytical performance, is able to generate a complete blood count with five-part differential on low blood volumes and has considerable back-up capacity.     

Improved automated leucocyte counting and differential in newborns achieved by the haematology analyser CELL-DYN 3500™

Summary Automated leucocyte counts in newborns generated by the impedance principle are artificially affected by the high osmotic resistance of some newborn RBC and possibly by the high normoblast numbers present during the neonatal period. Erroneously high WBC counts may result. The haematology analyser CKLL-DYN 3500^TM (Abbott Diagnostika GmbH, Wiesbaden-Delkenheim, Germany) has two different channels for the WBC count, an electrical resistivity (impedance) channel and a laseroptical channel. In combination with facultative extended lysis of resistant RBC before WBC count, this instrument is claimed to be very suitable for newborn blood analysis. We measured the WBC count and differential of 165 blood samples from newborns and cord blood on the CELL-DYN 3500^TM. Reticulocyte count and manual differential including normoblasts were determined. Furthermore, some technical aspects of neonatal blood analysis were evaluated: precision, cell stability, effect of incorrect blood-anticoagulant ratio of small blood collecting tubes. The internal decision making process of the CELL-DYN 3500^TM selects the result either from the optical channel (identifies and excludes normoblasts) or from the resistivity channel (eliminates resistant RBC). This instrument gives a reliable and accurate WBC count and differential of neonatal samples even in blood samples with normoblasts and lytic resistant RBC. The result given by the CELL-DYN 3500^TM can be confirmed by a subsequent run in extended lyse mode.     

Simultaneous differentiation and quantitation of erythroblasts and white blood cells on a high throughput clinical haematology analyser

After the neonatal period, the presence of nucleated red blood cells (NRBC) in peripheral blood is indicative of pathology. Despite the clinical utility of such measurements, automated NRBC counting has hitherto not been available on routine automated blood cell counting analysers. To address this, an automated method for the analysis of NRBC was developed and incorporated into the Abbott Cell Dyn 4000 (CD4000) haematology analyser. The system white blood cell (WBC) reagent was specifically formulated to preserve concomitantly white blood cell (WBC) morphology, rapidly lyse red blood cell and NRBC membranes, and subsequently stain NRBC nuclei with a nucleotide specific fluorochrome dye (Kim et al. 1996a). The fluorochrome itself does not permeabilize the membrane of intact viable white blood cells. The sample is processed by flow cytometry and the signals generated from an argon-ion laser light source are analysed. Axial light loss (AxLL), intermediate angle light scatter (IAS) and red fluorescence (FL3) are used to discriminate between particles of various types. By using these discriminators in a three-dimensional approach, NRBC form a discrete cluster which can easily be separated from leucocytes and enumerated as a distinct cell population during the optical WBC differential analysis. Consequently, accurate absolute WBC counts and differentials can be obtained even in the presence of NRBC. Background ‘noise’ (both fluorescent and non-fluorescent) from platelets, Howell-Jolly bodies, basophilic stippling, RNA from lysed reticulocytes, and DNA from leucocyte and megakaryocytic fragments are essentially eliminated (Kim et al. 1996b). While the membranes of intact and viable leucocytes remain impermeable to the passage of the fluorochrome stain, leucocytes with damaged membranes are permeable to the dye and generate FL3+ signals. Such cells, which are commonly seen as a consequence of sample ageing as well as in some distinctive pathologies, are identified by the algorithm (using their AxLL signal size) and are labelled as non-viable. Moreover, because non-viable leucocytes are retained in the WBC count and differential analyses, the CD4000 is further able to provide both numerical and graphical data regarding the relative frequency of viable and non-viable components. This additional information can serve as valuable ‘decision-drivers’ in the laboratory data review process.     

Performance evaluation of BC-3200 hematology analyzer in a university hospital

The BC-3200 automated hematology analyzer was evaluated and compared with the Beckman-Coulter AcT (Ac.T diff 2) 3-part differential hematology analyzer. The BC-3200 was evaluated according to guidelines published by the International Committee for Standardization in Hematology (ICSH), Clinical and Laboratory Standards Institute (CLSI), and Department of Food and Drug Administration (FDA). The results demonstrated no background, minimal carryover (<0.5%), and excellent linearity for hemoglobin (Hb) level, white blood cell (WBC), red blood cell (RBC), and platelet (PLT) counts (>0.998). Precision was generally acceptable for all complete blood count (CBC) parameters; coefficients of variation (CVs) were within the manufacturer's claims and CVs of CBC parameters, including WBC, RBC and PLT counts, Hb and mean corpuscular volume, were <6%. Correlation between the BC-3200 and Ac.T diff 2 was excellent (r > 0.98) for all major CBC parameters (WBC, RBC, and PLT counts and Hb). We conclude that the overall performance of the BC-3200 is excellent and compares well with that of the Coulter Ac.T diff 2.     

Automated leucocyte differentials in 292 patients with leucopenia: an evaluation of the Abbott CELL-DYN 3500 (CD3500) haematology analyser

Summary The widespread use of intensive therapies and the need to haematologically monitor patients on a frequent basis means that the proportion of blood samples with moderate to severe leucopenia is significant and increasing. From a laboratory perspective, particularly because of the need to spend significant amounts of time in obtaining manual differentials from stained smears with low leucocyte numbers, these clinical trends have created additional pressures on what is often a limited manpower resource. Moreover in such situations, differentials obtained from examination of only 20 or 50 cells are not uncommon and the statistical consequences of this will be clearly apparent. Currently, there is general user confidence for automated leucocyte differentials for blood samples with normal WBC parameters, but there has been some reluctance to extend this to samples with leucopenia. In order to explore this further, we examined the efficiency of a modern automated five-part differential analyser (Abbott CELL-DYN 3500) in an unselected series of 292 samples with leucopenia (WBC count range range; 0.28–2.48 × 10⁹/l). Of these, 49 were from leucopenic sero-positive HIV patients with the remaining 243 samples originating from haematological oncology clinics, patients receiving radiotherapy for non-haemopoietic malignancies, and from patients with various chronic diseases. Morphologically, 204 of these samples did not show any blast cells or NRBC, 48 had blast cells but no NRBC, 29 had NRBC but no blasts, and the remaining 11 showed both blasts and NRBC. For 277 cases with less than 5% blasts, there was an excellent correlation between the manual and CD3500 automated differential, with no obvious bias between manual and automated subpopulation estimates at any percentage level. Linear regression analyses comparing absolute neutrophil, eosinophil, lymphocyte and monocyte counts for these same samples further revealed impressive correlations (r > 0.92) for all leucocyte populations and the absolute neutrophil count in particular (r = 0.986). Manual and CD3500 leucocyte differential comparisons for 11 cases with > 5% blasts showed good correlations for absolute neutrophil and eosinophil counts although, when the blast cell percentage was high, correlations for lymphocyte and monocyte counts were less consistent (an operator alert in the form of a ‘Blast Flag’ was, however, given in 10/11 of these particular cases). Four additional cases where manual differentiation between lymphoid cells and monocytes was recorded as difficult also showed consistently good correlations for manual vs automated neutrophil and eosinophil estimates. Not surprisingly, and essentially as a result of the low confidence noted for the manual differential itself, correlations for lymphoid and monocytic cells were relatively poor. In conclusion, this study has demonstrated that the CD3500 provides reliable and accurate absolute neutrophil and eosinophil counts in leucopenic samples irrespective of the presence of blasts or NRBC. These observations are particularly important in terms of monitoring patients who are liable to develop neutropenia as a result of chemotherapy and radiotherapy, and provide evidence that the routine use of automated leucocyte differentials may be confidently extended to the analysis of leucopenic samples.     

Comparison of the reticulocyte mode of the Abx Pentra 120 Retic,Coulter® General‐S™, Sysmex® SE 9500, Abbott CD 4000 and Bayer Advia® 120 haematology analysers in a simultaneous evaluation

The Abx Pentra 120 Retic, Coulter^® General‐S?, Sysmex^® SE 9500, Abbott Cell Dyn^® 4000 and Bayer Advia^® 120 were evaluated simultaneously in a general hospital laboratory. Linearity, precision, sample stability, carry‐over and comparability of the reticulocyte mode were determined following International Council for Standardization in Haematology guidelines for the evaluation of blood cell analysers. All analysers showed good results for dilution, stability and carry‐over testing. The between‐batch coefficient of variation of the General‐S? was high compared to the other analysers evaluated. Multiple correlation studies showed good agreement for all analysers in the normal and high reticulocyte range, with correlation coefficients above 0.7. Multiple correlation studies for reticulocytopenic samples (< 15.10⁹/l) were less satisfactory, with a wider range of correlation coefficients (r‐values 0.0–0.9). Overall, the General‐S?, SE 9500 and Advia^® 120 gave lower reticulocyte counts than the Pentra 120 Retic and CD 4000. Reagent costs were also evaluated. Reagent consumption was close to the manufacturers’ specifications for the SE 9500 (Search reagent), CD 4000 (CD Retic) and Advia^® 120 (Retics) but was higher than stated for the Pentra 120 Retic (Retix), General‐S? (Retic kit) and SE 9500 (Sheath reagent). Our results show that these new generation haematology analysers will meet the needs of hospital laboratories for reliable and cost‐effective reticulocyte counting.     

Development and clinical application of nucleated red blood cell counting and staging on the automated haematology analyser XE‐2100TM

We initially developed a new flow cytometric (FCM) reference method for the enumeration and staging of nucleated red blood cells (NRBC) in 1997 [ Wang et al., 1998 (XIth International Symposium on Technological Innovations in Laboratory Haematology, Banff, Canada, 1998); Tsuji et al., 1999 (Cytometry, 1999)]. The method used CD45 antibody and propidium iodide staining to separate NRBCs from other cells. Accuracy and precision were enhanced because larger numbers of cells were counted than was possible with the manual method. We also developed a method for automated NRBC counting on a haematology analyser, the XE‐2100 (Wang, 1988). NRBC were separated from other cells using a special lysing buffer and a fluorescent dye. The XE‐2100 was found to detect peripheral and cord blood NRBC accurately and precisely when compared with cell morphology or FCM control methods. The FCM NRBC staging method was established through the identification of different NRBC populations following the novel staining and lysing method. To evaluate the method further, we sorted samples containing NRBCs using a FACSort and investigated NRBC staging on the Sysmex XE‐2100^TM based on the cell sorting results. Data were analysed using special software (ida ). First, we used the data in various parameter combinations. We then established gates to classify the NRBC populations. Finally, we analysed blood specimens from patients with different types of diseases to explore possible clinical applications.     

Performance of the SE-9000 automated haematology analyser in a laboratory serving a haematological oncology unit

The performance of the SE-9000 automated haematology analyser in a laboratory receiving a high number of abnormal specimens from haematological oncology patients was assessed according to formal protocols for the evaluation of blood cell counters. Linearity over a useful working range, precision in clinically important ranges and negligible carry-over were demonstrated in this group of patient samples confirming the results of previous investigators. The comparability of instrument derived differential leucocyte counts from both normal and distributionally abnormal samples with those obtained by visual microscopy using the NCCLS H-20 A protocol was very good. The sensitivity of flags for the detection of immature granulocytes and myeloid blast cells was high and this can be attributed to the incorporation of a new measuring channel (Immature Myeloid Information or IMI channel). The number of unrecognized abnormalities was low and when compared with the poor sensitivity of the routine 100-cell visual differential leucocyte count, the analyser was judged suitable for monitoring patients with haematological malignancies. The performance of flags such as ‘left shift’ and ‘atypical lymphocytes’ can be improved by taking into consideration distributional abnormalities such as neutrophilia and lymphocytosis. The trigger level for these flags should be adapted to the clinical need particularly in cases of neutropenia following chemotherapy, and in lymphoproliferative disorders and infection.     

The new haematology analyzer DxH 800: an evaluation of the analytical performances and leucocyte flags,comparison with the LH 755

Introduction: The analytical performance and the abnormality messages on differential (flags) of the new analyzer Beckman Coulter DxH 800 were compared with those of the LH 755. Methods: First, we evaluated the accuracy of the results of the DxH 800, in comparison with the LH 755, in 125 samples without alarm using unflagged sample results on both analyzers. Second, flagged samples on the LH 755 but not flagged by the DxH 800 were evaluated by flow cytometry for accuracy of the DxH 800 results. Finally, we evaluated the sensitivity and specificity of abnormality messages on differential given by the analyzers, in comparison with manual blood smears. Results: The correlation coefficients (R) for complete blood count parameters and differential demonstrated that the DxH 800 results were similar to that of LH 755. Excellent correlation coefficients between DxH 800 and flow cytometry results were found for white blood cell count (R = 0.985, n = 31), platelet count (R = 0.976, n = 51) and nucleated red blood cells (R = 0.966, n = 37). The overall performance showed an increased sensitivity (0.892) and specificity (0.864) of the flags on DxH 800 when compared to the LH 755 (0.846 and 0.733, respectively). Conclusion: The DxH 800 provides reliable results and increases laboratory efficiency by reducing working time and costs associated with the optical validation of the results.     

Diagnostic performance of the variant lymphocyte flag of the Abbott Cell‐Dyn 4000 haematology analyser

Background: In addition to differential cell counts, modern haematology analysers generate suspect flags if abnormal cells are detected. Reports on validation of suspect flags are scarce. We have routine experience with the Abbott Cell‐Dyn 4000 analyser for over 5 years and have previously demonstrated the utility of the blast flag. Here we report a similar study on the performance of the analyser's Variant Lymphocyte (VL) flag. Aim of the study: Evaluation of the diagnostic performance of the Cell‐Dyn 4000 VL flag, as compared with lymphocyte morphology in blood smears. In addition, we investigated the usefulness of the numerical VL flag confidence index as provided by the analyser. Materials and methods: All samples generating a VL flag were reviewed over a 5‐month period. We also reviewed smears from patients with known lymphoid disorders, even if the analyser did not flag the sample. Two experienced investigators assessed lymphocyte morphology independently. Results: In total, 187 samples were included in the study, of which 183 had a VL flag and four had not. Of the 183 flagged samples, 83 appeared to have abnormal lymphocyte morphology and 100, normal lymphocyte morphology. The sensitivity of the VL flag for detecting abnormal lymphocytes was 0.95 and the positive predictive value was 0.44. Using ROC analysis of the VL flag confidence index, the area under the ROC curve was 0.58 (95% confidence interval 0.50–0.65). Conclusions: The Cell‐Dyn VL flag has reasonable sensitivity but a high false‐positive rate. In addition, its performance is insufficient for detecting clinically relevant abnormal lymphocytes. As the VL flag appeared to rely mainly on numerical criteria, it has no added value over numerical criteria defined by the laboratory.     

Evaluation of the Roche Cobas Argos 5Diff automated haematology analyser with comparison to a Coulter STKS

Summary. The performance of the Roche Cobas Argos 5Diff (Argos) automated haematology analyser was evaluated by comparison to manual blood film examination and a Coulter STKS (STKS) analyser. The Argos demonstrated excellent between and inter-batch imprecision for all parameters, except the MCHC, and good linearity for Hb, WBC and platelet count (PLT). After an initial fall the PLT, results were stable for up to six h at 18°C in EDTA(K3) after which an increasing proportion of cells were classified as lymphocytes. Results of 239 patient samples analysed on both instruments, compared by linear regression, gave excellent correlation (r² > 0.90) for most parameters with the exceptions of the MCHC (0.317), eosinophils% (0.756), monocytes% (0.48) and basophils% (0.002).‘Flagging’of cellular abnormalities by the Argos resulted in excellent sensitivity (97.5%), specificity (93.2%) and efficiency/agreement (93.2%), with fewer false positive and negative results than the STKS, although these differences were not statistically significant. The performance characteristics of the Argos were comparable to those of the STKS with a possible improvement in its flagging abilities.     

Performance evaluation of the Abbott CELL‐DYN Ruby and the Sysmex XT‐2000i haematology analysers

Two mid‐range haematology analysers (Abbott CELL‐DYN Ruby and Sysmex XT‐2000i) were evaluated to determine their analytical performance and workflow efficiency in the haematology laboratory. In total 418 samples were processed for determining equivalence of complete blood count (CBC) measurements, and 100 for reticulocyte comparison. Blood smears served for assessing the agreement of the differential counts. Inter‐instrument agreement for most parameters was good although small numbers of discrepancies were observed. Systematic biases were found for mean cell volume, reticulocytes, platelets and mean platelet volume. CELL‐DYN Ruby WBC differentials were obtained with all samples while the XT‐2000i suppressed differentials partially or completely in 13 samples (3.1%). WBC subpopulation counts were otherwise in good agreement with no major outliers. Following first‐pass CBC/differential analysis, 88 (21%) of XT‐2000i samples required further analyser processing compared to 18 (4.3%) for the CELL‐DYN Ruby. Smear referrals for suspected WBC/nucleated red blood cells and platelet abnormalities were indicated for 106 (25.4%) and 95 (22.7%) of the XT‐2000i and CELL‐DYN Ruby samples respectively. Flagging efficiencies for both analysers were found to be similar. The Sysmex XT‐2000i and Abbott CELL‐DYN Ruby analysers have broadly comparable analytical performance, but the CELL‐DYN Ruby showed superior first‐pass efficiency.     

Immature granulocyte detection by the SE‐9000 haematology analyser during pregnancy

The objective of this study was to determine the nature of the alarm for immature granulocytes appearing in haemograms from pregnant women, as detected by the immature cell information channel (IMI) of the SE‐9000^TM automated haematology analyser. Of all tests run on pregnant women in a 4‐month period (n = 698), the first 100 haemograms with immature granulocyte alarms (14.33%) were collected. Each of these samples was then stained with Wright‐Giemsa stain. The following variables were also analysed: age of the mother, trimester and days of gestation, type of delivery, weight and sex of the baby, and Apgar score. Most pregnant women were in the third trimester of gestation (82%) when an alarm was noted on the IMI channel. Of the patients, 62% had normal deliveries. The most frequent complication was obstructed delivery (23%). Mean percentages by microscopic counts of band cells, metamyelocytes, and myelocytes were 2.99, 0.45, and 0.19%, respectively. There was a statistically significant correlation for all cell types between the SE‐9000^TM and the manual count method. No association was observed between the presence of immature granulocytes and the clinical variables analysed. The SE‐9000^TM analyser shows high sensitivity in the IMI channel for detection of immature forms.     

Rule based processing of the CD4000, CD3200 and CD Sapphire analyser output using the Cerner Discern Expert Module

The latest version of our Laboratory Information System haematology laboratory expert system that handles the output of Abbott Cell-Dyn Sapphires, CD4000s and a CD3200 full blood count analyser in three high-volume haematology laboratories is described. The three hospital laboratories use Cerner Millennium Version 2007.02 software and the expert system uses Cerner Millennium Discern Expert rules and some small Cerner Command Language in-house programs. The entire expert system is totally integrated with the area-wide database and has been built and maintained by haematology staff members, as has the haematology database. Using patient demographic data, analyser numeric results, analyser error and morphology flags and previous results for the patient, this expert system decides whether to validate the main full blood count indices and white cell differential, or if the analyser results warrant further operator intervention/investigation before verifying, whether a blood film is required for microscopic review and if abnormal results require phoning to the staff treating the patient. The principles of this expert system can be generalized to different haematology analysers and haematology laboratories that have different workflows and different software.     

Quantification of red blood cell fragmentation by automated haematology analyser XE‐2100

The extent of red blood cell fragmentation in peripheral blood is useful for diagnosis and follow‐up in many diseases, e.g. haemolytic uremic syndrome, transplantation‐associated thrombotic microangiopathy (BMT‐TMA). However, this quantification still relies on manual counting of fragmented red cells on blood smears. We have developed a quantification system by gating a fixed area of fragmented red blood cells (Gate 1) on an automated haematology analyser (XE‐2100, Sysmex Co., Kobe, Japan). The fragmented red cell percentage (FRC%) calculated with this system, from 100 samples, was highly correlated with the manual count (r=0.902, P < 0.0001). Because microcytic anaemia specimens usually occupy a lower position on the XE‐2100 scattergram, with microcytic cells overlapping Gate 1 and causing a spuriously high FRC% calculation, a supplementary gate (Gate 2) was added. Using the particle number in this gate as well as in Gate 1, a revised method for such samples was developed and its validity confirmed (revised FRC% correlated with a manual count for 10 subjects (P < 0.001). Because this gating system can be programmed on any XE‐2100, it is likely to prove useful for accurate quantification of red blood cell fragmentation and for the monitoring of the development of BMT‐TMA.