How to use image analysis for islet counting

AIM: Assessment of islet mass before islet transplantation requires a reliable technique to enable exact analysis of islet volume. This study aimed to test the applicability of digital image analysis (DIA) for evaluation of samples of purified and non-purified islets. METHODS: Pancreatic islets were isolated from 10 Lewis rats. Samples of purified (n = 10) and non-purified islets (n = 30) were counted conventionally and by using a computerized method. The equipment for the computerized counting consisted of a digital camera installed on a stereomicroscope and connected to a personal computer. Images of 2272x1704 pixels were processed using a previously described non-commercial program originally developed for this purpose. Islets were converted to equivalents using globe and ellipsoid models. The insulin content of purified islets was assessed using radioimmunoassay and was correlated to the absolute and standardized islet number. RESULTS: Mean absolute numbers of purified islets ± SD were 908 ± 130 and 1049 ± 230 (manually and DIA respectively). Mean insulin content ± SD obtained from purified islets was 161 ± 45 mU. The mean equivalents of purified islets (1589 ± 555 for globe and 1219 ± 452 for ellipsoid) significantly correlated with insulin content. However, this correlation was not significant when absolute islet numbers were used, counted using either method. There was no significant difference in absolute non-purified islet numbers assessed by manual and computerized methods (average ± SD in 50 µl samples; 12.6 ± 4.1 and 13.3 ± 5.3 respectively; p = 0.22). The manual method showed a significantly higher yield of islet equivalents (IE; p < 0.001 for both globe and ellipsoid). CONCLUSION: The computer-based system for islet counting correlated better to insulin content than conventional islet estimation and prevented overestimation. Reproducibility and ease of assessment make it potentially applicable to clinical islet transplantation.     

Automatic differential blood counting in plasma cell leukemia

The authors report a case of primary plasma cell leukemia and two cases of multiple myeloma with a large number of circulating plasma cells. The display of Technicon H-1 shows a very high "cloud" in the area of large unstained cells. A massive invasion of the basophil area was not found. Differences between these results and those found in different lymphoid leukemias are discussed.     

A comparison of differential white cell counting on the Coulter VCS and the Technicon H1 using simple and multiple regression analysis

Summary This study uses the statistical methods of simple and multiple regression to compare the differential white blood count on the Coulter VCS and Technicon HI analysers. The results demonstrate that both are good at distinguishing lymphocytes, neutrophils and eosinophils. Monocyte differential counts show disappointing correlation, both by simple and multiple regression techniques. Basophils, though less frequently a clinical problem, also correlated poorly in this study.     

A comparison of differential white cell counting on the Coulter VCS and the Technicon H1 using simple and multiple regression analysis.

This study uses the statistical methods of simple and multiple regression to compare the differential white blood count on the Coulter VCS and Technicon H1 analysers. The results demonstrate that both are good at distinguishing lymphocytes, neutrophils and eosinophils. Monocyte differential counts show disappointing correlation, both by simple and multiple regression techniques. Basophils, though less frequently a clinical problem, also correlated poorly in this study.     

Pseudobasophilia as an erroneous white blood cell differential count with a discrepancy between automated cell counters: report of two cases

We report two cases that showed erroneous white blood cell differential counts by automated cell counters. Each case showed an interesting discrepancy of differential count between cell counters, and marked pseudobasophilia was observed by one of the two counters. The first patient was a 44-year-old female who suffered from multiple myeloma for more than one and a half years. Increased myeloma cells (43%) in peripheral blood were counted as basophils by the ADVIA 120, and as monocytes by SE-9000, respectively. The second patient was a 72-year-old female diagnosed as having chronic myelomonocytic leukemia. Dysgranulopoietic neutrophils (50%) and monocytes (31%) were increased in the peripheral blood. Dysgranulopoietic neutrophils were counted as basophils by STKS. In contrast, about half of the increased monocytes were counted as neutrophils by the ADVIA 120. These interesting findings highlight the importance of microscopic examination of the blood film in routine laboratory practice, and automated cell counters, especially for the hematologic patients, cannot completely substitute for it. These results also imply that at least some subpopulations with different membrane or cytoplasmic properties may exist even in the similarly classified cells.     

The Haematology Analyser SF-3000: performance of the automated white blood cell differential count in comparison to the Haematology Analyser NE-1500

The present study evaluates the performance of automated white blood cell (WBC) differential counts by the new Haematology Analyser SF-3000. Five hundred and sixty-six WBC differential counts performed by the SF-3000 were compared with WBC differential counts of the well established analyser NE-1500 and to manual reference counts. Numerical results of the WBC differential counts were correlated to each other by regression analyses. The efficiency of instrument flagging for the presence of abnormal WBC was expressed as per cent of subjects correctly classified. Neutrophil and lymphocyte counts correlated well between analysers and to manual reference counts. Monocyte counts for the SF-3000 correlated significantly better with the microscopic counts, whereas correlations of eosinophils and basophils were better for the NE-1500. The efficiency rates of flagging for the presence of ≥1% abnormal WBC were 80% for the NE-1500 and 70% for the SF-3000. This difference was exclusively due to low specificity of the SF-3000 in flagging cells of the `Left Shift' category, especially in samples with elevated WBC counts. The flagging efficiencies for blasts, promyelocytes, myelocytes, atypical lymphocytes and nucleated red cells were identical for both analysers. Thus, with regard to the performance of automated WBC differential counts the SF-3000 seems comparable with other, well established haematology analysers.     

Interference of Hb-H disease in automated reticulocyte counting

Hb-H disease is a form of alpha-thalassemia commonly found in south-east Asia. In this condition, numerous Hb-H bodies are found in erythrocytes using reticulocyte staining preparations. This is the first study addressing the possible interference of Hb-H inclusions in automated reticulocyte counting. In this study, seven Hb-H disease samples were tested. Results obtained by the visual method and the Technicon H*3 automated method agreed relatively well. This was in contrast to the Coulter STKS automated method which generally gave lower results. Furthermore, when the incubation time was extended to 180 min in the Coulter STKS method, two Hb-H disease samples gave results several-fold higher than those obtained using the 60 min incubation time. These discrepant results were likely to have been caused by the analyser using the wrong threshold to separate reticulocytes and mature erythrocytes. High and low interference in Hb-H disease samples was observed in the Coulter STKS automated reticulocyte method. Laboratories should be aware of this potential problem, particularly when samples are from patients of Asian origin.     

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.     

Evaluation and use of the white blood cell differential provided by the Coulter® STKS in a children's hospital

Summary. The Coulter® STKS was evaluated in a children's hospital, in order to (a) compare the WBC differential given by the instrument to a 400 cell visual differential (reference method); (b) evaluate the sensitivity and specificity of the alarm system, and (c) provide data concerning the use and interpretation of results in children. 653 blood samples were collected. The Coulter® STKS results were studied in 523 patients having no morphological abnormalities in the blood smears, separated into subgroups according to the presence of STKS alarms and according to age. The results were found accurate both in STKS negative and STKS positive patients (i.e., those with alarms:‘Blasts', Imm Gran 2, Variant Lymph, NRBC, review slide). Negative STKS results had the same accuracy in all age groups, except in neonates where slide review must be systematically performed. The instrument exhibited a good sensitivity of the suspect flags studied (91.4%), with a lower specificity (72%) reflecting the number of false positive results found in our group, probably due to the cytological features particular to children. However, it was shown that the numerical results given by the Coulter® STKS in positive patients could be taken into account, provided that a scan of the blood smear was negative for morphological WBC abnormalities.     

Artefactual elevation of an automated white cell count following femoral vein puncture

We report the novel occurrence of an erroneous automated total and differential white blood cell count on a blood sample obtained by traumatic femoral venepuncture, caused by contamination of the blood sample with subcutaneous adipose tissue. The erroneous counts were observed on a Bayer-Technicon H2 analyser while counts on a Coulter GenS, were much less affected. Characteristic scatter plots from both instruments are illustrated.     

Automated counting of white blood cells in synovial fluid

OBJECTIVES: To evaluate the performance of automated leucocyte (white blood cell; WBC) counting by comparison with manual counting. METHODS: The number of WBC was determined in heparinized synovial fluid samples by the use of (i) a standard urine cytometer (Kova) and a microscope (reference method) and (ii) a haematology analyser (Sysmex XE-2100; WBC/BASO and DIFF channels). Imprecision within and between days was determined by replicate analysis of a low (level A; WBC approximately 0.560 x 10(9)/l) and a high (level B; WBC approximately 1.081 x 10(9)/l) dedicated synovial fluid control (Quantimetrix). RESULTS: The WBC count of the DIFF channel was highly correlated with the WBC count of the microscopic reference method (r = 0.99; WBC analyser = 0.870 x WBC reference method + 0.413). In contrast, no agreement existed between WBC counts generated by the WBC/BASO channel of the analyser and the reference method (r = 0.52; WBC analyser = 0.008 x WBC reference method + 0.079). Within-day imprecision (4-7%) and between-day imprecision (10%) of the haematology analyser were smaller than the within-day imprecision (12%) and the between-day imprecision (20-22%) of the manual reference method. For manual counting, inter-observer coefficients of variation were 35.9% (control level A) and 21.0% (control level B). CONCLUSIONS: The WBC count in synovial fluid can be reliably determined using the DIFF channel of the Sysmex XE-2100. Automated counting of WBC in synovial fluid offers more precise and faster results than manual counting.     

Automated counting of white blood cells in synovial fluid

SIR, We were interested to read the article on synovial fluidwhite count estimations by de Jonge and colleagues [1]. It isclear from data presented in that paper that the total cellcount can be accurately estimated by automated methods ratherthan by more labour-intensive manual counting. The literature, as de Jonge et al. explain, states that theestimation of total and differential white     

Adaptation of the May-Grünwald-Giemsa staining method for automated differential counting of blood leucocytes by a Hematrak analyzer

The selection of exact pH (6.85/6.90) concentrations of the stains and staining times made it possible to use the May-Grünwald-Giemsa (MGG) staining method for smears studied with a Hematrak model 240 automated differential leucocyte counter. The differences between the smears stained by the method of Wright according to the recommendations of the manufacturer and the present MGG-modification resulted in non-significant differences in the recognition of various types of leucocytes. The differences between the manual method and those obtained by Hematrak for the MGG-stained samples with normal total leucocyte counts were nonsignificant with all types of leucocytes except basophils, which were underestimated by the instrument. The present staining procedure was adequate in studying of neutrophilias. When samples with lymphocytosis or with immature cells were examined, too few lymphocytes and immature cells and too many monocytes were obtained by the instrument, as compared to the manual method.     

Adaptation of the May-Grünwald-Giemsa staining method for automated differential counting of blood leukocytes by a Hematrak analyzer

The selection of exact pH (6.85/6.90) concentrations of the stains and staining times made it possible to use the May-Grünwald-Giemsa (MGG) staining method for smears studied with a Hematrak model 240 automated differential leucocyte counter. The differences between the smears stained by the method of Wright according to the recommendations of the manufacturer and the present MGG-modification resulted in non-significant differences in the recognition of various types of leucocytes. The differences between the manual method and those obtained by Hematrak for the MGG-stained samples with normal total leucocyte counts were nonsignificant with all types of leucocytes except basophils, which were underestimated by the instrument. The present staining procedure was adequate in studying of neutrophilias. When samples with lymphocytosis or with immature cells were examined, too few lymphocytes and immature cells and too many monocytes were obtained by the instrument, as compared to the manual method.