Automated systems within clinical laboratories aim to increase the efficiency of reporting patient results. The results from analysers used for blood cell counting and white blood cell differentiation may sometimes need to be verified by a manual blood film review especially, if there are atypical, immature cells or nucleated red blood cells present.Figure 1. CellaVision DM96

Manual blood film examination is costly both in a financial and time sense. With patient result turnaround times of utmost importance to any clinical laboratory, the time taken to perform manual differentials means the biomedical scientist is unable to perform other routine duties within the laboratory. This study was undertaken to compare the speed, efficiency and accuracy of an automated film reader, namely the CellaVision DM96 (CellaVision AB Lund Sweden) to biomedical scientists. The CellaVision DM96 consists of a slide-scanning unit and a computer with the CellaVision Blood differential Software (Version 1.5). The scanning unit consists of a motorised microscope (10x, 50x, and 100x objective) and a digital CCD camera. The instrument can be loaded with eight racks of twelve slides, continuously fed into the analyser, processing them immediately. The DM96 scans the slides, identifies potential WBCs, takes digital images of them and uses an artificial neural network-based software to analyse the cells. Digital images of the pre-classified cells are presented to the operator on a user definable computer display. The operator is then able to re-classify the cells by double clicking or dragging the images to the group of cells to which it belongs. Cells can also be magnified with simple mouse movements to allow finer cellular examination. All pre-classified cells have to be examined by the user and re-classified if necessary before data is saved and authorisation is allowed. The DM96 also partially classifies red blood cell morphology; polychromasia, hypochromia, macrocytosis, anisocytosis and poikilocytosis reported on a scale of 0 to 3. Figure. 2 Screenshot of evaluation of red blood cells on DM96

This study aims to evaluate the accuracy of the DM96 in identifying all classes of leucocytes including abnormal and immature cells. Results from five different laboratory scientists’ manual differentials and those from the DM96 were compared to a standard reference manual differential (Clinical and Laboratory Standards Institute H20-A2, 2007) and the time taken for the slides’ analysis, recorded. A limited evaluation of the red cell morphology from the DM96 was also undertaken.

REFERENCE METHOD The reference method was a 200 cell manual differential count performed by two experienced laboratory scientists using light microscopy. The DM96 was set to count 105 cells to counter the effects of artefacts, although 100 cells was not always achieved due to some samples having very low WBC counts. Two thirds of the reference films were abnormal with various clinical conditions such as HIV, chemotherapy, infections, leukaemia's and lymphomas, iron deficiencies and haemaglobinopathies, and diseases of the liver and kidney. The WBC counts ranged from 0.5x109 to 73. 09/L (analysed on Sysmex XE-2100). Red blood cell analysis was compared with the microscopic method.

The laboratory scientists were selected with differing experience in blood cell morphology; two were highly experienced, one moderately experienced, and two newly qualified whom may still require assistance in the laboratory with very abnormal blood films.

METHOD

Thirty films were presented to the scientists, firstly for classification on the DM96, then to perform their own 100 cell manual differential on a light microscope. No clinical information or history, nor haematology analyser results were available to the scientists. The 100 cell manual differentials for each individual were compared to the result for the pre-classified, and then their re-classified cells from the DM96, and additionally all results compared to the reference cell differential. The time taken for the differentials from the CellaVision and manual method were recorded for each individual.

RESULTS

The resulting percentage of the various cell classes pre-classified correctly by the DM96 was highest for mature cells and lowest for metamyelocytes and promyelocytes. There was good classification of nucleated red blood cells (table 1)..

Table 1.

Assessment of the accuracy of the DM96’s pre-classification versus reclassification of results was carried out by determining the correlation coefficient (R2) between the reference manual differential and the pre- and re-classified results from the DM96 for all cell classes. Correlation coefficients were higher after the cells had been re-classified, where R2 values were greater than 0.9 for all cells except monocytes (0.81) and eosinophils (0.67). Due to the low number and subjectivity of classification of metamyelocytes, myelocytes and promyelocytes, the results for these cells were also reported as a total immature granulocyte count. Poor correlation for the individual immature granulocytes was attributed to the low number of cells counted and the subjectivity of classification into each maturity class. Correlation for the total immature granulocytes was excellent. Figure 3 demonstrates correlation graphs for the immature granulocytes, nucleated red blood cells and blasts..

Figure 3.

The classification of the red blood cells was less successful, with the instrument reporting macrocytosis and anisocytosis on many samples that had normal red blood cells. However in 95% of samples, the quality of red cell images was sufficient for the operator to clearly identify morphological features and reclassify the results. (Figure 2) In this evaluation, the default red cell pre-characterisation setting was used, which may have contributed to the disagreement; settings can be adjusted by the operator to achieve a level of classification equivalent to that by manual microscopy. The overall accuracy of the manual differentials by the five different operators did not exceed that of the DM96. This was demonstrated by the comparison of each individual’s manual differential as well as their pre- and reclassified differentials from the DM96 to the reference method.

For some cell types and for some operators, the accuracy of the DM96 compared to the reference method was better than that of the individuals’ 100 cell manual differential. Correlation with the reference method for the lymphocyte count from the reclassified results from the DM96 is very poor for one of the inexperienced operators this is due to a single sample where the DM96 pre-classified lymphocytes were not correctly reclassified as blasts.. Correlation for the eosinophils on the DM96 was not as good as the manual method (R2 – 0.60) even after reclassification. This may be attributed to a staining issue, where eosinophils appear more pink on the DM96 than under a microscope.Statistically, the pre-classification results on the DM96 for lymphocytes, immature granulocytes, myelocytes and to a lesser extent basophils, had significantly higher values than those by the reference method, reclassified results or each operators manual differential. The DM96 had a tendency to classify other cells into these classes. For reclassified cells there were very few statistically significant differences identified between the specific users’ manual differential and the DM96 when compared to the reference method. The greatest number of statistically significant differences occurred again with lymphocytes and basophils; basophils attributed to by the low number of cells counted.For lymphocytes the median range for reclassified cells on the DM96 is 1.20 – 1.42 x109/L, for the manual differentials 1.07 – 1.52 x109/L and for the reference method count was 1.42 x109/L. The highly experienced operators showed better statistical agreement between the manual and DM96 differentials.

The total time required for the pre-classification and reclassification on the DM96 for the thirty blood films was similar for all operators, averaging 80 minutes for all scientists, regardless of their experience. The manual differential required a greater amount of time, the quickest being 100 minutes, with the inexperienced scientists taking significantly longer (table 3).

Table 3.

Figure 4. Screen shot of cells presented for reclassification on the DM96

So can the an instrument ever replace the laboratory scientist’s manual differentiation?

For any automated image system to be introduced in the haematology laboratory it would have to demonstrate that it could reliably and correctly identify WBCs. Though the DM96 is capable of morphological classification of cells, its accuracy depends on both the blood pathology and the experience of the scientist. For some cell types and for some operators, the DM96’s accuracy was better than the individual’s 100 cell manual differential. It is also a quicker alternative. The DM96 certainly has a place in the routine haematology laboratory where it would improve workflow, and assist in the training and competency testing of scientists.