Automated differential counting

Automated Differential Counting

K. G. Patterson, A. B. Carter

The Need for Automation

Technical Problems

ofManual DtjFerential Counts

The standard manual differential count involves the recognition, counting and classification of 100 leucocytes on a stained blood film. Technical difficulties in the counting method are well recognised.‘*2~3 Uneven distribution of the different leucocyte types in the wedge blood film is usual, with the larger and stickier cells (monocytes, neutrophils) carried to the tail of the him by the spreader, leaving a relative excess of lymphocytes in the body of the film. To overcome this difficulty special counting techniques have been recommended such as the ‘battlement’4 or ‘strip’2 techniques. Instruments have been produced to make a monolayer of blood cells by rapidly spinning a slide bearing a drop of blood.5 These avoid the problems of uneven distribution of leucocytes but may cause aerosols of blood droplets thrown from the edge of the slide. When the blood is leucopenic there may be insufiicient cells on the monolayer film to do a 100 cell differential count.

for basophil counts has been stated as 0.02-0.1 x log/ 1.’ If one basophil is seen on a routine 100 cell differential count in a patient having a total leucocyte count of 11 x log/1 then a basophilia is present. If that cell is not seen, then there is a basophilopenia. It was only with the development of automated flow cytometric methods for basophil counting on the Hemalog D automated differential counter that the changes in basophil count in thyrotoxicosis and myxoedema were appreciated.8 This instrument counted 10 000 cells in its basophil channel alone. The larger the number of cells counted, the more reliance can be placed on the result. Modern automated differential counters count 1000-20 000 leucocytes to derive the differential count. This may be particularly useful for neutropenic patients recovering from combination chemotherapy, when the appearance of granulocytes heralds clinical recovery. ’ Manual methods are available for obtaining more accurate absolute basophil and eosinophil counts by using a specific supra-vital staining technique on lysed whole blood in a counting chamber”*” but these are relatively tedious and time consuming.

Statistical Considerations Assuming a uniform distribution of leucocytes on the blood film, the accuracy of the differential count is dependant on the number of leucocytes counted. Statistical evaluation of the 100 cell differential count demonstrates the potential inaccuracy of such counts, particularly for minor components of the differential.‘*6 At the commonsense level, the normal range K. G. Patterson, A. B. Carter, Department of Haematology, University College Hospital, Gower Street, London WClE 6AU, UK. Blood Reviews (1991) 5, 78-83 0 1991 Longman Group UK Ltd

Methods For Automated Differential Counting The majority of automated

differential counters also perform standard full blood counts including red cell indices and platelet counts. Only aspects of the differential counting process are covered in this review. The Coulter VCS” is the only dedicated differential counter on the market at present that does not perform a full blood count.

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Pattern Recognition

The earliest automated differential counters employed the principles of image analysis.” A television camera scanned a monolayer stained blood film on an automated, computer controlled microscope. The computer was programmed to recognise common leucocyte types by their size, colour and staining pattern. The two important limitations of pattern recognition methods of differential counting are speed and inability to classify rare types of cell and distinguish them from artefact. Even with very fast computer processors, speed is limited by the use of a mechanical stage, scanning the film and stopping to allow analysis of recognised leucocytes. No pattern recognition instruments dedicated solely to automated differential counting are currently marketed. Flow Cytometry

All current commercial automated differential counters employ variations of flow cytometry.‘3 Cells in a rapidly flowing solution pass in front of various optical and electronic detectors. The pulses from these detectors are analyzed by a computer which derives the differential count. The measurement of a single parameter provides insufficient information to provide a five part differential and several parameters are usually assessed at once. To prevent clumps of cells blocking the detection area the stream of solution containing the cells to be analyzed is enclosed in an outer cylinder of transparent fluid (sheath flow). Technical Methods Employed in Flow Cytometry OriJice Impedance Measurement (Coulter principle)

This is an enhancement of the most commonly used method for counting red and white cells in laboratories.‘4*1s The red cells in diluted blood are lysed, leaving the leucocytes in suspension. The lysing agent causes a release of the fluid part of the cell cytoplasm, leaving the nucleus and granules tightly enclosed by the cell membrane. As the leucocytes pass through the counting area (orifice) they interrupt an electric current passing through the orifice, causing an electronic pulse. The pulses can be counted to derive the leucocyte count and sized to determine a three population differential count (lymphocytes, granulocytes, monocytes). This method of obtaining a three-population differential count is employed in the Coulter S Plus IV, STKR, Sysmex KlOOOand related blood counters. Figure 1 shows examples of the leucocyte peaks obtained from a STKR. Although monocytes are the largest cell seen on the blood film, in this system the release of cytoplasmic fluid renders the monocytes smaller than granulocytes, which retain their granules. The areas under each peak are counted to provide the cell counts. Individual types of granulocytes are not distinguished by this system but eosinophils lie in the area E in the figure. Elevation of this area due to a

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high eosinophil count is used to trigger a warning of eosinophilia on the count. High Frequency Impedance

The electric currents employed in orifice impedance counting are of low frequency or direct current. As the frequency of the current used is increased the cell membrane becomes increasingly transparent to the electric current until at radio frequency the impedance is related to the nuclear size rather than to overall cell size. The Coulter VCS and STKS blood counters and the Sysmex NE800016 are among the differential counters that employ a combination of low frequency (cell volume) and high frequency (nuclear volume) measurements to classify different leucocyte populations. Further enhancements are required to generate a 5 part differential. In the Coulter systems measurement of laser light scatter as an index of granularity is used. The Sysmex systems use pH specific lysing agents, which lyse all leucocytes except for either eosinophils or basophils. Light Scatter

Light from a laser (Coulter VCS, STKS) or conventional (Technicon) source is directed at the stream of cells. This is reflected by the cell surface and intracellular organelles. Light scatter at low angle is related to cell size, and at high angle to complexity of structure. By measuring the scattered light at different angles measurements of cell size and granularity can be made. Measurement of light absorbance is of particular use in methods involving the cytochemical staining of cells employed by Technicon instruments. Cytochemical Staining

Several differential counting instruments from the Technicon company employ specific cytochemical stains for the detection of different leucocyte populations, l7 including the H6UO0,H* 1l8 and H*2. Peroxidase staining is employed to distinguish eosinophils, neutrophils monocytes and lymphocytes. At the alkaline pH employed eosinophils stain strongly, neutrophils moderately and monocytes weakly. Lymphocytes remain unstained. Detectors for light scatter (size) and light absorbtion (staining intensity) assess each leucocyte and these are displayed on a scatterplot (Fig. 2). Basophils are detected on their own separate channel either after specific staining using alcian blue (H6000) or by stripping the cytoplasm off all leucocytes except basophils and assessing their light scatter signals (H* 1, H*2). Data Processing

Scatterplots-pictorial representations of cell populations enclosed by counting thresholds-are widely

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Fig. 1 Leucocyte volume distribution histograms from the Coulter STKR three-population differential counter. The left hand peak represents lymphocytes (small size) and the broader right-hand peak granulocytes (medium and large size). The approximate positions of normal leucocytes are shown above the histograms. A normal leucocyte distribution is shown (A) with a neutrophilia (B) and a lymphocytosis (C). E indicates the approximate position of eosinophils.

CELL STAINING Fig. 2 The peroxidase scattergram display from the Technicon H’l The vertical axis shows cell size (light scatter), the horizontal axis staining for peroxidase (light absorption). The display is divided by vertical and horizontal counting thresholds enclosing the major cell populations: eosinophils (eos), neutrophils (neuts), lymphocytes (lymphs), monocytes (mono) and large unstained cells (LUC).

employed in differential counters. Each dot on the display represents a single leucocyte and its position on the display is related to its size, light absorbtion, granularity, or other complex parameter. Thresholds divide the display into boxes enclosing each cell population. Some thresholds are self adjusting, hunting for the valleys between separate cell populations. Others, which enclose cell populations of low variability, are adjusted to fit normal cell populations at the time of instrument calibration. Alarms are provided to indicate the presence of atypical cell populations overlying counting thresholds. Figure 2 shows a scattergram from a Technicon H*l counter, which displays cell size vs. cell staining for myeloperoxidase. Figure 3 shows a scattergram from a Sysmex NE 8000 counter, which displays cell volume vs. nuclear volume.

Raw data from the various detectors is processed by a computer using a series of algorithms (equations) provided from a software program. This program is regularly changed and updated as improved algorithms are developed. One of the major problems of evaluating differential counters is that the software issue must be specified, and if any instrument is found wanting a new set of software is then produced requiring instrument re-evaluation. Performance Assessment of Differential Counters The majority of assessments performed on automated differential counters follow the guidelines of The United States National Committee for Clinical Laboratory Standards. lg This standard recommends a

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CELL VOLUME

CELL VOLUME Fig. 3 Scattergram display from the Sysmex NE 8000 differential counter. The horizontal axis represents cell volume (low frequency conductivity) and the vertical axis nuclear volume (high frequency conductivity). The display is divided by thresholds separating the cell populations: granulocytes (gran), lymphocytes (lymph), and monocytes (mono). Debris (noise) is separated from the cell populations by the major oblique threshold T. Below the scattergram is displayed the same count in a different format. Again the horizontal axis reoresents cell volume but the vertical axis reoresents cell numbers. The two graphs show cell numbers above (A) and below (B) the major oblique threshold (T).

reference differential method based on the examination of a Romanovsky stained wedge blood film by two expert examiners counting 200 leucocytes each. For the evaluation of a differential counting method at least 200 samples are required, of which 100 are normal. Detailed recommendations are given on the method of film examination, classification of leucocytes and statistical procedures. Performance

of Three-population

Dlyerential

Counters

We have had the opportunity of assessing the Coulter STKR” three-population differential counter which is has been in routine use in our laboratory for 3 years. This showed good correlation (r =0.9 or better) of total granulocytes, lymphocytes, and monocytes when compared with an 800 cell manual differential count. The major anxiety of laboratories considering the use of three population differential instruments is that they may miss small numbers of primitive cells or eosinophilias/basophilias. The STKR indicates the presence of abnormal cells in response to various abnormalities in the shape of the leucocyte histogram. Some of these indications are narrative reports in plain English such as ‘suspect atyp lymphs, or blasts’ triggered by a

particular histogram abnormality. Samples were flagged as ‘positive’ by manual methods if one or more immature cells were identified by any of the four observers on a 200 cell manual differential. Disagreement between manual and STKR results was found in 24% of samples. False positive STKR results were found in 7% of samples, and false negative STKR results in 18%. Closer examination of the false negative STKR results showed that only 18% of them were associated with an immature cell count of over 0.5% (so may well have been missed by a standard 100 cell manual differential). 14% had clear abnormalities of the blood count that would in any case have prompted manual film examination. This left 4% of false negatives or 0.75% of all samples analyzed in the study in which immature cells may have been missed. Some of these, from patients known to have haematological disorders, would have had manual film examinations performed as a routine in any case. The STKR does not provide a count for eosinophils or basophils, but is able to indicate that they are lower than 0.7 x 109/1for eosinophils or less than 0.2 x 109/1 for basophils. Values above these ranges are flagged by narrative statements on the report. Six out of 7 samples found to have a manual eosinophil count over

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0.7 x log/1 were flagged as possible eosinophilias, the 1 sample not flagged having an eosinophil count of 0.7 x log/l. Ten out of 14 samples having a manual basophil count greater than 0.2 x log/1 were flagged as possible basophilias, the 3 not flagged having manual basophil counts of less than 0.3 x log/l. The introduction of the STKR differential counter allowed a 24% reduction in the number of manual film examinations in this laboratory, indicating the potential usefulness of such relatively simple differential counters in reducing laboratory workload. Performance of Five-population Dtflerential Counters

All commercially available five-part differential counters currently available in the UK provide satisfactory correlations with manual methods for neutrophils, lymphocytes, monocytes and eosinophils. Correlations for basophils with manual methods is universally poor with correlation coefficients rarely exceeding 0.3. It is, however, generally accepted that current statistical methods for the assessment of basophil correlation are difficult to apply to a cell type that is present in such small numbers in the majority of blood samples. The Coulter VCS counter, in our laboratory, failed to flag 12% of samples found to be abnormal by manual reference methods, but flagged as abnormal 13% of samples considered normal by reference methods. In the same study the Technicon H*l found a false negative rate of 18%, and a false positive rate of 7%. As with any highly automated laboratory apparatus the information produced by automated differential counters must be interpreted in the light of the patients clinical status and likely results. Any automated differential system can be ‘fooled’ by abnormal leucocyte populations. We have seen several examples of mobile thresholds selecting the wrong leucocyte population for counting, and mention is made below of the difficulty of counting leukaemic cell populations. Additional Useful Clinical Information Generated by Automated D@erential Counters

One of the parameters measured by Technicon differential analyzers (H6000, H*l, H*2) is the proportion of large unstained cells in the peroxidase channel (Fig. 2). These may be blast cells, reactive lymphocytes, or the hairy lymphocytes of hairy cell leukaemia. Binet” found a correlation between clinical stage of chronic lymphocytic leukaemia (and hence prognosis) and numbers of LUc’s in the blood. In this laboratory we found that LUC numbers generally reflected modal lymphocyte size in chronic lymphocytic leukaemia but were not related to Rai clinical stage in our patients. Peroxidase staining is the major method of leukocyte differential counting on Technicon systems. This is a useful cytochemical stain in the diagnosis and classification of acute myeloid leukaemias and characteristic

scattergram displays can be produced.” The larger size and variable peroxidase activity of myeloblasts compared to lymphoblasts tends to push them towards the top left of the display (into the ‘neutrophil’ and LUC boxes) whereas in lymphoblastic leukaemia the blast cells tend to occupy the ‘lymphocyte’ and LUC boxes. When assessing the instrument blood counts from patients with acute myeloid leukaemia it is important to appreciate that the ‘neutrophil’ count merely represents cells with peroxidase positivity, not necessarily neutrophils. During regeneration of bone marrow after ablative chemotherapy there is an increase in the percentage of LUC’s in the blood which is closely followed by the appearance of the ‘blast cell’ flag.” These features may be used to predict bone marrow recovery. Peroxidase enzyme activity persists within leucocytes longer than recognisable morphological features. When delay in analysis is expected (e.g. postal samples) cytochemistry systems may produce a meaningful differential when conventional morphology cannot. Neutrophil peroxidase deficiency, found as a congenital abnormality or in myelodysplasia/AMLz3 can be quantitated in the Technicon system, and has allowed recognition of increased as well decreased activity in AML.24 Minor modification of the H*l peroxidase staining system allow it to be used for the detection of cell surface antigens by an immunoperoxidase technique.2sy26 This is used particularly in the counting of lymphocyte subsets (pan T, pan B, CD4, CD8). A primary antibody is coupled to the antigen being detected e.g. mouse anti-T. This is then coupled with a species specific (anti-mouse) antibody which has been labelled with an avidin-biotin-peroxidase complex. The peroxidase staining is then quantitated by the instrument, and the display thresholds used to separate labelled from unlabelled lymphocytes. The relatively strong staining of the neutrophils and monocytes allows them to be distinguished from positive staining lymphocytes. Methods for lymphocyte subtyping using Coulter systems are being developed, and the ability to perform these immunological techniques may make 5 population differential systems economically viable for the smaller laboratory. Conclusion Automated differential counters will continue to handle an increasing proportion of the routine laboratory workload. The indications from preliminary clinical and research studies are that useful information, not currently available from manual film examination may, in the future, assist in patient management. References 1. England .I M 1976 Total and differential leucocyte count (Annotation). British Journal of Haematology 33: l-7 2. Dacie J V, Lewis S M 1991 Differential leucocyte count. In:

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17. Mansberg H P, Saunders A M, Groner W 1974 The Hemalog D white cell differential system Journal of Histochemistry and Cytochemistry 22: 71 l-724 18. Ross D W, Bentley S A 1986 Evaluation of an automated haematology system (the Technicon H* 1) Archives of Pathology Laboratory Medicine 110: 803-808 19. National Committee for Clinical Laboratory Standards 1989 Reference leucocyte differential counting and evaluation of methods: Approved Standard H20-A, Villanova, Pennsylvania 20. Barnard D F, Barnard S A, Carter A B et al 1989 Detection of important abnormalities of the differential using the Coulter STKR blood counter. Journal of Clinical Pathology 42: 772-776 21. Binet J L, Vaugier G, Dighiero G et al 1977 Investigation of a new parameter in chronic lymphocytic leukaemia: the percentage of large peripheral lymphocytes determined by the Hemalog D 22. A comparison of automated cytochemical analysis and conventional methods in the classification of acute leukaemia 1980 Patterson K G, Cawley J C, Goldstone A H et al Journal of Laboratory and Clinical Haematology 2: 281-29 1 23. Catovsky D, Galton D A G, Robinson J 1972 Peroxidase deficient neutrophils in acute myeloid leukaemia. Scandinavian Journal of Haematology 9: 142-148 24. Patterson K G, Goldstone A H, Richards J D M, Cawley J C 1982 Increased neutrophil peroxidase activity in acute myeloid leukaemia. Acta Haematologica 68: 242-248 25. Kim Y R, Martin G, Paseltiner I et al 1985 Subtyping lymphocytes in peripheral blood by immunoperoxidase labelling and light scatter/absorption flow cytometry Clinical Chemistry 31(9): 1481-1486 26. Mason D Y. Cordell J L, Abdulaziz Z et al 1982 Preparation of peroxidase : antiperoxidase (PAP) complexes for immunohistological labelling of monoclonal antibodies Journal of Histochemistry and Cytochemistry 30(11): 11141122 27. Kinsey S E, Carter A B, Watts M J et al 1988 The use of the H* 1 in predicting marrow recovery following ablative chemotherapy for leukaemia or lymphoma. Clinical and Laboratory Haematology 10(l): l-5