Anal. Chem. 1999, 71, 363R-365R
Clinical Analyzers. Advances in Automated Cell Counting Thomas S. Kickler
Johns Hopkins Hospital, 600 North Wolfe Street, Meyer B 121, Baltimore, Maryland 21287 One of the most frequently performed laboratory test panels is the complete blood count, consisting of the white count, hemoglobin, red cell count, and platelet count. In addition, with each white count a white cell differential is frequently needed. The white cell differential consists of identifying individual white cell types, consisting of neutrophils, lymphocytes, monocytes, eosinophils, and basophils. For decades the differential was done by microscope examination of the peripheral smear. Automated cell counters now offer electronic “five-part” differentials. The most recent advancement in differential cell counting is the ability of recognizing blasts, atypical lymphocytes and nucleated red cells with accuracy. Other new parameters include counting of reticulocyte fractions, detection of blasts, immunoplatelet counting, and surrogate stem cell counting. In selecting a cell counter, one may need an instrument that analyzes a relatively low number of specimens to the high-end instruments that analyze 100-140 specimens/h. The purpose of this review will be to focus on the high-capacity instruments that measure all hematologic parameters. In selecting a cell counter in a setting where one encounters many abnormal patients, the rate of flagging results for manual review is a critical consideration if one wants to reduce labor-intensive peripheral smear review. Some instruments historically had a high rate of flagging, not only for normal but also for abnormal results. This was particularly true for platelet counting and immature leucocytes (K1). Because of the interest in reducing manual review, a variety of new technologies and user-defined cutoff levels for different parameters have been developed. Despite technologic advancement, microscopic review is still a necessity. To reduce the labor involved, automated slide markers are available from most manufacturers. In addition, automated scanning of slides, laboratory image analysis, and projection on a high-resolution color monitor permit “walkaway microscopy”. The following review discusses new developments in the measurement of hematologic parameters and describes the technologies available for each parameter. HEMOGLOBIN CONCENTRATION Between the different manufacturers, the methods for counting total blood hemoglobin concentration are the most similar of all (K1). Table 18 summarizes the comparison between manufacturers for the method of hemoglobin measurement. The reference method for determination of hemoglobin is the cyanomethemoglobin method (K1). Concerns with hazards posed by cyanide in laboratory waste have led to noncyanamide methods. These changes promise to improve calibration and validation of new 10.1021/a1999911a CCC: $18.00 Published on Web 05/20/1999
© 1999 American Chemical Society
hemoglobin instrument measurements. The sodium laurel sulfate hemoglobin method introduced by Sysmex is a significant advance in reducing turbidity interference and in allowing rapid analysis on a highly automated instrument (K2, K3). This method appears equal to the reference method. RED CELL INDEXES The classification of anemia is based upon the red cell indexes, including MCV, MCHC, and MCH (L4). With the exception of Bayer’s cell counter, each of the manufacturers obtains a sizedependent signal with aperture impedance detection after preparing the cells by diluting them in isotonic diluent (K1). Bayer spheres the red cells at constant volume and measures the size with a two-angle optical scatter system. Conversion from the signal size in the two-optical sensing detectors into red blood cells size is made by using a special algorithm based on the optical scattering of spherical dielectric particles. Shape changes of abnormally deformed red cells in aperture impedance analyzers are known to affect MCV, MCHC, and hematocrit estimation (K5). The introduction of hydrodynamic focusing into impedance detection instruments such as the Sysmex SE9500 appears to reduce the effect of red cell shape change and deformity. The Beckman-Coulter instruments yield red cell indices, especially MCHC values, that are affected by using an impedance aperture not equipped with hydrodynamic focusing. Clinicians may not be aware of the differences between hematology analyzers in the estimation of the MCV, the hematocrit, and MCHC in samples with abnormal red cells. RETICULOCYTE COUNTING Measurement of reticulocytes is important in assessing bone marrow erythroporetic activity. Thus, reticulocyte analysis has been an accepted parameter in differentiating hypoproliferative anemias (low reticulocytes from hemolytic anemias (elevated reticulocytes). Dividing reticulocyte scattergram into three sectors according to fluorescent intensity, reticulocytes can be fractionated into maturity stage (K6). The very young, highly flourescent reticulocytes are a very early indicator of marrow recovery. These appear before the total reticulocyte count is elevated. Because of this, the reporting of high fluorescent reticulocyte may be useful in determining marrow recovery following marrow transplantation or detecting the response to exogenous erythropoietin administration. Reticulocyte measurement can now be done without manual addition of reagents in full automated mode by all manufacturers. Sysmex was the first to introduce a reticulocyte count for the clinical laboratory which uses a fluorescent stain (Auromune-0) Analytical Chemistry, Vol. 71, No. 12, June 15, 1999 363R
Table 18. Hemoglobin Determination instrument
identification method detection method
Beckman-Coulter
Sysmex
Bayer
Abbott
ABX
cyanomet-Hb absorb at 525 nm
SLS-met Hb absorb at 555 nm
cyanomet-Hb with direct cellular x Hb absorb at 546 nm
cyanomet-Hb absorb at 546 nm
cyanomet-Hb absorb at 540 nm
Table 19. Platelet Counting Abbott platelet counting method linearity (109/L) discrimination between nonplatelet events
Coulter-Beckman
optical/impedance reflexive immunoplatelet counting 0-2000 optical, impedance, and reflexive immunoplatelet counting
Sysmex
Bayer
fixed impedance threshold
nonfixed impedance
optical
0-999 fixed thresholds limit accuracy of single-parameter detection
0-999 nonfixed thresholds allow discrimination
0.005-3000 optical and refractive index allows discrimination
Table 20. WBC Counting and Differential WBC method differential method linearity (109/L)
Abbott
Coulter-Beckman
Sysmex
Bayer
optical scatter/fluorescence optical scatter/fluorescence 0-250
impedance Coulter 3-D VCS technology 0-99.99
direct current direct current and radio frequency 0-99.9
flow cytometry peroxidase and nuclear density 1-99.9
detected by fluorescence flow cytometry. Beckman-Coulter uses traditional new methylene blue and counts reticulocytes. This method is less than ideal since because of its relative imprecision, caused by difficulty in standardizing methylene blue, and interference caused by Howell Jolly bodies is possible. Bayer instruments use the optical absorption of an Oxazine-750 dye in the helium neon laser channel. On the Bayer system, reticulocyte measurement may be integrated into the distribution of RBC volume and Hb content. This permits direct measurements of reticulocyte staining intensity and provides direct measurement of reticulocyte cellular indexes. These newest parameters may be used to estimate reduction in red cell survival and may allow for improved monitoring of a variety of treatments for anemias (K1, K7). PLATELET COUNTING Table 19 summarizes current platelet counting approaches by different manufacturers (K1). Platelet counting using a single parameter such as impedance sizing with fixed thresholds to identify platelets has a significant tendency to overestimate the true platelet count in samples containing nonplatelet particles. Quantitation of platelets in flood samples is important in clinical management of thrombocytopenic patients and for the recognition of thrombocytopenia. In addition, being able to measure high levels of platelets is important for patients with elevated platelet counts and in the quality control of platelet products. A limitation of the current methods is the inability to discriminate platelets from interfering particles such as RC fragments and cellular debris. In addition, the ability to count large platelets is important, which is routinely excluded from platelet-counting algorithms. Manufacturers have addressed these concerns differently. Abbott provides both optical and impedance platelet-counting capability. If there is no concordance between the optical and impedance platelet count, the platelet count can be validated on Abbott instruments by a completely automated immunoplatelet-counting techniques using a monoclonal antibody to platelet glycoprotein IIIa. This glycoprotein is not on any other circulating blood cell. Sysmex uses an impedance method for platelet counting. The thresholds are not fixed, and the resultant values highly correlate with the reference method, but without a high incidence of 364R
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flagging as seen with particle counters, such as the BeckmanCoulter, that have a fixed threshold (K8, K9). Bayer has developed a two-dimensional platelet count in which both volume and refractive index are determined on a cell by cell basis. This permits a high level of discrimination among platelets, red cells, and interfering particles having similar sizes. This technology is extremely reliable and perhaps one of the most ingenious methods for platelet count. A variety of other platelet parameters on the Bayer instrument are possible including MPV platelet volume distribution width and platelet crit. The clinical utility of these parameters is still under investigation. WHITE CELL COUNTING AND DIFFERENTIAL ANALYSIS Table 20 summarizes different aspects of WBC counting and WBC differential analysis of cell types (K1, K10). WBC differentials have traditionally been done manually by microscopic review of the peripheral smear. The advantages of automated WBC differentials include the following: improved precision because of the larger number of cells countedsseveral thousand compared to a 100-cell manual countsand improved turnaround time because of the speed at which instruments can perform WBC cell typing compared to a technologist. Included as new parameters are nucleated red cells, immature granulocytes, and blasts. Although there is much interest in being able to identify bands, there is no technology available now in the foreseeable future. STEM CELLS Stem cells are the progenitor cells from which other blood cells develop. While their concentration is greatest in the bone marrow, they may be harvested from peripheral blood when modulated after cytotoxic chemotherapy and the administration of growth factors. When harvesting them by apheresis, it is necessary to be able to quantitate their presence in the peripheral blood. Normally this can be done by immunofluorescence using FITC labeled and CD 34 monoclonal. This testing is timeconsuming and not always readily available. Because stem cells contain less lipid and are resistant to lysis by detergents, stem
cells can be readily identified using impedance and radio frequency as developed by Sysmex. This testing requires only a few minutes compared to 2-3 h for flow cytometry. SUMMARY Recently, several hematology analyzers have been developed to improve accuracy and to reduce manual work. The new analyzers use traditional impedance technology or optical detectors alone or in combination. The introduction of argon laser technology to clinical instruments is one new development that may permit integrated flow cytometry parameters leading to a whole new horizon in routine hematologic analyses. Thomas S. Kickler, M.D., is the Director of Hematology and Coagulation Laboratory at Johns Hopkins Hospital, Baltimore, MD. He is a professor of pathology, medicine, and oncology is the School of Medicine. Doctor Kickler’s research is in platelet immunology and coagulation.
LITERATURE CITED (K1) Groner, W.; Simson E. Modern Hematology Analyzers; Wiley: West Sussex U.K.; 1995; pp 51-93. (K2) Karsan, A.; Maclaren, I.; Conn, D.; Wadsworth, L. Am. J. Clin. Pathol. 1993, 100, 123-126. (K3) Cantero, M.; Conegjo, J. R.; Jimenez, A. Clin. Chem. 1996, 42, 987-988. (K4) Savage, R. A. Clin. Lab. Med. 1993, 13, 773-785. (K5) Paterakis, G. S.; Aoutaris, N. P.; Alexia, S. V. Clin. Lab. Hematol. 1994, 16, 235-245. (K6) Corberand, J. Hematol. Cell Ther. 1996, 38, 487-94. (K7) Brugnara, C.; Zelmanovic, T.; Sorette, M.; Ballas, S. K.; Platt, O. Am. J. Clin. Pathol. 1997, 108, 133-142. (K8) Ault, K. Laboratory Hematol. 1996, 2, 139-143. (K9) Kickler, t. S.; Rothe, M.; Blosser, L.; Schisano, T.; van Hove, L. Laboratory Hematol. 1998, 4, 80-87. (K10) Thalhammer-Scherrer, R.; Knobl, P.; Korninger, L.; Schwarzinger, I. Arch Pathol Lab Med. 1997, 121-573-577. (K11) Takekawa, K.; Yamane, T.; Suzuki, K. Acta Hematol. 1997, 98, 54-55.
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