Monocyte counting: a comparison of manual and automated counting methods

Sysmex Xtra Online | April 2011

When comparing manual and automated cell differentiation, the following questions keep cropping up in respect of monocyte numbers: Why are the monocyte results obtained in manual differentiation lower than those from automated ones? Why are there different reference values for monocyte results obtained using manual and automated methods? The reasons for the different monocyte results can be found in the methods used, in statistical principles and in the adherence of monocytes.

Methods Manual differentiation The quality of smears prepared according to the Pappenheim or Wright methods is affected by many known factors, such as e.g. grease-free carrier slides, the quality of the spreader slide, the smear angle and staining. Moreover, monocytes especially are adherent cells. Thus, the glass surface of the carrier slide and the ground edge of the spreader slide provide excellent adhesion surfaces for this leukocyte subpopulation. As a result, the monocytes tend to accumulate more around the edge of the smear [1].
Since differentiation normally takes place at the central region of the smear, where predominantly smaller cells (e.g. lymphocytes) are present, the monocytes present near the edge are not included in the differentiation. This factor alone suffices to explain the often lower monocyte count when using this differentiation method.

Sysmex Xtra Online | April 2011 | 9 pages Monocyte counting: a comparison of manual and automated counting methods

X-CLASS technology: fluorescence flow cytometry

Fig. 1 DIFF scattergram, XS-series

Fig. 2 DIFF scattergram, XT- and XE-series

In the DIFF channel of X-class systems, four cell populations (neutrophils, eosinophils, lymphocytes and monocytes) are determined in the XE- and XT-series of instruments and five (four populations + basophils) in the XS-series. The measurement relies on a reagent system consisting of a combination of a lysis reagent and fluorescent dye, which is diluted with the blood. During this process, the first component, the lysis reagent (Stromatolyser-4DL), lyses all the erythrocytes and perforates the cell membranes of the leukocytes. The fluorescent dye (Stromatolyser-4DS) can then penetrate the cells and bind to the nucleic acids present in the nucleus and cytoplasm. This allows inferences to be drawn about cell activity and the maturity of the leukocytes. The cells remain largely intact during this process. Following the incubation period, the sample is analysed by means of flow cytometry, using the semiconductor laser. The cells fluorescence intensity and laterally scattered light are measured during their passage through the flow cell. The measured fluorescence intensity is proportional to the cells RNA/DNA content and provides information about cell maturity and activity. The intensity of the laterally scattered light depends on the cells granularity and on the size or lobularity of the nucleus, thus being a function of the cells internal structure. For additional cell analysis, these measured signals relating to each individual cell are recorded simultaneously and represented in scattergrams. Single points in the scattergrams represent the measured data of the relevant cells. These points can then be assembled or assigned to populations, and finally the number of cells in each population is calculated [2]. Monocyte adherence is unimportant in automated counting, since the cells are recorded individually as a result of the dilution and the subsequent analysis by flow cytometry. Given these methodical differences, the literature quotes the following reference ranges which are adjusted for the relevant method:

Sysmex Xtra Online | April 2011 | 9 pages Monocyte counting: a comparison of manual and automated counting methods

Monocyte reference values for manual differentiation

Source Female Monocytes Cell number/ L 80600 80540 Male Monocytes Cell number/ L 80600 80540 Female Monocytes % 28 26 Male Monocytes % 28 26 Total WBC number/L 4,0009,000 4,0009,000

F. Heckner [3] R. Mahlberg, A. Gilles, A. Lsch[4]

Table 1 Summary of reference study results for manual differentiation

Monocyte reference values for automated counting in X-class systems

Source Female Monocytes Cell number/ L 01000 250840 Male Monocytes Cell number/ L 01000 290950 Female Monocytes % 314 4.211.8 Male Monocytes % 314 5.215.2 Total WBC number/L 2,6007,800 3,90012,700

J. Pfaeffli [5] J. M. PekelHaring [6]

Table 2 Summary of reference study results for X-class differentiation

Statistics The number of differentiated cells also plays an important part in connection with the accuracy of the various methods. The effect of the number of counted cells on the reliability of the results is explained below, using an example. Manual differentiation: Usually, 100 leukocytes are evaluated in routine lab tests when manual differentiation is used (n = 100 cells). Automated differentiation: The number of cells actually differentiated is calculated according to the following formula, using the XE-2100 or XE-5000 as an example:
WBC 51 (dilution)

n=

x 40 L (counted volume)

Therefore, if for example a leukocyte value of 12,000/L is shown, in actual fact 9,421 cells are analysed through flow cytometry (n = 9,421).

Sysmex Xtra Online | April 2011 | 9 pages Monocyte counting: a comparison of manual and automated counting methods

The Rmke table [7] permits a statistical prediction to be made about the accuracy of counting parameters:
A 0 1 5 10 15 20 30 40 50 70 80 90 n=100 03.6 0.05.4 1.611.3 4.917.6 8.623.5 12.729.2 21.240.0 30.350.3 39.860.2 60.078.8 70.887.3 82.495.1 n=200 01.8 0.13.6 2.49.0 6.215.0 10.420.7 14.726.2 23.736.9 33.247.1 42.957.1 63.176.3 73.885.3 85.093.8 98.2100 n=500 00.7 0.32.3 3.37.3 7.513.0 12.018.4 16.623.8 26.034.2 35.744.4 45.554.5 65.874.0 76.283.4 87.0-92.5 99.3100 n=1,000 00.4 0.51.8 3.76.5 8.212.0 12.817.4 17.622.6 27.232.9 36.943.1 46.953.1 67.172.8 77.482.4 88.091.8 99.6100 n=10,000 00.1 0.81.3 4.55.5 9.410.7 14.315.8 19.220.8 29.131.0 39.041.0 49.051.0 69.070.9 79.280.8 89.390.6 99.9100

100 96.4100

Table 3 95% confidence limits for the actual of leukocytes. A = % leukocytes in a cell population.

With an assumed figure of 5% monocytes in a blood sample and a differentiation of n = 100 cells in the smear, this means that the counting result can vary between 1.6 and 11.3 cells. There is, therefore, a very wide underlying confidence interval here, because the number of differentiated cells is very low. In comparison, the XE-2100 differentiates almost 10,000 cells in the same sample (see calculation example above). With this number of differentiated cells, the result would fluctuate at most between 4.5 and 5.5 cells. In statistical terms, therefore, a larger number of cells leads to a more reliable result.

Studies Studies that compare the various methods with each other (manual differentiation, flow cytometry and/or automated differentiation using X-class instruments), show the following results:

Evaluation of the 5-Part Differential Leukocyte Count obtained with the Sysmex XE-2100 Automated Hematologic Cell Analyzer as compared to Flow Cytometry and Microscopy; L.I. Sanches Abarca, M.D. et al, Service of Cytometry Laboratory, University Hospital of Salamanca, Salamanca, Spain; ISLH XIVth International Symposium [8]

Sysmex Xtra Online | April 2011 | 9 pages Monocyte counting: a comparison of manual and automated counting methods

In this study, 50 normal blood counts in K3EDTA blood-drawing tubes were analysed within 4 hours of taking the sample, using 3 different methods: 1: 100 L of the relevant sample were mixed with monoclonal antibodies (HLADR-FITC/CD33PE/CD45PC/CD14APC) and analysed in a FACSCalibur. 2: Sample measurement in an XE-2100 3: Producing a smear, followed by May-Grnwald-Giemsa staining and examining a 400-cell differential under a microscope. Monocyte results in a comparison between FACSCalibur and XE-2100: r = 0.89; slope = 1.06; y-intercept = 1.30 Monocyte results in a comparison between 400-cell differential and XE-2100: r = 0.60; slope = 0.55; y-intercept = 5.1 As expected, the automated differentiation agrees more closely with the flow cytometry than with the manual method (400-cell differential).
Precision

and Accuracy of the Leukocyte Differential on the Sysmex XE-2100; R. Herklotz et al; Centre for Laboratory Medicine, Aarau Canton Hospital, Switzerland; Sysmex J Int 11: 821, 2001 [9] Two spun smears (Horizonter, Hettich) were prepared from each sample within 2 hours of sample collection and stained using the May-Grnwald-Giemsa method. These spun smears were counted using a 500-cell differential and compared with the XE-2100 result. Monocyte results, absolute: r = 0.8360; slope = 1.121; y-intercept = 0.009 Monocyte results, percentage: r = 0.8629; slope = 1.044; y-intercept = 0.610

Sysmex Xtra Online | April 2011 | 9 pages Monocyte counting: a comparison of manual and automated counting methods

Performance of the XE-2100 leucocyte differential; G. Stamminger et al., Clin Lab Haem 2002; 24: 271280 [10] In this study, the 194 measured patient samples were divided into 2 groups and differentiated according to the total leukocyte number: a) NCCLS group: n = 131; leukocytes > 4.0 x 109/L without morphological flags b) Leukopenia group: n = 63; leukocytes > 0.1 and < 4.0 x 109/L, also without morphological flags The absolute values from the XE-2100 were compared with the averaged results of four microscopically assessed smears per patient sample (200 cells per smear), converted into absolute values.

Analyte NCCLS group Neutrophil Gran. Lymphocyte Monocyte Eosinophils Basophils Leukopenia Neutrophil Gran. Lymphocyte Monocyte Eosinophils Basophils

Microscope (x 109/L) mean (range)

XE-2100 (x 109/L) mean (range)

5.30 (4.1124.42) 1.50 (0.13.94) 0.50 (0.011.67) 0.175 (07.31) 0.03 (00.16)

3.49 (0.9922.37) 1.36 (0.073.45) 0.40 (01.41) 0.17 (06.62) 0.03 (00.18)

0.9961 0.9374 0.9119 0.9984 0.6721

1.37 (03.32) 0.59 (01.51) 0.34 (00.93) 0.05 (00.58) 0.03 (00.24)

1.49 (0.013.45) 0.56 (0.021.56) 0.27 (00.85) 0.05 (00.54) 0.02 (00.15)

0.9931 0.9745 0.9370 0.9570 0.5439

Table 4 Study results: Performance of the XE-2100 leucocyte differential; G. Stamminger et al., Clin Lab Haem 2002; 24: 271280 [10]

Sysmex Xtra Online | April 2011 | 9 pages Monocyte counting: a comparison of manual and automated counting methods

In summary, one can see that the results of the cited studies demonstrate precise results from automated differentiation both in comparison with flow cytometry and with the manual method, once at least 400 cells have been differentiated. This comparison between the methods, and also the Rmke table, permit the conclusion that automated differentiaFig. 3 Digital morphology analytical system: tion should be the method of choice with CellaVision DM1200 regard to determining cell numbers. However, in the case of a pathological automated differentiation with warnings such as e.g. Blasts?, assessment of cell morphology should be based on a manual smear. Nowadays, this microscopy work can be facilitated considerably with the help of automated image analysing systems such as e.g. CellaVision DM96 or DM1200.

Automated differentiation The CellaVision DM96 (throughput ca. 35 smears/h) and the DM1200 (throughput ca. 20 smears/h) are automated image analysing systems, which pre-classify the blood cells by using a high-resolution camera in conjunction with a high-quality microscope and advanced image processing technology (artificial neuronal network technology based on an extensive cell database). Both systems locate nucleus-containing cells automatically in a stained blood smear. Images of cells are recorded, analysed, preclassified and categorised into various cell types (with an accuracy of up to 95%)[11].

Pre-classification for mature leukocytes: lymphocytes, monocytes, neutrophils, eosinophils, basophils Pre-classification for immature leukocytes: bands, metamyelocytes, myelocytes, promyelocytes, blasts Pre-classification for non-leukocytes: NRBC, giant platelets and other non-classifiable cells

The results and the cell images from this automated analysis of the smear are displayed on the screen and evaluated by the technician. Reclassification can be performed if necessary. Evaluation of erythrocyte morphology (also pre-assessed automatically by the systems) is followed by validation of the complete differential blood count, with subsequent transfer to the LIS (including all comments).

Sysmex Xtra Online | April 2011 | 9 pages Monocyte counting: a comparison of manual and automated counting methods

Fig. 4 Cell images produced by CellaVision DM1200

Sysmex Xtra Online | April 2011 | 9 pages Monocyte counting: a comparison of manual and automated counting methods

Literature
[1] The progresses in the determination of blood monocytosis and its perspectives of clinical application in pathology; G. Flandrin et al.; Ann Biol Clin (Paris) 1993; 51: 78791 [Article in French] [2] Fluorescence flow cytometry in haematology; Sysmex Xtra Online Vol. 0, 2005 [3] Practical Microscopic Hematology: F. Heckner; Lea & Febiger, USA, 1994 [4] Hmatologie; R. Mahlberg, A. Gilles, A. Lsch; 2. vollstndig berarbeitete und erweiterte Auflage 2005, Wiley-VCH [5] Reference Limits for the Automated Haematology Analyser Sysmex XE-2100; J. Pfaeffli; Sysmex J Int 12: 1823, 2002 [6] Haematology reference intervals for established and novel parameters in healthy adults; J.M. Pekelharing et al., Diagnostic Perspectives 2010; 1: 0111 [available online] [7] The statistically expected variability in differential counting, Ruemke, C. L.; in: Koepke, J. A., Differential leukocyte counting. College of American Pathologists, Sokie, Illinois, 1979 [8] Evaluation of the 5-Part Differential Leukocyte Count Obtained with the Sysmex XE-2100 Automated Hematologic Cell Analyzer as Compared to Flow Cytometry and Microscopy; L.I. Sanches Abarca et al.; Lab Hematol 7;44 (Abstracts by ISLH, Montpellier) Poster Abstracts No. 20, 2001 [9] Precision and Accuracy of the Leukocyte Differential on the Sysmex XE-2100; R. Herklotz and A.R. Huber; Zentrum fr Labormedizin, Kantonsspital Aarau; Sysmex J Int 11: 821, 2001 [10] Performance of the XE-2100 leucocyte differential; G. Stamminger et al.; Clin Lab Haem 2002; 24: 271280 [11] Examination of peripheral blood films using automated microscopy; evaluation of diffmaster Octavia and CellaVision DM96; H. Ceelie et al., Journal of Clinical Pathology 2007; 60: 7279

CellaVision DM96 and DM1200 are made by CellaVision AB, Lund, Sweden.

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