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ISSN : 1225-1577(Print)
ISSN : 2384-0900(Online)
The Korean Journal of Oral and Maxillofacial Pathology Vol.44 No.4 pp.113-120
DOI : https://doi.org/10.17779/KAOMP.2020.44.4.002

Mixed Cell Analysis of Blood Samples Using the Sysmex K-4500 Hematology Analyzer

Eun Ju Oh1), Young Ki Lee2), Ga-Yeon Kim3)*
1)Department of Medical Laser Cooperative Curriculum, Dankook University Graduate School of Medicine, Korea
2)Department of Biomedical Laboratory Science, Dankook University College of Health Sciences, Korea
3)Department of Dental Hygiene, Dankook University College of Health Sciences, Korea
†

Contributed equally to this work


*Correspondence: Ga-Yeon Kim, Department of Dental Hygiene, Dankook University College of Health Sciences, Dandae-ro, Dongnam-gu, Cheonan 3116, Korea Tel: +82-41-550-1493, Fax: +82-41-550-1490 E-mail:
sysnhj77@gmail.com

May 30, 2020 June 5, 2020 August 7, 2020

Abstract


General blood tests and leukocyte differential count are very useful in diagnosing, treating and managing patient’s diseases. This study was conducted to reduce errors caused by the manual method as much as possible by using an automated hematology analyzer (Sysmex K-4500), and to alleviate work burden of the laboratory. The T1 and T2 values calculated from leukocyte differential count that was obtained from the automated hematology analyzer Sysmex K-4500, and the results measured from the manual method were compared and analyzed by using the blood samples of 400 patients who were commissioned in the diagnostic laboratory of the hospital with 120 beds. Upon the results of the study, T1 and T2 obtained from lymphocyte differential count of the automated hematology analyzer Sysmex K-4500 demonstrated high correlation compared to manual method, and the analysis of monocyte, eosinophil, basophil and band cell obtained from T1, T2 and manual method enabled percentile calculation, even though it was difficult to predict the exact monocyte, eosinophil, basophil and band cell. Therefore, it is regarded that the optimal patient results can be reported if manual method observation is selectively combined while referring to T1 and T2 obtained from lymphocyte differential count of the automated hematology analyzer Sysmex K-4500.


Automated hematology analyzer , Mixed cell , Lymphocyte , Neutrophil

Sysmex K-4500 혈액 분석기를 이용한 혈액 검체의 Mixed cell 분석

오 은주1), 이 영기2), 김 가연3)*
1)단국대학교대학원 의학레이져 협동과정
2)단국대학교 보건복지대학 임상병리학과
3)단국대학교 보건복지대학 치위생학과

초록

    Ⅰ. INTRODUCTION

    The lymphocyte differential count is one of the basic and essential test items in the hematologic assessment of the healthy person and the patient, and has been used to calculate 100 leukocyte with naked eyes 1-5). These manual method is best to directly identify abnormal cells, but there are disadvantages such as time consuming, additional cost, and need to be carried out by skilled professionals, which puts a heavy burden on the laboratory6). Therefore, since it takes a lot of time and manpower to examine all samples with the manual method in the hospitals with large sample numbers, most hospitals are conducting lymphocyte differential count by using the automated hematology analyzer. A varie- ty of lymphocyte differential count methods have been developed: Sysmex SE-9000 (TOA Medical Electronics Co., Japan), which differentiates leukocyte by using high frequency and direct current method and displays flag to recommend confirmation with the manual method if presence of abnormal leukocyte is suspected7-9) Coulter STKS (Coulter Co., USA), which differentiates 5 types of leukocyte by measuring volume, conductivity and light scattering using low frequency direct current, high frequency electromagnetic wave, and laser ray while maintaining the original form of leukocyte; and other methods 10-13).

    To date, clinical trials to objectively evaluate patient conditions for dental treatment are very rare, but blood tests can be optionally conducted in patients with high probability of systemic complications before and after treatment to identify systemic diseases related to bleeding and infection and to more objectively check patient conditions14). By conducting a basic blood test, the CBC test, the possibility of complications related to treatment can be reduced through a close understanding of the patient's condition. This study is an evaluation of small CBC automatic blood cell analyzer, and we hope to help with more effective and safe dental treatment through this researcher. Also, While the use of an automated hematology analyzer has advantages of reducing the burden of time and manpower compared to the manual method, having improved accuracy as lymphocyte differential count is performed by calculating much more number of cells, and allowing faster process of many samples, it is irrational to report the leukocyte differential count results of the automated hematology analyzer for all samples as it lacks the ability to detect immature granulocyte or abnormal cells and has false-negative and false-positive. Although it is most ideal to confirm with the manual method for all samples, it can imply a section of the graph of the automated hematology analyzer, namely LD is the platelet size when a predetermined small size is as a frequency standard, UD is the platelet size when a predetermined large number is set as the frequency standard( Sysmex Corporation (Hyogo, JP). T1 (one point in mixed cell graph among leukocyte graph obtained from Sysmex K-4500) and T2 (one point in mixed cell graph among leukocyte graph obtained from Sysmex K-4500). This study aims to assist in calculating mixed cell without using the manual method by identifying the location of T1 and T2 and comparing this result with the result obtained with the manual method. Furthermore, to allow direct reporting of the results of the automated hematology analyzer, this study was conducted to identify the usefulness of T1 and T2, to reduce the external factors such as errors due to manual method, and to reduce the work burden of the laboratory.

    Ⅱ. MATERIALS AND METHODS

    1. Collection of Samples

    The Institutional Review Board Deliberations of Dankook University approved this study (IRB No. DKU 2020-08-005) and does not use the subject's personally identifiable information.

    Among the inpatients and outpatients commissioned for blood count and leukocyte differential count from April 2005 to August 2005 in the diagnostic laboratory of the hospital with 120 beds, Okcheon St. Mary's Hospital, located in Okcheon-gun, Chungcheongbuk-do, the subjects of this study were a total of 400 patient samples, including 200 samples within the normal reference range (hemoglobin of 10.0~17.0g/㎗, leukocyte count of 4,000~10,000/㎕ and platelet count of 100,000 ~ 600,000/㎕) and 200 samples outside the normal reference range. The study was conducted in 2005. The samples were collected in EDTA 2K bottle, stored in room temperature, and conducted blood count and leukocyte differential count using automated hematology analyzer (Sysmex K-4500; Sysmex, Japan) within 6 hours from the collection.

    2. Manual method using microscopic analysis

    While the results were obtained from blood count and leukocyte differential count using an automated hematology analyzer (Sysmex K-4500 ; Sysmex, Japan), 100 leukocytes were differentially calculated with a microscope of 400 magnification after preparing a peripheral smear and staining with wright-giemsa dye to calculate leukocyte differential count using the manual method. For the use of the result analysis, the calculated metamyelocyte, myelocyte, promyelocyte and myeloblast were included in neutrophil, and atypical lymphocyte and lymphocyte were included in lymphocyte. The observation errors between individuals were prevented by allowing one person to prepare all sample slides and interpret the manual method 15).

    3. Analysis Using the K-4500

    An automated hematology analyzer Sysmex K-4500 analyzes the particle sizes of leukocyte, erythrocyte and platelet, calculates the results, and produces the graphs for each leukocyte, erythrocyte and platelet. The leukocyte graph is calculated by classifying into 3 sections: neutrophil, lymphocyte and mixed cell for the rest of the cells. On this graph, the T1 and T2 (Fig. 1) indicate mixed cell (Fig. 2). The location of this two lines were divided into when T1 is higher and when T2 is higher, and compared with eosinophil, basophil, band cell and monocyte, which are mixed cell calculated from the manual method.

    4. Statistical Analysis

    The paired t-test was used to verify whether there is any difference in the automated leukocyte differential count using Sysmex K-4500 and the manual leukocyte differential count using manual method, and One-Way Anova test and Chi-Square test were used to compare and analyze T1, T2 and mixed cell. The correlation between the mixed cell percentile of automated hematology analyzer in the normal and abnormal samples and the graph results was compared and analyzed using One-Way Anova test.

    Ⅲ. RESULTS

    1. Comparison of leukocyte differential count using automated hematology analyzer and manual method in normal and abnormal blood samples

    For the normal range samples, the mean neutrophil value was 57.21 ± 13.5 in Sysmex K-4500 and 57.43 ± 13.62 in manual method, the mean lymphocyte value was 33.85 ± 12.71 in Sysmex K-4500 and 33.96 ± 12.5 in manual method, the mean mixed cell value was 8.58 ± 4.88 in Sysmex K-4500 and 8.56 ± 4.885 in manual method, demonstrating no significant difference between Sysmex K-4500 and manual method. For the abnormal range samples, the mean neutrophil value was 70.80 ± 17.24 in Sysmex K-4500 and 70.82 ± 17.01 in manual method, the mean lymphocyte value was 20.92 ± 14.21 in Sysmex K-4500 and 21.24 ± 14.4 in manual method, the mean mixed cell value was 7.79 ± 5.51 in Sysmex K-4500 and 7.78 ± 5.51 in manual method, demonstrating no significant difference between Sysmex K-4500 and manual method (Table 1).

    2. Comparison of automated hematology analyzer results from T1 and T2 values in normal and abnormal blood samples

    The mean mixed cell value of the 200 normal range samples was 8.56%, in which 4.76% was monocyte mean value, 3.43% was eosinophil mean value, 0.30% was basophil mean value, and 0.13% was band cell. The mean mixed cell value of the 200 abnormal range samples was 7.78%, in which 4.80% was monocyte mean value, 2.65% was eosinophil mean value, 0.10% was basophil mean value, and 0.25% was band cell.

    Upon the analysis results, the distribution in T1＜T2 group of the normal range samples was in the order of monocyte (5.29%), eosinophil (4.43%), basophil (0.39%), band cell (0.22%), while the distribution in T1＞T2 group of the abnormal range samples was in the order of monocyte (5.37%) and eosinophil (3.50%). The basophil demonstrated more high distribution in T1＞T2 group with 0.16% and T1＜T2 with 0.36% (Table 2).

    3. Frequency of T1 and T2 in normal and abnormal blood samples

    For the normal range samples, 28.5% was T1＞T2, 24.5% was T1＜T2, and 47.0% was T1 = T2. For the abnormal range samples, 19.0% was T1＞T2, 36.5% was T1＜T2, and 44.5% was T1 = T2. The distribution was highest in T1 = T2 group for both normal range blood samples and abnormal range blood samples. With a x² value of 8.658 and a statistical significance of 0.013, there was a significant association between T1 and T2 of normal and abnormal range (Table 3).

    4. Correlation of leukocyte count between automated hematology analyzer and manual method in normal and abnormal blood samples

    Upon the correlation analysis results, all corelation coefficients of leukocyte differential count from Sysmex K-4500 and manual method in normal and abnormal samples were above 0.9 for all neutrophil, lymphocyte and mixed cell, showing high correlation. Among this, mixed cell demon-strated the highest correlation with a correlation coefficient of 0.999 (Table 4).

    5. Analysis result of monocyte, eosinophil, basophil and band cell by comparing the correlation of mixed cell percentile value and graph result in the automated hematology analyzer in normal blood samples

    For the group 1, which was classified based on mixed cell percentile and graph T value of the automated hematology analyzer, the mean value of mixed cell from automated hematology analyzer was 3.33%, and this can be converted into percentile of 95% monocyte and 5% eosinophil. For the group 2, the mean value of mixed cell from automated hematology analyzer was 3.30%, and this can be converted into percentile of 79% monocyte, 12% eosinophil, 7% basophil and 2% band cell.

    The below Table 5 indicates the analysis results of monocyte, eosinophil, basophil and band cell by comparing the correlation between mixed cell percentile value and graph result of the automated hematology analyzer in normal blood samples of each 2~12 groups. The groups 1~8 and group 12 had the highest monocyte percentile, the groups 9~11 had the highest eosinophil percentile. The percentiles given for each group can be referred when calculating mixed cell values using an automated hematology analyzer Sysmex K-4500 (Table 5).

    6. Analysis result of monocyte, eosinophil, basophil and band cell by comparing the correlation of mixed cell percentile value and graph result in the automated hematology analyzer in abnormal blood samples

    The mean value of mixed cell from automated hematology analyzer for the group 1 was 3.0%, which can be converted into percentile of 90% monocyte, 5% eosinophil, 0% basophil and 5% band cell. For the group 2, the mean value of mixed cell from automated hematology analyzer was 2.96%, and this can be converted into percentile of 92% monocyte, 4% eosinophil, 0% basophil and 4% band cell.

    The below Table 6 indicates the analysis results of monocyte, eosinophil, basophil and band cell by comparing the correlation between mixed cell percentile value and graph result of the automated hematology analyzer in abnormal blood samples of each 2~12 groups. The groups 1~6, groups 8~10, and group 12 had the highest monocyte percentile, the group 7 had the same percentile of monocyte and eosinophil, and the group 11 had the highest eosinophil percentile. The percentiles given for each group can be referred when calculating mixed cell values using an automated hematology analyzer Sysmex K-4500 (Table 6).

    Ⅳ. DISCUSSION

    The Evaluation of Instrumental Methods; Approved Standard16,17) of 800 cells, presented by the National Committee for Clinical Laboratory Standards of U.S. is accurate in identifying the morphological abnormalities, but has time, manpower and economic limitations in efficient process of the rapidly growing number of requests in large-scale hospitals. An automated hematology analyzer, which can supplement the problems of the manual method and allows 3 types (granulocyte, lymphocyte, monocyte) of leukocyte differential count, was developed in the 1980s, and since the mid-1980s the analyzer was developed to allow 5 types (neutrophil, lymphocyte, monocyte, eosinophil, basophil) of leukocyte differential count.

    While the difference in lymphocyte differential count in the automated hematology analyzer and in the manual method is variously reported depending on the model and the researchers18-20), this study aims to compare and analyze the T1 and T2 graph of leukocyte differential count obtained from an automated hematology analyzer Sysmex K-4500 and the results observed from a manual method, to identify the differences in significance between the value measured from an automated hematology analyzer and the results observed from a manual method, and to determine the clinical usefulness of the use of T1 and T2. In comparison with the manual method, the leukocyte differential count obtained from an automated hematology analyzer Sysmex K-4500 was the highest in mixed cell with a correlation coefficient of 0.999, and mostly demonstrated high correlation, implying that there would be no problem in clinical comparison and analysis.

    When studying the correlation between the leukocyte differential count measured by an automated hematology analyzer Sysmex K-4500 and a manual method, the differential ability for netrophil can be regarded as very remarkable, considering that neutrophil demonstrated high correlation coefficient of 0.973, and neutrophil measured by other models (coulter MAXM, Bayer H 6000, H3) also demonstrated correlation higher than 0.9018). Also in the case of lymphocyte, Sysmex K-4500 demonstrated a correlation coefficient of 0.972 and most literature references reported correlation higher than 0.9819,21) or demonstrated correlation, exhibiting very remarkable differential ability. Mixed cell showed a correlation coefficient of 0.999, demonstrating a higher correlation coefficient than neutrophil or lymphocyte. Therefore, since neutrophil, lymphocyte and mixed cell demonstrated high correlation coefficient in an automated hematology analyzer Sysmex K-4500 and a manual method, the results of Sysmex K-4500 and the manual method can be trusted. Moreover, it can be regarded that Sysmex K-4500 calculates relatively accurate results as the general manual method observes 100 leukocytes but Sysmex K-450 measures 8,000 leukocytes and produces statistics.

    As a result of analyzing the result value of mixed cell according to T1 and T2 of an automated hematology analyzer and the distribution of monocyte, eosinophil, basophil and band cell in normal samples and abnormal samples, monocyte, eosinophil, basophil, band cell had high distribution in T1 < T2 group of the normal samples, monocyte and eosinophil had high distribution in T1 > T2 group of the abnormal samples, basophil had high distribution in T1 = T2, and band cell had high distribution in T1 < T2. Even though it is difficult to predict the exact monocyte, eosinophil, basophil, band cell using T1 and T2 by analyzing T1 and T2 obtained from an automated hematology analyzer Sysmex K-4500 and monocyte, eosinophil, basophil, band cell obtained from the manual method, percentile can be derived. Based on these results, it is regarded that the optimal patient results can be reported if manual method observation is selectively combined while referring to T1 and T2 obtained from lymphocyte differential count of the automated hematology analyzer Sysmex K-4500. It is also believed that the government will have to establish the standards suitable for the circumstances to accumulate a lot of experience and reduce the work of the laboratory to the maximum.

    Figure

    KAOMP-44-4-113_F1.gif

    Graph according to WBC size distribution in Sysmex k-4500 (The unit of WBC on the x-axis is ‘/㎕’)

    Table

    Comparison of Leukocyte differential count between Sysmex K-4500 and manual method in normal and abnormal blood sample

    Comparison of mean values between Sysmex K-4500 and manual method from results of T1, T2 in normal and abnormal blood sample

    Unit: %

    Frequency of T1, T2 in normal blood sample and abnormal blood sample

    Unit: case (%)

    Correlation coefficient of Leukocyte differential count between Sysmex K-4500 and manual method in normal and abnormal blood sample

    **P＜0.001

    Mean of group 1~12 between mixed cell in normal blood specimen

    Unit: Mean (%)

    Mean of group 1~12 between mixed cell in abnormal blood sample

    Unit: Mean (%)

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