Mononuclear cell evaluation: A correlation study between manual and analyzer-based estimation

Rehan Ahmed; Prosenjit Ganguli; UD Gupta; YK Jaiswal; Velu Nair; Satyaranjan Das; Ajay Sharma; Sana Khan; Rekha Singh Ganguli

doi:10.4103/bbrj.bbrj_133_19

ORIGINAL ARTICLE

Year : 2020 | Volume : 4 | Issue : 1 | Page : 34-40

Mononuclear cell evaluation: A correlation study between manual and analyzer-based estimation

Rehan Ahmed¹, Prosenjit Ganguli², UD Gupta³, YK Jaiswal⁴, Velu Nair⁵, Satyaranjan Das⁶, Ajay Sharma⁶, Sana Khan⁷, Rekha Singh Ganguli⁸
¹ Department of Clinical Haematology and Centre for Stem Cell Therapy and Research, Army Hospital (Research and Referral), New Delhi, India
² Department of Pathology, Command Hospital (EC), Kolkata, West Bengal, India
³ Former Director, National Jalma Institute of Leprosy and Other Mycobacterial Diseases-ICMR, Agra, Uttar Pradesh, India
⁴ Department of Biochemistry, Faculty of School of Studies, Biochemistry, Jiwaji University, Gwalior, Madhya Pradesh, India
⁵ Former DGMS Army (Director General Medical Sciences-Army), Gurgaon, Haryana, India
⁶ Military Hospital, Allahabad, Uttar Pradesh, India
⁷ Department of Mathematics, Aligarh Muslim University, Aligarh, Uttar Pradesh, India
⁸ Ex-DGMS-Army (Director General Medical Services), Bhagirathi Neotia Woman and Child Care Centre, Kolkata, West Bengal, India

Date of Submission	25-Sep-2019
Date of Acceptance	18-Nov-2019
Date of Web Publication	17-Mar-2020

Correspondence Address:
Dr. Prosenjit Ganguli
Department of Pathology, Command Hospital (EC), Kolkata, West Bengal
India

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/bbrj.bbrj_133_19

Abstract

Background: Bone marrow transplantation (BMT) is being routinely done at the Army Hospital for the past 15 years. Our experience with Stem Cell Transplantation and clinical trial on stem cell efficacy, our Centre had brought out the issue of trial deviates (<2 × 10⁸ mononuclear cells [MNCs]) based on low yield of MNCs. PubMed search did not reveal any comparative data of analyzers versus manual counts. Thus, we decided to investigate the cause for the discrepancy calculated on 3-part and 5-part analyzers and manual estimation. This study has institutional relevance for hospitals involved in hematopoietic stem cell transplantation, therapy, and research. Introduction: MNCs are widely being used as a morphologic analog of precursor cells in hematopoietic stem cell transplantation and BMT units. The aim of the present study was, therefore, to evaluate the validity of the automated blood cell counter with gold standard of a manual count. Methods: The samples of peripheral blood (n = 80) and bone marrow (n = 80) were included in the study. Informed consent was obtained, and approval was taken from the IEC (Institutional Ethics Committee), Army Hospital Research and Referral, New Delhi, India. Results: Evaluation of MNC with 5-part hematology analyzer and gold standard manual count shows the correlation in the group of total leukocyte count samples: <4000, 4000–12,000, and 12,000–20,000 with no significant difference and >20,000 showing significant difference. Conclusion: The good correlation of 5-part hematology analyzer with manual gold standard method of MNC count is indicating equivalent performance. MNC can be performed by 5-part with nonsignificant difference and can be used as good as manual count.

Keywords: Bone marrow transplantation, hematology analyzer, lymphocyte, mononuclear cells, monocytes

How to cite this article:
Ahmed R, Ganguli P, Gupta U D, Jaiswal Y K, Nair V, Das S, Sharma A, Khan S, Ganguli RS. Mononuclear cell evaluation: A correlation study between manual and analyzer-based estimation. Biomed Biotechnol Res J 2020;4:34-40

How to cite this URL:
Ahmed R, Ganguli P, Gupta U D, Jaiswal Y K, Nair V, Das S, Sharma A, Khan S, Ganguli RS. Mononuclear cell evaluation: A correlation study between manual and analyzer-based estimation. Biomed Biotechnol Res J [serial online] 2020 [cited 2021 Sep 19];4:34-40. Available from: https://www.bmbtrj.org/text.asp?2020/4/1/34/280858

Introduction

Mononuclear cells are the white blood cells (WBCs) with one lobed nucleus (may be folded). There are predominantly two types of mononuclear cells, namely monocytes and lymphocytes. Monocytes are phagocytic cells having single nucleus, partially lobulated, deeply indented.^[1],[2] In the human hematopoietic system, the development of mononuclear cells is termed as monocytopoiesis.^[3] In addition, the precursor cells, which are termed as promonocytes, constitute 3% of total cells in the bone marrow.^[1],[4],[5] Differentiation occurs rapidly with morphological maturation by progressive lobulation of the nucleus.^[6] In promonocytes, the nucleus is less lobulated, chromatin finer, and the cytoplasm not as gray as in monocytes.^[7]

Lymphocytes are mononuclear cells without specific cytoplasmic granules. “These are the workhorses of the immune system” (Sutherland, 2000)^[8] and are responsible for both humoral and cellular immunity. These cells vary in size and shape in normal peripheral blood and bone marrow. They can be arbitrarily divided into small and large lymphocytes. Functional and immunological subsets cannot be reliably distinguished by morphology; however, small lymphocytes contain regular nuclear contours with condensed chromatin. The “mature-appearing” nucleus is slightly larger than the size of a normocytic red blood cell (RBC) and is surrounded by a thin rim of cytoplasm. In children, normal small lymphocytes may have nuclear clefts. Large granular lymphocytes normally comprise 10%–20% of the total lymphocyte [Supplementary Table 1] which has pale blue moderate to abundant cytoplasm, less condensed nuclear chromatin than small lymphocytes, small indistinct nucleoli, and round to slightly irregular nuclear contours.^[9]

Routine laboratory evaluations of mononuclear cells require the use of cell counter. They may be done using 3-part or 5-part cell counter. Cells diluted in a conducting solution are counted and their volume is determined by measuring the change in electrical resistance as they flow through a narrow aperture and interrupt a direct electrical current. Software analysis defines RBCs, WBCs, and platelets based on volume limits. Manual total WBC count is routinely performed using the traditional hematological Neubauer chamber method with bright field microscopy. A Nageotte chamber count can be done additionally for <2000/cumm.

The Sysmex KX21 (3 part, Sysmex Corporation, Japan) and COULTER HmX Hematology Analyzer (5 Part, Beckman Coulter, USA) provide size distribution (cell volume in fL) versus relative number or frequency of cells for WBCs, RBCs, and platelets. A Sysmex/Coulter proprietary computerized algorithm is used to classify the cells into three or five categories. Both percentage and absolute counts are determined. The percentage for each cell type is multiplied by the total leukocyte count (TLC) to obtain absolute number of cells. Particles between 35 and 90 fL are considered lymphocytes [Supplementary Table 2], those between 90 and 160 fL are “mononuclear” (normally primarily monocytes), and those from 160 to 450 fL are granulocytes.^[11] Sysmex KX21 gives the monocytes within the mixed population instead of differential count.

Mononuclear cells (MNCs) are widely being used as a morphologic analog of precursor cells in hematopoietic stem cell transplantation (HSCT) and bone marrow transplantation (BMT) units to decide the cell dose to treat all hematological malignancies globally. In a trial setting wherein HSCTs are used in modified or novel roles, they are used to estimate the dose/yield of stem cells. An automated cell counter, as a quantitative method, if estimating the true MNC value would be useful in calculating MNC cell dose during BMT. The aim of the present study was therefore to evaluate the validity of the automated blood cell counter with gold standard of a manual count.

Methods

Patients

The samples of peripheral blood (n = 80) were included in the study. Samples of bone marrow (n = 80) were obtained from patients who were evaluated as part of hematology workup. Informed consent was obtained according to the Declaration of Helsinki. Approval was taken from the IEC (Institutional Ethical Committee), Army Hospital Research and Referral (R and R), New Delhi, India. TLC was performed for each of the sample. Four groups of twenty samples were designed with TLC ranging from <4000, 4000–12,000, 12,000–20,000, and >20,000 in each variable. TLC was routinely encountered in transplant setting, i.e., <4000, 4000–12,000, 12,000–20,000, and >20,000 of different groups. All the procedures were carried in accordance with the institutional guidelines. The samples were included only if the patient's vital parameters were in the normal physiological range.

Mononuclear cell obtaining from peripheral blood

Peripheral venous blood was drawn from patients of variable TLC (<4000, 4000–12,000, 12,000–20,000, and >20,000). Blood samples were collected in 2 mL ethylene diamine tetra-acetic acid anticoagulation tubes BD/Kriesen (5.4 mg/spray dried). The tubes were then gently inverted to mix the content (8–10 times) and analyzed on a 3-part (Sysmex KX21, Japan) and from 5-part (Beckman Coulter HMX, California) analyzer which require sample volume of 375 μL. Blood films were made within twenty 4 h of received in the laboratory. Blood films were prepared using a Wright–Giemsa stain. The relative numbers of the different types of mononuclear cells present were counted by three independent observers and the results were averaged. At least hundred cells were counted on each slide by each observer with randomized selected fields. All slides were picked randomly from any of four groups and counted differentially by observers. The 5-Part HMX analyzer differentials were correlated against the average of these two differentials done manually. In the event of a discrepancy between the differentials of the two trained observers, a third blinded hundred cells differential was used for comparison found by the observers and not flagged by the analyzer and as differences between the two observers in quantitation of results or qualitative detection of abnormal leukocyte population.

Mononuclear cells obtaining from bone marrow

Samples were drawn from the iliac crest. Bone marrows were drawn following aseptic precaution and standard procedure. The slides were dried for 1 h. Fixed for 30 min in methanol and then dried again. Subsequently, stained slides were examined by three independent observers. Fourth observer obtained the automated hematology analyzer counts from a BM sample drain during the procedure simultaneously.

Statistical analysis

Statistical analysis was performed using SPSS software package (Statistical Package for the Social Sciences version 20.0, Chicago, IL, USA). Hematological parameters were compared between three groups of different TLC range using ANNOVA and independent samples t-test. The level of significance for all analyses was set at 0.05.

Results

Correlation of 3-part versus manual count

In this study, morphological identification of MNCs [Figure 1] was performed with correlation of hematology analyzer for different ranges of TLC group. We correlated mean value for lymphocytes in 3-part and manual using ANOVA. No interdependence of mean value for monocytes and mononuclear cell (monocytes and lymphocytes) were seen in 3-part, because differential count for mixed population cannot be determined in 3-part and was excluded from the study. Hence, only lymphocytes were correlated [Figure 2] and [Table 1].

Figure 1: Morphological analysis of mononuclear cell. (a) Unidentified mononuclear cell in × 5. (b) Unidentified mononuclear cell in × 10. (c) Identified mononuclear cell with certainty in × 40. (d) Identified mononuclear cell confirmatory in × 100

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Figure 2: All the plots (a-d) represent the comparison of mean between three groups; Gp1 = Sysmex KX21 (3 Part), Gp2 = HMX Coulter (5 Part) and Gp3 Manual for Lymphocyte in different ranges of total leukocyte count.(a) Compare mean of lymphocytes for Gp1, Gp2, and Gp3 in total leukocyte count range of <4000. (b) Compare mean of lymphocytes for Gp1, Gp2, and Gp3 in total leukocyte count range of 4000–12,000.(c) Compare meanof lymphocytes for Gp1, Gp2, and Gp3 in total leukocyte count range of 12,000–20,000. (d) Compare mean of lymphocytes for Gp1, Gp2, and Gp3 in total leukocyte count range of >20,000

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Table 1: Lymphocyte count in various range of TLC with 3 part haematology analyser (n=20)

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In <4000, the mean value for lymph in 3-part and manual shows significant difference.

(P = 0.250 and mean difference = −11.2).

In TLC 4000–12,000, the mean value of lymph in 3-part and manual shows less significant difference.

(P = 0.76, mean difference between = −2.91).

In TLC range 12,000–20,000, the mean value for lymph in 3-part and manual shows significant difference (P = 0.187, mean difference = −7.35).

In TLC range >20,000, the mean value for lymph in 3-part and manual shows significant difference, and the mean value for both was less than the normal range (P = 0.776, mean difference = −3.40).

Correlation of 5-part versus manual count

The average differences in mean and standard deviation (SD) are listed [Table 2], [Table 3], [Table 4]. The correlation between 5 Part haematology analyser and manual observation were performed [Figure 3].

Table 2: Lymphocyte, monocyte and MNCs count in various range of TLC with 5 part haematology analyser

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Table 3: Lymphocyte, monocyte and MNCs count in various range of TLC with manual count

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Table 4: Monocyte count between five part haematology analyser and manual count in various range of TLC

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Figure 3: Correlation of bone marrow-mononuclear cell between 5-part and manual counts

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In TLC <4000 range, the mean value for lymph in 5-part and manual count [Figure 4] shows less significant difference (mean difference of 3.85, P = 0.250). Mononuclear cell count (monocytes and lymphocytes) for 5-part was found to be 50.06%, while for manual count was 49.10%, reflects no significant difference (P = 0.072) [Figure 5].

Figure 4: All the plots (a-d) represent correlation analysis between HMX Coulter (5 Part) and manual for lymphocyte in different ranges of total leukocyte count. (a) Correlation of lymphocytes between 5-part and manual count in total leukocyte count range of <4000. (b) Correlation of lymphocytes between 5-part and manual count in total leukocyte count range of 4000–12,000. (c) Correlation of lymphocytes between 5-part and manual count in total leukocyte count range of 12,000–20,000.(d) Correlation of lymphocytes between 5-part and manual count in total leukocyte count range of >20,000

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Figure 5: All the plots (a-d) symbolize correlation analysis between HMX Coulter (5 Part) and manual for mononuclear cell in different ranges of total leukocyte count. (a) Correlation of mononuclear cell between 5-part and manual count in total leukocyte count range of <4000. (b) Correlation of mononuclear cell between 5-part and manual count in total leukocyte count range of 4000–12,000. (c) Correlation of mononuclear cell between 5-part and manual count in total leukocyte count range of 12,000–20,000. (d) Correlation of mono nuclear cell between 5-part and manual count in total leukocyte count range of >20,000

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In TLC range 4000–12,000 [Table 5], the mean value for lymph in 5-part and manual count shows less significant difference (mean difference = −4.3, P = 0.551. The mean value for MNC for 5-part is 30.58%, while manual count is 30%, both values show no significant difference (P = 0.87).

Table 5: Mononuclear cell count between five part haematology analyser and manual count in various range of TLC

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In TLC range 12,000–20,000, the mean value for lymph in 5-part and manual count shows a significant difference (mean difference = −7.85, P = 0.149).

The mean value for MNC in 5-part is 23.10%, while manual count is 25.85%, which is less than the reference range, shows no significant difference (P = 0.098).

In TLC range >20,000, the mean value for lymph in 5-part and manual count shows a significant difference, both the values are less than normal range having mean difference = −7.89, P = 0.149.

The mean value for mononuclear cell (monocytes and lymphocytes) for 5-part is 16.60%, while manual count is 22.80%, having a SD of 11.5, P = 0.05.

When the TLC ranges are increasing, the MNCs count is decreasing in 5-part and even in manual counting. If the TLC count is low or high than the normal range, MNC is indirectly proportional to TLC, while lymphocyte values are inversely proportional to TLC.

Correlation of bone marrow mononuclear cell in 5-part and manual

Eighty samples were included in the study. We excluded the 3-part values as it was not showing differential counts for mixed population. The correlation of MNCs in bone marrow samples between 5 Part haematology analyser and manual analysis were performed [Table 6].

Table 6: Bone marrow mononuclear cell in 5 part and manual

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Discussion

Published literature has favored the utility of these instruments in performance of a screening leukocytes differential count. Agreement between manual counts and instruments has been especially good for lymphocyte and granulocyte counts (Cox, 1985; Miers, 1987).^{[11],[12],[13]} Manual mononuclear counts are gold standard, however are time-consuming, more laborious, require specific trained technician, require standardized staining, reliable and estimation by single/two trained observers.

The correlation in “mononuclear” count has been less satisfactory, with an r value of about 0.5 because of their lower numbers in the blood. Many other less frequent or unusual cell types (eosinophils, basophils, atypical lymphocytes, plasma cells, blast cell, and immature granulocytes) also appear in the mononuclear or granulocyte region and affect the correlation between the two methods. Missed abnormalities (false-negative reports) from use of the instrument differential appear to be equal (9%-Kalish, 1986, or less frequent 6%-Griswold, 1985) when compared with the manual\visual procedure using the National Committee for Clinical Laboratory Standards reference methods (Pierre, 1987).^[13],[14] Most laboratories using instrument differential results have action limits based on the complete blood count and leukocyte differential results that determine whether the counts can be accepted or require verification by scanning the blood film or performing a manual differential count (Koepke, 1985; Payne, 1987).^{[13],[14],[15],[16]}

We manually count only 200–300 cells in peripheral blood smear film which requires 20 μL volume (4000–12,000 cells/μL × 20 μL) or 80,000–240,000 cells. In a routine normal sample of blood count which contains approximately total 80,000–240,000 cells in 20 μL of volume. Cell into 5-part analyzer acquires 50 μL volumes (400–12,000 cells/μL × 50 μL) or 200,000–600,000 cells. Statistically the chance of error and standard error is inversely proportional to the number of observant. Manual and 5-part cell counter give count for lymphocyte and monocytes together which can be used to determine MNCs. The stem cells also lie within this population. The study highlights the use of well calibrated and quality controlled 5-part differential counter; this can provide a reliable substitute to manual estimation of MNCs for a transplant settings. Inadequate MNC cases were excluded from the study as trial deviate. All SCs may MNCs, but all MNCs are not the SCs; they may be progenitor cell/committed cells/differentiated cells.

Conclusion

MNC is an important tool for cell dose in BMT setting. The good correlation of 5-part hematology analyzer with manual gold standard method of MNC count is indicating equivalent performance. MNC can be performed by 5-part with nonsignificant difference and can be used as good as manual count. MNC cannot be performed by 3-part due to mixed differential count of monocytes, basophils, and eosinophils.

Acknowledgments

The authors are grateful to the Directorate General Armed Forces Medical Services, Armed Force Medical Research Cell, New Delhi, India, and commandant Army Hospital R and R, Dhaula Kuan, Delhi Cantonment, New Delhi. I would like to thank Lt. Col (Dr) Barun Chakrabarty for his support during writing this article.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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