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 Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 9  |  Issue : 2  |  Page : 145-149

Automated versus manual method for reticulocyte count: A comparative study in rural central India


Department of Pathology, Mahatma Gandhi Institute of Medical Sciences, Sevagram, Wardha, Maharashtra, India

Date of Submission22-Aug-2020
Date of Acceptance16-Sep-2020
Date of Web Publication10-Nov-2020

Correspondence Address:
Dr. Nitin M Gangane
Department of Pathology, Mahatma Gandhi Institute of Medical Sciences, Sevagram - 442 102, Wardha, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijh.ijh_42_20

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  Abstract 


BACKGROUND: Reticulocytes are immature red blood cells that contain remnants of ribonucleic acid. Various reticulocyte parameters can help in the proper diagnosis of different anemias. It can be measured by manual as well as by automated method.
OBJECTIVE: The aim is to compare between the manual and automated methods of reticulocyte count (RC) in anemia.
MATERIALS AND METHODS: It was a laboratory-based cross-sectional study in which the comparison of RC by manual and automated method along with various reticulocyte parameters was done in 300 patients with anemia and 300 control samples matched for age and sex.
RESULTS: The study cases included 146 females and 154 males. No statistically significant difference was found between the automated and manual count among the male (P = 0.77) as well as female patients (P = 0.61). No statistically significant difference was found in mean RC among the infants (P = 0.71), children (P = 0.59), and adults (P = 0.66) between automated and manual count. The difference between mean manual and automated RC was statistically significant only in the case of males in the macrocytic anemia group (P = 0.092). Among reticulocyte indices, mean immature reticulocyte fraction (IRF), mean reticulocyte volume (MRV), and reticulocyte hemoglobin cellular content (RHCC) was found to be statistically significant among all types of anemia (P = 0.001, 00001 and 0.0001 respectively) while it was insignificant in case of mean corrected RC (P = 0.89). A significant positive correlation was found between manual and automated RC method by using Pearson's correlation coefficient (r = 0.985, P = 0.0001).
CONCLUSION: There was no significant difference between the automated and manual methods for reticulocyte counting. However, the manual method may be preferred as it is cost-effective; yet, it is laborious, time-consuming, need efficient technique, not suitable for heavily loaded laboratories and may be suitable for under-resourced laboratories. However, the automated method is preferred as it is fast, highly precise, and it is mandatory for certain diseases where reticulocyte parameters are required as a statistically significant difference was found among the different parameters such as IRF, MRV, and RHCC.

Keywords: Anemia, automated reticulocyte count, manual reticulocyte count, pentra XLR, reticulocyte parameters


How to cite this article:
Gorte TR, Deshmukh AV, Gangane NM. Automated versus manual method for reticulocyte count: A comparative study in rural central India. Iraqi J Hematol 2020;9:145-9

How to cite this URL:
Gorte TR, Deshmukh AV, Gangane NM. Automated versus manual method for reticulocyte count: A comparative study in rural central India. Iraqi J Hematol [serial online] 2020 [cited 2020 Nov 24];9:145-9. Available from: https://www.ijhonline.org/text.asp?2020/9/2/145/300420




  Introduction Top


Careful assessment of the blood is the first step in the assessment of hematological function and diagnosis of the related diseases.[1] Reticulocytes are young or immature red blood cells that are released from bone marrow and that contain remnants of ribonucleic acid (RNA) and ribosomes.[2] Reticulocyte count (RC) is the index of erythropoietic activity within the bone marrow and its measurement provides an initial assessment of anemia.[1],[2] It is having great diagnostic and prognostic value in hemolytic anemias, in acute hemorrhage, to study the response to iron, folic acid and Vitamin B12 therapy as well as after chemotherapy.[3]

The RC can be done by either by manual or by an automated method. Manual RC by microscopy has been considered as the standard method since 1940 because of its simplicity and low-cost.[4] However, it presents some inconvenience and limitations such as lack of accuracy, more time required for analysis, lack of quality of the stain, and inappropriate blood films.[5] Automated RC, through continued instrument, software, and reagent developments, provides an improved precision for the RC. It is a rapid and simple investigation which assists in providing a sensitive approach to the diagnosis and therapeutic monitoring of the anemic patients.[6]

Automated reticulocyte counting makes the determination fast, specific, and efficient by counting a large number of red blood cells.[7] It can provide information about individual cell characteristics, such as hemoglobin content of reticulocytes, hemoglobin content of mature erythrocytes, percentages of microcytic erythrocytes and hypochromic cells, mRNA content and of cellular indices such as volume, hemoglobin concentration, and content. All these novel parameters are useful in reporting and interpretation in the diagnosis of specific anemia.[7]

There are very few reports in the literature regarding this aspect, especially in Central India. Hence, the present study was carried out to compare between the automated and manual method of RC in different anemias.


  Materials and Methods Top


It was a laboratory-based cross-sectional study conducted in the Department of Pathology at Mahatma Gandhi Institute of Medical Sciences, Sevagram, Wardha, a rural tertiary care hospital in Central India over a period of 24 months (October 2016 to September 2018) after clearance from Ethics committee of the Institute. Both the cases and control samples were obtained from the Haematology section, from patients who were already being investigated for their illnesses. In this study, comparison of RC by the manual and automated method was done in 300 patients with anemia and 300 control samples which were matched for age and sex. The cases were selected randomly by systematic randomization (every alternate sample) with hemoglobin (Hb) <11 g/dL and control samples were collected from the same laboratory with Hb >11 g/dL. All the samples (both cases and controls) were processed for RC by manual as well as automated method within 4 h of sample collection.[8],[9] We divided our cases into three different age groups according to age, which included infants (up to 1 year), children (>1 year to 14 years) and adults (>14 years) for comparison of mean RC. We also classified 300 patients of anaemia according to morphological types of anemia; mean corpuscular volume (MCV) <80 fL as microcytic, MCV = 80–100 fL as normocytic and MCV >80 fL as macrocytic.[9] The cases with microcytic anemia were 177, normocytic were 93, and macrocytic were 30 in numbers.

Method

For manual RC,[8] fresh ethylene diamine tetra-acetate (EDTA) anticoagulated blood samples were used. For each sample, aliquots of 50 μL of EDTA anticoagulated blood and 50 μL of Azure B stain were added to the polystyrene tube. The mixture was incubated for 30 min at 37°C protected from light. After remixing, blood films were prepared on clean glass slides for microscopy. The slides were allowed to dry for 10 min. Counting was performed using a microscope with ×100 objective lens in oil immersion field. The number of reticulocytes was calculated by counting the number of these cells in 1000 erythrocytes evaluated on each smear of all samples. The counting was performed by the two pathologists for control samples. The results were expressed as a percentage of cells containing stained RNA (reticulocyte).

For automated RC,[2]Horiba Pentra XLR was used. It was a fully automated hematology analyzer used for the in vitro diagnostic testing of whole blood specimens. Horiba Pentra XLR gives the various parameters like complete blood count along with differential counts and different reticulocyte parameters. The differential reticulocyte parameters include immature reticulocyte fraction (IRF), corrected RC (CRC), mean reticulocyte volume (MRV) and reticulocyte haemoglobin cellular content (RHCC).

Ethics

The study protocol was approved by the Institute Ethics committee of Mahatma Gandhi Institute of Medical Sciences, Sevagram, letter number MGIMS/IEC/PATH/100/2016, dated October 5, 2016. Patient confidentiality was maintained during all research procedures.

Statistical analysis

Statistical analysis was done by using descriptive and inferential statistics using Z-test for the difference between two means and Cronbach's alpha, Z-test, F test, and Pearson's correlation coefficient test. Software used was SPSS17.0 (IBM Corp. Released 2011. IBM Statistics for Windows, Version 20.0: Armonk, New York, United States) and graph pad PRISM 5.0 version (GraphPad PRISM, Version 5.0: San Diego, California, USA) and P < 0.05 was considered as the minimum level of significance.


  Results Top


Basic demography in study cases showed 146 females and 154 males. Out of 300 cases, 22 cases were infants (up to 1 year), 62 cases were children (>1 year to 14 years) and 216 cases were adults (>14 years).

The mean automated RC for males was 4.71 ± 4.18 and it was 4.85 ± 4.34 by the manual method. In the case of females, the mean automated RC was 3.62 ± 3.44 and that by the manual method, it was 3.82 ± 3.66. No statistically significant difference was found between the automated and manual count among the male (P = 0.77) as well as female patients (P = 0.61) by z-test for the difference between the mean automated RC and mean manual RC [Table 1] upper half].
Table 1: Comparison of mean reticulocyte count between male and female by automated and manual method and comparison according to age

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The mean automated RC among infants (up to 1 year) was 2.31 ± 1.47 and it was 2.13 ± 1.72 by manual method. In the case of children (>1 year-14 years), the mean automated RC was 3.89 ± 2.89 and 4.17 ± 3.02 by manual method. For adults (>14 years), the mean automated RC was 4.45 ± 4.22 and 4.63 ± 4.40 by manual method. Z-test for difference between two means was applied. No statistically significant difference was found in mean RC among the infants (P = 0.71), children (P = 0.59), and adults (P = 0.66) between automated and manual count [Table 1] lower half].

When we divided cases as per morphologic classification of anemia in both sex, the difference between mean manual and automated RC was statistically significant only in the case of males in the macrocytic anemia group (P = 0.092) while it was insignificant in rest cases and sex groups in other groups [Table 2].
Table 2: Mean reticulocyte count according to morphologic classification of anaemia in study cases

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When compared for reticulocyte indices, mean IRF, Mean MRV, and mean RHCC was found to be statistically significant among all types of anemia (P = 0.001, 00001, and 0.0001, respectively) while it was insignificant in the case of mean CRC (P = 0.89) [Table 3].
Table 3: Comparison between mean immature reticulocyte fraction, corrected reticulocyte count, mean reticulocyte volume and reticulocyte haemoglobin cellular content for normocytic, macrocytic and microcytic cases by automated method in study cases

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A significant positive correlation was found between manual and automated RC method by using Pearson's correlation coefficient (r = 0.985, P = 0.0001) [Table 4] and [Figure 1].
Table 4: Correlation between manual and automated method of reticulocyte count by Pearson's correlation coefficient

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Figure 1: Correlation between manual and automated method of reticulocyte count

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  Discussion Top


Our study showed no statistically significant difference between the mean automated RC and mean manual RC amongst males as well as females (P = 0.77 and P = 0.61). The literature shows studies with varied results which compared RC between males and females,[10],[11] but not the methods. Thus, both methods are suitable for the determination of mean RC, but the manual method can be more preferred as it is cost-effective. We also did not find any significant difference among different age groups, i.e., infants, children, and adults (P = 0.71, 0.59, and 0.66) [Table 1]. The comparison of mean RC value by both manual and automated method showed varied results in the literature. Osgood et al.[12] found no significant difference in average RC percentage in the age group of 4–13 years while; Jain P et al.[13] found a significant decrease in the elderly group. Bukhari and Zafar [14] found a significant difference in RC in (<27 days) age group in their study on infants (P < 0.05). Tarallo P et al.[10] found no statistical difference between boys and girls aged 4–19 years. However, it was significantly higher in men than in women over 20 years of age.[10]

The highest mean RC was observed in macrocytic anemia by both automated and manual methods followed by microcytic anemia in males in our study. The lowest mean RC was seen in microcytic anemia in female patients [Table 2]. The literature did not show such an association between the morphological type of anemia and mean RC. This study was mainly focused on finding out the difference between the manual and automated methods of reticulocyte counting. No statistically significant difference was observed between these two methods in microcytic, normocytic and macrocytic anemia. However, considering that most cases of macrocytic anemia will be having megaloblastic or hemolytic etiology,[15],[16] it is expected that there will be reticulocytosis because of hemolysis and subsequent erythroid production. While, in females, the most common cause for not so significant increase in RC is iron deficiency anemia and as expected the mean RC was lowest in microcytic anemia in the present study. The most important cause of microcytic anemia is iron-deficiency anemia.[17] All these above findings indicate that there is no significant difference between mean RC obtained by automated and manual methods by age, sex, and morphology of anemia.

Regarding reticulocyte indices, we observed that the mean IRF was higher in macrocytic anemia as compared to microcytic and normocytic anemia (P = 0.001) [Table 3]. Our findings are consistent with Lacombe et al.,[18] Sindhu et al.[19] and Sunkara and Kotta,[20] all of which found a highly significant difference in IRF values. Rastogi et al.[21] found that a significant difference in the values of CRC obtained by manual versus automated method. The findings of our study showed that there was not much difference in manual and automated counts in both controls and cases, and the difference was also not statistically significant (P = 0.89). The reported differences between manual and automated RC may reflect on many variations in the staining technique, use of dye, and reliability by the observers. Furthermore, the deviation in the study by Rastogi et al.[21] was huge and it varied from minimum 2.2%–211%. In our study, we observed that MRV was higher in macrocytic anemia as compared to microcytic anemia (P = 0.0001). Our findings are consistent with Butthep et al.[22] who found a highly significant decrease in MRV in iron deficiency anemia patients versus normal (MRV = 95.89 ± 8.57 FL, P ≤ 0.0001). Hence, it adds to the peripheral smear observation that the size of the reticulocytes observed in iron deficiency anemia is smaller than macrocytic anemia. We also found that RHCC was higher in macrocytic anemia as compared to microcytic anemia (P = 0.0001). Our findings are consistent with Butthep et al.,[22] Mast et al.,[23] Ceylan et al.,[24] and Ageeli et al.[25] (P < 0.001).

In this study, a significant positive correlation was found between manual and automated RC method by using Pearson's correlation coefficient (r) (r = 0.985, P = 0.0001) [Table 4] and [Figure 1]. Our study is consistent with the studies of Lacombe et al.,[18] Simionatto et al.[26] and Ali et al.[27] who found a high degree of correlation and an excellent agreement between the two methods.


  Conclusion Top


We conclude that there was no significant difference between automated and manual methods for reticulocyte counting in any gender for microcytic, normocytic, or macrocytic patients.

However, the manual method may be preferred as it is cost-effective; yet, it is laborious, time-consuming, need efficient technique, not suitable for heavy loaded laboratories and may be suitable for under-resourced laboratories. However, the automated method is preferred as it is fast, highly precise and it is mandatory for certain diseases where reticulocyte parameters are required as a statistically significant difference was found among the different parameters such as IRF, MRV, and RHCC.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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Sindhu R, Behera SK, Mishra DP. Role of immature reticulocyte fraction in evaluation of aplastic anemia in cases of pancytopenia. Indian J Basic Appl Med Res 2016;5:619-24.  Back to cited text no. 19
    
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Mast AE, Blinder MA, Lu Q, Flax S, Dietzen DJ. Clinical utility of the reticulocyte hemoglobin content in the diagnosis of iron deficiency. Blood 2002;99:1489-91.  Back to cited text no. 23
    
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26.
Simionatto M, Paula JP, Nascimento AJ, Leonart MSS, Cicchetti D. Analysis of manual reticulocyte counts in the clinical laboratories of ponta-grossa and campos gerais, PR, Brazil. Rev Bras Hematol Hemoter 2009;31:315-20.  Back to cited text no. 26
    
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    Figures

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    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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