Comparison of three methods for enumeration of residual white blood cells in single donor apheresis platelets: A pilot study from Eastern India as a part of quality monitoring process for leukoreduction

Nasir Nabi Naikoo; Sabita Basu; Animesh Bajpayee; Deepak Kumar Mishra; Suvro Sankha Datta

doi:10.4103/gjtm.gjtm_30_22

ORIGINAL ARTICLE

Year : 2022 | Volume : 7 | Issue : 2 | Page : 174-177

Comparison of three methods for enumeration of residual white blood cells in single donor apheresis platelets: A pilot study from Eastern India as a part of quality monitoring process for leukoreduction

Nasir Nabi Naikoo¹, Sabita Basu¹, Animesh Bajpayee¹, Deepak Kumar Mishra², Suvro Sankha Datta¹
¹ Department of Transfusion Medicine, Tata Medical Center, Kolkata, West Bengal, India
² Department of Laboratory Hematology, Tata Medical Center, Kolkata, West Bengal, India

Date of Submission	12-Apr-2022
Date of Decision	18-Aug-2022
Date of Acceptance	07-Oct-2022
Date of Web Publication	5-Nov-2022

Correspondence Address:
Suvro Sankha Datta
Department of Transfusion Medicine, Tata Medical Center, Kolkata, West Bengal
India

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/gjtm.gjtm_30_22

Abstract

Background and Objectives: The aim of this study was to compare three platforms side-by-side: Nageotte hemocytometer, flow cytometry (FC), and standard hematology analyzer for enumeration of residual white blood cells (rWBCs) in single donor platelets (SDPs) apheresis. Materials and Methods: This was a prospective observational study conducted in a tertiary care oncology center by evaluating 36 units of SDP that were collected and tested in parallel by three different methods for the enumeration of rWBCs. All tests were performed within 24 h of collection according to the manufacturers' recommended methods. Counting by the Nageotte hemocytometer was done by: rWBC/μl = (cells counted × dilution/gridded area volume [50 μl]) and FC calculation was performed by: rWBC/μl = (total beads × total number of leukocyte events/total beads acquired × total sample volume). Results: The number of rWBCs detected by FC was between 1 white blood cell (WBC)/μl and 8 WBCs/μl; whereas, those detected by Nageotte chamber were between 3 WBC/μl and 6 WBCs/μl. The range of rWBC detected by hematology analyzer was 100 WBC/μl to 270 WBCs/μl. There was no correlation observed between the results obtained in standard hematology analyzer with any of the other two methods. The concordance correlation coefficient was measured by kappa analysis and found to be 0.71 between hemocytometry and FC. Linear regression analysis also showed a moderate correlation (R² = 0.42) between the two methods. However, the coefficient of variation was found to be 49.32% in Nageotte method compared to the 17.55% in FC (P < 0.001). Conclusion: FC followed by Nageotte chamber counting is a better method compared to the standard hematology analyzer for the enumeration of rWBCs. To overcome the cost of FC it is high time to explore the scope and feasibility of a centralized quality monitoring system in India for all leukoreduced blood components.

Keywords: Blood components, flowcytometry, leukoreduction, Nageotte hemocytometer, quality control

How to cite this article:
Naikoo NN, Basu S, Bajpayee A, Mishra DK, Datta SS. Comparison of three methods for enumeration of residual white blood cells in single donor apheresis platelets: A pilot study from Eastern India as a part of quality monitoring process for leukoreduction. Glob J Transfus Med 2022;7:174-7

How to cite this URL:
Naikoo NN, Basu S, Bajpayee A, Mishra DK, Datta SS. Comparison of three methods for enumeration of residual white blood cells in single donor apheresis platelets: A pilot study from Eastern India as a part of quality monitoring process for leukoreduction. Glob J Transfus Med [serial online] 2022 [cited 2023 Mar 28];7:174-7. Available from: https://www.gjtmonline.com/text.asp?2022/7/2/174/360479

Introduction

Leukoreduction in cellular blood components has become an integral part of modern transfusion medicine. The proven benefits of leukoreduction include the prevention of febrile transfusion reactions, alloimmunization to HLAs, and transmission of infectious agents such as cytomegalovirus. According to the Drug and Cosmetic Act 1940 and quality council of India, it is required that the platelet (PLT) concentrates collected by apheresis (single donor platelet [SDP]) should contain <5 × 10⁶ white blood cells (WBCs), and each month minimum 4 units should be checked for residual leukocytes (residual WBCs [rWBCs]).^[1] A residual number of 5 × 10⁶ leukocytes per unit, corresponding with approximately 17 leukocytes/μl for a 300-ml SDP unit, is currently widely used as the standard cut off for leukoreduction in India. At least 75% of the tested units must conform to this level. The lowest concentration that can be quantified reliably by a standard hematology analyzer is 100 leukocytes/μl; much higher than the rWBCs present in an SDP unit. To overcome this technical challenge, dedicated methods have been designed for rWBCs detection, i.e., the Nageotte hemocytometer; flow cytometry (FC); microfluorometry, etc.^[2],[3],[4] Although these techniques have a theoretical lower limit of detection of 0.1 leukocytes/μl, the level of accurate detection is considered to be 10–20 times higher.^[5] There is a gray zone in the process control of leukoreduced blood components in India regarding the method of testing for the enumeration of rWBCs as a part of quality monitoring system. The aim of this study was to compare three platforms side-by-side: Nageotte hemocytometer, FC, and standard hematology analyzer for enumeration of rWBCs in SDP units.

Materials and Methods

Study design

This was a prospective, observational, pilot study conducted in 2020–2021 at a tertiary care oncology center in Eastern India. A total of 36 SDP units were evaluated and rWBCs were counted side-by-side by three different methods as already mentioned. The number of samples tested was according to the pack size of the kit used in FC.

Study cohort

PLTs were collected by apheresis from healthy blood donors according to the guidelines stated in the Drug and Cosmetic Act 1940 and the Standards for Blood Banks and Blood Transfusion Services.^[1] The same apheresis machine (Trima Accel, Terumo BCT, US) was used for all procedures. SDP units with a volume ranging from 200 mL to 300 mL containing at least 3 × 10¹¹ PLTs were included in the study. Procedures showing WBC flags at the end of the PLTs collection were excluded from the study as outliers.

Sampling

Apheresis PLT concentrates were sampled into integral sample packs after a minimum rest period of 30–45 min following the donation process. Samples were not fixed and were tested within 24 h of the collection. Plateletpheresis as well as sampling was done by one technologist to avoid the interpersonal variability. Tests were performed in duplicate at three different platforms by three different persons at the same point of time without knowing the results of each other to ensure the proper blinding. The study was conducted in a single center to avoid inter-laboratory variation and sample variation or deterioration.

Leukocyte counting by three methods

A complete blood count for all samples from the integral sample pack was performed by a D×H 500 hematology analyzer (Beckman Coulter India Pvt. Ltd, Mumbai, India).

The Nageotte hemocytometer is a large-volume (50 μl) hemocytometer, which, after a 1-in-5 dilution of the sample with a counting solution, has a theoretical limit of detection of one cell in a 10 μl neat sample. Therefore, 100 μl of the PLT sample was mixed well with 400 μl of Turk's fluid in a clean test tube by pipetting multiple times. Calibrated pipettes were used, with an accuracy of ± 5% of the set volume and a coefficients of variation (CV) <5%. The mixed sample was loaded on the hemocytometer with a cover slip. The chamber was placed in a moist petridish with a damp filter paper for 15 min, allowing the WBCs to settle over the counting chamber. Then, the hemocytometer was scanned back and forth under a microscope using a ×20 objective. Leukocytes appeared as gray-blue refractile cells bearing a nucleus. All the WBCs present in the 50 μl volume of the counting chamber were counted including 40 rectangles and both the upper and lower grids. The following formula was applied to know the final WBC count.

WBC/μl = Number of WBC/50 μl × 5 (dilution factor).

Total rWBC count in the SDP = WBC/μl × 1000 × product volume (mL).

Flow cytometric methods use a nucleic stain such as propidium iodide (PI) or 4', 6-diamino-2-phenylindole to detect the leukocytes. The BD leukocount reagent contains PI which, when used with RNAse, stains only cellular DNA. White blood cells are nucleated cells that contain DNA and are therefore stained with the dye. Nonnucleated particles like PLTs do not stain with this reagent. In this study, BD Leucocount kit was used for enumerating rWBCs in SDP products (cat. no. 340523, BD Biosciences, San Jose, CA, US). A volume of 100 μl of PLT sample was mixed with 400 μl of BD Leucocount reagent in each TruCount tube, which contains a lyophilized pellet with a predefined number of counting beads. The tubes were gently vortexed for 15s and then incubated for 5 min in the dark at room temperature till they were ready for acquisition. The BD Leucocount PLT control cells (cat. no. 341002, BD Biosciences, San Jose, US) were used during each run as controls. Finally, the samples were acquired using an FC (BD FACSCanto II, US) in low flow rate, and a minimum of 10,000 events were acquired in Region-1 (R1). Tests were performed according to the manufacturer's instructions. For the analysis of list mode data, using the Fluorescent label 1 (FL1) versus FL2 dot-plot, gates were set around the population of beads (R1) and the population of leukocytes (R2) using a sample with a relatively high leukocyte concentration. These gates were used throughout the study. The number of WBC/μl was calculated with the below equation:

WBC/μl = total beads × total number of leukocyte events/total beads acquired × total sample volume.

Statistical analysis

Data were primarily collected and analyzed using Microsoft Excel statistics software package (Microsoft Corporation, Redmond, WA,US). Further statistical calculation was performed by R software (version 3.4.2.) and P < 0.05 was considered statistically significant. Continuous variables were expressed as mean with standard deviation and range. To assess the precision of each method, intra-assay CV were calculated. Linear regression analysis was performed to calculate the concordance correlation coefficient between results obtained by three different methods. We evaluated the agreement between two methods using Cohen's kappa statistic. For interpreting kappa values, we used the criteria set by Landis and Koch: <0 = poor agreement; 0.01–0.20 = slight agreement; 0.21–0.40 = fair agreement; 0.41–0.60 = moderate agreement; 0.61–0.80 = substantial agreement; and 0.81–0.99 = nearly perfect agreement.

Results

A total of 36 SDP units were included in this study with a mean volume of 272 ± 15.6 mL. The mean age of donor was 32.5 ± 6.1 years and mean body weight was 64.5 ± 4.8 kg. All were repeat apheresis donors. Among them, 3 were female and the rest 33 were male. The mean hematocrit of these donors was 44.06 ± 2.26, mean total leukocyte count was 8768.94 ± 1569.25 per μl, and mean PLT count was 257368.42 ± 17811.38 per μl. The number of rWBCs that were detected by FC ranges between 1 WBC/μl and 8 WBCs/μl; whereas, those detected by the Nageotte chamber were from 3 WBC/μl to 6 WBCs/μl. The range of rWBC detected by the hematology analyzer was 100 WBC/μl to 270 WBCs/μl. All control results were within the normal limit. There was absolutely no correlation observed between the results obtained in the standard hematology analyzer with any of the other two methods. The kappa value was found to be 0.71 between hemocytometer and FC which showed a substantial agreement. Linear regression analysis also demonstrated a moderate correlation (R² = 0.42) between the two methods. However, CV was found to be 49.32% in the Nageotte method compared to the 17.55% in FC (P < 0.001). Results are summarized in [Table 1].

Table 1: Results of testing by each of the three methods

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Discussion

As an ever greater proportion of the blood supply is now being leukoreduced in India, more units are subject to quality control (QC) testing. However, it is clear that there is very little agreement on the structure of a leukoreduction monitoring program between blood centers that includes (1) appropriate level of conformance to the adopted specification, (2) identification of the outliers, (3) contingency in the event of a process failure, (4) determination of a proper control within a process stream, and (5) determination of an appropriate method of leukocyte testing required by QC process. In this study, we ought to compare the method of testing for rWBCs in leukoreduced apheresis PLTs. Among the published methods, Nageotte hemocytometer has been the most widely accepted.^[6],[7] Although it is suited to process control testing, microscope-based counting techniques are labor-intensive, time taking, and require both technical training and considerable experience. Because microscopic recognition of stained WBCs can be subjective, Nageotte chamber counting may also suffer from variability between laboratories as well as among individuals. On the contrary, automated techniques though costly, have a faster throughput and are less labor-intensive. In addition, the automated methods are more sensitive and do not appear to be subjected to inter-observer variability found in the Nageotte method.^[8] In our study, the Nageotte counting enumerated slightly but not significantly higher rWBC results than the FC which is in concordance with published literature.^[9],[10] This might be because of artifacts as the same chamber and slide cover was repeatedly used and also because of the FC gating that was set up to encompass only intact WBCs. Any component which has an increased prestorage leukoreduction time has more WBC fragments in the leukoreduced products that may cause false elevation of leukocyte counts by the Nageotte method. In the present study, all SDP units were analyzed within 24 h of preparation to avoid such misinterpretation. However, in the majority of studies, Nageotte method results were lower than those obtained by FC due to the poor sensitivity.^[11],[12] Compared to these two methods standard hematology analyzer was found to be far inferior for the enumeration of rWBCs. In India, till date only two studies compared the results between hemocytometer and FC. In a recent study, Pandey et al.^[13] demonstrated poor correlation between two methods when rWBCs in the leukoreduced red cell units were counted. The Nageotte hemocytometer had the lowest accuracy, especially when tested with leukoreduced PRBCs which might be due to the incomplete lysis of red cells with lysis solution, preventing proper sedimentation of leukocytes.^[11] However, a comparative study by Javed et al.^[14] on random donor buffy-coat derived PLTs showed a good correlation between two methods (r = 0.9122) which is similar to our results where a moderate correlation was observed between the two methods. As the mean concentration of WBCs present in the buffy coat derived PLTs is significantly high compared to the SDPs and hemocytometer is known for the better precision when WBC count is more than 30/μl;^[11] therefore, a strong correlation is commonly observed with FC in the presence of a higher concentration of rWBCs. In fact, Lutz and Dzik^[7] also noted a good correlation comparing Nageotte's method and FC results in apheresis PLT concentrates which is in line with our findings. However, our study results demonstrated that testing by FC resulted in much better precision than did the Nageotte hemocytometer (P < 0.001). With FC the CV was <20% which is within the normal range. In contrast, the CV for the Nageotte hemocytometer method was > 45% which is far away from the acceptable range. That might be due to the subjectivity of the manual counting. Again, these results are in concordance with the process validation and process control report of the Biomedical Excellence for Safer Transfusion network.^[15]

A process failure for apheresis PLTs constitutes two specification failures from the same machine in a 2–3-day period.^[16] In these circumstances, the specific machine should not be used until checked by the manufacturer. The nonconforming apheresis units might be secondary filtered before use to ensure adequate leukoreduction. In our case, all SDP units that were tested had fulfilled the quality requirements. There were no outliers in our study.

Before the selection of a leukocyte counting method for a blood center, the following parameters such as the access to the testing platform, technical expertise, sample throughput, cost per test, and customer support have to be taken into account. Because these logistic items are different from center to center; a one-size model does not fit into all. Automated methods require expensive equipment and disposables, unlike the inexpensive hemocytometer and are financially challenging to adopt in a country like India. With increased emphasis on process control of leukoreduced blood components; there is growing interest in centralized testing services which could mitigate some of these limitations. However, sample deterioration during shipment and storage represents an important consideration for centralized testing services. Finally, maintaining an appropriate testing interval from the component preparation, performance assessment by introducing an external quality assessment scheme or standardization of FC for the stabilized blood samples are the major challenges in resource-constrained health-care facilities.

Our study does have some limitations including a small sample size. As inter-site variability can be assessed only in a multicenter study; therefore, such study including large sample size is needed to define the most appropriate method for testing of rWBCs in leukoreduced blood components in Indian settings along with a cost analysis.

Conclusion

Finally, we conclude that FC followed by Nageotte chamber counting is a better method compared to the standard hematology analyzer for the enumeration of rWBCs. To overcome the cost of FC, it is high time to explore the scope and feasibility of a centralized quality monitoring system in India for all leukoreduced blood components by initiating a well-designed national survey.

Statement on ethics

Institutional review board approval was obtained for the study. Since both Nageotte hemocytometry and flow cytometric enumeration are acceptable methods for quality control of leukodepletion, the ethical committee approval was waived off by the institution.

Financial support and sponsorship

Intramural funding received from Tata Medical Center, Kolkata.

Conflicts of interest

There are no conflicts of interest.

References

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