|Ahead of print
|Comparative study of quality of leukoreduced packed red blood cell units as assessed by nageotte hemocytometry and flow cytometry
Mohd Anas Sheikh1, Amit Kumar Biswas1, Ajay Kumar Baranwal1, Neerja Kushwaha2, Santosh Karade3, Joseph Philip4
1 Department of Immunohematology and Blood Transfusion, Armed Forces Medical College, Pune, Maharashtra, India
2 Department of Immunohematology and Blood Transfusion, CH(CC), Lucknow, Uttar Pradesh, India
3 Department of Microbiology, Armed Forces Medical College, Pune, Maharashtra, India
4 Department of Transfusion Medicine, Bharati Vidyapeeth University, Pune, Maharashtra, India
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|Date of Submission||17-Jul-2021|
|Date of Decision||10-Sep-2021|
|Date of Acceptance||19-Sep-2021|
|Date of Web Publication||26-Sep-2022|
| Abstract|| |
PURPOSE: Assessment of residual white blood cell (rWBC) count is vital to ascertain the quality of leukodepleted (LD) blood components. Automated cell analyzers lack the sensitivity for the assessment of very few leukocytes as found in LD blood components. Flow Cytometry (FC) based methods and Nageotte hemocytometer are the most commonly used techniques for this purpose. The objective of this study was to compare the use of Nageotte hemocytometer and FC for quality control of LD red blood cell units.
MATERIALS AND METHODS: A prospective, observational study was conducted in the Department of Immunohematology and Blood Transfusion of a tertiary care center from September 2018 to September 2020. About 303 LD-packed red blood cell units were tested by FC and Nageotte hemocytometer for rWBCs.
RESULTS: The number of rWBC (mean) detected by flow cytometer and Nageotte's hemocytometer was 1.06 ± 0.43 white blood cell (WBC)/μL and 0.67 ± 0.39 WBC/μL, respectively. Coefficient of variation was 58.37% by Nageotte hemocytometer method and 40.46% by FC. Linear regression analysis did not show any correlation (R2 = 0.098, P = 0.001) whereas Pearson's correlation coefficient showed a weak relation (r = 0.31) between the two methods.
CONCLUSION: Flow cytometric technique provides a more precise and accurate objective tool compared to Nageotte hemocytometer which is labor intensive, time consuming, and prone to errors arising out of subjectivity along with reported underestimation bias. In the absence of adequate infrastructure, resources, and trained workforce, Nageotte hemocytometer method is a reliable alternative. Nageotte's chamber could be best used in the resource-constrained setup as it offers a relatively inexpensive, simple, and viable means to enumerate rWBCs.
Keywords: Alloimmunization, flow cytometry, leukodepletion, nageotte hemocytometer, quality control, residual white blood cell count
|How to cite this URL:|
Sheikh MA, Biswas AK, Baranwal AK, Kushwaha N, Karade S, Philip J. Comparative study of quality of leukoreduced packed red blood cell units as assessed by nageotte hemocytometry and flow cytometry. Asian J Transfus Sci [Epub ahead of print] [cited 2022 Dec 4]. Available from: https://www.ajts.org/preprintarticle.asp?id=356848
| Introduction|| |
As leukoreduced blood components have become the standard of medical care and more countries are adopting Universal Leukoreduction, it becomes imperative to adopt quality standards that assure supply of efficient and safe blood to patients.
Leukocyte contamination in blood components is known to cause a plethora of transfusion-associated deleterious effects. These include transfusion-related immunomodulation, febrile nonhemolytic transfusion reaction, transfusion-related acute lung injury, allergic reactions, transfusion-transmitted hepatitis, and new variant Creutzfeldt-Jacob disease.,,,, Besides these, many short and long-term adverse transfusion reactions such as platelet refractoriness, human leukocyte antigen alloimmunization, and cytomegalovirus transmission can be prevented or substantially reduced by performing leukodepletion below a specific level.,,,
Various techniques to perform leukoreduction include saline washing, buffy coat removal, glycerolization-freeze-thaw deglycerolization, differential centrifugation, and leukofiltration., Out of all the mentioned techniques, leukofiltration is known to give the best results in terms of log reduction. By performing leukofiltration, leukocyte count log reduction of the order of 3 logs or 4 logs can be easily achieved.
Leukofilters work by multiple mechanisms such as leukocyte blocking, leukocyte bridging, interception, and adhesion.,, These employ mechanical sieving as well as electrostatic adhesion for leukodepletion and are able to fulfill American Association of Blood Banks (AABB), European council criteria along with Directorate General of Health Services (DGHS) (India) guidelines for leukodepleted (LD) blood units (<5 × 106, <1 × 106 and <5 × 106 leukocytes per unit, respectively).,, Automated cell analyzers have poor sensitivity and accuracy for such low counts. Flow cytometry (FC) and Nageotte hemocytometer are among the most widely approved and validated techniques for assessing residual white blood cells (rWBCs) in LD blood component.,
The primary objective of this study was to compare the use of Nageotte hemocytometer and FC for quality control of LD red cell units.
| Materials and Methods|| |
Settings and design
We conducted a prospective, observational study between September 2018 and September 2020 in the Department of Immunohematology and Blood Transfusion of a tertiary care healthcare setup in India. A total of 303 components were assessed for leukoreduction by both FC and Nageotte hemocytometer.
Whole blood collection
Whole blood was collected from voluntary blood donors at the Department of Immunohematology and Blood Transfusion using 450 ml and 350 ml triple or quadruple bag system without integral filter for LD (Polymed, India). All components including packed red blood cell (PRBC) units were prepared in accordance with the standard operating procedures of the department and National guidelines.
Collection of samples and complete blood count
PreLD, two mL blood samples were aseptically collected in EDTA vacutainers (BD Sciences, Mumbai, Maharashtra, India) from the segment of PRBC units prepared and complete blood count for all samples was done by XT-2000i analyzer (Sysmex Transasia, Mumbai, Maharashtra, India). The PRBC units were then subjected to leukofiltration within 48 h of collection using leukofilter with attached transfer bag (Bio-Rad Laboratories, India). PostLD samples from LD units were then assessed for rWBC count by FC and Nageotte hemocytometry.
Determination of residual white blood cell count by Nageotte hemocytometer
PostLD, sample dilution by adding 40 μL of blood sample to 160 μL of Turk's solution followed by thorough mixing using vortex for 10-15 s to reach a final dilution of 1:5. The test tube was then left undisturbed at room temperature for 15 min to allow red cell lysis.
A coverslip was placed over the Nageotte hemocytometer (Marienfeld, Germany), and the diluted sample was carefully loaded on the chamber till the chamber was completely charged. The chamber was subsequently kept in a moist petri dish for 15 min to allow the leukocytes to settle. The rWBCs were then counted by viewing to and fro under the microscope across the gridded area using ×10 objective lens. Counting was performed in all 40 rectangles and in both upper and lower grids (for duplicate counting). rWBC counts were obtained using the following formula:-
Leucocytes/μL = (Cells counted × Dilution)/Gridded area volume (50 μL).
Determination of residual white blood cell counts by flow cytometry
Flow cytometric analysis was done on BD FACSCalibur (BD Biosciences, India) using BD Leukocount Kit. The assay uses BD Trucount tubes to assess absolute cell counts of rWBCs in a single tube. BD Leukocount controls were run in parallel with test samples. BD CellQuest Pro software was used for data analysis and interpretation.
Hundred microliter of test sample was taken in a labeled BD Trucount tube. 400 μL of BD Leukocount reagent was then added to each tube followed by gentle vortex for not more than 15 s. The tubes were then incubated in the dark at room temperature for 5 min. Following incubation in dark, sample acquisition was done on BD FACSCalibur flow cytometer.
Data were computed in SPSS software (IBM Corp. Released 2011. IBM SPSS Statistics for Windows, Version 20.0. Armonk, NY:IBM Corp.). Mean values were calculated. To assess the precision of each method, intraassay coefficients of variation were calculated. For all tests, a probability <0.05 was used to indicate statistical significance. Linear regression analysis, Pearson's correlation coefficient, and coefficient of variance were calculated using the SPSS for Windows, version 20.0 (SPSS Inc. Chicago, Illinois, USA). Bland–Altman plot was used to study the agreement between the two methods.
| Results|| |
Donor demographic data
A total of 303 whole blood donations were included in the study. This included 126 units of 450 ml bags and 177 units of 350 ml bags. Among the 303 donations included in the study, 37 were from female donors (12%), rest were from male donors (88%). Fifty-five of the 303 collections were donated by repeat donors, out of which 13 were regular, repeat voluntary donors. The mean age of donors who donated these units was 41.20 ± 09.60 years. Donor demographic data have been represented in [Figure 1].
|Figure 1: Donor Demographics-Gender distribution and frequency distribution of donors|
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Donor packed red blood cell units' hematological data
The hematological data as obtained for 350 ml units and 450 ml units have been summarized in [Table 1].
Results obtained by flow cytometry-based estimation
For 350 ml packed red blood cell units
All control results were as expected. The mean rWBC count in LD PRBCs was 1.07 ± 0.44 WBC/μL. Coefficient of variation was 40.93%.
For 450 ml packed red blood cell units
All control results were as expected. The mean rWBC count in LD PRBCs was 1.05 ± 0.41 WBC/μL. Coefficient of variation was 39.90%.
Results obtained by Nageotte chamber-based estimation
For 350 ml packed red blood cell units
The mean rWBC count in LD PRBCs was 0.68 ± 0.40 WBC/μL. Coefficient of variation 59.20%. Thirteen samples (7.34%) failed to give a result (count zero). Thirty-one samples (6.78%) gave rWBC count more than that given by FC.
For 450 ml packed red blood cell units
The mean rWBC count in LD PRBCs was 0.64 ± 0.36 WBC/μL. Coefficient of variation 56.90%. Ten samples (7.93%) failed to give a result (count 0). Twelve samples (9.52%) gave rWBC count more than that given by FC.
Comparison of both methods
Linear regression analysis showed no correlation between the two techniques for 350 ml or 450 ml units (R-squared = 0.0929 and 0.1069, respectively) [Figure 2] and [Figure 3]. Coefficient of variation for 350 ml and 450 ml units was 59.20% and 56.90%, respectively, in Nageotte hemocytometer method. It was 40.93% and 39.90%, respectively, in FC method. Pearson's correlation coefficient was calculated for the two methods. The value of “R” was 0.30 and 0.33, respectively, signifying a weak relation between the results obtained by the two methods.
|Figure 2: Scatter diagram of paired measurements of residual white blood cells (rWBCs) per μL as determined by Flow cytometry and Nageotte haemocytometry, for all 350 ml leucoreduced PRBC units (n=177)|
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|Figure 3: Scatter diagram of paired measurements of residual white blood cells (rWBCs) per μL as determined by Flow cytometry and Nageotte haemocytometry, for all 450 ml leucoreduced PRBC units (n=126)|
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Taking P < 0.05 as significant (alpha = 0.05), the P values obtained in our study were 0.0000371 and 0.000187 for 350 ml and 450 ml units, respectively. Thus, there is a statistically significant variation in values obtained by both methods. This is suggestive of poor precision and wide intraassay variation by both Nageotte hemocytometer and FC.
The Bland-Altman plot method was used for comparing two measurements of rWBC as assessed by Nageotte chamber and FC. The widespread of counts on plot revealed no consistent bias of one approach versus the other [Figure 4]. In our graph, many points lie outside the limits. The graph depicts no agreement between the two techniques for assessing rWBCs.
|Figure 4: Bland-Altman plot for measuring agreement between Nageotte hemocytometry and Flow cytometry|
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| Calculation of log reduction|| |
Log reduction in the number of leukocytes was calculated using the given formula-
a. For 350 ml bags (by Nageotte method)
= ([198 × 6771 × 1000] − [0.68 × 1000 × 198])/(198 × 6771 × 1000) ×100
=log 4 reduction
b. For 450 ml bags (by Nageotte method)
= ([259 × 6910 × 1000] − [0.64 × 1000 × 259])/(259 × 6910 × 1000) ×100
= log 4 reduction
c. For 350 ml bags (by Flow cytometry method)
= ([198 × 6771 × 1000] − [1.07 × 1000 × 198])/(198 × 6771 × 1000) ×100
= log 4 reduction
d. For 450 ml bags (by Flow cytometry method)
= ([259 × 6910 × 1000) − [1.05 × 1000 × 259))/(259 × 6910 × 1000) ×100
= log 4 reduction.
| Discussion|| |
As an ever-greater proportion of the blood supply is WBC reduced, greater numbers of units will be subject to QC testing. Because of insufficient precision in detecting low levels of WBCs, high investment cost, and requirement of highly trained staff, standard hematology analyzers such as the XT-2000i are not suitable for use.
The Nageotte hemocytometry was the first technique used for the assessment of rWBCs counts in LD blood components. It was found to be suitable for routine quality control testing. The drawbacks of the method being time consuming, low accuracy, and requirement of both technical expertise and considerable experience. The experience with dilution studies has shown that of the Nageotte hemocytometer performs poorly in terms of accuracy and precision at the rejection concentration of LD blood components and showed a bias to underestimation when compared to automated methods.,
The underestimation of rWBCs counts in the red cell concentrates samples by Nageotte hemocytometry as compared to FC might be due to the following reasons:-
- Incomplete lysis of red cells by Turk's solution thus preventing leukocyte sedimentation hence yielding a falsely low count
- Leukocytes may be destroyed or lost during sample processing.
On the other hand, 43 out of 303 (14%) samples run gave a Nageotte count more than FC count. This can be attributed to the following reasons:-
- There are indications that in freshly produced blood components' samples, the manual methods may demonstrate higher rWBCs counts
- Delay in sample processing and acquisition for FC analysis leading to leucocyte lysis and disintegration which is missed by FC but counted as leukocytes by the observer in Nageotte chamber
- Variations in time period between WB collection, PRBC preparation, leukofiltration, sample processing, and analysis may give false low or high count because of artifact production and cell lysis.
However, all of the above results were within the prescribed quality control range. In our study, rWBCs counts obtained by Nageotte hemocytometer were persistently lower than those obtained by FC, which is in agreement with a number of other similar results in the literature.,,,,,
Nageotte chamber usually produce results with a negative bias and has lower accuracy. Limiting the period of time between the collection, component production, leukoreduction, sample recovery, and treatment with preservative should help to minimize the source of error that might occur in some settings.
Appropriate steps were obtained to ensure that all products were processed and analyzed on the day of preparation. In our study, manual method failed to find leukocytes in 23 samples (7.6%) of the samples analyzed. This resulted in sample count of zero by Nageotte method. The causes of such results could be as follows:-
- High lower detection limit of Nageotte hemocytometer compared to the flow cytometric method
- Subjective error by the observer
- Misinterpretation of leukocytes for artifacts or debris causing their omission from final counts
- Failure of sedimentation of leukocytes because of improper or incomplete lysis of red cells by the Turk's solution.
Since Nageotte method is a manual method of rWBC enumeration, it is heavily dependent on the observer's technique, experience, and observation. Duplicate reading of a sample preferably by two independent observers is therefore recommended to improve upon the accuracy and the precision of test results. In our study, duplicate reading by one observer was obtained. Results can be improved upon if analysis is undertaken by multiple observers with adequate experience and skills.
Correlation between the two methods was weak in all the samples. Several studies have listed that for low concentrations of WBCs in leucoreduced blood products, counts assessed by Nageotte chamber and microscopy provided results with either comparable precision or less favorable than those observed by automated methods such as microfluorometry or FC.,,,
Furthermore, FC and Nageotte hemocytometry were poorly correlated, although they are assumed to measure the same quantity. The Nageotte method is still considered a reliable technique for processes validation. Even though the Nageotte's method is time consuming, only one percent of the LD products prepared in a blood centre is to be analyzed in a month for quality control purpose. Hence, its use could be implemented effectively. However, there are certain precautions that need to be exercised in a resource-poor blood centre, while using Nageotte hemocytometer for rWBC enumeration. These are as follows:-
- Meticulous technique of sample processing and acquisition on Nageotte chamber for analysis
- Regular training of staff and skill augmentation through workshops on quality control
- Duplicate reading preferably by two independent technicians
- Adequate time for leukocyte sedimentation after sample dilution and charging of chamber. This allows for the prevention of false labeling of PRBC unit as LD
- Variations in time taken for whole blood collection, component preparation, leukoreduction, sample recovery, and treatment with preservative should be minimized to reduce the source of error.
| Conclusion|| |
The absence of any strong correlation between Nageotte hemocytometer and FC implies that the two methods cannot be used interchangeably at a blood centre for quality control purposes. In addition, the Nageotte chamber counting seems to be unsuitable for quality control of the LD PRBC unit under real working blood bank conditions owing to underestimation of rWBCs counts. However, in the absence of adequate infrastructure, resources and trained workforce and to reduce the processing cost of leukoreduced PRBC units, Nageotte hemocytometer can be used as an alternative to FC in resource-constrained settings of developing countries.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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Department of Transfusion Medicine CH(CC), Lucknow - 226 002 Uttar Pradesh
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
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