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 Table of Contents  
ORIGINAL ARTICLE
Year : 2016  |  Volume : 1  |  Issue : 2  |  Page : 43-45

Evaluation of two methods for counting residual leukocytes in leuko-reduced platelets: Nageotte's method and flow cytometry


1 Department of Transfusion Medicine, TATA Medical Centre, Kolkata, West Bengal, India
2 Department of Laboratory Hematology, TATA Medical Centre, Kolkata, West Bengal, India

Date of Web Publication6-Sep-2016

Correspondence Address:
Rizwan Javed
Department of Transfusion Medicine, TATA Medical Centre, Kolkata, West Bengal
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2455-8893.189854

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  Abstract 


Introduction: Leukoreduced (LR) blood components are used for the prevention of several transfusion adverse effects. Advancement in technology has led to newer methods to count residual leukocytes (rWBC) which miss detection on most standard automated hematology analyzers.
Materials and Methods: Samples from thirty eight platelet concentrates (prepared by Buffy-coat method) were randomly taken on the day of preparation for rWBC count using Nageotte's chamber and flowcytometer.
Results: The rWBC count on Nageotte's ranged from 2.5 WBC/μL to a maximum of 600 WBC/μL where as the flowcytometric count had a lowest of 1.97 WBC/μL to a highest of 740 WBC/μL. We found that the WBC counts using the Nageotte's method and flowcytometeric method are highly correlated. The concordance correlation coefficient or intraclass correlation coefficient which is a measure of reliability was 0.78
Conclusion: In view of the high concordance in correlation coefficient between the two methods, Nageotte's method could be skillfully performed for assessing leukoreduction in LR platelet concentrates of resource constrained blood banks of developing nations.

Keywords: Flow cytometry, leukoreduced, Nageotte's


How to cite this article:
Javed R, Basu S, Mishra DK. Evaluation of two methods for counting residual leukocytes in leuko-reduced platelets: Nageotte's method and flow cytometry. Glob J Transfus Med 2016;1:43-5

How to cite this URL:
Javed R, Basu S, Mishra DK. Evaluation of two methods for counting residual leukocytes in leuko-reduced platelets: Nageotte's method and flow cytometry. Glob J Transfus Med [serial online] 2016 [cited 2019 Jun 20];1:43-5. Available from: http://www.gjtmonline.com/text.asp?2016/1/2/43/189854




  Introduction Top


Leuko-reduced (LR) blood components are widely used for the prevention of several transfusion adverse effects. Advancement in technology has led to newer methods to count residual white blood cells (rWBC) which miss detection on the most standard automated hematology analyzers.

To ensure good quality of LR products, it is imperative to routinely monitor the efficacy of leukoreduction in blood products. New enumeration methods such as flow cytometry [1],[2],[3],[4],[5],[6] and microvolume fluorometry [1],[2],[3],[6] have replaced manual methods such as Nageotte hemocytometer and microscopy.[1],[3],[4],[6]

We undertook this study at our center to weigh the pros and cons of a newer method over the contemporary ones in measuring rWBC in LR platelet products.


  Materials and Methods Top


Segments of 38 platelet concentrates (prepared by Buffy-coat method) were randomly taken on the day of preparation, from October 1, 2015 to October 15, 2015. After stripping the segments of platelet concentrates, the segments were sealed into two parts for measurement using the Nageotte's chamber and flow cytometer which were performed by different persons.

Automated method for counting rWBCs in buffy-coat platelet concentrates: BD Leucocount reagent, USA, was used for Leucocount assay. A volume of 100 μL of platelet sample was mixed with 400 μL of BD Leucocount reagent in each Tru-count test tube. The tubes were gently vortexed (maximum up to 15 s) and then, incubated for 5 minutes in dark at room temperature. Finally, the samples were acquired using Flow cytometer (BD FACSCanto II, USA) in low flow rate, and a minimum of 10,000 events were acquired.

After acquisition, analysis was performed by a predefined template. The template was prepared by processing samples of ten healthy individuals. WBC events (R2) and Beads events (R1) were used for final calculation from the acquired data.

The absolute number of rWBC was then calculated by using the formula given below:



Manual method of counting residual white blood cell (Nageotte's)

One hundred microliter of the platelet sample was mixed well with 400 μL of Turk's fluid in a clean test tube by pipetting several times. The mixed sample was loaded on the hemocytometer with a cover slip by using a pipette. The charged hemocytometer was placed in a  Petri dish More Details with a damp filter paper for 10–15 minutes so as to allow the WBCs to settle over the counting chamber. Then, the hemocytometer was focused under a microscope using a 20× objective. WBCs appeared as gray-blue refractile cells bearing a nucleus. All the WBC present in 50 μL volume of the Nageotte's counting chamber were counted and the following formula was applied to know the final count.

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

Total WBC count in the LR product = WBC/μL ×1000 × product volume (ml).


  Results Top


A total of 38 platelet concentrates were analyzed. The rWBC count on Nageotte's ranged from 2.5 WBC/μL to a maximum of 600 WBC/μL whereas the flow cytometry count had a lowest of 1.97 WBC/μl to a highest of 740 WBC/μl. The mean rWBC count was higher in flow cytometry than Nageotte's method [Table 1].
Table 1: Comparison of measurements by Nageotte's and flow cytometry methods

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Statistics

The WBC counts using the Nageotte's and flow cytometric methods are highly correlated (Pearson's correlation coefficient: r = 0.9122). Using the Bland–Altman method, mean difference between the two methods was 15.3 units. Ninety-five percent limits of agreement is −10 to +41. The concordance correlation coefficient or intraclass correlation coefficient which is a measure of reliability was 0.78.


  Discussion Top


In several studies, Nageotte's method results were lower than those obtained by flow cytometry.[1], 2, [7],[8],[9],[10] However, in some studies, WBC counts in LR red blood cells (RBCs) were higher when measured by Nageotte's method than by flow cytometry.[4],[11],[12]

In our study, many of our platelet products had a high count on Nageotte's method than that on flow cytometry. This could be because of artifacts as the same Nageotte's chamber, and slide cover was repeatedly used and also because the flow cytometry gating was setup to encompass only intact WBCs. It is believed that products having an increased pre-storage leukoreduction time increases the number of WBC fragments and cell-free DNA in the LR products,[5],[13],[14],[15],[16],[17],[18] which may interfere with rWBC counts. Accurate values of rWBC could be achieved by minimizing the time between collection, component preparation, leukoreduction, sample recovery, and addition of Pallfix preservative. Hence, we ensured that all our products were analyzed on the same day of preparation to avoid such inadequacies. Other studies have found no significant difference in the values of rWBC counts for platelet products prepared from citrated or ethylenediaminetetraacetic blood collections, irrespective of the enumeration method.[19]

In fact, Van der Meer et al.[18] did not use any WBC preservative to stabilize samples in spite of doing leukoreduction after 24 hours. Many studies have reported that rWBC counts by Nageotte's hemocytometer and microscopy in LR RBC and platelet products were not as precise as the results obtained from the automated methods such as microfluorometry or flow cytometry.[1],[9],[10]

Lutz and Dzik [8] noted a correlation coefficient of r = 0.9995 comparing Nageotte's method and flow cytometry results in the range of 0.3–18.4 WBCs per mL for LR apheresis platelet concentrates. rWBC counts on stabilized samples measured by EPICS XL-MCL flow cytometer gave results which were precise, specific, reliable, accurate, and sensitive. Nageotte's method provided similar sensitivity (0.1 WBC/mL), precision, and reliability, when done by experienced technologists, yet was less acceptable in terms of specificity and accuracy.[20] In our study, the two methods were equally good, and in resource-constrained settings of developing countries, Nageotte's chamber could be best used to reduce the cost. Even though the Nageotte's method is time-consuming, only 1% of the LR products prepared are to be analyzed in a month for quality control purpose. Hence, it could be practiced conveniently.


  Conclusion Top


Our study demonstrated a good correlation between the Nageotte's and flow cytometry methods for counting rWBC in LR platelet concentrates. Given the high concordance in correlation coefficient between the two methods, Nageotte's method could be skillfully performed for assessing leukoreduction in LR platelet concentrates of resource-constrained blood banks of developing nations.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
  References Top

1.
Dzik S, Moroff G, Dumont L. A multicenter study evaluating three methods for counting residual WBCs in WBC-reduced blood components: Nageotte hemocytometry, flow cytometry, and microfluorometry. Transfusion 2000;40:513-20.  Back to cited text no. 1
    
2.
Van der Meer PF, Gratama JW, van Delden CJ, Laport RF, Levering WH, Schrijver JG, et al. Comparison of five platforms for enumeration of residual leucocytes in leucoreduced blood components. Br J Haematol 2001;115:953-62.  Back to cited text no. 2
    
3.
Krailadsiri P, Seghatchian J, Rigsby P, Bukasa A, Bashir S. A national quality assessment scheme for counting residual leucocytes in unfixed leucodepleted products: The effect of standardization and 48 hour storage. Transfus Apher Sci 2002;26:73-81.  Back to cited text no. 3
    
4.
Conte R, Bontadini A, Cirillo D, Fruet F. Process control of filtered red blood cells: Which counting method? Transfus Med 1997;7:217-9.  Back to cited text no. 4
    
5.
Bashir S, Cardigan R. The origin and identification of unknown events associated with low-level leucocyte counting by flow cytometry. Vox Sang 2003;85:190-8.  Back to cited text no. 5
    
6.
Seghatchian J, Krailadsiri P, Chandegra B, Beard M, Beckman N, Bissett L, et al. National evaluation of IMAGN 2000 for quality monitoring of leucodepleted red cell and platelet concentrates: Comparison with flow and Nageotte. Transfus Sci 2000;22:77-9.  Back to cited text no. 6
    
7.
Dzik WH, Ragosta A, Cusack WF. Flow-cytometric method for counting very low numbers of leukocytes in platelet products. Vox Sang 1990;59:153-9.  Back to cited text no. 7
    
8.
Lutz P, Dzik WH. Large-volume hemocytometer chamber for accurate counting of white cells (WBCs) in WBC-reduced platelets: Validation and application for quality control of WBC-reduced platelets prepared by apheresis and filtration. Transfusion 1993;33:409-12.  Back to cited text no. 8
    
9.
Backteman K, Ledent E, Berlin G, Ernerudh J. A rapid and reliable flow cytometric routine method for counting leucocytes in leucocyte-depleted platelet concentrates. Vox Sang 2002;83:29-34.  Back to cited text no. 9
    
10.
Barclay R, Walker B, Allan R, Reid C, Duffin E, Kane E, et al. Flow cytometric determination of residual leucocytes in filter-depleted blood products: An evaluation of Becton-Dickinson's LeucoCOUNT system. Transfus Sci 1998;19:399-403.  Back to cited text no. 10
    
11.
Bontadini A, Tazzari PL, Manfroi S, Tassi C, Conte R. Apoptosis in leucodepleted packed red blood cells. Vox Sang 2002;83:35-41.  Back to cited text no. 11
    
12.
Sheckler VL, Loken MR. Routine quantitation of white cells as low as 0.001 per microL in platelet components. Transfusion 1993;33:256-61.  Back to cited text no. 12
    
13.
Dijkstra-Tiekstra MJ, van der Schoot CE, Pietersz RN, Reesink HW. White blood cell fragments in platelet concentrates prepared by the platelet-rich plasma or buffy-coat methods. Vox Sang 2005;88:275-7.  Back to cited text no. 13
    
14.
Hérault O, Binet C, Rico A, Degenne M, Bernard MC, Chassaigne M, et al. Evaluation of performance of white blood cell reduction filters: An original flow cytometric method for detection and quantification of cell-derived membrane fragments. Cytometry 2001;45:277-84.  Back to cited text no. 14
    
15.
Dijkstra-Tiekstra MJ, van der Schoot CE, Pietersz RN, Huijgens PC, van der Meer PF, Reesink HW. Development of white blood cell fragments, during the preparation and storage of platelet concentrates, as measured by using real-time polymerase chain reaction. Vox Sang 2004;87:250-6.  Back to cited text no. 15
    
16.
Wagner T, Guber SE, Stubenrauch ML, Lanzer G, Neumueller J. Low propidium iodide intensity in flow cytometric white blood cell counting as a marker of cell destruction? Transfusion 2005;45:228-33.  Back to cited text no. 16
    
17.
Janatpour K, Paglieroni TG, Schuller L, Foley K, Rizzardo T, Holland PV. Interpretation of atypical patterns encountered when using a flow cytometry-based method to detect residual leukocytes in leukoreduced red blood cell components. Cytometry 2002;50:254-60.  Back to cited text no. 17
    
18.
Van der Meer PF, de Wildt-Eggen J. The effect of whole-blood storage time on the number of white cells and platelets in whole blood and in white cell-reduced red cells. Transfusion 2006;46:589-94.  Back to cited text no. 18
    
19.
Seghatchian J, Krailadsiri P, McCall M. Statistical process monitoring of WBC-reduced blood components assessed by two types of software. Transfusion 2001;41:102-5.  Back to cited text no. 19
    
20.
Palmer DS, Birch P, O'Toole J, Henderson D, Scalia V. Flow cytometric determination of residual white blood cell levels in preserved samples from leukoreduced blood products. Transfusion 2008;48:118-28.  Back to cited text no. 20
    



 
 
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