| Abstract|| |
BACKGROUND: Leukocytes are responsible for producing both immune and nonimmune adverse reactions, and therefore, various methods have been developed to remove them from the blood components before transfusion.
AIM: The aim of this study was to analyze the quality parameters in leukoreduced red cell concentrates (RCCs) and investigate the efficiency of leukocyte removal and red cell recovery in the leukoreduction methods followed in our center.
MATERIALS AND METHODS: The study evaluated the quality parameters in 112 RCCs prepared using buffy-coat reduction by the Terumo automatic component extractor II+ system, manual saline washing, and leukofiltration using the Leucolab filter system.
RESULTS: With analysis, leukofiltration was found to be the most efficient in reducing leukocyte content in RCCs, achieving a mean leukoreduction of 99.99%. Buffy-coat reduction and saline washing achieved a leukoreduction of 78.54% and 82.67%, respectively. While filtration showed the least red cell recovery of 81.93% compared to 90.57% in buffy-coat reduction and 91.87% in saline washing methods. An analysis of hemoglobin content showed that none of the buffy-coat removed RCCs processed from 350-ml collections and underwent poststorage leukofiltration could meet the European Standards for minimum hemoglobin content.
CONCLUSION: Filtration is found to be the better method for leukoreduction of RCCs. It is suggested to perform a single method of leukoreduction preferably leukofiltration for maximum red cell recovery.
Keywords: Buffy coat, leukofiltration, leukoreduction, Nageotte, quality control, red cell washing
|How to cite this URL:|
Chand S, Rudrappan RB, Gupta D. Leukoreduced red cell concentrates: Are they meeting the quality standards?. Asian J Transfus Sci [Epub ahead of print] [cited 2022 Dec 4]. Available from: https://www.ajts.org/preprintarticle.asp?id=356856
| Introduction|| |
Leukocytes are a constituent of human blood that forms the body's primary defense against bacterial pathogens. However, the role of donor leukocytes in transfused blood products is limited. Rather they are responsible for various transfusion reactions in the recipient such as febrile nonhemolytic transfusion reaction (FNHTR), human leukocyte antigen alloimmunization, and platelet refractoriness, as well as transmission of infectious agents like the cytomegalovirus. As leukocytes are a major reason for producing immune and nonimmune transfusion reactions, there have been several attempts to remove them from the blood components before transfusion. The concept of leukocyte removal from the blood was introduced by Fleming as early as 1920 when he passed blood through a bent glass tube having a cotton wool plug and a constricted limb. Leukoreduction can be defined as the process by which white blood corpuscles are removed from the blood components, and the leukoreduction technologies are constantly evolving to provide the standard of care treatment.
There are several acceptable techniques for leukoreduction that vary in their complexity and equipment required. In our center, buffy-coat reduction, saline washing, and leukofiltration methods are performed as per the requirement of the patient. The objective of this study was to analyze the quality parameters in leukoreduced red cell concentrates (RCCs) prepared by buffy-coat reduction, washing, and filtration and to determine the efficacy of these leukoreduction methods. The study was approved by the institutional ethics committee.
| Materials and Methods|| |
Data collected as part of the quality control program for a period of 1 year from October 2019 to September 2020 were included in this study. As per the recommendations from the Directorate General of Health Services (DGHS), Government of India (GoI), the frequency of quality control for leukoreduced red cells is 4 units/month. Hence, 48 buffy-coat reduced, and 48 leukofiltered RCCs were sampled. Only 16 RCCs were washed during the 1-year study period and were included in the study.
Preparation of leukoreduced RCC
Under sterile precautions, 350- (±10%) or 450 (±10%)-ml whole blood (WB) was collected from eligible donors into conventional blood collection bags (Terumo Penpol Private Limited, Puliyarakonam, Trivandrum, India) containing anticoagulant solution with or without an additive solution.
A total of 48-WB units collected in 350-ml (n = 16) or 450-ml (n = 32) quadruple blood collection bags with top and bottom configuration, containing 49 or 63 ml of citrate-phosphate-dextrose (CPD), respectively, were buffy-coat reduced on the day of collection using a semi-automated blood component extractor (Terumo automatic component extractor [T-ACE] II+, Automatic Component Extractor, Terumo BCT). The leukoreduced RCC was separated into a secondary bag containing additive solution saline-adenine-glucose-mannitol (SAGM), and plasma was extracted out, leaving behind buffy-coat in the parent bag which was discarded.
A total of 16 RCC processed from WB collected in 450-ml (n = 5) or 350-ml (n = 11) double bags anticoagulated with CPD adenine and stored were washed irrespective of the storage age using 0.9% saline. Saline was manually added, mixed, and centrifuged to remove the supernatant, and the whole process was repeated three times. The leukoreduced RCC was suspended in saline and issued within 24 h from the time of opening the bag.
Poststorage leukofiltration at room temperature was performed in the blood center before issue in 48 RCCs <10 days old using the Leucolab (Maco Pharma, France) filters which are suitable for filtering RCCs suspended in CPD-SAGM. We filtered buffy-coat reduced CPD-SAGM suspended RCCs processed from both 450-ml (n = 14) and 350-ml (n = 13) collections as well as CPD-SAGM suspended RCCs processed from 350-ml WB collected in triple bag system (n = 21).
Red blood cell indices were determined using an automated hematology analyzer (MEK-6420, Nihon Kohden). A manual counting method using the Nageotte chamber was used to determine the residual leukocytes in filtered RCCs. Red cell supernatants were prepared by centrifugation at 3000 g for 5 min (Megafuge 1.0, Terumo Fisher Scientific). The supernatant of the centrifuged sample was used for measuring plasma hemoglobin using a plasma/low hemoglobin system (HemoCue India) and extracellular potassium using a biochemistry analyzer (Dimension RxL Max Integrated Chemistry System, Siemens). Sterility testing was performed in all the units tested using an automated microbial detection system (BacT/Alert, Organon Teknika Corp.).
To compare pre- and postprocedure continuous variables, the paired t-test was used. The ANOVA test was used to compare the three leukoreduction methods, and the post hoc analysis was performed to do multiple comparisons. Quantitative variables were expressed as mean and standard deviation. P < 0.05 was considered statistically significant. Data analysis was performed using SPSS version 16.0.
| Results|| |
Leukocyte counts were significantly reduced by all three methods of leukoreduction, however, only the filtration technique could consistently achieve leukocyte levels <1× 106 in the RCCs [Table 1] and [Table 2]. As shown in [Table 3] and [Figure 1], maximum leukoreduction was achieved with the filtration and least with the buffy-coat reduction method. The leukoreduction ranged from 78.54 ± 10.39% with buffy-coat reduction to 82.67 ± 7.44% (P > 0.05) and 99.99 ± 0.01% (P < 0.05) with washing and filtration, respectively.
|Table 1: Leukocyte count and hemoglobin content in red cell concentrates processed from 350 ml collections|
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|Table 2: Leukocyte count and hemoglobin content in red cell concentrates processed from 450-ml collections|
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|Figure 1: Leukoreduction of red cell concentrates. Mean results obtained with buffy-coat reduction, washing, and filtration. The standard deviations are represented by vertical bars. Bar graphs followed by different letters indicate significant differences (P < 0.05)|
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Red cell recovery
The red cell recovery was found to be least with the filtration procedure as shown in [Table 3] and [Figure 2]. Filtered RCCs gave a red cell recovery of only 81.93 ± 3.78% compared to 90.57 ± 4.05% (P < 0.05) and 91.87 ± 3.73% (P < 0.05) with buffy-coat reduction and washing methods, respectively.
|Figure 2: Red cell recovery in leukoreduced red cell concentrates. Mean results obtained with buffy-coat reduction, washing, and filtration. The standard deviations are represented by vertical bars. Bar graphs followed by different letters indicate significant differences (P < 0.05)|
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Hemoglobin content in the RCCs was significantly reduced following buffy-coat reduction and filtration procedures [Table 1] and [Table 2]. Buffy-coat reduced RCCs processed from 450ml to 350ml collections had mean hemoglobin content of 57.38 ± 5.29 g and 45.06 ± 4.71 g, respectively. Mean hemoglobin content of 43.86 ± 2.92 g was seen in the filtered RCCs processed from 350-ml collections. However, following filtration of the buffy-coat reduced RCCs, we obtained mean hemoglobin content of 47.36 ± 5.80 g in the RCCs prepared from 450-ml collections and only 33.23 ± 3.68 g in those prepared from 350-ml collections.
Plasma hemoglobin and potassium
A significant reduction of the plasma hemoglobin and potassium content in the RCCs was noted with buffy-coat reduction and saline washing procedures. However, in the filtered RCCs, there was a significant rise in potassium levels immediately following filtration, although the plasma hemoglobin content did not alter significantly. The plasma hemoglobin and potassium levels in the leukoreduced RCCs are listed in [Table 4].
|Table 4: Plasma hemoglobin and potassium levels in leukoreduced red cell concentrate|
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| Discussion|| |
Leukoreduction methods have become a major part of blood transfusion services in delivering optimum patient care. This study focused on quality parameters in 112 leukoreduced RCCs prepared by buffy-coat reduction using the T-ACE II+ system, manual saline washing, and leukofiltration using the Leucolab filter system. When the three methods of leukoreduction were compared, filtration provided the best results in terms of leukocyte removal, achieving mean absolute leukocyte count of <1 × 106, accounting for leukoreduction of 99.99%, and the new generation leukofilters were shown to achieve this mark.
Quality standards for leukoreduced red cell preparations are laid down by various National and International Organizations and are adapted by the transfusion services across the world. The data on quality parameters in the leukoreduced RCCs from the study were compared with the quality recommendations from the Association for the advancement of Blood and Biotherapies (AABB), Council of Europe (CoE), and DGHS, GoI which are listed in [Table 5].,,
|Table 5: Current guidelines relating to buffy-coat reduced, washed, and filtered red cell concentrates|
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The RCCs leukoreduced by buffy-coat removal using the T-ACE II+ system satisfied both CoE and DGHS, GoI guidelines. However, when intended to prevent febrile transfusion reactions, leukocytes in the final component shall be <5 × 108 as per the recommendations which were achieved in only <46% of the buffy-coat reduced RCCs in this study. A similar study using the T-ACE system also showed a mean leukocyte count >5× 108. Hence, buffy-coat reduction using the T-ACE system may not be effective to prevent FNHTR. Nevertheless, a significant reduction in leukocyte content was observed following buffy-coat reduction method.
For washed RCCs, the minimum hemoglobin content as per the CoE guidelines was achieved only in 87% of the units tested. The two units which had low hemoglobin levels after washing were those processed from 350-ml collections. The CoE guidelines for hematocrit in washed RCCs were met only in 81% of units tested. However, the hematocrit levels can vary with the volume of saline used for resuspending the erythrocyte sediment after the last wash., Manual saline washing achieved a good red cell recovery, but DGHS recommendation for leukoreduction was met in <40% of the units tested. Still, leukoreduction obtained in our study was consistent with the values from previous studies on manual washing by other investigators.,
The washing procedure comes with its limitations such as the time consumption, availability of the equipment as well as handling and expertise of the technical staff. If an open method of washing is performed, it comes with an additional disadvantage of early expiry, the chance of contamination, and also washing techniques cannot bring down the leukocyte content in the RCCs to a level enough to prevent certain transfusion reactions. Even then, red cell washing is a method of choice in conditions like IgA deficiency because of its ability to remove plasma content, thereby preventing plasma protein-associated transfusion reactions.,
When the methods of leukoreduction were compared in terms of their ability to reduce leukocyte content, filtration provided the best results. Even though the mean absolute leukocyte count was lower in the buffy-coat reduced RCCs that were filtered compared to the filtered RCCs from which buffy-coat was not removed, all the RCCs filtered using the Leucolab filter system achieved absolute leukocyte count <1 × 106, which met the AABB, CoE, and DGHS recommendations. However, when red cell recovery was considered, only <25% of the filtered RCCs achieved 85% red cell recovery thereby not satisfying the AABB or DGHS criteria. However, red cell loss is inherent with the filtration procedure which represents the dead space volume in the filter set used, and the Leucolab filter showed the lowest red cell yield when compared with seven other leukofilters in a study by Musuraca and colleagues., Leukofiltration was also shown to significantly increase potassium levels which can be attributed to the mechanical force generated by filtration. However, we did not observe a significant change with plasma hemoglobin levels probably because sampling was performed immediately after the procedure, and the unit was not stored for analysis.
In our study, the buffy-coat-reduced red cell units processed from 350-ml collections and underwent filtration did not meet the CoE standards for minimum hemoglobin content compared to the filtered RCCs from which buffy-coat was not removed. This signifies the red cell loss that can happen when buffy-coat reduction and leukofiltration methods are combined, as previously reported. There was another report of increased hemolysis in RCCs during storage when buffy-coat reduction and filtration were combined, showing no definite advantage of the combination method over filtration. The findings in our study led us to the conclusion that performing filtration using the available new generation leukofilters is more beneficial than combining buffy-coat reduction and filtration procedure in a red cell unit. This is especially important when RCCs processed from 350-ml WB collections are considered as the volume of the bag itself is a significant factor affecting hemoglobin content in the final product. Furthermore, the red cell recovery and hemoglobin content can vary depending on the filter characteristics. Further studies are necessary using other leukofiltration systems available in the market and should also focus on the effect of prestorage leukofiltration in the buffy-coat reduced RCCs.
| Conclusion|| |
The current generation of leukofilters could achieve 4 log reductions in leukocyte content. Various countries have adopted universal leukoreduction policies to leukoreduce all the blood components prepared., While for resource-poor countries, selective leukoreduction of blood components using new generation leukofilters is a viable option to prevent leukocyte-associated adverse transfusion reactions in the recipients and improve the outcome, particularly of the multitransfused patient population. However, performing poststorage leukofiltration of the buffy-coat reduced red cell units should be done with precaution as they may not meet the quality recommendations and can result in increased transfusion requirements in recipients and this should be taken care of when the leukoreduction protocols are being formulated by the blood transfusion services.
| References|| |
Hervig T, Seghatchian J. Leukocyte-reduced blood components: Laboratory and clinical aspects. In: Simon TL, McCullough J, Snyder EL, Solheim BG, Trauss RG, editors. Rossi's Principles of Transfusion Medicine. Chichester, WestSussex: John Wiley & Sons, Ltd; 2016. p. 278-85.
Williamson LM. Leucocyte depletion of the blood supply – How will patients benefit? Br J Haematol 2000;110:256-72.
Sharma RR, Marwaha N. Leukoreduced blood components: Advantages and strategies for its implementation in developing countries. Asian J Transfus Sci 2010;4:3-8.
] [Full text]
Saran RK, editor. Transfusion Medicine Technical Manual. 2nd
ed. New Delhi: DGHS, Ministry of Health and Family Welfare, Government of India; 2003.
Musuraca V, Santilli E, Leonardo P, D'ettoris AR, Vecchio S, Geremicca W. Quality control of leucodepleted products. A comparative analysis through interleukin assays and residual leucocyte count. Blood Transfus 2005;3:144-56.
Fung MK, Grossman BJ, Hillyer CD, Westhoff CM, editors. Technical Manual. 18th
ed. Maryland: AABB; 2014.
Council of Europe. Recommendation NO.R (95) 15 Guide to the Preparation, use and Quality Assurance of Blood Components. 20th
ed. Strasbourg: Council of Europe; 2020.
Sonker A, Dubey A, Chaudhary R. Evaluation of a red cell leukofilter performance and effect of buffy coat removal on filtration efficiency and post filtration storage. Indian J Hematol Blood Transfus 2014;30:321-7.
Tóth CB, Kramer J, Pinter J, Thék M, Szabó JE. IgA content of washed red blood cell concentrates. Vox Sanguinis 1998;74:13-4.
Cardigan R, New HV, Tinegate H, Thomas S. Washed red cells: Theory and practice. Vox Sang 2020;115:606-16.
Polesky HF, McCullough J, Helgeson MA, Nelson C. Evaluation of methods for the preparation of HL-A antigen-poor blood. Transfusion 1973;13:383-7.
Langfelder M, Jakschitz M, Jánossy A. Comparison of different methods used in the preparation of leucocyte-free whole blood and erythrocyte concentrates. Vox Sang 1970;19:57-63.
Aston A, Cardigan R, Bashir S, Proffitt S, New H, Brown C, et al
. Washing red cells after leucodepletion does not decrease human leukocyte antigen sensitization risk in patients with chronic kidney disease. Pediatr Nephrol 2014;29:2005-11.
Tobian AA, Savage WJ, Tisch DJ, Thoman S, King KE, Ness PM. Prevention of allergic transfusion reactions to platelets and red blood cells through plasma reduction. Transfusion 2011;51:1676-83.
Müller-Steinhardt M, Hennig H, Kirchner H, Schlenke P. Prestorage WBC filtration of RBC units with soft-shell filters: Filtration performance and impact on RBCs during storage for 42 days. Transfusion 2002;42:153-8.
Ran Q, Hao P, Xiao Y, Zhao J, Ye X, Li Z. Effect of irradiation and/or leucocyte filtration on RBC storage lesions. PLoS One 2011;6:e18328.
Müller-Steinhardt M, Janetzko K, Kandler R, Flament J, Kirchner H, Klüter H. Impact of various red cell concentrate preparation methods on the efficiency of prestorage white cell filtration and on red cells during storage for 42 days. Transfusion 1997;37:1137-42.
Rudrappan RB, Gupta D, Mohan L. A comparative analysis of factors influencing haemoglobin content in RBC units. Transfus Apher Sci 2021;60:103214. [doi: 10.1016/j.transci. 2021.103214].
Yazer MH, Podlosky L, Clarke G, Nahirniak SM. The effect of prestorage WBC reduction on the rates of febrile nonhemolytic transfusion reactions to platelet concentrates and RBC. Transfusion 2004;44:10-5.
Paglino JC, Pomper GJ, Fisch GS, Champion MH, Snyder EL. Reduction of febrile but not allergic reactions to RBCs and platelets after conversion to universal prestorage leukoreduction. Transfusion 2004;44:16-24.
Department of Transfusion Medicine, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Thiruvananthapuram - 695 011, Kerala
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]