Need to reconsider national quality standards for red cell components: Evidence from a retrospective observational analysis

Material & Methods

This study was retrospective and observational, conducted at the Department of Transfusion Medicine, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, a tertiary care referral centre in northern India with due approval of the Institute Ethics Committee (IEC). At the study hospital, WB units (450 ml/350 ml) are routinely subjected to the preparation of RCCs (RCC₄₅₀/RCC₃₅₀) in accordance with departmental protocols. Data of such routine QC testing of RCCs over a period of 10 years (September 2009 to August 2019) was analyzed in this study QC criteria/standards published by three national^3,4,17 and three international^15,18,19 regulatory or accreditation agencies were used as reference to assess the QC results of RCCs prepared during the study period.

Results

During the study period, 236,255 RCCs (RCC₃₅₀=89,254, RCC₄₅₀=147,001) were prepared, out of which 5,861 (2.48%) units were subjected to routine QC testing. Data from 643 units was excluded due to incomplete records/missing donor data (n=603) and unit modifications after preparation (n=40). Thus, data of 5,218 (RCC₃₅₀=2,433, RCC₄₅₀=2,785), 2.21 per cent of the units prepared, were included in the analysis. This accounted for 2.73 per cent and 1.89 per cent, respectively, of the total RCC₃₅₀ and RCC₄₅₀ units prepared during this period. According to the component processing method, there were 297 units of RC₃₅₀, 2,136 units of RCC₃₅₀-AS(TAT), 2,411 units of RCC₄₅₀-AS(TAT) and 374 units of RCC₄₅₀-AS(TAB). The donor characteristics for the different types of RCC are shown in Table I. The unit characteristics (QC parameters) are also summarized in Table II.

Effect of component processing method on the quality of RCC₄₅₀

The proportions of units meeting the three national and three international standards were comparatively higher for RCC₄₅₀-AS(TAT) than RCC₄₅₀-AS(TAB), except for WBC content, which met the EDQM criteria in 37.5 per cent of the former and 83.4 per cent of the latter (Table III). RCC₄₅₀-AS(TAT), had significantly higher volume as compared to RCC₄₅₀-AS(TAB), Hct, Hb content and WBC content (Table II). The WBC content had a weak positive correlation with Hb content (ρ=0.2, P<0.001) and a weak negative correlation with days of storage (ρ=–0.054, P=0.004).

The variation in QC parameters across the four different types of RCC are shown in Figure.

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Figure.

Variation in QC parameters across different types of red cell concentrates (RCC). (A) Volume of PRBC (B) Haematocrit of PRBC (C) Hb content of PRBC (D) WBC content of PRBC; 1=RC₃₅₀, 2=RCC₃₅₀-AS(TAT), 3=RCC₄₅₀-AS(TAT), 4=RCC₄₅₀-AS(TAB). Black horizontal lines and vertical error bars represent the mean and one standard deviation, respectively. Dots represent individual observations. Dotted lines and shades represent the proposed standards in Table V. Data were expressed as mean±standard deviation P* <0.05, **<0.001; Mann-Whitney U test. PRBC, packed red blood cell.

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Discussion

Stored blood and its components, including RCCs, are considered as drugs whose therapeutic benefit for the recipient depends on their quality as determined by QC testing. In this study, we compared our QC data to Indian as well as international standards which are widely accepted. We observed that RCCs prepared at our institute complied better with the Hct criteria as described by EDQM rather than the criteria as mentioned in the national standards (NABH, D&C Rules or DGHS) (Table III).

Hb content of ≥43 g/unit (EDQM)¹⁵ was observed in 93.9 per cent of RCC₄₅₀-AS(TAT) and 60.2 per cent of RCC₄₅₀-AS(TAB). Currently, there are no predefined criteria for Hb content of RCC₄₅₀/RCC₃₅₀ in the Indian standards. In India, for WB donation, the donor must have a minimum Hb 12.5 g/dl³. Therefore, theoretically, the least expected Hb content of WB collected from such donor is 56.25 g/43.75 g for 450 ml/350 ml. If 43 g/unit is taken as the minimum acceptable Hb content of RCC₄₅₀ processed by the BC method (RCC₄₅₀-AS), the loss in Hb during processing is ∼13.25 g (23.56%). Assuming a similar (23.56%) loss of Hb during processing of RCC₃₅₀ by the BC method, the estimated minimum expected Hb content is ∼33.4 g/unit, which was satisfactory for 94.3 per cent of our tested RCC₃₅₀ units: 83.5 per cent for RC₃₅₀ and 95.8 per cent for RCC₃₅₀-AS(TAT). A cut-off of 33.4 g/unit is above 30 g/unit¹⁹, which is the minimum acceptable Hb content of RCC that may be used for transfusion. With no standards available for the Hb content of RCC₃₅₀, 33.4 g/unit may be considered in our setting for RCC₃₅₀-AS. Similarly, 35 g/unit is estimated as the minimum Hb content of RCC₃₅₀ prepared by the PRP method (RC₃₅₀), taking a reference of 45 g/unit as the minimum requirement as per EDQM¹⁵ criteria for red cells.

Among RCC₃₅₀ units, RC₃₅₀ had lower Hct and Hb content, possibly due to a significantly higher proportion of female donors with expected lower Hb as compared to RCC₃₅₀-AS(TAT) (Table I). Another reason could be the difference in processing (manual for PRP vs. semi-automated for BC method) leading to higher red cell losses. Since a significant number of WBCs from the WB is removed (near one-log reduction)⁴ by the BC method, we observed lower WBC content in RCC₃₅₀-AS(TAT) than in RC₃₅₀.

Among RCC₄₅₀, lower Hct, Hb content and WBC content were observed in RCC₄₅₀-AS(TAB) than in RCC₄₅₀-AS(TAT), implicating higher red cell loss in B-TAB as compared to the B-TAT technique. This finding was in concordance with that of Cid et al¹⁶. We observed that units having lower WBC content also had lower Hb content, which may suggest that the method was more effective in reducing WBCs from WB also results in more red cell losses. A high demand/supply ratio of RDPs for our Hemato-Oncology patients has been observed in recent years, and therefore, the priority is to prepare RDPs from most of the WB collections. Hence, after implementation of the B-TAB technique, the component preparation method was recalibrated to maintain adequate volume and platelet yield with minimum cellular contamination of RDPs, which resulted in a lower volume of RCC₄₅₀, reflecting as a lower Hb content of RCC₄₅₀-AS(TAB).

We also observed a higher variation in volumes of our RCC₄₅₀-AS(TAB) units (SD ∼40 ml) as compared to that of our RCC₄₅₀-AS(TAT) units (SD ∼19 ml), which was contrary to that observed by Cid et al¹⁶ (SD of 21 ml and 25 ml, respectively, for B-TAB and B-TAT methods). Our findings could be explained by the frequent calibration of the B-TAB method in accordance with the institutional requirements as stated above.

The current DGHS⁴ criteria requires the WBC content of BC reduced RCCs to be <5×10⁸ cells/unit; there are no specific criteria for the WBC content of RCC₃₅₀ in the Indian standards. The maximum estimated WBC content of RC₃₅₀ was found to be ∼3.09×10⁹ cells/unit (mean+3SD) in this study. Since no RC₄₅₀ was prepared at our centre during the study period, considering the WBC content of RC₃₅₀ as a reference, the maximum WBC content of RCC₄₅₀ before processing is expected to be ∼3.97×10⁹ cells/unit. The EDQM¹⁵ criteria for WBC content (<1.2×10⁹ cells/unit) is ∼30 per cent of this estimated WBC content in RCC₄₅₀ processed by the BC method (RCC₄₅₀-AS). Since the BC method is known to remove ∼70–80 per cent of WBCs, the WBC content found in our study units seems to be justified.

The current criteria for the volume of RCCs as defined by the Indian regulatory or accreditation agencies are not specified according to the method of preparation. However, 450 ml±10 per cent of WB collected in our settings is comparable to that laid down in both JPAC¹⁹ (470±50 ml) and EDQM¹⁵ (450±50 ml) standards. The JPAC criteria for the volume of red cell units was met by 97.3 per cent of RCC₄₅₀-AS(TAT) and 82.9 per cent of RCC₄₅₀-AS(TAB).

Considering the example of a 450 ml WB unit processed by the BC method, as per DGHS⁴ criteria, the total volumes of all the components that can be prepared from one unit of WB (RCC: 350±35 ml, RDP: 70–90 ml and plasma: 220–300 ml) plus the volume lost during component preparation (∼40–50 ml for BC discarded and the transfer tubes) comes out to be 645–825 ml, which is much higher compared to total blood volume actually available for processing (568–658 ml: WB 450±45 ml, CPDA 63 ml and SAGM 100 ml). This discrepancy in total volumes questions the practical applicability of present volume criteria for RCC in our national standards. Therefore, there is a need to reconsider the criteria for volume requirements in the Indian scenario with redefined specific ranges according to different methods of component preparation. In Table V, we propose a criterion for redefining the RCC QC standards according to the method of preparation. The criteria have been mathematically derived based on our study results as well as statistical process control (SPC) defined by mean±2SD for each category rounded off to the nearest multiple of five.

The unit characteristics of QC of red cell components which have an impact on the therapeutic outcome of the recipient are Hb content⁵ and WBC content^6, where the former is directly related to the correction of anaemia and the latter is responsible for certain adverse effects of transfusion. Strict compliance with the NABH¹⁷ criteria of volume and Hct for RCC₄₅₀ would result in modifications of the component preparation methods in such a manner that a lot of red cells are lost during processing, or there could be a possibility of preparing diluted red cell units with larger volumes, part of which may get wasted or such units may pose a risk of volume overload to the patient. Considering D&C Rules³ criteria of minimum volume and Hct for both RCC₄₅₀ and RCC₃₅₀ would result in RCC units with low Hb content not beneficial to the patient, while maximum limits of these QC characteristics would result in red cell loss during processing. Considering the DGHS⁴ standards for RCC₄₅₀-AS with minimum volume of 315 ml with Hct 50 means that the Hb content in the RCC should be minimum 52.5 g, which is practically impossible to achieve from a donor with Hb 12.5 g/dl, and 450 ml volume of WB collected, which would result in only 56.25 g of Hb collected from the donor, followed by further red cell losses during processing; maximum volume of 385 ml is a large volume for transfusion. BC method is a cost-effective method of component preparation with some benefit of WBC removal in the Indian scenario, but only a low proportion of RCC units prepared by the BC method in this study had compliance with the current DGHS criteria (Table III). Thus, meeting the present reference national criteria for QC of red cell components is not only difficult to achieve by the blood centres across the country, but also RCC units produced in compliance with the existing national criteria may often result in transfusion of many units with such characteristics, which may not benefit or may produce adverse outcome in the recipient. Our proposed QC criteria are aimed at achieving an acceptable proportion of quality with more emphasis on Hb content and WBC content for the therapeutic benefit of patients.

Apart from the method of component preparation, the other significant factors influencing the Hb content were the age and gender of the donor, while the WBC content was influenced by days of storage and the ABO group of donors.

RCC₃₅₀ prepared from blood donated by female donors was observed to have lower Hb content and Hct compared to male donors. A similar finding was reported by Jordan et al¹¹. It is commonly known that females in the general population have lower Hb due to dietary deficiency of iron and menstrual blood losses. Males have higher Hb due to higher levels of the hormone testosterone that stimulates the red cell production²⁴.

Our observation of decreasing WBC content of RCCs with increasing duration of storage may be attributed to the senescence, loss of membrane integrity and subsequent lysis of WBC during storage²⁵. Higher WBC content was observed in units of female donors. Gwak et al²⁶ in a study on postoperative patients, found females to have higher neutrophils. However, with no differential leucocyte count data being documented in routine QC at our centre, we are not able to generalise the effect of gender on the WBC content of RCC. We observed higher WBC content in O and A group RCCs. This finding correlated with a study on 2,864 patients with acute coronary syndromes by Johansson et al²⁷.

Similar to Sawant et al²⁸, we observed higher haemolysis in RCCs prepared by the PRP method as compared to the BC method. RCC₄₅₀-AS(TAB) had the minimum percentage of haemolysis, suggesting the B-TAB method to have lesser physical stress on the RBC membrane as compared to the B-TAT technique. RCCs of female donors were previously¹¹ observed to have lesser haemolysis than that of male donors, possibly due to the antioxidant effect of oestrogen in females²⁹ and the propensity of testosterone in males to induce instability in RBC membrane³⁰. However, such a finding could not be ascertained in this study.

This study had certain limitations. Throughout the study period, the pre-donation Hb of the donors was checked by either quantitative or qualitative methods, resulting in non-uniformity in records of this parameter, which could not be considered for analysis. During the study period, no RC₄₅₀ (RCC prepared from 450 ml WB collection by the PRP method) units were prepared at our centre and hence were not available for analysis. Also, RCCs were prepared using the blood bag system from a single manufacturer; therefore, the possibility of better compliance with QC standards with the use of blood bag system from another manufacturer(s) cannot be ruled out.

Overall, this study shows that the quality of RCCs is significantly influenced by the processing method and certain donor factors which may be considered in upgrading our current quality standards. Evidence generated from this retrospective study alone, however, is not sufficient for redefining the QC criteria of red cell components. A multi-centric study with a larger sample size is required to confirm these observations.