| Abstract|| |
Background: As a part of continuous quality initiatives, while moving from triple bags to quadruple bags, we undertook a study to compare platelet-rich plasma (PRP) and buffy-coat (BC) methods with respect to all blood components (red blood cells [RBCs], random donor platelet concentrate [RDPC], and fresh frozen plasma [FFP]) prepared by PRP and BC methods.
Materials and Methods: It was a prospective analysis of different physical and quality parameters of RDPC, RBC, and FFP prepared out of 100 whole blood (WB) donations. Of these, 50 WB units were processed by PRP method using Triple bags and 50 WB units by BC method, using quadruple (top and bottom) bags, with an attached integral filter.
Results: RBC prepared by BC method had higher hematocrit (61.3 ± 1.91% vs. 56.03 ± 3.37%; P < 0.05) and lower white blood cell (WBC) contamination (6.3 × 104 ± 6.1 vs. 5.41 × 105 ± 2.5; P < 0.05) in comparison to prepared by PRP method. Higher PLT yield (7.67 × 1010 ± 1.8 vs. 6.47 × 1010 ± 1.5; P < 0.05) and lower WBC count (8.24 × 103 ± 1.1 vs. 1.5 × 104 ± 2.1; P < 0.05) was observed in RDPC prepared by BC method than PRP derived. CD62P expression was lower in RDPC prepared by BC method (31.46 ± 9.7%; P < 0.05) as compared to PRP method (43.35 ± 12.5%; P < 0.05). The BC method also resulted in increased plasma yield (210.56 ± 18.54 ml vs. 187.92 ± 12.93 ml; P < 0.05) in FFP in comparison to PRP method.
Conclusion: The blood components produced from WB by the BC method have laboratory variables suggestive of superior quality than those produced by the PRP method.
Keywords: Buffy-coat, fresh frozen plasma, platelet-rich plasma, random donor platelet concentrate, red blood cell, white blood cell contamination
|How to cite this article:|
Bhardwaj G, Tiwari AK, Aggarwal G, Pabbi S, Sharma J, Luthra A, Upadhyay A. Evaluation of quality matrix when practice changed from triple bags to quadruple (top and bottom) bags: In vitro analysis of blood components!. Asian J Transfus Sci 2021;15:30-6
|How to cite this URL:|
Bhardwaj G, Tiwari AK, Aggarwal G, Pabbi S, Sharma J, Luthra A, Upadhyay A. Evaluation of quality matrix when practice changed from triple bags to quadruple (top and bottom) bags: In vitro analysis of blood components!. Asian J Transfus Sci [serial online] 2021 [cited 2022 Aug 11];15:30-6. Available from: https://www.ajts.org/text.asp?2021/15/1/30/318237
| Introduction|| |
In past few decades, there have been major strides in transfusion safety with improvements in the entire transfusion chain of events in the process of whole blood (WB) and apheresis donations, including pre- and post-donation counseling, donor recruitment, donor safety, testing for transfusion transmitted infections, optimization and quality assurance (QA) of blood components and pre-transfusion testing.
Processing of blood donation into separate components is key link of the transfusion chain. There are two common methods of component preparation; platelet-rich plasma (PRP) method and buffy-coat (BC) method. Although both methods produce a platelet (PLT) product, the products have somewhat different in vitro characteristics. There is noticeable quality improvement in laboratory markers of BC-produced PLTs (BC-PCs) compared to PRP-produced PLTs (PRP-PCs). This improvement is characterized by a higher proportion of discoid PLTs, lower CD62 expression, and better hypotonic shock response (HSR) at the end of storage. BC preparation of PLT is the common method of preparation of random donor platelets (RDP) in countries such as Canada, the UK, and Europe.
In India, there is a mix of processing methods; PRP and BC method of component preparation across various blood banks. Few studies comparing the PLT components prepared by these two methods have been done, in India. However, there is paucity of data on comparison of components other than random donor platelet concentrate (RDPC), such as red blood cells (RBCs) and fresh frozen plasma (FFP). As a part of continuous quality initiatives (CQI) when we were moving from triple bags to quadruple bags, we undertook a study to compare PRP and BC methods, their compliance to published quality standards with respect to not just PLT concentrate; rather all blood components (RBC, RDPC, and FFP) prepared by PRP and BC methods.
| Materials and Methods|| |
It was a prospective analytical study measuring different physical and quality parameters of RBC, RDPC, and FFP prepared from WB donated by eligible donors over a period of 2 months (June 2019 to July 2019) undertaken in the Department of Transfusion Medicine in a large tertiary care hospital in the National Capital Region (NCR).
Sample size calculation
For sample size calculation, the variable considered in two methods (PRP vs. BC) was difference in volume of PLTs made by these two different methods. The volume was calculated as the average from three different studies,,, and this was 5.8 ml considered as (m1-m2) in the formula.
- Group 1 = PRP Group
- Group 2 = BC Group
- Parameter of Interest: RDPC Volume (ml)
- Mean value of the parameters for group 1(m1) = 53.6
- Mean value of the parameters for group 2 (m2) = 59.4
- Standard deviation of the parameters for group 1 (σ1) = 7.0
- Standard deviation of the parameters for group 2 (σ2) = 8.9
- Difference in the parameters for two groups (m) = (m1 – m2) = 5.8.
The formula used was as follows:
n = (σ12+ σ22) (Zα + Zβ)2/(m1 – m2)2
Zα = Value of standard normal variate corresponding to α level of significance
Zβ = The standard normal deviate for desired power
m = Average
σ = Standard deviation
With the above assumptions, the minimum sample size for 95% confidence level and 95% power worked out 50 blood units per group.
An analysis of blood components (RBC, RDPC and FFP) prepared out of consecutive 100 WB donations was done. Of these, 50 WB units were processed by PRP method and 50 WB units by BC method. The WB donations were collected from donors after they were administered health history questionnaire, underwent physical examination as per departmental standard operating procedures (SOP) and were found suitable for donation. Informed consent of each donor is part of the donor selection process. Few study-specific inclusions were applied; such as only male blood donors of age group between 20 and 40 years, with hemoglobin (Hb) level between 14 g/dl to 16 g/dl were included in the study. This was done to minimize the confounding effect of donor related variables (e.g., gender, age, and Hb level) on the comparative analysis of components made from PRP method and BC method.
Whole blood collection and component preparation
Four hundred and fifty milliliters of WB were collected from medically screened, apparently healthy donors in quadruple (Top and Bottom) bags (CompoFlow, Fresenius Kabi AG, Bad Homburg, Germany) with an attached integral filter and Triple bags (CompoFlex, Fresenius Kabi AG, Bad Homburg, Germany), for BC method and PRP method, respectively. Both type of bags (i.e., Top and Bottom bags and Triple bags) contained citrate, phosphate and dextrose (CPD) as anticoagulant solution in the primary collection bag (63 mL) and 100 ml of saline, adenine, glucose, and mannitol (SAGM) additive solution for RBC. The WB collection completed within 7–9 minutes from the starting time was included in the study. Blood units were given a minimum resting period of 1 hour (h) at room temperature (20°C–24°C), before further processing and blood components. All components were prepared within 6 h of collection, using centrifuge, Cryofuge 6000i (Heraeus, Germany) and automated component separator, CompoMat G5 (Fresenius Kabi, Bad Homburg, Germany).
Sampling of units and measurements
Different physical and quality parameters were analyzed as shown in [Table 1] and [Table 2].
|Table 2: Physical and quality parameters of different blood components by platelet-rich plasma and buffy-coat method|
Click here to view
For all units including WB, subjected to testing of quality parameters, sampling was done only after proper homogenization of the bag to make sure that sample in the segment represents the actual content of the bag. Samples from WB were collected 2 h from the time of collection (before separation). For RBC, samples were drawn and tested, 1 h after preparation. For RDPC and FFP, samples were drawn and tested on day-1.
Volume of WB, RBC, PLTs, and FFP was calculated from the net weight of the product divided by specific gravity (1.053 for WB; 1.09 for RBCs; 1.03 for PRP-PC and 1.06 for BC-PC; 1.02 for plasma).
Hematological values and cell counts
All hematological values such as Hb, hematocrit (HCT), PLT count, white blood cells (WBCs) for WB and RBC were obtained using a routinely calibrated automated hematology analyzer, Sysmex XE 2100 analyzer (Sysmex Corporation, Japan). The RBC count per bag for WB or RBC was calculated by multiplying RBC count/μl with product volume. The Hb per bag for WB or RBC was calculated by multiplying Hb count/dl with product volume. The WBC count per bag for WB was calculated by multiplying WBC count/μl with WB volume. The PLT count per bag for WB or PLT concentrate was calculated by multiplying PLT count/μl with product volume.
All the blood components (RBC and PLTs) used in our center are leukoreduced pre-storage using leukocyte reducing filters. Filtration time was accessed between buffy coat method derived RBC (BC–RBC), which were filtered using integral filters and PRP method derived RBC (PRP–RBC), which were filtered using BIO– R filters (Fresenius Kabi, Bad Homburg, Germany). For PRP method “Filtration Time” included total time taken from connecting the blood bag to leukoreduction filter plus the time taken for blood bag to filter. We analyzed the filtration time for RBC only, since filtration procedure differ for PRP-RBC (where an additional filter has to be attached) and BC prepared RBCs (having integral filter).
White blood cell count and log reduction
Ten samples each of RBC and RDPC (10 each for PRP method and BC method) were analyzed for WBC contamination pre- and post-leukocyte filtration using flow-cytometry. RDPC were leukoreduced using BIO– P filters (Fresenius Kabi, Bad Homburg, Germany). WBC log-reduction was calculated by using pre- and post-filtration WBC count. WBC count was analyzed pre- and post-filtration on flow-cytometer ([BD FACS VerseTM], Bacton, Dickinson and Company, BD Biosciences, San Jose, CA). WBC count per bag for RBC and RDPC was calculated by multiplying WBC count analyzed through flow cytometry by product volume.
Randomly selected 10 samples each for PRP-PC and BC-PC and corresponding RBC units were sent for aerobic and anaerobic cultures to the microbiology department of the hospital, as per the department protocol. Sterility testing samples were drawn on day-1 postcollection and were performed as part of routine QA.
Swirling in all the units of PRP-PCs and BC-PCs units were assessed visually against the light source and was documented as “present” or “absent.”
Lactate dehydrogenase and HCO3 levels of platelets
Fifty samples each for PRP-PCs and BC-PCs were analyzed for lactate dehydrogenase (LDH) and bicarbonate (HCO3). The samples were sent to biochemistry laboratory of the hospital and were measured on immunoassay analyzer (Vitros immunoassay analyzer [Ortho-Clinical Diagnostics, Unites States]).
pH of platelet units
PLTs, pH was measured by a calibrated portable pH meter ([OAKTON pH 700], Oakton Instruments, IL, USA], following the departmental SOP.
Twenty samples each of RDPC prepared by PRP method and BC method were analyzed for amount of activation by calculating their CD62P expression. PLT surface CD62P expression were measured using flow cytometry ([BD FACS VerseTM], Bacton, Dickinson and Company, BD Biosciences, San Jose, CA). PLTs were stained by phycoerythrin monoclonal mouse anti-human antibodies and were analyzed according to the manufacture's instruction.
Red blood cell contamination of platelets
For RBC contamination, the red cell count was multiplied with the mean corpuscular volume (MCV) of one red cell (90 fL) and PLT volume;
Prothrombin time/activated partial thromboplastin time/international normalised ratio
Ten samples each of FFP prepared by PRP (PRP-FFP) and BC (BC-FFP) were sent to hematology laboratory for analysis of prothrombin time (PT), activated partial thromboplastin time (APTT), and international normalized ratio (INR).
Fibrinogen and factor VIII
Ten samples for calculating fibrinogen level and factor VIII levels of PRP-FFP and BC-FFP were sent to hematology laboratory for analysis. The fibrinogen per bag was calculated by multiplying fibrinogen count (mg/dl) with product volume.
We used the Student's t-test for comparative analysis. P < 0.05 was considered statistically significant. SPSS software version 23 (Statistical Package for the Social Sciences; IBM Bengaluru, India) was used for analysis.
The study was approved by the institutional ethics committee.
| Results|| |
An analysis of 100 WB units (50 each for separation by PRP and BC method, later), before separation, was done and was similar in terms of their volume, erythrocyte content, Hb, hematocrit, PLT content, and leukocyte content for both PRP method and BC methods. The mean volume of WB was 452.2 ± 10.8 ml and 458.74 ± 10.9 ml for PRP and BC method, respectively. The other parameters are summarized in [Table 1].
Analysis of physical and quality parameters of blood components
Red blood cell
The mean volume of BC-RBC was lower as compared to PRP-RBC (293 ± 9.13 ml and 322.12 ± 11.31 ml, respectively, P < 0.05). The total time taken for filtration for leukoreduction of was higher for PRP-RBC which was 23.84 min as compared to BC-RBC, which took 19.58 min.
Significant difference (P < 0.05) was observed between RBC count per bag, Hb per bag, and HCT per unit. The mean RBC count per bag and mean Hb per bag was more by PRP-RBC, which was 2.08 × 1012 ± 0.13 and 60.08 ± 4.26 g as compare to BC-RBC, which was 1.93 × 1012 ± 0.14 and 52 ± 3.7 g, respectively. The mean Hb/dl for PRP-RBC was 18.53 ± 0.97 and for BC– RBC was 18.92 ± 0.47 and was not significant. The mean HCT per unit was significantly more for BC-RBC (61.3 ± 1.91%) then PRP-RBC (56.03 ± 3.37%). These results are summarized in [Table 2].
The mean prefiltration mean-WBC count per unit for PRP-RBC was 9.1 × 108 ± 1.3 and for BC-RBC was 6.2 × 108 ± 3.2, respectively, and was not significant. The postfiltration mean-WBC count per unit was significant (P < 0.05) between PRP– RBC having count of 5.4 × 105 ± 2.5 and BC-RBC with count of 6.7 × 104 ± 6.1. For BC-RBC, in eight out of 10 units, a leucocyte reduction of 99.99% (4-log reduction) and in remaining two units a 99.9% (3-log reduction) reduction was observed. In PRP-RBC, in seven out of 10 units a 3-log reduction and in remaining three units a 4-log reduction was observed.
Microbiological cultures of randomly selected samples processed by both PRP method and BC method were found to be sterile.
Random donor platelet concentrate
The mean volume of PRP-PCs and BC-PCs was 70.54 ± 5.315 ml and 72.14 ± 5.70 ml respectively and was not significant.
The mean PLT count per bag was significantly higher (P < 0.05) for BC-PCs with mean count of 7.67 × 1010 ± 1.8 as compare to PRP-PCs having mean count of 6.47 × 1010 ± 1.5.
The prefiltration mean WBC count per bag for PRP-PCs and BC-PCs was 4.6 × 107 ± 1.5 and 0.93 × 107 ± 5.7, respectively, and was significant (P < 0.05). The postfiltration mean WBC count per bag for PRP-PCs and BC-PCs was 1.5 × 104 ± 2.1 and 0.82 × 104 ± 1.1 respectively and were significant (P < 0.05). For RDPC prepared each by PRP and BC method a 3-log-reduction was observed in all the 10 units analyzed.
The CD62P expression was significantly higher (P < 0.05) for PRP-PCs in comparison to BC-PCs, with values of 43.35 ± 12.5% and 31.46 ± 9.7%, respectively.
The other quality parameters such pH, HCO3, LDH, and RBC contamination of PLTS which were analyzed are shown in [Table 2].
Swirling was present in all the units derived by PRP and BC methods. Randomly selected RDPC units were sterile and none showed any cultivable microbial growth.
Fresh frozen plasma
The mean volume of PRP-FFP was significantly (P < 0.05) lesser as compared to BC-FFP (187.92 ± 12.93 ml and 210.56 ± 18.54 ml, respectively). The mean fibrinogen value for PRP-FFP units was 486.27 (±141.13) mg and for BC-FFP units were 611.71 (±137.3) mg.
Other quality parameters such as FVIII levels, PT, APTT, and INR were also analyzed, and no significant difference was observed. The results are depicted in [Table 2].
| Discussion|| |
In India, studies,, carried out in the past regarding PRP method and BC method were confined to comparing the quality parameters of PLTs only. The main objective of the present study was to analyze and compare the quality of all blood components (RBC, RDPC, and FFP) made by PRP method and BC method in our setting.
Quality assessment of red blood cell
In the present study, the physical and quality parameters such as volume, RBC count per bag, Hb per bag, and HCT of BC-RBC units, were similar, with the study done by Hurtado et al. In general, the results of the quality control analysis of RBC derived by BC method showed a high degree of compliance with the Council of Europe (CE) quality recommendations for the production and storage of blood components. The BC method are defined better by CE, since European nations predominantly prepare blood components using BC method. The DGHS does not outline any specific guidelines pertaining to BC-RBCs. The RBC units prepared by PRP method were in conformance to the DGHS and AABB guidelines, since in India and the United States, component preparation is mainly done by PRP method.
Significant difference was observed between volume, Hb per bag and HCT of RBC prepared by PRP and BC method, as shown in [Table 2]. PRP–RBC had higher volume and Hb per bag whereas BC-RBC had higher HCT. However, on analyzing Hg/dl, which was 18.53 (±0.97) g/dl for PRP-RBC and 18.92 (±0.47) g/dl for BC-RBC, no significant difference was seen. Thus, the higher Hb per bag for PRP-RBC can be attributed only to their higher volume. Hematocrit or packed cell volume is a measurement of the ratio of the volume occupied by RBCs to the total volume of the blood sample. Unit volume is the total volume of the RBC unit following processing. The administration of high volumes of transfusion products can lead to circulatory overload. In order to avoid these adverse effects but still administer enough RBC for restoring the oxygen carrying capacity of the recipient, it is important to have consistent products with a high RBC mass to volume ratio (i.e., HCT).
The volume of residual plasma in RBC does not have any clear specification or standard and is not subjected to any routine quality control. However, the residual plasma volume could be important for allergic reactions or TRALI. Case studies, have shown that RBC-associated TRALI remains a significant cause of transfusion-related morbidity and mortality. A study carried out by Canadian Blood Services on preventive measures of TRALI, have also observed a decrease in RBC-associated TRALI cases and it was attributed to decrease in plasma content in RBC components that was a result of introduction of the buffy coat method of component production.
The mean post- leukoreduction WBC count per unit was significantly (P < 0.05) higher for PRP-RBC (5.4 × 105 [±2.5]) in comparison to BC-RBC (6.7 × 104 [±6.1]). This was expected since WBC separate better (move to BC layer) in BC method as compared to PRP method, where most of the WBCs remain entrenched in RBC layer. Hirosue et al. also reported higher WBC contamination in PRP-RBC units than in BC-RBC units.
The time taken for leukocyte filtration was significantly lesser for BC-RBC as compare to PRP-RBC. In top and bottom bags, since an in-line integral filter was already present, no extra-time and no additional workforce was required to connect RBC bags to filters, using sterile connecting device. In contrast, PRP method required both extra time and manpower and there are distinct chances of errors while labeling and then connecting the blood bags to leukocyte reduction filters.
Quality assessment of random donor platelet concentrate
The physical and quality parameters of both PRP-PCs and BC-PCs in the present study, fulfilled the quality criteria recommended by DGHS.
The volume of BC-PCs was more than PRP-PCs, but not statistically significant. The volume obtained were comparable with other Indian studies by Singh et al., Raturi et al., and Talukdar et al., also showing higher volume for BC-PCs then PRP-PCs.
The present study results showed significantly higher (P < 0.05) PLT count for BC-PCs as compare to PRP-PCs with values of 7.67 (±1.8) ×1010 and 6.47 (±1.5) ×1010 respectively. The lesser PLT yield can be attributed to the fact that, the centrifugation conditions used with the PRP method results in an average of 21% of the plasma and 19% of the PLTs remaining with the infranatant red cells. Studies done by Raturi et al. and Taludkar et al., also analyzed a comparable but not statistically significant increase in PLT count by BC method then by PRP method.
Leukocytes found in blood components are responsible for most transfusion reactions and exert adverse effects on the blood component quality and stability (storage lesion) and are also responsible for transfusion associated virus transmission (CMV). The WBC count obtained in BC-PCs was lower in comparison to PRP-PCs in the present study in both prefiltration and postfiltration stages. Since the preleukoreduction WBC counts were significantly lower in BC-PC as compared to PRP-PC, this difference was carried forward in the post-leukoreduction stage, as well. The filtration was constant in both the groups. Post-leukoreduction WBC in the present study was in concordance with studies conducted by Fijnheer et al., Singh et al. and Raturi et al., since they also reported higher post- leukoreduction residual WBC count per unit in PRP-PCs than in BC-PCs.
CD62P expression measures PLT secretion and it indicates the level of activation of the PLTs. An increase in CD62P expression associated with PLT storage lesions as well and it can be used as a sensitive quality marker. In our study, the expression of CD62P, was significantly higher for PRP-PC in comparison to BC-PC, as analyzed on Day one. A study by Ali, displayed, during storage for up to 5 days' PRP-PCs units displayed a significant increase in the CD62P, as compared with BCs units (P < 0.05).
Recommendations for erythrocyte contamination as proposed by DGHS standards is supposed to be < 0.5 mL in each bag, in order to prevent RBC-associated alloimmunization. The PLT units prepared in the present study by both PRP and BC method had RBC values well below the desired limits.
A simpler technique to determine the quality of PLTs in the laboratory is by using the “Swirling test.” The discoid morphology of PLTs correlates with the in vivo survival and can be demonstrated by the swirling phenomenon. In the present study, all freshly prepared PRP-PCs and BC-PCs, exhibited swirling, indicating their intact discoid morphology.
Biochemical tests such as decrease in pH, LDH accumulation, and changes in bicarbonate levels can be employed to assess PLT viability. These in vitro tests are more economical and rapid. All the PRP-PCs and BC-PCs were found within the acceptable limit (pH > 6.0 units) as per DGHS standards. No significant difference was observed between HCO3 and LDH levels between the PLTs prepared by these two different methods. Although studies have shown difference in biochemical changes between PRP-PCs and BC-PCs, with storage, in our study, we analyzed the samples only on day of component preparation.
Quality assessment of fresh frozen plasma
The different physical and quality parameters of FFP, prepared by both PRP and BC method, met the DGHS guidelines.
The volume of BC-FFP was significantly higher (P < 0.05) in comparison to PRP-FFP. The lesser volume of PRP-FFP is due to the centrifugation conditions used with the PRP method resulting in an average of 21% of the plasma remaining with RBCs. The fibrinogen level per bag was considerably more for BC-FFP as compared to PRP-FFP, though not statistically significant. This can be attributed to the higher volume obtained in BC plasma bags.
No significant difference was observed between other quality parameters such as Factor VIII, PT, APTT, and INR. A study by Sheffield et al., analyzing the coagulation variables in plasma during the transition period in the Canadian Blood Services, when they switched from PRP method to BC method of component preparation, observed that there was no reduction in frozen plasma (FP) quality made by the BC method (FP-BC) in the characteristics they tested such as fibrinogen, factor (F) V, ABO-matched FVIII, and antithrombin levels in comparison FP made by the PRP method (FP-PRP). They also concluded that, FP-BC supported global clotting, as measured by prothrombin time or activated partial thromboplastin time.
| Conclusion|| |
The blood components produced from WB by the BC method have laboratory variables suggestive of a superior quality than those produced by the PRP method, such as better HCT of RBCs, higher PLT count, and lesser WBC contamination in both RBC and PLTs. The BC method gives an increased plasma yield in FFP. The kinetics of CD62P expression are influenced by the method used to prepare the PLTs, with activation in PRP-PCs exceeding BC-PCs.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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Dr. Aseem Kumar Tiwari
Department of Transfusion Medicine, Medanta-the Medicity, Sector-38, Gurgaon - 122 001, Haryana
Source of Support: None, Conflict of Interest: None
[Table 1], [Table 2]