The comparison of efficacy and safety of different platelet concentrates in patients with hematologic malignancies

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1 LITHUANIAN UNIVERSITY OF HEALTH SCIENCES DEPARTMENT OF HEMATOLOGY The comparison of efficacy and safety of different platelet concentrates in patients with hematologic malignancies Final Master s Thesis Faculty of Medicine By Henning Hilgendorff Supervised by MD, Diana Remeikienė Kaunas,

2 TABLE OF CONTENTS 1. SANTRAUKA SUMMARY ACKNOWLEDGEMENTS CONFLICT OF INTEREST CLEARANCE ISSUED BY THE ETHICS COMMITTEE ABBREVIATIONS TERMS INTRODUCTION AIM AND OBJECTIVES TERMS LITERATURE REVIEW Methods of efficacy evaluation Comparing efficacy of whole blood and apheresis derived platelet concentrate Factors affecting efficacy of platelet transfusions Frequency of transfusion reaction in different platelet concentrates RESEARCH METHODOLGY AND METHOD Method Study design Patients Platelet concentrates Clinical Assessment Statistics RESULTS General characteristics Platelet increments of apheresis and whole blood derived platelet concentrates Transfusion intervals of apheresis and whole blood derived platelet concentrates Incidents of transfusion related reactions of apheresis and whole blood derived platelet concentrates

3 12.5. Transfusion intervals of men and women Platelet increments of men and women DISCUSSION Comparison of efficacy of apheresis and whole blood derived platelet concetrates Longer Transfusion intervals for apheresis than whole blood derived platelet concentrate transfusions Men have higher platelet increments than women Longer transfusion intervals for men Incidence of transfusion reactions CONCLUSION Compare the platelet increment of apheresis and whole blood derived platelet concentrate transfusions Compare the duration of transfusion intervals of patients receiving apheresis and whole blood derived platelet concentrate transfusions Compare the frequency of adverse reactions of apheresis and whole blood derived platelet concentrates REFERENCES

4 1. SANTRAUKA Autorius: Henning Hilgendorff. Vadovė: MD, Diana Remeikienė. Darbo tema: Skirtingų trombocitų koncentratų transfuzijų veiksmingumo ir saugumo palyginimas hematologinėmis piktybinėmis ligomis sergantiems pacientams. Tyrimo tikslas: Palyginti aferezės būdu paruoštų ir sukauptųjų trombocitų saugumą ir veiksmingumą hematologinėmis ligomis sergantiems pacientams. Tyrimo uždaviniai: 1.Palyginti trombocitų kiekio padidėjimą po paruoštų aferezės būdų ir sukauptųjų trombocitų transfuzijų. 2. Palyginti trukmę tarp transfuzijų pacientams, kuriems perpilami paruošti aferezės būdu ir sukauptųjų trombocitų koncentratai. 3. Palyginti nepageidaujamų reakcijų dažnį perpilant paruoštų aferezės būdu ir sukauptųjų trombocitų koncentratus. Tyrimo objektas: Lietuvos sveikatos mokslų universiteto ligoninės Kauno klinikų Onkologijos ir hematologijos klinikos pacientai, sergantys hematologinėmis ligomis, kurių gydymo metu atliekamos dažnos trombocitų transfuzijos. Atranka ir tyrimo metodai: Bus analizuojami Lietuvos Sveikatos Mokslų Universitetas Kauno Klinikos Onkologijos ir hematologijos klinikos pacientų, sergančių hematologinėmis ligomis, klinikiniai (trombocitų transfuzijų efektyvumas, nepageidaujamos transfuzijų reakcijos) ir laboratoriniai (bendras kraujo tyrimas) tyrimų duomenys. Rezultatai: Parodė, kad po sukauptųjų trombocitų transfuzijų trombocitų padidėjimo mediana buvo didesnė nei po aferezės būdu paruoštų trombocitų, tačiau skirtumas buvo statistiškai nereikšmingas. Po aferezės būdų būdu paruoštų trombocitų perpylimo intervalai tarp transfuzijų buvo ilgesni už sukauptųjų trombocitų, bet statistiškai nereikšmingai. Intervalai tarp transfuzijų vyrams buvo statistiškai reikšmingai didesni negu moterims. Trombocitų padidėjimas vyrams buvo didesnis, tačiau statistiškai nereikšmingai. Nebuvo transfuzijų reakcijų. Maža statistinė tyrimo reikšmė gali būti dėl mažo imties dydžio. Išvados: Aferezės būdu paruoštų ir iš sukauptųjų trombocitų koncentratų, veiksmingumo rezultatai buvo panašūs ir gali būti lygūs. Visi sukauptųjų trombocitų koncentratai paruošti iš leukocitųtrombocitų sluoksnio. 2 nėštumų faktorius gali turėti įtaką mažesniems transfuzijų intervalams moterims nei vyrams. 4

5 2. SUMMARY Author: Henning Hilgendorff. Supervisor: MD, Diana Remeikienė. Research title: The comparison of different platelet concentrates by their safety and efficacy in patients with hematologic malignancies. Aim: The aim was to compare the efficacy and safety of whole blood derived and apheresis derived platelet concentrates transfusions in patients with hematologic malignancies. Objectives: 1. Comparison of platelet increments from apheresis and whole blood derived platelet concentrates. 2. Comparison of transfusion intervals of apheresis and whole blood derived platelet concentrates. 3. Comparison of frequency of transfusion reactions in apheresis and whole blood derived platelet concentrates. Patients: The study was based on patients from the hematology and oncology department of the Lithuanian university of health sciences suffering. All patients were suffering from hematologic disease and were treated with platelet transfusions. Method and methodology: We analyzed patients of the hematology and oncology department in the Lithuanian University of Health Sciences Kauno Klinikos with hematologic malignancies clinically (platelet increments and the occurrence of transfusion reactions) and laboratorial tests (complete blood count). Results: The results showed higher medians of platelet increment for whole blood derived than for apheresis derived platelet concentrates, but the difference was statistically not significant. The transfusions intervals were longer for apheresis than for whole blood derived platelet concentrate transfusions but were not statistically significant. The platelet increment and transfusion intervals seemed to be more effective in men than in women, due to statistically significant longer intervals and higher increments for men. There was no incidence of transfusion reaction. Low statistical significance of the study may be due to low sample size. Conclusion: Apheresis and whole blood derived platelet concentrates showed comparable results and might be equal, assuming all whole blood derived platelet concentrates are made by the buffy coat method. The factor 2 pregnancies has more impact on decreasing transfusion intervals than factor men. 5

6 3. ACKNOWLEDGEMENTS Appreciations are going to my supervisor MD, Diana Remeikienė from the hematology department of Kauno Klinikos and Professor Algimantas Kriščiukaitis from the biophysics department of the LUHS for their time and patience investing in the success of my scientific project. 4. CONFLICT OF INTEREST There is no conflict of interest to proclaim. 5. CLEARANCE ISSUED BY THE ETHICS COMMITTEE Research title: The comparison of efficacy and safety of different platelet concentrates in patients with hematologic malignancies. This research was approved by Center of Bioethics of Lithuanian University of Health Sciences, Kaunas, Lithuania. Date: Document Number: BEC-MF-56 6

7 6. ABBREVIATIONS TERMS Following abbreviations are used consciously in the following text: APC: Apheresis derived platelet concentrate CBC: Complete blood count CCI: Corrected increment count Fig.: Figure IQR: interquartile range LUHS: Lithuanian university of health sciences PAS: Platelet additive solution PC: Platelet concentrate PI: Platelet increment PLC: Platelet count PRP: Platelet rich plasma TI: Transfusion interval WBD: Whole blood derived 7

8 7. INTRODUCTION For the treatment of thrombocytopenia in patients with hematologic malignancies are different platelet concentrates (PC) used[1]. There are apheresis PC (APC), which are made of a single donor products and there are whole blood derived (WBD) PCs, which are made of 4-6 different donor products and is produced by either the buffy coat (BC) or platelet-rich plasma (PRP) method[2]. For the production of BC either the chain method or the pooling kit can be used[1]. Generally speaking the BC method is more prevalent in Europe and the PRP method is more commonly used in the USA for the production of whole blood derived PCs[1]. From July first of 2015 universal leukoreduction was introduced into Lithuania and into the Kaunas Klinikos blood bank, in which period the PRP method was predominantly used in opposite to the Vilnius blood bank, where the buffy coat method was more prevalently used. This period is the time of our study. Both whole blood derived (WBD) PC methods differ by their centrifugation steps[1]. BC PC production starts with high speed centrifugation[3] and afterwards is centrifuged by low speed, whereas PRP is first centrifuged by slow speed[4]. Apheresis blood products is gained by direct access to the patient s vein by a catheter. Most of the apheresis system work by the PRP method, gaining platelets by centrifugation and then reintroducing left over plasma, leukocytes and erythrocytes back into the human cardiovascular system. No waste products emerge. More efficient systems produce today 2-3 doses of apheresis platelets in one collection session, with one dose containing at least 3x10*11 platelets in 200 ml of human plasma, which approximates 6 whole blood donations for the production of one therapeutic unit of WBD PC[1,5].On 2013 in Germany 61.5% of the used platelet products were apheresis derived PCs and 38.5% were whole blood random donor derived PCs. In the UK on 2013 the distribution along the usage of platelet products was 80,7% for APCs and 19.3% for WBD PCs[6]. On 2015 in Kauno Klinikos 62% of platelet products were apheresis derived and 37% were from leukodepleted WBD PC from either BC method or PRP method. On 2016, in Kauno Klinikos 22.5% of all PCs were apheresis derived products and 77.5% were WBD PCs. On the ground of these numbers the trend is going towards leukodepleted WBD products. Most likely due higher availability of blood products for the production of WBD PCs and more economical production than for APCs[1]. Still the question is on-going which type of procedure is the better than the other, WBD or APCs in terms or efficacy or safety. This is the question want to answer with our research or at least contribute to the answer, which is often not obvious and divided. The division and the current scientific status towards this topic we want to elucidate in the following literature review. 8

9 8. AIM AND OBJECTIVES 8.1. Aim: The aim of the study was to compare the safety and efficacy of the apheresis derived and whole blood derived platelet concentrate hematologic patients Objectives: 1. Compare the platelet increment of APC and WBD PC transfusion. 2. Compare the duration of transfusion intervals of patients receiving APC and WBD PC transfusions. 3. Compare the frequency of adverse reactions of APC and WBD PCs. 9

10 9. TERMS Corrected count increment Leukodepleted Leukoreduced Thromboelastography 10. LITERATURE REVIEW Methods of efficacy evaluation The efficacy of the platelet transfusion is determined by 2 factors[7], the viability and functionality of the platelet. Both can be tested in vivo and in vitro. The viability can be evaluated either in vitro by the corrected count increment (CCI) where CCI-1, 1-4h after the transfusion, reflects the early recovery of the PLC and the CCI-2, 18-24h after the transfusion, the later platelet survival[7 10]. In vivo, the viability can be evaluated by transfusion intervals or by the platelet survival in circulation[11]. The transfusion interval together with the CCI-2 reflect the platelet survival. The functionality integrity of the platelet can be evaluated in vivo by the 5 point WHO bleeding score[5] or in vitro by performing thromboelastography (TEG), which is a measurement of hemostasis[7,12]. The prophylaxis treatment strategy is based on CCI and the therapeutic treatment goals are oriented on bleeding assessment[7]. But treating a bleeding like counting variables is difficult and makes the usage of the WHO bleeding score for evaluating difficult. Therefore Apelseth et al. conclude the necessity of the reevaluation of the WHO derived bleeding scheme[7]. Further findings of their study concludes no correlation between clinically bleeding status, calculated bleeding status and PI values[7]. For example as an more progressive way to measure bleeding, Gniadek et al. hypothesize an evaluation of an in vivo assay for the evaluation of blood in oral salivary pre- and post- transfusion to determine the efficacy of the PC transfusion[13]. The CCI is used in many studies to measure efficacy in terms of the responsiveness to the transfusion to determine if a transfusion is successful or not. A CCI -1 of more than 7.5x10*9/L is seen as a successful transfusion and a CCI-2 of more than 4.5x10*9/L as well[9,14]. It is calculated by subtracting from the post-transfusion the pre-transfusion count, divided by the number of platelets transfused multiplied by the body surface in square meters. 10

11 Here the scientific opinion is divided if APCs are reaching better CCIs or WBD PCs. Some authors are seeing clear advantage in reaching higher CCIs for APCs than for WBD PCs[9,15] other studies suggest better CCIs for WBD PCs[2]. But the increase in circulating platelet count does not necessarily mean that the function of the transfused platelet in preserved. Despite an increase of platelet count the hemostatic abilities may be impaired[7]. A study from 2006 from Arnold et al compared the in vivo survival and recovery of apheresis and PRP LR platelets. Platelets of both methods were traced with Indium-111 or Chromium-51 in equal proportions. In vivo the APC showed prolonged survival hours compared to PRP leukoreduced products and higher percentage of recovery (Table 1). It s of great interest that in this study autologous PCs were transfused back to the same healthy donor. Participants with underlying febrile condition, recent platelet transfusion and splenectomy were excluded from this study. Some factors resulting in decreased platelet increments, were excluded and should not have influence on the recovery and survival values of the platelets. But even then, in good, non-diseased, circumstances for the platelet transfusion to succeed, APC showed more effective recovery and γ-survival, which are shown in table 1[1,11,15,16]. In addition to that APCs showed superior storage related features after 5 days of storage compared to the PRP leukoreduced products. APCs had higher morphology scores and lower P-selectin expression at 5 days of storage, which indicates higher viability of the product[11,17]. Worse storage related factors for PRP PCs than for BC PC products was as well backed by Costa et al. s study[17]. Apheresis might have collected younger, larger platelets, compared to PRP method. The PRP method has an inline leukocytes filters, which might have led to the loss of younger, larger platelets. Another factor might be harder G-forces in PRP method affecting the cell integrity by this the quality of the cells, leading to lower recovery and gamma survival counts[11]. Table 1 Values of recovery and γ-survival along APCs and PRP PCs in circulation (Arnold et. al.)[11] APC PRP PC p Recovery (percent) 51.1 ± ± γ-survival (in hours), mean ± ± 46.2 <

12 10.2. Comparing efficacy of WBD PC and APC Anderson et al. conclude in a study from 1997 that there are no differences statistically of the CCI comparing PRP, BC and single donor APC. Means of CCI-1 and CCI-2 are seen in table 2. Important to mention is that this study was performed before universal leukoreduction. In this study 3 types of platelet concentrates are compared: Single donor APC, PRP PC (non-leukoreduced) and BC PC in patients with hematologic malignancies. 80% of APC and 30-50% of BC PC units seemed to have less than 5x10*6 WBCs. APC showed superior CCI-1 and CCI-2 compared to PRP PC and BC PC. But still the increment values of APC transfusions are similar to BC PC transfusions. Maybe this is due to higher WBC count in non-leukoreduced PRP PCs[17,18]. Table 2 CCIs results along APCs, BC and PRP PCs (Anderson et al.)[8] APC BC PC PRP PC P CCI-1 at 1-6h, 12.00± ± ± mean CCI-2 at 18-24h, mean 8.63± ± ± Heddle et al. found superior 1h CCI-1 for APC, than in WBD PCs (means respectively, 6.60±7.04 vs. 4.40±5.31), this data was obtained as raw data from Heddle et al s older study[10] and reanalyzed for its metaanalysis[9]. The CCI was expressed as successful transfusion of more than 4.5x10*9/L an unsuccessful transfusion as less than 4.5x10*9/L. 53% of APC, 35% of leukoreduced PRP PC and 38% of plasma reduced PRP PC transfusions were successful. What translates into a superior CCI-2 for APC transfusions. In consequence, CCI-1 and CCI-2 of APC transfusions were shown in this study superior against leukoreduced PRP PC and plasma removed PRP PC[10], what reflects Anderson et al. s results towards better CCI-1 and CCI-2 for APC transfusions. But it may suggest that even leukoreduction of PRP products makes them not equal effective to BC PC products with comparable CCI-1 and CCI-2 to APC transfusions[9,10]. But still we can see that results are divided along different studies. For example, Baolan et al. found in their study higher 1h CCI-1 and 24h CCI-2 for BC PC than for APC transfusions. But no statistical significance was found (p>0.05). Results of his study are presented in table 3. They discussed that maybe patients factors were different in the compared groups leading to different responses to platelet therapy and that low quality of PCs result in quick refractoriness[2,14]. In their study the refractoriness was 36-47%[2]. 12

13 Table 3 CCIs and PLCs (x10*9/l) along APC and BC PC transfusions (Baolan et al.)[2] APC BC PC CCI-1 at 1h, 13.56± ±4.65 mean CCI-2 at 24h, 8.67± ±3.36 mean Effective rate 53% 64% PLC, after 24h 31.28± ±12.34 PLC, before trans 15.90± ±8.34 Some other authors even suggest there might be no differences between product type and maybe other factors influencing the platelet increment. Akkök et al. found in their study no significant differences between APC and WBD PC in CCI- 1 and CCI-2. Only the storage is affecting the effectivity of platelet products for both transfusion types leading to significant reduction of CCI-1 and CCI-2 (respectively, p=0.001, p=0.03). The percentage of successful transfusion for CCI-1 and CCI-2 (CCI-1, >7.5x10*9/L, CCI-2, >4.5x10*9/L), was decreasing for both, APC and WBD PC transfusions by increased storage time of the platelet product. No statistical difference in terms of successful CCI-1 between APC and WBD PC was found. In the proportion of as successful measured CCI-2 was a statistical difference established between BC PC and APC transfusions. The proportion of successful transfusions of BC PC was decreasing significantly (p=0.002) in opposite to APC transfusions. The proportion of successful APC transfusions was not decreasing significantly over prolonged storage time. Despite the authors conclusion of no difference of APC and BC PC, it may show a little advantage in maintaining quality of the PC, which results in better values for longer stored products[14]. In terms of interval to next transfusion, as measurement of efficacy, no difference between the two platelet products was established, just again storage of the product resulted in significant (p= 0.001) shortening of transfusion intervals[14]. 13

14 10.3. Factors affecting efficacy of platelet transfusions Heim et al. found in their study that higher increments of platelet transfusion are statistical significant associated with longer transfusion intervals. Transfusions which resulted in a transfusion interval of 2 days had significantly higher CCI means than, transfusion resulted in a TI of 1 day (Table 4). As a consequence, the product resulting in higher increment is also more effective in terms of resulting in longer TI. Like prior discussed the data is different on which platelet product is reaching higher increments. Yet longer transfusion intervals would translate into more patient safety[1]. Table 4 CCI results compared according to TI, in days (Heim et al.)[16] CCI, mean 11.33± ± ± ±8.78 P < Transfusion interval, in days. 1 d. 2 d. 3 d. 4 d. The question is to ask which features are influencing the product quality and its efficacy. Akkök et al. concluded that storage had a significant influence on the transfusion interval (P=0.001). The longer the product was stored the more the TI decreased. Heim et al. found decreasing CCI means from 1 st till 5 th day of storage[16]. Slichter et al. found a significant increase in platelet increments with products stored for 48h or less and the platelet dose (P<0.0001). [15]. Therefore we can conclude that storage is influencing the efficacy of platelet products by diminishing PI and TIs and through this the safety of the patient due to the need of more transfusions[1,16]. Another result of Heim et al.`s, which was as well backed by Slichter et al. s study, was the finding of ABO mismatched PCs resulted in significant lower CCI, compared to ABO matched platelet transfusion[15,16]. Bidirectional mismatch resulted in much lower mean increments than matched (11.1±8.0 vs. 15±9.5)[16]. However not just product related factors influencing the efficacy of transfusion. Patient factors have a big influence on the efficacy of a transfusion. Patient factors that improved platelet responses were splenectomy and increasing patient age, ATG treatment. Patient younger than 10 and older than 40 years showed better platelet responses than other age groups[16]. In contrast, at least two prior pregnancies, male gender, splenomegaly, bleeding, fever, increasing height and weight, lympho- cytotoxic antibody positivity, an increasing number of platelet transfusions or receiving heparin or amphotericin or allogenic HSCT treatment were associated with decreased post-transfusion platelet responses [1,7,15,16]. 14

15 Subsequently we can conclude that not only the product type is responsible for the success of the transfusion, more like the success of transfusion is depending on several factors and the product type is one of them. But the product quality, in terms of storage related effects and patient factors might have big influences, too[5]. Finally, there are many screws to screw on to achieve optimal transfusion results or at least it is important to be aware of these factors, when transfusing PCs Frequency of transfusion reaction in different PCs In the prospective, randomized study from Anderson et al. was reported a reduction in the probability of a non-febrile hemolytic reaction, from approximately 17% to 3%, by using platelets derived from BC or apheresis when compared with random donor PRP-PC. 1 out of 6 transfusions were associated with transfusion reaction with the PRP method. With the buffy coat and apheresis method 1 out 30 transfusions were related to transfusion reaction [8]. The occurrence of a transfusion related reaction was statistically significant (p=0.0001) higher in PRP PCs transfusion than in APC and BC PC transfusions[8]. That shows clear the safety disadvantage of the PRP PCs. On the other hand, it shows the comparable safety of the apheresis and buffy coat method.[8] Higher frequency of transfusion reaction again is be related to higher amount of WBCs in PRP[18] and higher IL-6 level, which were found in plasma reduced non-leukodepleted PCs than in prestorage leukoreduced PCs[19]. This result is based on a study by Heddle et al. in which they further established higher frequencies of transfusion reactions for non-leukodepleted PRP products than for leukoreduced BC PC or APC. In their study, they compared plasma removed platelet PRP PCs with pre-storage leukoreduced APCs and PRP PCs with 129 patients and 1190 performed transfusions. Pre-storage whole blood derived PCs had severe transfusion reaction of 1.7% and pre-storage leukoreduced APCs 1.4 %. The general frequency of transfusion reaction for leukoreduced APCs was 13.3% and 11% for WBD leukoreduced products. The overall frequency of transfusion reaction was 13.6%[19]. Despite the low statistically significance of the study[19] it shows the equality of safety of the leukoreduced PCs, be it apheresis or WBD PCs and that maybe other factors play into the equation of safety of platelet products, e.g. the duration of storage, which influences cytokine level in both types of PCs[17][15]. Also platelet additive solution are known to reduce the incidence transfusion reactions like the reduction of allergic reaction, hemolysis and ABO mismatched reactions by the reduction of plasma contents in PC products[20]. Azuma et al. compared plasma reduced in plasma additive solution suspended APCs with APCs suspended in their own plasma. The PAS used is glucose containing and reduced the plasma content to 15

16 less than 10%. It showed a reduction of allergic reactions from 42% with plasma containing products to 0.63% with the non-plasma containing products in 12 patients with recurrent allergic reactions[21]. Kerkhoffs et al.[22] compared plasma reduced BC PCs suspended in PAS 2 with non-plasma reduced BC PCs. BC PCs suspended in PAS 2 appeared to be mild, infrequent and significantly lower transfusion related reactions compared non-suspended BC PCs (respectively, 2.4% vs 5.5%). The BC PC was suspended in 35% rest plasma and PAS 2[22], which is non-glucose containing[20]. Glucose is a crucial point in cell functioning, which is contained in the plasma. That s why nonglucose containing PAS are to a lesser extent able to reduce the plasma to a lower amount compared with glucose containing PAS[20]. Generally seen in both PC products are leading plasma reduction together with PAS to less amount of transfusion related reactions[20][22]. 16

17 11. RESEARCH METHODOLGY AND METHOD Method Study design The investigation was designed as a retrospective study, based on data from patients case history of the 2 nd half of 2015 from the oncology and hematology department of the Lithuanian university of health sciences hospital Kauno Klinikos. There were evaluated CBCs, and status reports and transfusion documents of the patients Patients Patients for this study were chosen by the number of transfusions and primarily by the disease type. All patients must have hematologic malignancies as the main diagnosis and at least 5 platelet transfusions to be part of the study. All patients were from the same time period, from July till December 2015 and were treated in the oncology and hematology department of the LUHS hospital Kauno Klinikos Platelet concentrates Generally, it was not to specify if the WBD PC was produced by PRP or BC method, because many of the transfused products are coming from the national blood center in Vilnius But when the product derived from Kauno Klinikos blood center, the PCs were produced with following specifications: Single donor APCs are derived from Haemonetics-MCSR+ (Haemonetics Co., Braintree, Massachusetts, USA). WBD PCs were either produced by the BC or PRP method. They were centrifuged by the Thermo Scientific KR4i Centrifuge (Thermo-Fischer Scientific, Waltham, Massachusetts, USA). Buffy coat products were centrifuged by 2732g for 8 minutes with a temperature of 22 C. PRP PCs are centrifuged by 533 g for 8 min with a temperature of 22 C. Platelets were separated from plasma either manually or by an automatic blood component processing system. CompoMatRG5 (Fresenius Kabi, Cheshire, United Kingdom) was used as automatic blood component processing system. All products are leukoreduced by filters included in the pooling or Apheresis kit. Not all products were irradiated. No additive solution was used for the PCs. It was stored maximum for 5 days in platelets incubators with their plasma. One unit for transfusion has at least 200x10*9 platelets. 17

18 Clinical Assessment The difference of the pre- and post-transfusion platelet count was calculated as a measurement of efficacy. The post-transfusion CBC was evaluated 12-36h after the transfusion. A transfusion was defined successful when the platelet increment is higher than 4x10*9. A transfusion reaction was defined as any one of a rise in temperature of 1 C, shivering/rigors, urticaria, bronchospasm or anaphylaxis within 4 h of the transfusion by the responsible doctor either on the transfusion document or on the status report of the patient Statistics Discrete variables are presented as absolute numbers (n) and percentages (%); continuous variables are expressed as median with the interquartile range (IQR) stated in brackets for not normally distributed data and mean ± standard deviation (SD) for normally distributed data. Kolmogorov- Smirnov test and Shapiro-Wilk test were used to assess normality of data distribution: according to these tests results, our data was not following the normal distribution, therefore non-parametrical tests were used for statistical analysis. Wilcoxon Signed Rank test was used to compare equality of distributions of two variables from related samples (e.g. pre- and post-transfusion CBC), equality of more than two distributions of variables was compared using Kruskal-Wallis test for independent samples. The Mann Whitney-U test was used to compare the equality of distributions two variables from independent samples (e.g. Increment and transfusion type) The median test was used to compare the equality of distribution of two variable from independent samples (e.g. Medians of time to next transfusion and gender). For the calculation of time to next transfusion 0 days and more than 15 days were excluded, because more than 15 days to next transfusion reflect not the efficacy of the transfusion more the duration to next hospitalization, which is a disease related factor not a transfusion related factor. 0 means treatment ended. No further transfusion was performed. New hospitalization was counting as a new interval of transfusions. For the evaluation of efficacy, PI of 4x10*9 and less were excluded. We determined them as failed transfusions. For the calculation of medians of the pre- and post-transfusion platelet counts all transfusions were included. All analyses were performed with SPSS statistics (version ; IBM, Armonk, New York, USA). 18

19 12. RESULTS General characteristics We included in our study 159 transfusion, 103 APC and 56 WBD PC performed to 17 patients, 10 men and 7 women from various age groups. All were suffering from hematologic malignancies Platelet increments of APC and WBD PCs We defined a successful transfusion as a post-transfusion PI of more than 4x10*9/L. In a total number of 159 transfusion, 103 APC and 56 WBD were transfused. 57%(n=91) of the transfusion were successful and achieved a PI over 4x10*9/L in the following CBC, 12-36h after the transfusion. 64.1% (n=66) of the APC and 44.6%(n=31) of the WBD PC transfusions were efficient. 42% (n=68) of all transfusion failed, 35.9% (n=37) of the APC and 55.4% (n=31) of the WBD PC transfusion. Failed transfusions with an PI of 4x10*9/L and less were excluded from the evaluation of efficacy. The increment values of APC and WBD PC were distributed not according normal distribution (Kolmogorov-Smirnov test, APC p=0.000, WBD PC p=0.121, Shapiro-Wilk APC p=0.000 WBD PC p=0.004). So, we used non-parametric tests to compare their values in regard to various factors. We found that generally pre- and post-transfusion count were not equal for APC and WBD PC (Wilcoxon signed rank test, p<0.01). That means both transfusion types are effective. Further we found that both transfusion types were equally effective (Mann Whitney U-test, P=0.698). Though medians of platelet increments calculated from our samples were different for APC and WBD PC (15 (IQR=11) vs. 16 (IQR13)) (here and after values are given as median and interquartile range in brackets), but statistically there were no significant difference seen (Mann Whitney U- test, P=0.698). Slightly higher platelet increment medians for WBD PC were achieved and lower for APC transfusions. The results are seen again in table 5 and fig. 1. The distribution of pre- and post-transfusion platelet counts for APC and WBD PC were not distributed according normal distribution, so parametric analysis was applied. The pre- transfusion PLCs medians were the same and statistical not significant different (median test, p=0.734, Mann- Whitney U test, p=0.413). The post-transfusions PLCs were different with higher medians for APC than for WBD PC transfusions (respectively, (20 (IQR=15) vs. 17 (IQR=19.50)). However, the results were statistically not significant (Median test, p=0.734; Mann-Whitney U test p=0.413) (Table 5). 19

20 Fig. 1 The boxplot displays PI according to PC Table 5 Various factors regarding the efficacy of APC and WBD PC PI, medians Pre-transfusion PLC, medians Post-transfusion PLC, medians Successful transfusion, In % Failed transfusion, In % APC 15 (IQR=11) 9 (IQR=9) 20 (IQR=15) 64.1% 35.9% WBD PC 16 (IQR=13) 9 (IQR=9) 17 (IQR=19.50) 44.6% 55.4% p Mann Whitney Median test, Median test, U- test, p=0.948; p=0.734; Mann- P=0.698 Mann-Whitney Whitney U test U test, p=0.839 p=

21 12.3. Transfusion intervals of APCs and WBD PCs Transfusion intervals (in days) of APC and WBD PC are distributed not according normal distribution (Kolmogorov-Smirnov p< 0.01, Shapiro-Wilk p<0.01), so non-parametric evaluation was necessary. The medians of the TIs of APC and WBD PC are different, (3 (IQR=2) vs. 2 (IQR=1)) (Fig. 2; Table 6). The results weren t statistically significant (Mann Whitney- U test, p=0.058, Median test p=0.141). Fig. 2 The Boxplot displays TIs in days according to type of PC 21

22 12.4. Incidents of transfusion related reactions of apheresis and WBD PCs There were no incidents of transfusion reaction Transfusion intervals of men and women Values of time to next transfusion (transfusion interval) in men and women were not distributed according normal distribution. In consequence we used non-parametric tests. The medians of time to next transfusion in men and women were different (3 (IQR=2) vs. 2 (IQR=1.25)) (Table 6; Fig. 3). The difference was statistically significant (Median test p<0.001, Wilcoxon sign Rank test p=0.002). Table 6 Comparison of transfusion intervals (medians, in days) along gender and type of PCs TI, medians p APC 3 (IQR=2) WBD PC 2 (IQR=1) Mann Whitney- U test, p=0.058, Median test p=0.141 Men 3 (IQR=2) Women 2 (IQR=1.25) Median test, p<0.001, Wilcoxon sign rank test, p=

23 Fig. 3 The boxplot displays the TI in days according to men and women Platelet increments of men and women Men received a total of 62 transfusion of PCs, women received 97 transfusions. A successful transfusion was defined as a PI over 4x10*9/L in the following CBC, 12-36h post-transfusion. 61.8% of men s transfusions were successful, 38.2% failed. 53.6% of women s transfusions were successful, 46.4% failed. Not successful transfusions with an PI of 4x10*9/L and less were excluded from the evaluation of the PI of women and men. Values of platelet increments in men and women were not distributed according normal distribution. Subsequently, we used parametric analysis. The medians of the PI were different in men and women (16.5 (IQR=12.5) vs. 12 (IQR=10.75)) (Fig. 4). But the differences are not statistically significant (Median test, p=0.320, Wilcoxon sign rank test, p=0.068). The values of pre- and post-transfusion platelet count (PLC) of men and women were not distributed according normal distribution. So parametric analysis was applied. The medians of pre- and post-transfusion PLC were statistically significant different (both, Wilcoxon sign rank test, p<0.0001) (Table 7). 23

24 Table 7 Efficacy of transfusions in men and women PI, medians Pretransfusiotransfusion Post- Successful Failed transfusion transfusion PLC, medians PLC, medians Men (IQR=8) % 38.2% (IQR=12.5) (IQR=16.75) Women 12 6 (IQR=8) % 46.4% (IQR=10.75) (IQR=16.25) P Median test, Wilcoxon sign Wilcoxon sign p=0.320, rank test, rank test, Wilcoxon p< p< sign rank test, p=0.068 Fig. 4 The boxplot displays the PI according the men and women 24

25 13. DISCUSSION Comparison of efficacy of APC and WBD PC Our numbers differ to results of other authors like Nancy H.. In our data the percentage of successful APC and WBD PC were higher. In her study 52% percent of APC and 35% of WBD PC transfusions were successfully. The tendency of a higher rate of successful transfusion of APC than for WBD PC is similar to our study (64% vs. 44.6%). The differences might be due to higher and more even number of transfusion in both groups. Or due to higher platelet counts in their transfusion bags leading to higher increments and more successful transfusions. Another factor might be the more accurate assessment of efficacy by the CCI of 4h after transfusion and 18-24h after transfusion. Instead we used just the difference pre- and post-transfusion CBC to evaluate the increment neglecting body surface and number of transfused platelets in the calculations. Most recently Baolan et al.[2] found higher means of CCI-1 and CC-2 for BC PC transfusion compared to APC transfusions. Though again a statistical significant difference was not found (P>0.05), what is similar to our study, where no statistical significant difference between the increments of APC and WBD PC transfusion was found (P=0.698). Baolan at al. established further higher means for the WBD BC PC than for the APC transfusions (means respectively, 15.83±4.65 vs ±4.45) in CCI-1 and lower mean increments in CCI-2 for WBD BC PC than for APC (respectively, 8.67±4.21 vs. 9.57±3.36). In our study we see higher medians for WBD PC than for APC (medians respectively, 16 (IQR=13), 15 (IQR=11)). Our increment values are difficult to compare with the CCI because of differences in calculations. But most likely our increments values have to be compared with the 18-24h CCI-2, because the post transfusion CBC was most often taken the next day after transfusion with a range of 12-24h or occasionally 36h. The large range of our medians are maybe due to low sample size compared to Baolan et al.`s study[2] in which 72 patients were treated with APC and 83 were treated with WBD BC PC. 234±0.37x10*9 of platelets were transfused per APC unit, 61±0.23x10*9 are in one bag derived from one donor of WBD BC PC. 4 bags are approximately corresponding to one APC unit. The numbers of platelets of one transfused unit in the Affiliated Cancer Hospital of Zhengzhou University are higher but still similar to the unit (at least 200x10*9 platelets) transfused in Lithuania. This corresponds to similar PIs of their study to our study. Though an additional finding of our study is showing higher post-transfusion platelet counts for APC transfusions than for WBD PC transfusions by equal medians of pre-transfusion PLC (Table 5). This finding might be paradox to the finding of lower PI medians for APCs. This is different to Baolan et al. s result, who established higher post-transfusion PLCs (after 24h) and higher CCIs for 25

26 APCs than for WBD BC PC (Table 3). But our results are explained due to the inclusion of PLCs from transfusion, which actually failed, but still were included into the calculation of post-transfusion PLCs. In consequence this value cannot be used for evaluation of efficacy Longer Transfusion intervals for APC than WBD PC transfusions Akkök et al. found in their study no statistical difference of transfusion intervals of APC and WBD BC PC. Just longer storage time had significant influence on shortage of transfusion intervals in both PC types and as well significant lower CCI-1 and CCI-2 (p=0.03, both) in the group of patients with no infection and no bleeding. But as well fever and infection had a significant influence on CCI-1 but not on CCI-2. But in bleeding patients storage time had no influence on CC-1 and CC-2, but resulted in significant shorter transfusion intervals[14]. Apelseth et al. found no correlation between platelet increment and clinical bleeding status before or after a PC transfusion[7]. Even though our medians for APC were longer than the medians for WBD PC (3 (IQR=2) vs. 2 (IQR=1)), we might be able to conclude that there might be no differences in in the transfusion intervals because of statistical insignificance. Though longer transfusion intervals for APC than for WBD PC may be due to the usage of different WBD PC products. BC PCs have proven better CCIs than PRP PCs and resulted in higher increments[9], which in turn results in longer transfusion intervals[16]. This result is found in the study of Slichter et. al., finding longer transfusion intervals for APCs than for leukoreduced PRP PCs transfusions (means in days, 1.81±1.31 vs. 1.72±1.25) and as well lower CCI-1 and CC1-2 for PRP PCs than for APCs (respectively, CCI ± 18.8 vs ±24.6; CCI-2, 31.6 ± 16.0 vs ±19.7). 26

27 13.3. Men have higher platelet increments than women Another finding in our study is that men have higher median increments than women (16.5 (IQR=12.5) vs. 12 (IQR=10.75)), though not statistically significant, but almost (p=0.068). Heim et al. found in his study that sex has definitive influence on platelet increments[16]. Though his finding contradicts with our data. Male sex had significantly (p=0.01) lower CCI compared to women (mean, 13.8 vs. 14.2)[16], but on the other hand Slichter et al. found that women with at least 2 pregnancies are associated with reduced lower CCI-1 and CCI-2, but this finding was not progressive with higher numbers of pregnancies. He found as well the male sex as a significant (p<0.001) factor for lower CCI-1 and CCI-2 (means, 8.9x10*9 vs. 5.7x10*9)[15]. One reason for higher increments for men might be higher number of men than women in our study (10 vs. 7). But maybe our female patients have a history of at least 2 pregnancies, which might have resulted in lower platelet increments. Men showed also statistical significant higher post-transfusion PLCs than women. Women had lower medians of pre-transfusion PLCs than men (Table 5). This finding could reflect the higher PI for men than women Longer transfusion intervals for men An additional result of our study is the significant (p<0.001) higher intervals of transfusion for men than for women (medians, 3 (IQR=2) vs. 2 (IQR=1)). This might be due to higher platelet increments found for the male sex in our study than for the female sex (medians, 16.5 (IQR=12.5) vs. 12 (IQR=10.75)). Such as Heim et al. found in their study that transfusions resulted in longer transfusion intervals, had significantly bigger platelet increments than transfusion resulted in shorter transfusion intervals (Table 8)[16]. For that reason, in our study bigger platelet increment of male patients than for female patient leading to longer transfusion intervals for men than for women. Our result is still paradox to Slichter et al. s, like before with increments discussed, male gender is a factor for significant lower transfusion intervals, but on the other hand female with at least two pregnancies too. If you would consider all our female patients (7) having a history of 2 or more pregnancies, it would maybe show that the factor at least two pregnancies for platelet increments is more crucial in our case, than the factor male gender leading to lower platelet increments. But other factors play a role in lower platelet increments, too, which are not researched in our study, but might played a role: DIC, Heparin usage, positivity to lymphocytotoxic antibody, bleeding, usage of amphotericin, fever and palpable spleen[1,15]. 27

28 Table 8 Relation of longer transfusion interval to higher CCIs (Heim et al.)[16] CCI, mean Transfusion interval, in days p d d. p< d. p= d p= Incidence of transfusion reactions In our researched patients, we didn t find the occurrence of transfusion reactions. No occurrence of transfusion reaction differs to the general scientific opinion regarding to frequency of transfusion reactions. Heddle et al. found slightly lower incidence of transfusion reactions for pre-storage leukoreduced APCs than for pre-storage WBD PRP PCs (11.4% vs. 13.3%). The incidence of severe reaction was 1.4% in the APC group and 1.7% in the WBD PRP PC group. Maybe the low incidence of transfusion reaction in our study is due documentation error of transfusion reaction, due to failure of recognition of transfusion reaction or due to high quality platelet products used in Kauno Klinikos. 28

29 14. CONCLUSION Compare the platelet increment of APC and WBD PC transfusions We have two conclusions, either our data (Table 5; Fig. 1) shows superior platelet increments for WBD PC than for APC transfusions or there is no difference between our researched APC and WBD PC transfusions due no statistical significance. It is not clear in our study how many of our WBD products were produced by the BC or PRP method. Therefore, assuming all WBD PC transfusions were BC derived, which could show the non-inferiority of BC to APCs. PC transfusions Compare the duration of transfusion intervals of patients receiving APC and WBD The duration of transfusions intervals seems to be longer in apheresis derived PC transfusions, than in whole blood derived PC, which were found in our study (Table 6; Fig. 2). We can conclude either that the longer transfusion intervals for APC may reflect the possible high usage of PRP products in this time, which are shown to be inferior in terms of platelet increment and TI to APC products, or that due to statistical insignificance we have no difference between the two products and we could assume that we used more BC products. Our data showed significant longer transfusion interval and non-significant higher PI for men (Table 6, Table 7). In consequence, we may conclude that the factor 2 prior pregnancies resulting in shorter transfusion intervals is more weighted then factor men Compare the frequency of adverse reactions of APC and WBD PCs Our study had no incidence of transfusion reactions. It may be due to human error of documentation or due to the high quality of the platelet products used in Kauno Klinikos. Or it might be an incidence of small sample size. 29

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