3. INTRODUCTION AND BACKGROUND:

Apheresis is simply removal of desired component and returning the remaining components to the individual. According to literature ‘Apheresis’ is described as a process by which blood being removed from healthy voluntary donors is continuously separated into component parts, usually to allow a desired component (or components) to be retained while the remainder is returned to the healthy voluntary donors (1). ‘Plateletpheresis’ refers to a procedure in which portion of donor’s platelets and some plasma is removed with the return of donor’s red blood cells (RBC), white blood cells (WBC) and remaining plasma (2). The component prepared from apheresis system is called as a Single Donor Platelet (SDP) contains minimum of 3 x 10¹¹ platelets. Platelet concentrates can be prepared from whole blood donations called as Random Donor Platelets (RDP) contains minimum of 5.5 x 10¹⁰platelets (3). Hence six units of RDP will give equivalent platelet count increment as one SDP (3). Multiple RDP units’ transfusions there is a risk of allogenic donor exposure, transfusion transmissible infection exposure and transfusion reactions (4). For few platelets refractory cases those developed anti Human Leucocyte Antigen (HLA) antibodies, needs HLA matched platelet transfusion that can be done by the apheresis platelets transfusion (5). In oncology centre platelet transfusion is important.

Plateletpheresis procedure includes donor screening, eligible donor will be connected to the apheresis cell separator. Blood drawn from donor undergoes centrifugation in the cell separator which helps in settling of blood components into layers according to their specific gravity. Platelets with some amount of plasma will be collected and remaining blood components will be returned to the donor. Factors plays role in plateletpheresis procedure are donor blood volume, donor hematological parameters, time, anticoagulant etc.,

Platelets forms from megakaryocytes, plays an important role in hemostasis and thrombosis with life span of 5-7 days (6). Aster RH et al found that normal spleen contains one third of platelets which is splenic pool whereas circulating pool contains two third of platelets. Both splenic pool and circulatory pool are in equilibrium. Suggests circulating pool platelets will be continuously replenished during the plateletpheresis procedure (7). This replenishment is the main reason for achievement of high-quality platelets. Repeated platelet donation is easily managed because of the platelet replenishment from splenic pool to the circulatory pool. In context of physiology of plateletpheresis, Recruitment Factor (RF) is the constant replenishment of platelets to the circulatory pool.

In the Department of Transfusion Medicine, different cell separators based on Intermittent Flow Centrifugation (IFC) and Continuous Flow Centrifugation (CFC) principle are available. Most new cell separators are CFC based. Examples of cell separators which works on IFC principle include Haemonetics MCS plus LN9000. Examples of cell separators which works on CFC principle includes Baxter/Fenwal CS-3000 plus, Amicus, Cardian BCT COBE Spectra, Spetra Optia, and Fresenius AS-104. Collection efficiency (CE) of different cell separators varies. Collection efficiency is which compares the number of platelets that were in the collection bag verses the number of platelets that passed through the machine (8).

As per the Association for the Advancement of Blood and Biotherapies (AABB) mandates that SDPs should be minimum 3 x10¹¹ platelets per one unit. As platelets storage temperature is 20-24⁰C with 5 days shelf-life, there is a risk of bacterial growth (3). With escalation of platelet requirement day by day in the oncology centre, managing inventory with shrinking plateletpheresis donor pool is challenging issue. Double dose platelet (DDP) with available modern machines with stringent inclusion criteria will be helpful. Along with general plateletpheresis donor eligibility criteria given by Drugs and Cosmetics rule (second amendment) 2020 (9), additional criteria as for Director General of Health and Services (DGHS) for DDP pheresis are weight of the donor should be 60 kgs or more, platelet count should be 250 x10¹¹/ul, not a first-time donor, donating DDP with minimum 4 weeks interval. DDP target platelet count (yield) should meet the AABB standard criteria for DDP (10). Platelet count in DDP should be minimum 6 x 10¹¹ platelets (3,10). One DDP unit will be separated into two SDPs. Each SDP should pass the quality control (QC) of minimum 3 x10¹¹ platelets.

With increasing haemato-oncology patients, it is mandatory for platelets inventory maintenance to manage medical emergencies associated with bleeding. Multiple platelet transfusions will be required in such patients for prophylactic and therapeutic management of bleeding patients. In the Department of Transfusion Medicine (DTM), Tata Memorial Centre, oncology centre with annually more than 5000 plateletpheresis procedures, 700- 800 DDP plateletpheresis procedures are collected to meet the platelet requirement in the centre. There is a limited literature on DDP and RF for the donor safety.

Recruitment of platelet for plateletpheresis facilitates collection good yield of platelet in single donation. Thus, helps in splitting of single donation product into two products. This way will reduce multiple donor exposure risk. Recruitment of platelets during donor plateletpheresis may prevent post-procedure thrombocytopenia, thereby increasing the safely achievable maximal platelet yield (8). Cell recruitment can be calculated as recruitment factor (RF), following the formula of Knudsen and co-workers (11). A recruitment factor of 1 (RF = 1) would suggest that no platelet mobilization occurred during the plateletpheresis.  RF values lower than 1 (RF < 1) would represent a platelet loss, whereas values greater than 1 (RF > 1) allude to a platelet mobilization after plateletpheresis.

 Recruitment of platelets facilitates harvesting of double dose platelets in a single donation procedure which may be influenced by the donor and cell separator parameters. Additionally, the purpose of this study is to ensure plateletpheresis donors’ safety and wellbeing.    

4. AIM AND OBJECTIVES:

4.1. AIM

To study the platelet recruitment in platelet donors during double dose plateletpheresis on two different cell separators.

4.2. OBJECTIVES:

4.2.1. Primary objective: To calculate and compare the Recruitment Factor (RF) in Double Dose plateletpheresis donor across two different cell separators.

4.2.2. Secondary objective:

1.To compare collection efficiencies (CE) and collection rates (CR) in Double Dose Plateletpheresis (DDP) procedure across two cell separators.

2. To assess the platelet yield of Single Donor Platelets (SDP) prepared from Double Dose Platelets product. 

3. To analyze the adverse donor reactions in Double Dose Plateletpheresis donors if any.

4. To compare pre and post procedure hematological parameters of the double dose plateletpheresis (DDP) donors.

5. STUDY DESIGN AND METHOD:

5.1. TARGET POPULATION:

The present study will be a prospective observational pilot study of 30 voluntary plateletpheresis donors undergoing double dose plateletpheresis procedure on two different cell separators during study period, in the Department of Transfusion Medicine, Tata Memorial Hospital, Parel over a period of 6 months.

5.2. INCLUSION CRITERIA:

Donors who fulfil the plateletpheresis donor eligibility criteria laid down by Drugs and Cosmetic (second amendment) Rule, 2020 and consent to participate in the study, will be selected.

General donor eligibility criteria for plateletpheresis are as follows (3,9)

1. Age- 18-60 years

2. Weight of donor- 50 kgs and more (3)

3. Blood pressure-100 to 140 mm Hg systolic, 60 to 90 mm Hg diastolic with or without medication.

4. Pulse-60- 100/min

5. Temperature- Afebrile; 37⁰C or 98.4⁰F

6. Haemoglobin- 12.5 g/dl and more

7. Platelet count- 150,000/ul and more (3)

8. Donation interval- Between procedures should be at least 48 hours. A donor should not undergo the procedure more than 2 times a week or 24 times a year. After the whole blood donation plateletpheresis, the donor should be accepted only after 28 days of interval.

9. Skin- Free from any skin disease

10. Transfusion transmitted infections- Nonreactive status

However, for Double dose plateletpheresis procedure (DDP), the donors should fulfil the additional criteria according to Directorate General of Health Services (DGHS) 3^rd edition 2022: (12)

1.Weight of the donor should be > 60 kgs,

2. Platelet count > 250 x 10¹¹/ul,

3. Not a first-time donor,

4. Interval between two plateletpheresis procedure- at least 1 month

5.3. EXCLUSION CRITERIA:

Donors who do not fulfil the plateletpheresis donor criteria laid down by the Drugs and Cosmetics (Second amendment) rules, 2020 (9) and not consent to participate in the study will be excluded.

5.4. MATERIAL AND METHODS:

This study will be a prospective observational pilot study with DDP collection in 30 voluntary plateletpheresis donors across two different cell separators in the Department of Transfusion Medicine over a period of 6 months. All the eligible donors will be explained about the procedure including pre, post procedure sampling and informed consent will be taken. All eligible participants (platelet donors) enrolled for the study will donate DDP on two different cell separators on two different occasions at least 4 weeks interval. DDP will be separated into SDP- 2 units as per Departmental SOP and will be labelled as A and B. Platelet yield will be set as 6 x 10¹¹ for both cell separators during all the DDP procedures. Pre and post procedure Complete Blood Count (CBC) of blood samples of participants will be done. DDP procedure parameters will be used to calculate Recruitment Factor (RF) and Collection Efficiency (CE).

5.4.1. EQUIPMENT:

a) Cell separators: The plateletpheresis procedure will be conducted as per Departmental SOP on two different cell separators available in the department.

1. AMICUS version 4.4 (Fresenius Kabi)

2. SPECTRA OPTIA (Terumo BCT)

b) Automated cell counter: SYSMEX XN10

This cell counter will be used for assessing the donor hematological parameters from pre and post procedure samples and platelet count (yield) assessment of DDP and SDP.

CBC will be done on SYSMEX XN 10 in the Hematopathology laboratory of Tata Memorial Hospital, Mumbai.

5.4.2. SAMPLE COLLECTION:

Two times sample will be collected from the donor, pre-procedure and post-procedure samples.

a) Pre-procedure:

About 2 ml Blood sample will be collected in Ethylene Diamine Tetra Acetate (EDTA) tube at the beginning of procedure from the diversion pouch and will be considered as pre-procedure sample. This sample will be used for assessment of pre-procedure hematological parameters of donors.

b) Post-procedure:

About 2ml of blood sample will be collected 10-15 minutes after completion of procedure in EDTA tube from different phlebotomy site of the donor as per standard phlebotomy guidelines. This sample will be considered as post-procedure sample and will be used for assessment of hematological parameters of donors.

c) Product sample:

1. DDP sample of 1ml will be collected into clean test tube from sample pouch (for maintaining sterility), for assessing the platelet count (yield) of DDP unit (that is product).

2. After separation of SDP from DDP, sealed segment will be taken from each part (to maintain sterility), 1ml SDP sample will be collected into clean test tube for assessing the platelet count (yield). All the SDPs will be utilized for the patients

5.4.3. PARAMETERS TO BE ANALYSED:

1) Donor parameters:

a) Demographic details: Donor demographic parameters such as age, gender, blood group, weight, height and total blood volume will be documented.

b) Hematological parameters: Donor hematological parameters as hemoglobin (HB), hematocrit (HCT), platelet count (PLT), Red Blood Cell count (RBC), Mean platelet volume (MPV) and White blood cell count (WBC) obtained while screening the donor will be documented before the procedure.

Platelet Recruitment Factor (RF) after the procedure will be analyzed by using formula (13,14)

RF = (post procedure platelet count + platelet yield) / pre procedure platelet count

2) Procedural parameters:

a) Plateletpheresis procedure details: Such as Total blood volume (TBV) processed, anticoagulant (acid-citrate-dextrose-A, ACD-A) volume used, procedure time, target platelet yield, collection rate, inlet rate, return rate, etc., will be documented for each procedure.

b) Equipment related parameters: Collection efficiency (CE) of both AMICUS and SPECTRA OPTIA calculated by using formula (13,15)

CE = Platelet yield / Total platelets processed* x 100

*Total platelet processed = (pre-procedure platelet count + post-procedure platelet count/ml)/ 2 x Total blood volume processed* (ml) x conversion factor (1000 ul/mL)

*Total blood volume processed (ml) = Blood volume processed (ml) – ACD-A volume (ml)

3) Product related Parameters:

Platelet count (yield) and volume of DDP and SDP separated from DDP will be assessed.

4) Donor Adverse reactions: (16) list of apheresis adverse reactions

a) Citrate toxicity

b) Vascular access complications

c) Vasovagal reactions

d) Hypovolemia

e) Allergic reactions

f) Air embolus

g) Circulatory and respiratory distress

5.4.4. FORMULAE FOR CALCULATION OF PARAMETERS: (13,14,15)

1) Platelet yield = platelets in the unit (platelets count/ul) x unit volume [mL] x conversion factor (1000 ul/mL)

2) RF = (post procedure platelet count* + platelet yield)/pre donation platelet count

*Absolute number of platelets in blood = platelet count (x 10⁹/L) x total blood volume (L)

3) CE = Platelet yield / Total platelets processed* x 100

*Total platelet processed = (pre-procedure platelet count + post-procedure platelet count/ml)/ 2 x Total blood volume processed* (ml) x conversion factor (1000 ul/mL)

*Total blood volume processed (ml) = Blood volume processed (ml) – ACD-A volume (ml)

4) Collection rate (CR) = Platelet yield (Y)/separation time (hr)

5) Target collection time = Target platelet yield/CR.

A Recruitment Factor of 1 (RF = 1) would suggest that no platelet mobilization occurred during the plateletpheresis.  RF values lower than 1 (RF < 1) would represent a platelet loss, whereas values greater than 1 (RF > 1) suggests platelet mobilization after plateletpheresis (13).

6. STATISTICAL ANALYSIS:

Statistical analysis will be done by using SPSS software version 25. Comparison of Recruitment Factor (RF) for plateletpheresis and Collection Efficiencies (CE) across two different cell separators. All quantitative variables will be estimated by using, measures of central location (mean), measures of central deviation (standard deviation). Qualitative or categorical data will be described as frequencies and proportions. To verify the correlation between two variables Spearman or Pearson correlation coefficient will be calculated. Statistical significance will be considered with p<0.05.

7. POTENTIAL IMPACT OF THE STUDY:

With increasing daily platelet requirement due to increased yearly hospital admissions in oncology centre and with same donor pool it may be difficult to manage adequate inventory. This study will help to facilitate the harvest of double dose platelet product with a single donation procedure and ensure the post donation recovery in donors is adequate. The objective of this study is to analyse the RF to understand the recruitment of platelets in donors undergoing DDP procedure. Donors who are willing to participate in the study will be donating platelets on two different occasions on two different cell separators with 4 weeks interval between two donations, RF will be calculated with the help of CBC parameters. This will help in ensuring the post donation platelet recovery in donors, hence donor safety. DDP can be collected from the donors safely to manage the platelet inventory to fulfil the patient platelet transfusion requirements.

8. REFERENCES:

1.Apheresis: Principles and Practice, 3rd edition (CD-ROM) Edited by Bruce C. McLeod, MD; Zbigniew (Ziggy) M. Szczepiorkowski, MD, PhD, FCAP; Robert Weinstein, MD; and Jeffrey L. Winters, MD, aabb press, Bethasda ,maryland 2010.

2. Sahoo D, Mahapatra S, Parida P, Panigrahi R. Various aspects of plateletpheresis: Its impact on donor and patients.Glob J Transfus Med 2017;2:149-154

3. Apheresis. Ratti ram sharma, ed. Transfusion Medicine Technical Manual. Director General of Health Services, 3rd ed., New Delhi: Ministry of Health and Family Welfare, Government of India; reviewed and published-2022.

4. Grifols-Lucas JA (1952) Use of plasmapheresis in blood donors. BMJ 1(4763):854; 1952 Apr 19; PMCID:14916171.

5. Freireich EJ, Judson G, Levin RH. Separation and collection of leukocytes. Cancer Res. 1965 Oct;25(9):1516-20. PMID: 5861076.

6.Harrison P. Platelet function analysis. Blood Rev. 2005 Mar;19(2):111-23. doi: 10.1016/j.blre.2004.05.002. PMID: 15603914.

7.  Aster RH. Pooling of platelets in the spleen: role in the pathogenies of. The journal of clinical investigation. 1966 may 1;45(5):645-57.

8. Rajadhyaksha SB, Desai P, Navkudkar A, George M, Platelet recruitment in donors undergoing plateletpheresis procedures on two cell separators. AABB abstract 2022.

9. The Drug and Cosmetic rules 1945 (Second amendment). Ministry of Health & Family Welfare. Government of India; New Delhi, 11^th march 2020.

10. Apheresis Platelets- US Guidance-ICCBBA 2015. Available from https://www.iccbba.org/docs/tech.

11. Knudsen LM, Nikolaisen K, Gaarsdal E, Johnsen HE. Kinetic studies during peripheral blood stem cell collection show CD34+ cell recruitment intra-apheresis. J Clin Apher. 2001;16(3):114-9.

12. Directorate General of Health Services, Ministry of Health and Family Welfare, Government of India, Transfusion Medicine Technical Manual, 3^rd edition, 2022

 13. Fontana S, Kellerb P, Taleghanib MB (2011) Platelet recruitment during multiple donor plateletapheresis differs between cell separators. Transfus Med Hemother 38:195–198.

14. Chopra S, Kaur P, Bedi RK, Kaur G. Effect of double dose plateletpheresis on target yield and donor platelet recovery. Hematol Transfus Cell Ther. 2023 Jan-Mar;45(1):16-24. doi: 10.1016/j.htct.2021.05.002. Epub 2021 Jun 18. PMID: 34294601; PMCID: PMC9938487.

15. Tendulkar A, Rajadhyaksha SB. Comparison of plateletpheresis on three continuous flow cell separators. Asian J Transfus Sci. 2009 Jul;3(2):73-7.

16. Denis M. Harmening, Beth A. Hartwell, SBB and Mark D. Pool, modern blood banking and transfusion practices, seventh edition, philadelphia; F.A. Devis, 2019.