Buffy Coat Method of Component Preparation

Buffy Coat Method of Component Preparation

1. Buffy Coat Method - Component Dr. M. Mohandoss Additional Professor Transfusion Medicine Malabar Cancer Centre, Thalaserry

2. Blood In History Richard Lower is credited with performing, in 1665, the first authentic blood transfusion (animal to animal)

3. Human to Human Transfusion In 1818, James Blundell -Attempted human-to human transfusion

4. Modern Transfusion T herapy …….means component therapy

5. Whole blood in True sense…. MALABAR CANCER CENTRE

6. Whole blood is not Whole! After 24hrs of storage WB essentially becomes red cells suspended in a protein solution Changes in Platelets WB stored at 4°C – platelets rapidly lose viability Granulocytes & Monocytes – function reduced within 8hrs and disintegrates within 24hrs Microaggregates increase in number FVIII, FV – levels decreases

7. Component Therapy Optimal preservation of in vitro function of blood Efficient utilization of blood donations

8. MALABAR CANCER CENTRE COMPONENTS STORAGE TEMPERATURE Packed red blood cells (PRBC) +2 to 6°C Platelets (PLTS) +22 to +24°C under constant agitation Fresh frozen plasma (FFP) -30°C or below Cryoprecipitate (CRYO) -30°C or below

9. Blood Bags MALABAR CANCER CENTRE

10. MALABAR CANCER CENTRE

11. Methods of Component Preparation Whole Blood Apheresis Platelet Rich Plasma Method Buffy Coat Method Manual Method Automation

12. Platelet Rich Plasma (PRP) method Whole blood Soft spin within (1800 rpm 6 hrs x 9 min at 22 o C) Red cells Platelet rich plasma (PRP) Hard spin (3000 rpm x 7 min at 22 o C) Plasma Platelet Conc. (RDP) PRP RBC PRP PC FFP MALABAR CANCER CENTRE

13. PRP Method Manual plasma expresser MALABAR CANCER CENTRE

14. Buffy Coat Method Top and Bottom/ Top and top methods are available to produce Leuco -reduced blood components

15. Buffy Coat method Whole blood Hard spin within (3000 rpm 6 hrs x 9 min at 22 o C) Red cells Buffy coat plasma Soft spin (1800 rpm x 7 min at 22 o C) Platelet Conc. WBC with few RBC Plasma RBC

16. 1 st Spin Hard-spin centrifugation step lead to a buoyant density equilibrium, with red cells at the bottom followed by a layer of Granulocytes Lymphocytes Monocytes Platelets and Plasma on top

17. Buffy Coat & 2 nd spin Buffy coat had an average volume of 55 ml, and contained more than 80% of the platelets and 60% of the leukocytes After dilution with a small amount of plasma, this buffy coat of about 110 ml and a haematocrit of approximately 20% was centrifuged again under differential centrifugation conditions (soft speed), in which only the platelets were small enough to be pushed upwards in the centripetal streaming plasma

18. Automatic Component Extractor Two parallel pressure plates, one is stationary and other is pneumatically driven that simultaneously expel plasma in an empty satellite bag and red cells into another bag containing SAGM, an additive solution. The system is designed to leave a specific volume of buffy coat with plasma in the original collection pack. A light transmitter and two photocells control the flow of red cells and plasma into separate bags by the means of two automated clamping devices

19. Separation using component extraction Quadruple Bag (Top And Top Bags) Top and Bottom Bag (TAB)

20. Blood Component Extractor Sensors Sealers Sensor Sealer Press

21. Benefits of Buffy coat method Reduces the Leucocytes level by 70-80% and sacrifices 20% of the red cells Platelet yield improved a lot ( platelets at the top of bag will fall to the buffy coat but also that the platelets at the bottom of bags will rise to the buffy coat. So the buffy coat is good source of platelets) RBC contamination drastically decreased

22. During collection of blood Clean venepuncture with minimum tissue trauma and free flow of blood If any unit takes more than 10 minutes to draw, it is not suitable for preparation of platelet concentrate, fresh frozen plasma or cryoprecipitate If platelets are to be harvested, the blood bag should be kept at room temperature (20-24°C) until platelets are separated. Platelets should be separated within 6-8 hours from the time of collection of blood

23. In centrifugation Opposing cups with blood bag and satellite bags must be equal in weight otherwise excessive eccentric loads placed on rotor of centrifuge cause irregular wear and tear and eventual breakage The bags should be so placed that its broad side faces the outside wall of the cup.

24. QC of Modified Blood Components Leukoreduced Blood Components Irradiated Blood components Saline Washed Components (Red Cells/Platelets)

25. QC in Blood Bags Quality control of blood components is an integral part of the quality management systems in the Blood Centre Ensures Proper yield, Functionality Efficacy of components

26. Indian Standards for QC of Blood Components Drugs and Cosmetics Act 1940,Rules 1945, (Schedule F, Part XII B), Government of India Blood Bank Standards of NBTC, Ministry of Health and Family Welfare Government of India NABH Accreditation standards for Blood Banks

27. Essential Quality Elements Sampling techniques Frequency of QC Atleast of 1 % of all components per month (if >100 per month prepared ) Atleast 4 units per month (if < 100 per month prepared ) 75 % or more of components must meet specifications Washed Blood Components – on ALL BAGS

28. Volume of Blood/blood component

29. Quality control of Red Cells in additive solution (Prepared from 450 ml / 350 ml Whole Blood) Parameter   Quality Requirement Volume ( + additive solution) 250 ± 10% (+ 100 mL AS for 450 ml Bag) 150 ± 10% (+ 80mL for 350 ml Bag) PCV ( Hct ) 50-60% Sterility By culture  

30. Leukocyte Content of Various Blood Components Leukoreduction (LR) labeling requirements US < 5 x 10 6 /unit EU < 1 x 10 6 / unit

31. Levels of Leukoreduction % WBC removal 90% 99% 99.9 % 99.99 % Log Reduction Log 1 ( 10 8 ) Log 2 ( 10 7 ) Log 3 ( 10 6 ) Log 4 ( 10 5 )

32. Quality control of Leucocytes- Reduced red cells Method of Preparation Product Volume HCT Additional parameter Buffy Coat removed Red cells in additive solution from 450 ml WB   250 ml± 10% (+100 ml additive solution) 50-60%     Red cells in additive solution from 350 ml WB 150 ml ± 10% (+80 ml additive solution) 50-60%  

33. Quality control of Leucocytes- Reduced red cells Method of Preparation Product Volume HCT Additional parameter Washed using Normal saline   Washed Red cells Within locally specified volume range   50-60% RBC loss <20% Total protein content < 0.5 g / unit Leuco-filtration Leuco-filtered RBC (Leucocyte depleted) 250 ml ± 10% (+100 ml additive solution)  50-60% Hemolysis <0.8% or 1% WBC <5 x10 6 per unit Irradiation Irradiated RBC Within locally specified volume range Hemolysis <0.8%

34. Example : Vol. of Whole blood in a 350 mL QB = 356 mL WBC count in WB = 7.4 x 10 3 /µL HCT = 44% Therefore , WBC count in the bag = 7.4 x 10 3 x 356 x 10 3 1mL=10 3 µL = 2634.4 x 10 6 = 2.63 x 10 9

35. After leuco-reduction by buffy coat method: Volume of LR-RBC = 241 mL WBC count = 1 x 10 3 /µL HCT = 58 % Therefore, WBC count in the bag = 1 x 10 3 x 10 3 x 241 = 241 x 10 6 = 2.41 x 10 8 Volume – adequate WBC count <5 x10 8 /unit 1 log reduction obtained HCT – 50-60%

36. Quality control of Platelet Concentrate (RDP) from 350ml/450 ml of Whole Blood Parameter   Quality Requirement Appearance Swirling present, No visual RBC contamination, Volume 50-70 ml (PRP method) 50-90 ml ( Buffy coat method) Platelet count ≥3.5 x 10 10 platelets/unit from 350ml WB – PRP method ≥4.5 x 10 10 platelets/unit from 450 ml WB blood – PRP method >6X10 10 platelets/unit from 450 ml WB blood – BC method pH   >6.0 (at the end of permissible storage period) RBC contamination   Traces to 0.5 ml   ( < 5x10 9 RBCs) Sterility By culture  

37. When should QC be done ? As per SOP Platelet product done on expiry date On Installation and after repair of equipments If any Modification in procedure Recruitment of new personnel

38. Conclusion Component therapy has revolutionized transfusion medicine Standardized blood components help to assess the outcome of the component therapy. Procedures as leukofiltration , Irradiation of blood & should be standardized before putting in to use

39. MALABAR CANCER CENTRE Thank You