1. BLOOD COMPONENT PREPARATION
This presentation includes
1. The principle of component preparation
2. Procedure for preparation of all components
3. Equipments required to prepare components
4. Daily maintenance of equipments in component laboratory 1

2. LEARNING OBJECTIVES This presentation will enable participants to
Understand the basic principles and procedure of Component Separation
Know the different components that can be prepared in a blood bank 2

3. INTRODUCTION Transfusion service – certain patient goals
Cater to patient needs
Provide blood and components which are
Safe
Pure
Potent
Effective
Policies and procedures so that goals are met 3

4. Development of component therapy
• Earlier only whole blood
• Polyvinyl Chloride (PVC) bags
introduced in India in late ’80s
• Treatment in various diseases/conditions
Cancer : platelets
DIC : plasma, platelets, cryo
Haemophilia : cryo
The speaker will cover the developmental shift of blood containers from bottles to bags. The requirement of blood components is based on the clinical situation and indications 4

5. Definitions Blood product Any therapeutic substance prepared
from human blood
Whole blood Unseparated blood
containing anti-coagulant preservative
Blood component A constituent of blood, separated
(component from whole blood
separation) eg., red cells, platelets, FFP
Plasma derivative Human plasma proteins prepared
(fractionation) under pharmaceutical manufacturing
conditions eg., albumin
Different components that can be harvested from blood are defined.
The difference between blood component and blood derivative is highlighted 5

6. CARDINAL PRINCIPLES COMPONENT PREPARATION is “manufacturing”
SOPs - outline all details of procedures
Standardization
Quality assurance
Equipment for preparation and storage
- must be maintained and reassessed 6

7. BLOOD COMPONENTS STANDARD SPECIALIZED Whole blood
Packed Red Cells
Platelets
PRP, RDP, SDP
Fresh Frozen Plasma (FFP)
Cryoprecipitate
SPECIALIZED
Saline-washed Red Cells
Frozen Red Cells
Leucodepleted products
Irradiated products
The various blood components are enumerated. 7

8. Planning a component lab
Type of hospital and bed strength
Trained
manpower
Adequate
AC space
(+ 50 m2) QC
program
Equipment
Since component laboratory needs a separate licence from the regulatory authorities the various requirements are discussed
License from
Regulatory
authorities
Double, triple
or quadruple bags 8

9. Blood donors Fulfill criteria Hb ≥ 12.5 gm/dl Weight - 45 kg (350 ml)
No Aspirin < 3 days
Single bold venipuncture, free flow of blood
Collection - time < 10 minutes with frequent mixing
Eligibility of blood donors and various aspects that can influence the yield of components is discussed 9

10. Equipment for components
Additional
Sterile tubing welder
Gamma irradiator
Cell separator (apheresis)
Weighing balance
Two pan balance
Refrigerated centrifuge
Laminar air flow bench
Deep freezers (-40,-80oC)
Platelet shaker/ incubator
Refrigerated water bath
Plasma expressor
Tube sealer

11. Multiple integrated blood bags
For components separation multiple integrated bags provide a functionally closed system are used. Depending on the components to be harvested double, triple or quadruple bags can be used. In case of additive solution bags one of the satellite bags contain additive solution which can be ADSOL or SAGM 11

12. Quadruple Blood Bags
The quadruple bags could be either TOP and TOP or TOP and BOTTOM System 12

13. Preparation protocol Counter balancing Weighing Centrifugation
Blood bags are measured in grams (weight). This should be converted to ml. (volume) using the specific gravity formula.
Expression 13

14. } } } Centrifugation - Principle
Blood cells have different Sedimentation Coefficients }
Plasma }
Buffy Coat
On centrifugation whole blood gets separated into layers, shown in the illustration, due to the different sedimentation coefficients of its constituents }
Packed Red Cells

15. Protocol for preparation of Red Cells and FFP
Whole Blood
Soft spin at 4oC
Red cells Plasma freeze at -30oC
FFP
Within 8 hours
The centrifugal speed depends on the diameter of the rotor. The speed can vary based on the centrifuge and/or the rotor and has to be standardised.
Mention should be made about the refrigerated centrifuge attaining required temperature before centrifugation 15

16. Protocol for preparation of Platelets
Whole blood
Soft spin at 22oC within 8 hrs
Red cells Platelet rich plasma (PRP)
Hard spin at 22oC
Plasma Platelet Conc.
(RDP)
For preparation of RDP centrifugation involves a soft spin followed by a hard spin

17. PRECENTRIFUGATION CENTRIFUGATION
Allow for 2 Hours of Resting Time
Maintains pH of Platelets - Better Separation
CENTRIFUGATION
Proper balancing and Packing of Cups
- Consistent Yield - Good Interface
Speed and Time of Centrifugation
- Dictates yields - Cellular injury
Gentle Handling
- prevents mixing at interface
Resuspension of platelets before storage

18. Platelet Preparation Steps
The differences in the procedure for preparation of RDPs by PRP method versus Buffy Coat method are shown. The Buffy Coat method involves a hard spin followed by a light spin 18

19. Platelet Preparation Buffy Coat Method
The advantages of buffy coat method of platelet preparation including one log reduction of leucocytes and less platelet activation because of buffering of the platelets due to the first spin being a hard spin can be emphasized. 19

20. Triple Bag showing separated blood components
Packed Red Cells
Platelets
Plasma

21. Preparation of Cryoprecipitate
Fresh Frozen Plasma
Slow thaw at 4oC Thaw in cryobath at 4oC
in Cold Room or Hard spin at 4oC
Blood Bank Refrigerator
Cryopoor plasma Cryoprecipitate
Cryoprecipitate should be prepared from Fresh Frozen Plasma anytime during it’s storage period, but preferably as early as possible in it’s shelf life as coagulation factors slowly deteriorate over a period of time 21

22. Storage and shelf life of components
Component Storage Shelf Compatibility temp life
Red cells + 2o-6oC 35 days ABO / Rh
Red cells + 2o-6oC 42 days ABO / Rh
with additive solution
FFP oC 1 year ABO
CPP oC 5 year ABO
Platelets + 22oC 5 days preferably ABO match
Cryoprecipitate - 30oC 1 year any group
For calculating the expiry date the Day of collection should be considered as Day ‘0’. The Date of expiry on the manufacturer’s label on the blood bag should be the date by which blood should be collected from the donor. The date of expiry of blood or component unit is calculated from the date of collection of blood.
In quadruple bags with additive solution, the primary bag contains the anticoagulant CPD and not CPD-A. In case blood cannot be separated into components, the whole blood collected in the primary bag will have a shelf life of only 21 days. 22

23. Specialized equipment
Cell separator is used for preparation of Apheresis platelet concentrates i.e., Single Donor Platelet (SDP) and plasma by apheresis. Apheresis requires a separate licence from the regulatory authorities.
Sterile tubing welder is a device which enables preparation of aliquots in a functionally closed system for paediatric use
Sterile tubing welder
Cell separator 23

24. Leucodepleted blood (1)
Methods
Washing (saline washed red cells)
Freezing
Buffy coat removal
Microaggregate filtration
Specific leucodepletion filters
Low leucocyte apheresis devices (cell separators)
The various methods of reducing leucocytes in the blood are enumerated.

25. Leucodepleted Blood (2)
Of various methods
Leucocyte filters most efficient – 99.99% (4 log depletion)
Leucocyte filters can be Red cell filters or Platelet filters
Leucofiltration can be done
- At the bedside
- In the laboratory before issue
- Leucodepletion using inline filters

26. Concept of Log Reduction
1010
109
108
107
106
105
Whole Blood
1 log
Reduction
(90% reduction)
Buffy Coat Depleted Red Cells
Leucofiltered products
Whole blood has 109 leucocytes. Reduction of leucocytes in whole blood to one tenth (10%) of the original to 108 is 1 log reduction i.e., removal of 90% leucocytes. Reduction of leucocytes to one thousandth of the original to 106 is 3 log reduction i.e., removal of 99.9% leucocytes.
3 log
Reduction
(99.9%%) 26

27. IRRADIATED BLOOD
For Gamma irradiation (caesium or cobalt) dose is 25 Gy
Prevents transfusion associated graft-versus-host disease (TA GvHD)
INDICATION :
Bone Marrow Transplant
Congenital immunodeficiency
Premature infants
Intrauterine transfusions
First degree relatives

28. Plasma Derivatives
They are prepared in fractionation centres from plasma pools
Albumin
Plasma Protein Fraction
Factor VIII concentrate
Fibrinogen
Immunoglobulins
Other coagulation Factors
Emphasis should be placed on the fact that plasma derivatives are not blood components and are prepared in the fractionation centre using specialized equipment from plasma pools.
Plasma derivatives are not blood components, which are prepared in the blood bank 28

29. LEARNING OUTCOME
At the end of this presentation participants will have understood the process involved in separation of blood components and the various blood components that can be prepared 29