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

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

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

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

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

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

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

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

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

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

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

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

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

} } } 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

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

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

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

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

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

Triple Bag showing separated blood components Packed Red Cells Platelets Plasma

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

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 - 30oC 1 year ABO CPP - 30oC 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

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

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.

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

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

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

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

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