PREPARATION OF BLOOD COMPONENTS AND THEIR

THERAPEUTIC USES

PRESENTED BY :- MANTHAN

NIPS ,PGIMER

Introduction In the past whole blood was the only preparationBut it cause unnecessary administration of

unwanted cells or plasma constituents.A significant advance in transfusion medicine was

made when techniques became available for separation of blood in a closed system and patient could be administered specific replacement therapy.

Hence whole blood can be utilized for preparation of different components and thus can benefit multiple patients.

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

HISTORY 1926-THE BRITISH RED CROSS instituted the

firsthuman blood transfusion service in the world.

1940-the freeze dried plasma was developed. 1950-Glycerol cryoprotectant for freezing red

blood cells. 1950-Carl walter and W.P.Murphy introduced the

plastic bags for whole blood. 1979-A new anticoagulant preservative CPDA-

1,which extend shelf life of whole and red blood cells to35 days is introduced.

Contribution In a statistical study it is seen that total

no of blood donation in West Bengal- 713535 (Vol.

85.71%) Bihar – 47863 (Vol. 22.74%)

Jharkhand --- 73238 (Vol. 33.13%) Uttar Pradesh- 394699 (Vol. 17.3%)

Maharashtra- 377110 (Vol. 86.36%)

Blood donors

FULFIL CRITERIA Hb ≥ 12.5 gm/dl Weight

- 45 kg (350 ml)- 55 kg (450 ml)

No Aspirin < 3 days Single bold venipuncture, free flow of blood Collection - time < 10 minutes with frequent mixing

Equipment for components

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

Additional Sterile tubing welder Gamma irradiator Cell separator

(apheresis)

BLOOD COMPONENTSSTANDARD

Whole blood

Packed Red Cells

PlateletsPRP, RDP, SDP

Fresh Frozen Plasma (FFP)

Cryoprecipitate

SPECIALIZED

Saline-washed Red Cells

Frozen Red Cells

Leucodepleted products

Irradiated products

Plasma Expresser

Dielectric Sealer

Multiple integrated blood bags

Quadruple Blood Bags

Whole blood

Red cells

Plasma

Platelets

(Fresh) frozen plasma (F(FP)

Cryoprecipitate

Cryo supernatant

plasma (CSP) F

lX*

Immun

eGlobuli

nAlbumin

Fractionated products

F Vlla*

F Vlll*

Granulocytes

* Now available as recombinant products

Preparation protocolCounterbalancing

WeighingCentrifugation

Expression

Centrifugation

Principle Sediment of blood cells depend on their size as well as the difference of their density from that of the surrounding fluid, viscosity of medium, flexibility of the cells which are temperature dependent

Specific gravity of Blood components

Gravity separationIt is an old time, crude but cheaper method to separate plasma from whole blood

Steps 1. blood collected in a double or triple bag system. 2. Blood is kept hanging overnight at least for 12-16 hours.

There is clear plasma above and packed red cells below. 3. The supernatant plasma is expressed into the satellite

bag, leaving behind, 80-100 ml of plasma.4. Plasma thus collected is called single donor plasma and

can be stored for 24-26 days at 4 - 6°C or for 1 year at -20°C or below.

(b) Low speed refrigerated centrifugation:-It is used for preparing Platelet Rich Plasma (PRP)(c) High speed refrigerated centrifugation:-It is used for preparing Platelet Concentrate (PC), Fresh

Frozen Plasma (FFP) and Cryoprecipitate (CP).

Effect of centrifugation

•High speed of centrifugation causes much trauma to cells and also the breakage of plastic container.•Second hard spin in PRP-PC method causes platelet activation as they are forced against the wall of the container.

Whole Blood450 ml of blood 63 ml of anticoagulant solution.Hct-36-44%No components have been removed.Store at 1-6 oCShelf life-

Citrate-Phophate-Dextrose (CPD) - 21 daysCPDA-1 (adenine) - 35 daysSAG-M – 42 days

Administer through standard blood filter (150-280 micron)

Infuse within 4 hours of issue

Whole Blood Drawbacks:

› After storage for >24 hours, platelets and WBC are non-functional

› Factor V and VIII decrease with storage› Fluid overload

Indications:› Acute blood loss > 25% TBV› Exchange transfusion

Contraindication- Risk of volume overload : Chronic anemia Incipient cardiac failure

Packed Red Cell

units with red blood cells and some plasma

- with Anticoagulant ACD / CPD / CPDA – 1

- Hct ~ 75 – 80 %

Preparation of Packed RBCsPrinciple:

RBCs are obtained by removal of supernatant plasma from centrifuged whole blood.

 Preparation:

Centrifuge whole blood unit in refrigerated centrifuge containing the parameters RPM-3850,Time-5 Min, Temp- 40C

Express the supernatant plasma with the help of plasma expressor.Double seal the tubing between the primary and satellite bag.Check that the satellite bag has the same donor number as that on the

primary bag and cut the tubing between the two seal.

Advantages:Oxygen carrying capacity equal to that of whole blood in half the volume.Significantly decrease levels of isoagglutinins, metabolites and electrolytes.

 

Preparation of Packed RBCs

Shelf life: (If CPDA1 anticoagulant) -35 daysStorage temp. : 40C (Range is 2-60C)QC Requirements: PCV 80% (Range is 65-80%)Volume: 250- 300 ml.CONTENTS: Red cells- 65-80%

Plasma – 20-35%Some platelets, white cells storage lesion by products and anticoagulant preservative solutions.

Transfusion Criteria's: ABO/Rh specific and compatibleIndications: Restore oxygen carrying capacity

symptomatic anemia and surgical blood loss.Effect: 1 unit RBCs should raise HCT -3%, Hb 1 g/dl 

Preservation and storage of Blood

Since 1978 citrate-phosphate-dextrose with adenine (CPDA-1) is used as blood preservative for

35 days at 2-40C.

Action of ingredients of anticoagulant solution.

Citrate Prevents coagulation by chelating calcium

Sodium di-phospate

Prevents fall in pH

GlucoseSupports ATP

generation by glycolytic pathways

AdenineSynthesizes ATP,

increases level of ATP, extends the self life of RBC to 42 days.

Action of ingredients of anticoagulant solution.- Blood pH on day of collection is 7.5

and on 35th day become 6.84.- A fall in pH in the stored blood

results in a decrease in red cell 2, 3-DPG level, which results in increase in hemoglobin-oxygen affinity. CPDA-1 maintains adequate levels of 2,3-DPG for 10 -14 days.

- During storage Na+ and K+ leak through the red cell membrane rapidly. K+ loss is greater than Na+ gain during storage.

Centrifugation method- easiest and least cost- least efficient- reduce WBC only 70 - 80% - reduceRBC volume ~ 20%

Leukocyte poor red blood cells

30

Filtration method easy, quick, but more expensive high efficient remove WBC more than 99.9%

( third generation ) little loss of RBC volume

Leukocyte depleted red blood cells

Leukocyte reduced red cells

Indication - Minimizes white cell immunization in patients - Prevention of FNHTR (Febrile Non-Hemolytic Transfusion

Reaction ) - Reduces risk of CMV transfusionContraindication - Not prevent graft –vs- host diseaseDosage - same as Packed Red CellAdministration - same as Whole Blood

Granulocyte Concentrate Obtained by apheresis from family members

for administration to cancer patients. Contain 1.0 x 1010 granulocytes

Pre-treatment with recombinant G-CSF and dexamethasone can yield 4-8 x 1010 granulocytes

Stored at 24o C Infuse within 24 hours of collection

Criteria

ANC <500 Fever Documented infection (bacterial or

fungal) for 24-48 hours Unresponsive to appropriate antibiotics Reasonable hope of marrow recovery

Platelet Rich Plasma (PRP)

It is prepared from the whole blood within six-hours of collection, preferably stored at room temperature of 20-24°C.  

Steps 1. The blood is collected in-CPDA-1 double or triple bag system.  2. The blood bags are weighed on a weighing balance and bags

weighing equal are placed opposite to each other in the buckets of centrifuge.  

3. Temperature of the centrifuge is adjusted between 20 -24°C  4. The speed of the centrifuge is calculated according to the

radius of the arm of the centrifuge rotor.5. The calculated speed for platelet preparation is 1750 rpm for 11

min. in PGI  6. After centrifugation, the bags are taken out from centrifuge

chamber with minimal disturbance and the PRP is expressed from the primary bag into the satellite bag with the help of a plasma expresser. After placing proper knots, labeling the blood group and ensuring the screening status the satellite bag is detached from the primary bag.  

7. The PRP for storage can be kept at 22-24°C in a, platelet incubator with constant agitation for a maximum of 48-72.hours (at our centre in PGI).  

8. As a part of internal quality control 1% of the random components units over a month are tested for pH and yield. For PRP the pH should always be > 6.2 and yield 4.5 x 1010 / bag (Drugs and Cosmetics Act) and 5.5 x 1010/ bag (AABB Technical Manual) and one bag of PRP generally raises the platelet count in the recipient by 5000-10000 / µl.  

Note: Aspirin and related analgesics affect the platelet function, so the donor for platelets is accepted after 3 days of ingestion of these drugs.  

Platelets Concentrate (PC)This supplies the same amount of platelets as PRP, but in lesser volume (40 - 50ml).

Principle:Platelets are harvested from whole blood following ‘light spin‘ centrifugation. The platelets are concentrated by 'heavy spin' centrifugation with subsequent removal of supernatant plasma.

Steps1. Blood is collected in the triple bag system only2. PRP is prepared by following the above mentioned steps3. After detaching the satellite bags from the primary bag, the PRP in

again spun at 4000 - 5000 rpm (high spin) for 4 to 5 minutes.4. A platelet button is formed at bottom and platelet poor plasma is

expressed from the 1st satellite bag to the 2nd satellite bags, leaving behind 40 - 50 ml plasma for platelet button suspension.

5. The platelet concentrate in stored at 20°C-24°C for a maximum of 3 days (at PGI) with constant agitation.

Platelets Concentrate (PC)Shelf life: 3 days in platelet incubator & agitator.

24 hrs if no storage cabinet Storage temp.: 20°C - 24°C Q.C. Requirements: To be prepared within 8 hrs after

collection, pH should be 6.2 or more at the end of storage time. Platelet count > 5.5 x 1010 /unit.

Volume: 30 to 50 mlContents: Platelet - 5.5 x 1010 /bag

Plasma - 30 to 50 ml and some white cells Transfusion Criteria: ABO / Rh specific and compatible Indications: Severe thrombocytopenia,

qualitative platelet defects Effect: Increases in platelet count 10,000 / ul

per unit

PLATELET CONCENTRATE Dosage

1 unit of PC / 10 kg B.W. Increment will be less in

- Hypersplenism- DIC- Septicemia

1 unit of PC Platelet 5000-10,000 / ul

PLATELET CONCENTRATE Indications

Treatment of bleeding due to Thrombocytopenia Platelet Dysfunction Prevention of bleeding

Contraindication prophylaxis of bleeding in surgical

patients

Plasma Components

› Fresh Frozen Plasma› Frozen Plasma :- Aged plasma› Cryoremoved plasma› Cryoprecipitate

Fresh Frozen Plasma

Fresh Frozen Plasma Plasma along with anticoagulant preservative Volume ~ 250-300 ml Prepared from blood within 8 hrs of donation Maximum level of labile and non-labile clotting factors

(about 1 IU per ml) V & VIII, proteins C and S, complement, and immunoglobulin.

Good for 24 hours post thawThen it can be stored for 5 days as liquid plasma (labile factors V and VIII decreased)

Shelf life: 1 year

Fresh Frozen Plasma (FFP)  It is prepared from the whole blood collected in a CPDA-1 double or triple bag

system within 6-8 hours of its collection.

Principle: Plasma is separated from cellular blood elements and frozen to preserve the activity of labile coagulation factors. 

Steps

1. Blood is collected in a CPDA-1 double or triple bag system. 2. The blood bags are weighed in a weighing balance and bags weighing

equal are placed opposite to each other in the buckets of the centrifuge. 3. The temperature of the centrifuge is adjusted at 4 - 6°C. 4. The bags are subjected to a high spin (3850 rpm) centrifugation for 5 min. 5. After the centrifuge stops, the blood bags are taken out with minimal

disturbance and supernatant plasma is expressed into the satellite bag, with the help of a plasma expressor, leaving behind 80-90ml.  

6. After labeling the group and ensuring screening status, the plasma is stored at -20°C or below. The storage shelf life is one year at -20°C and 5 years at -70 to-80°C  

 

Fresh Frozen Plasma contains coagulation factors and other plasma protein (per unit or bag)

Volume - 200-250 ml Factor VIII - 0.6 IU / ml Factor IX - 0.9 IU / ml Fibrinogen - 250-300 mg / bag Proteins - Albumin, globulin, etc.1 IU / kg of factor VIII or factor IX raises the factor VIII levels

in plasma by 2% and factor IX levels by 1% respectively. Shelf life: One year Storage temp.: -20°C or belowQ. C, Requirements: The entire proems of preparation

and freezing should be completed within 8 hrs after collection.

Volume: 50 to 200 ml

Indications for FFP TransfusionClinically significant deficiency of Factors II, V, X, XI DICPlasma exchange ImmunodeficienciesMassive transfusion of stored blood.Liver diseaseUrgent reversal of warfarin therapyCorrection of known coagulation factor deficiencies for which

specific concentrates are unavailable Correction of microvascular bleeding in the presence of

elevated (> 1.5 times normal) PT or PTTCorrection of microvascular bleeding secondary to coagulation

factor deficiency in patients transfused with more than one blood volume and when PT and PTT cannot be obtained in a timely fashion

FFP Dose

› 10-15 ml/kg B.W› For warfarin reversal, 5-8 ml/kg of FFP

Contraindication› Volume expansion› Immunoglobulin replacement› Nutritional support› Wound healing

FRESH FROZEN PLASMA Precaution

› Acute allergic reaction are common › Anaphylactic reaction may occur› Hypovolemia alone is not an indication for use

DosageInitial dose of 15 - 20 ml / kg B.W

Administration› Must be ABO compatible› Infuse as soon as possible after thawing (

within 6 hrs )› using standard blood administration set

FROZEN PLASMAPlasma which separate from whole blood at any

time during storage.Contain all non-labile coagulation factors.

Indication Treatment of stable coagulation deficiencies

Contraindication same as FFP

CRYOPRECIPITATE

Cryoprecipitate is the cold – insoluble portion of plasma that precipitates when FFP is thawed between 1-60 C

Cryoprecipitate (C.P)Cryoprecipitate contains precipitated proteins of

plasma, rich in factor VIII and fibrinogen, obtained from FFP prepared within 6-8 hours of collection, subsequent thawing at 4- 60 C and removal of supernatant.

Also the advantage of Cryoprecipitate is that we can administer large amount of factor VIII without overloading the recipient, especially in pediatric patients.

Principle: Coagulation factor VIII can be concentrated by cryoprecipitation of freshly collected plasma. Cryoprecipitation is accomplished by rapid freezing of plasma and slow thawing at low temperature.

Cryoprecipitate (C.P)

Cryoprecipitate contains (1 unit)Volume - 10-20 mlFactor VIII-C - 80-120 IUFactor VIII R: Ag - high levelsFactor VIII vWF - high levelFibrinogen - 150-200mg / bagFactor XIII - 20-30% of original level

Cryoprecipitate (C.P)Shelf life: Frozen - 1 year

Thawed - 6 hours Storage temp: Frozen - -20°C or less Q. C. Requirements: Thaw at 37°C

Factor VIII: C-80 units/bag Volume: 10 to 20'mlContents: Factor VIII: C - 80 to 150 units/bag Fibrinogen -

150 to 250 mg/bag Factor Xlll - 20 - 30% of whole blood von Willebrand factor - 40-70% of whole blood

Transfusion Criteria: ABO compatibility not required Indications: Correction of factor VIII deficiency

(Hemophilia A, von Willebrand disease).

Cryoprecipitate Indication

› Quantitative and Qualitative Fibrinogen Deficiency : DIC

› von Willebrand Disease› Factor XIII deficiency› Uremic Coagulopathy › Fibrin Glue› Factor VIII ( haemophilia A )

CRYOPRECIPITATE Administration

› Dose of Cryo is based on the desired target level of the specific factor to be replaced

› ABO compatible if possible no compatibility testing required

› After thawing & pooling, infuse as soon as possible through blood admin. Set

› must be infused within 6 hours of thawing

Heavy spin,4oC(within 8 hrs)

Fresh Whole Blood

Packed Red Cells

Stored in 1- 6oC

Fresh Plasma

Freeze -80oC immediately

Stored at < -18oC

Fresh Whole Blood

Packed Red Cells

Light spin, 22oC(within 8 hrs)

Platelet Rich Plasma

Platelet Concentrate Fresh Plasma

Store at 22oC Freeze(FFP)

Heavy spin,22oC

Thaw at 4oC & heavy spin

Fresh Frozen Plasma

Cryoprecipitate

-Refrozen within 1 hr

-Store at < - 18oC

Cryoremoved Plasma

Freeze -80oC immediately

Stored at < -18oC

Component Storage Red blood cells: 1-6°C Platelets: 22-24°C, with continuous agitation

Plasma:FFP: -18°C (after thawing ~ 1-6°C for 24 hours)

FP: -18°C (after thawing ~ 1-6°C for 24 hours)

CSP: -18°C (after thawing ~ 1-6°C for 24 hours)

Cryoprecipitate: -18°C (after thawing ~ 20°C for 4 hours)

How long can Blood Components be Stored?

Red cells: 42 days, collected in CP2D/AS-3 35 days, collected in CPDA-1

Platelets: 5 days with continuous agitation

Cryo: 12 months at -18°C or 4 hours after thawing

Plasma: 12 months at -18°C or (FFP/FP/CSP) 24 hours after thawing

Apheresis (Hemapheresis)  

The word apheresis is derived from a Greek word which means separation. Apheresis also known as hemapheresis is removal of whole blood from donor / patient, separation into components, retention of desired or unwanted components and reinfusion of remaining constituents to the donor or patient.  

The AMICUS Separator (Baxter)

Single Needle Procedure

Adverse effects of Apheresis Citrate toxicity may occur in the form of

numbness and tingling sensation around the mouth if the amount of citrate infused exceeds the body's ability to metabolize it. This problem can be solved by decreasing the rate Of infusion of anti-coagulant or by giving exogenous calcium to the donor.  

The other side effects are similar to that of normal blood donation.