Preparation of various components of blood for transfusion

Dr. Topesh Patle

Introduction

  • Whole blood contains:

    • Red Blood Cells (RBCs)

    • White Blood Cells (WBCs)

    • Platelets

    • Plasma (with clotting factors, proteins, electrolytes)

  • Modern transfusion practice follows the principle:

    “Give only what the patient needs.”

  • Blood component preparation is performed in a licensed blood bank under sterile conditions.

  • It requires:

    • Blood collection bags with anticoagulant-preservative solution

    • Refrigerated centrifuge

    • Component extractor

    • Sealing device

    • Temperature-controlled storage units

 

Blood Collection

Types of Blood Donation

A. Whole Blood Donation

  • 350 mL or 450 mL collected

  • Most common method

  • Used for component preparation

B. Apheresis Donation

  • Specific component collected (platelets, plasma, RBCs)

  • Remaining blood returned to donor

  • Requires automated apheresis machine

 

Donor Selection Criteria

Proper donor selection is mandatory to prevent transfusion-transmitted infections and protect donor health.

A. Age

  • 18–65 years

B. Weight

  • Minimum 45 kg (for 350 mL)

  • ≥ 55 kg (for 450 mL)

C. Hemoglobin

  • ≥ 12.5 g/dL

D. Pulse

  • 60–100/min (regular)

E. Blood Pressure

  • Systolic: 100–180 mmHg

  • Diastolic: 50–100 mmHg

F. Temperature

  • ≤ 37.5°C

 

Donor Deferral

A. Temporary Deferral

  • Fever or infection

  • Recent vaccination

  • Recent surgery

  • Pregnancy

  • Recent blood donation (<3 months)

B. Permanent Deferral

  • HIV, HBV, HCV infection

  • Chronic renal disease

  • Malignancy

  • High-risk behavior

 

Pre-Donation Procedure

  1. Registration

  2. Medical history

  3. Physical examination

  4. Hemoglobin estimation

  5. Informed consent

 

Blood Collection Equipment

  • Blood collection bag (single, double, triple, quadruple bag)

  • Sterile needle (16G)

  • Blood mixer with weighing scale

  • Anticoagulant-preservative solution (CPDA-1, CPD, SAGM)

  • Tourniquet

  • Sterile swabs

  • Sealing device

 

Blood Collection Bags

Types:

  1. Single Bag

    • Used when whole blood is transfused directly

  2. Double Bag

    • For the preparation of PRBC + Plasma

  3. Triple Bag

    • For PRBC + Platelets + Plasma

  4. Quadruple Bag

    • For PRBC + Platelets + Plasma + Cryoprecipitate

 

Anticoagulant-Preservative Solutions

A. CPD (Citrate Phosphate Dextrose)

  • Shelf life: 21 days

B. CPDA-1

  • Shelf life: 35 days

  • Contains adenine for ATP maintenance

C. SAGM (Additive solution)

  • Shelf life: 42 days

  • Improves RBC survival

Mechanism of Action:

  • Citrate → Binds calcium (prevents clotting)

  • Dextrose → Energy source

  • Adenine → Maintains ATP

  • Mannitol → Prevents hemolysis

  • Phosphate → Buffer

 

Procedure of Blood Collection

Step 1: Donor Position

  • Donor lies comfortably on donation couch

Step 2: Site Selection

  • Median cubital vein preferred

Step 3: Skin Preparation

  • Clean with:

    • Spirit

    • Povidone-iodine

    • Spirit again

  • Allow to dry

Step 4: Venepuncture

  • Use 16G sterile needle

  • Insert smoothly

Step 5: Collection

  • Blood flows into bag

  • Blood mixer ensures:

    • Continuous mixing

    • Correct volume

  • Collection time: 8–12 minutes

Step 6: Completion

  • Clamp tubing

  • Remove needle

  • Apply pressure dressing

 

Volume Collected

Type Volume Collected
350 mL Bag 350 ± 10% mL
450 mL Bag 450 ± 10% mL

Anticoagulant ratio must be maintained properly.

Sample Collection for Testing

Small pilot tubes collected for:

  • ABO grouping

  • Rh typing

  • Crossmatching

  • Screening for:

    • HIV

    • HBsAg

    • HCV

    • VDRL

    • Malaria

Post-Donation Care

  • Donor rests for 10–15 minutes

  • Oral fluids given

  • Avoid heavy exercise for 24 hours

  • Remove bandage after 4–6 hours

 

Complications of Blood Donation

A. Local

  • Hematoma

  • Pain

B. Systemic

  • Vasovagal reaction

  • Dizziness

  • Hypotension

  • Rarely syncope

 

Transportation to Component Room

  • Blood labeled properly

  • Time of collection recorded

  • Transported at 20–24°C

  • Processed within 6–8 hours

Quality Control in Blood Collection

  • Correct volume

  • No clot formation

  • Proper labeling

  • Sterility maintained

  • Documentation completed

Principle of Component Separation

Blood component separation is based on the physical and biochemical differences between various elements of whole blood. The aim is to separate whole blood into its therapeutic components so that each patient receives only the required component.

1. Basic Concept

Whole blood contains:

  • Red Blood Cells (RBCs)
  • White Blood Cells (WBCs)
  • Platelets
  • Plasma

These components differ in:

  • Density
  • Size
  • Weight
  • Sedimentation rate

Separation is mainly achieved by centrifugation, which uses centrifugal force to separate components based on their density.

2. Principle of Centrifugation

Centrifugation works on the principle that:

When blood is rotated at high speed, heavier components move outward (bottom), and lighter components remain toward the top.

Order of settling (from bottom to top):

  1. Red Blood Cells (heaviest)
  2. Buffy coat (WBCs + platelets)
  3. Plasma (lightest)

3. Density of Blood Components

Component Approximate Density
RBCs 1.09 g/mL
WBCs 1.06–1.08 g/mL
Platelets 1.03–1.04 g/mL
Plasma 1.025 g/mL

Because RBCs are densest, they settle at the bottom during centrifugation.

4. Types of Centrifugation

A. Soft Spin (Light Spin)
  • Low speed centrifugation
  • Separates:
    • Packed RBCs (bottom)
    • Platelet-Rich Plasma (PRP) (top)

Used in:

  • Preparation of platelets

B. Hard Spin (Heavy Spin)

  • High speed centrifugation
  • Further separates PRP into:
    • Platelet concentrate (bottom)
    • Platelet-poor plasma (top)

Used in:

  • Preparation of platelet concentrate and plasma products

5. Methods of Component Preparation

Two major methods are used:

A. Platelet-Rich Plasma (PRP) Method

  1. Soft spin → PRP separated
  2. Hard spin → Platelet concentrate formed

B. Buffy Coat Method

  1. Whole blood centrifuged at hard spin
  2. Buffy coat layer separated
  3. Platelets prepared from buffy coat

Buffy coat method gives:

  • Higher platelet yield
  • Better standardization

6. Principle of Plasma Separation

Plasma is the lightest component.
After centrifugation, it remains at the top.

It can be:

  • Frozen immediately → Fresh Frozen Plasma (FFP)
  • Further processed → Cryoprecipitate

7. Principle of Apheresis

Apheresis uses:

  • Continuous flow centrifugation
  • Selective removal of one component
  • Remaining blood returned to donor

Principle:

  • Automated density-based separation in real time

8. Important Factors Affecting Separation

  1. Centrifuge speed (RPM)
  2. Relative centrifugal force (RCF)
  3. Duration of centrifugation
  4. Temperature
  5. Type of anticoagulant
  6. Time between collection and processing

Improper centrifugation may cause:

  • Hemolysis
  • Poor platelet yield
  • Reduced coagulation factor activity

9. Scientific Basis

Centrifugal force (F) depends on:

  • Speed of rotation
  • Radius of rotor
  • Mass of particles

Heavier particles experience greater outward force and sediment faster.

10. Advantages of Component Separation

  • Rational transfusion therapy
  • Reduced volume overload
  • Better inventory management
  • Optimal utilization of one donation for multiple patients

Preparation of Packed Red Blood Cells

Procedure

Step 1: Collection of Whole Blood

  • 350 mL or 450 mL collected

  • Anticoagulant used:

    • CPDA-1

    • CPD

    • SAGM (additive solution)

Step 2: First Centrifugation (Heavy Spin)

  • Whole blood placed in refrigerated centrifuge

  • Hard spin performed

Result:

  • RBCs settle at bottom

  • Plasma remains at top

  • Buffy coat layer in between

Step 3: Plasma Expression

  • Blood bag placed in plasma extractor

  • Plasma transferred into satellite bag

  • RBCs remain in primary bag

Step 4: Addition of Additive Solution (if used)

  • SAGM added to RBCs

  • Improves RBC survival

  • Extends shelf life

Characteristics of PRBCs

Parameter Value
Volume 250–350 mL
Hematocrit 55–65%
Hemoglobin content ~50–70 g/unit
Plasma content Minimal

 

Storage Conditions

  • Temperature: 2–6°C

  • Storage refrigerator with continuous monitoring

Shelf Life:

Anticoagulant Shelf Life
CPD 21 days
CPDA-1 35 days
SAGM 42 days

 

Biochemical Changes During Storage 

During storage, RBCs undergo:

  • Decrease in ATP

  • Decrease in 2,3-DPG

  • Increased potassium leakage

  • Membrane rigidity

  • Hemolysis (minimal if proper storage)

These changes may affect oxygen delivery capacity.

Preparation of Platelet Concentrates

Types of Platelet Concentrates

  1. Random Donor Platelets (RDP) – Prepared from whole blood

  2. Single Donor Platelets (SDP) – Prepared by apheresis

PART A: Preparation of Random Donor Platelets (RDP)

  • Based on difference in density between RBCs, platelets, and plasma.

  • Prepared by two-step centrifugation:

    • Soft spin

    • Hard spin

Equipment Required

  • Refrigerated blood bank centrifuge

  • Triple or quadruple blood bag system

  • Plasma extractor

  • Platelet agitator (20–24°C)

  • Tube sealer

Procedure (PRP Method)

Step 1: Collection of Whole Blood

  • 350 mL or 450 mL blood collected in CPDA-1/CPD bag

  • Blood processed within 6–8 hours

Step 2: First Centrifugation (Soft Spin)

  • Low-speed centrifugation

  • Purpose: Separate Platelet-Rich Plasma (PRP)

Result after soft spin:

  • Bottom: Packed RBCs

  • Top: Platelet-Rich Plasma (PRP)

Step 3: Transfer of PRP

  • Place bag on plasma extractor

  • Transfer PRP into satellite bag

  • RBCs remain in primary bag

Step 4: Second Centrifugation (Hard Spin)

  • High-speed centrifugation of PRP bag

Result:

  • Bottom: Platelet pellet

  • Top: Platelet-Poor Plasma (PPP)

Step 5: Expression of Plasma

  • Remove most of plasma

  • Leave about 40–70 mL plasma

  • Resuspend platelet pellet gently

Final product: Random Donor Platelet Concentrate

Final Characteristics of RDP

Parameter Value
Volume 40–70 mL
Platelet count ≥ 5.5 × 10¹⁰ per unit
WBC content Variable
pH (end storage) ≥ 6.2

Storage Conditions

  • Temperature: 20–24°C

  • Continuous gentle agitation

  • Shelf life: 5 days

Buffy Coat Method

  • Platelets concentrated from buffy coat layer

Steps

  1. Whole blood centrifuged at hard spin

  2. Buffy coat separated

  3. Buffy coats pooled (4–6 donors)

  4. Centrifuged again

  5. Platelets suspended in plasma

Advantages:

  • Higher platelet yield

  • Better standardization

  • Reduced plasma volume

PART B: Preparation of Single Donor Platelets (SDP)

  • Continuous flow centrifugation

  • Selective removal of platelets

  • Remaining blood returned to donor

Equipment

  • Automated apheresis machine

  • Anticoagulant (ACD-A)

  • Sterile disposable tubing kit

Procedure

  1. Donor selected and screened

  2. Venous access established

  3. Blood enters apheresis machine

  4. Centrifugal separation occurs

  5. Platelets collected

  6. RBCs and plasma returned to donor

Final Characteristics of SDP

Parameter Value
Volume 200–300 mL
Platelet count ≥ 3.0 × 10¹¹
Equivalent to 5–6 RDP units
Donor exposure Single donor

Storage Conditions

  • 20–24°C

  • Continuous agitation

  • Shelf life: 5 days

Quality Control Parameters

  • Platelet count adequate

  • pH ≥ 6.2

  • Swirling present (visual indicator of viability)

  • No clots

  • Sterility maintained

Preparation of Fresh Frozen Plasma

Procedure

Step 1: Collection of Whole Blood

  • 350 mL or 450 mL blood collected

  • Anticoagulant: CPD or CPDA-1

  • Time of collection recorded carefully

Step 2: Centrifugation (Heavy Spin)

  • Whole blood centrifuged at appropriate speed

  • After centrifugation:

    • Bottom layer → Packed RBCs

    • Middle layer → Buffy coat

    • Top layer → Plasma

Step 3: Plasma Separation

  • Blood bag placed in plasma extractor

  • Plasma transferred into satellite bag

  • RBCs remain in primary bag

Care must be taken not to disturb the buffy coat.

Step 4: Rapid Freezing

  • Plasma must be frozen within 6–8 hours of collection

  • Temperature: –30°C or lower

Rapid freezing preserves:

  • Factor V

  • Factor VIII

  • Other coagulation factors

If frozen after 8 hours → It is called Plasma Frozen within 24 Hours (PF24), not FFP.

Step 5: Labeling

Label must include:

  • Component name (Fresh Frozen Plasma)

  • Blood group

  • Volume

  • Date of collection

  • Expiry date

  • Unique donor number

Characteristics of FFP

Parameter Value
Volume 180–250 mL
Contains All coagulation factors
Fibrinogen 200–400 mg/unit
Plasma proteins Albumin, globulin

Storage Conditions

Condition Requirement
Temperature –30°C or below
Shelf life 1 year

If stored at –18°C → Shelf life is 3 months (depending on guidelines).

Preparation of Cryoprecipitate

Cryoprecipitate is prepared from FFP.

Procedure:

Step 1: Selection of FFP

  • Use properly stored FFP

  • Ensure it was frozen within 6–8 hours of collection

  • Maintain temperature at –30°C before use

Step 2: Controlled Thawing

  • Thaw FFP at 1–6°C

  • Slow thawing allows formation of cold-insoluble precipitate

  • Duration: Approximately 12–18 hours

Result:

  • White cloudy precipitate forms at bottom

Step 3: Centrifugation

  • Perform cold centrifugation

  • Precipitate settles at bottom

  • Supernatant (cryosupernatant plasma) remains above

Step 4: Separation

  • Using plasma extractor:

    • Remove most of supernatant plasma

    • Leave 10–15 mL plasma with precipitate

This forms Cryoprecipitate

Step 5: Refreezing

  • Immediately refreeze cryoprecipitate

  • Store at –30°C or below

Characteristics of Cryoprecipitate

Parameter Value
Volume 10–20 mL
Fibrinogen ≥ 150–250 mg/unit
Factor VIII ≥ 80 IU
Storage temperature –30°C
Shelf life 1 year

Storage Conditions

  • Temperature: –30°C or lower

  • Shelf life: 1 year

  • Must not be thawed and refrozen repeatedly

Thawing Before Transfusion

  • Thawed at 30–37°C

  • Should be transfused within 6 hours

  • After pooling → Use within 4 hours

 

Leukocyte-Reduced Blood Components

Method:

  • Filtration using leukoreduction filters.

  • Removes ≥ 99% leukocytes.

Benefits:

  • Prevents febrile non-hemolytic reactions

  • Reduces CMV transmission

  • Prevents HLA alloimmunization

Washed Red Cells

Method:

  • RBCs washed with normal saline.

  • Removes plasma proteins.

Indications:

  • IgA deficiency

  • Recurrent allergic reactions

Irradiated Blood Components

Purpose:

  • Prevent Transfusion-Associated Graft vs Host Disease (TA-GVHD)

Method:

  • Gamma irradiation (25 Gy)

Indications:

  • Immunocompromised patients

  • Intrauterine transfusion

  • Bone marrow transplant recipients

Storage Requirements 

Component Temperature Shelf Life
PRBC 2–6°C 35–42 days
Platelets 20–24°C 5 days
FFP ≤ -30°C 1 year
Cryoprecipitate ≤ -30°C 1 year

Quality Control of Blood Components

Each component must meet standards:

  • PRBC: Hematocrit 55–65%

  • Platelets: Adequate count per unit

  • FFP: Adequate factor levels

  • Sterility testing

Quality control is essential to ensure:

  • Safety

  • Potency

  • Efficacy

Advantages of Component Therapy

  • Rational use of blood

  • Reduced circulatory overload

  • Decreased transfusion reactions

  • Efficient inventory management

Complications if Improperly Prepared

  • Hemolysis

  • Bacterial contamination

  • Reduced factor activity

  • Platelet dysfunction

  • Transfusion reactions