Blood components and its Storage

Amalraj P
Basics about blood
• Blood composed of

CELLS
• RED BLOOD CELLS
• WHITE BLOOD CELLS
• PLATELETS
FLUID
• PLASMA

• Blood volume in adults: 75ml/kg

• Children: 80 ml/kg
• Neonates: 85 ml/kg (Term)
100 ml/kg (Preterm)
Collection basics

• Blood is collected in a primary bag that contains anticoagulant-

preservatives
• Anticoagulants prevent blood clotting
• Preservatives provide nutrients for cells
• Satellite bags may also be attached, depending on what components
are needed
• Anticoagulant-preservatives minimize biochemical changes and
increase shelf life
Separation of blood components are desirable because

1. Separation of blood components allows optimal survival for each

component
2. Allows transfusing specific blood components according to the
need of the patient
3. Allows use of unnecessary component which may be
contraindicated in a patient
4. Several patients can be treated from one unit of donated blood
5. Use of blood components supplements blood supply and adds to
the blood inventory
Red blood cells
• Most numerous of cells in the blood
• Occupies about 45% of the total blood volume.
• Produced in the bone marrow under the controlling influence of hormone - erythropoietin.
• Life span of 120 days
• Contains iron-containing pigment hemoglobin, whose primary function is to store and
transport oxygen.
 White blood cells
• Occupy < 1% of the total blood volume.
Consist of
Granulocytes
Lymphocytes
Monocytes

• Produced in the bone marrow and lymphatic tissue.

• Principal role in the blood is to identify, destroy and remove any foreign material that has entered
the body.
Platelets
• Small fragments of megakaryocytes produced in the bone marrow
• Function is to respond to any vascular wall damage by gathering together at the site of
injury to form an initial temporary platelet plug (Primary hemostatic plug)
• Platelets subsequently activate coagulation process by releasing their contents into blood.
Volume of blood collection

Donor Volume Type of Used for

Weight collected bag
45 -55 kg 350ml Single Whole
bag Blood

> 55 kg 450 ml Double Blood

/ Triple Component
s
bag
Components available
• Whole blood (35 days) • Cryoprecipitate (1yr)

• Red cells / packed red cells • Cryosupernatant

• Platelet rich concentrate

• Granulocytes (1 day)
(5 days)

• Irradiated products
• Fresh frozen plasma
(1 yr)
• Leuko depleted products
• Single donor platelets
 Donor selection
• Healthy donor
• Gender – All
• History of anti platelet drugs, alcohol,
regular medication
• Weight of donor
• Platelet count, albumin for apheresis donors
Steps of component preparation
• Prepared using closed system using multiple bags. (a sterile system of blood collection)
Precautions:
• Venipuncture should be smooth with good blood flow
• Duration of venisection should not be more than 8 mins to prevent activation of coagulation
system
• Adequate mixing of blood and anticoagulant should be monitored
• Appropriate volume of blood should be collected
• Clean thoroughly to minimize bacterial contamination
• Immediately strip and segment tubing
• Appropriate storage is essential before processing
Component preparation
Single
Designed for the Triple
collection Distributes whole blood into
and storage of three parts i.e. red cells,
whole blood platelets and plasma

Quadruple
Double Bags with inline
leukodepletion filters
Distributes whole blood Distributes whole blood into
into two parts i.e. red four parts i.e.Leuko
cells and plasma. Reduced Redcells, Buffy
coat, platelets and plasma
Indications for red cells:
• Decreased bone marrow production
 Luekemia
Aplastic anemia

• Decreased red cell survival

 Hemolytic anemia
Thalassemia

• In bleeding patients
Surgical bleeding
Traumatic bleeding
 Red cells preparation

• Using a plasma expresser, express plasma into the satellite bag leaving behind 50-75 ml of plasma in the
primary bag.
• Seal the main bag, detach and store at 2-60C in the Pre test/quarantine blood storage container in the
donor room till the infectious disease testing results are through.
 Red cells- Evidence based recommendations

• Transfusion trigger : < 7gm/dl

• 70 -100g/l – (limited evidence)

• Transfusion threshold - >100g/l – inappropriate – (level I)

• Storage: 2- 60C – 35 days (CPDA -1)

21 days (ACD or CPD)
42 days (Additive solution)

• Each unit of transfused red cells prepared from 450 ml of whole blood - increase
hemoglobin by about 1 g/dl (Hct 3%) in an adult and in infants by 2-3 gm/dl at 10-15 ml/kg
Percent blood volume loss as a guide to TX
therapy

• 15% - Transfusion not usually required

• 15-30% - Crystalloid Support usually adequate

• 30- 40% - Red cells likely to be required

• >40% - Red cells required ( BCSH – 2001)

FFP – Indications
• Fresh frozen plasma (FFP) is plasma that is separated from whole blood and is frozen within 6-8
hours of collection.
• Contains all coagulation factors – volume about 200-250 ml including labile factors V and VIII
• Storage: - 800 C: 1 year

Single coagulation factor deficiency – Fresh-frozen plasma should only be used to replace single
inherited clotting factor deficiencies for which no virus-safe fractionated product is available.
Active bleeding and multiple coagulation factor deficiency as in
• Liver disease
• Disseminated intravascular coagulation
Thrombotic thrombocytopenia purpura (TTP)
Reversal of warfarin effect - Should never be used for the reversal of warfarin anticoagulation in the
absence of severe bleeding.
Surgical bleeding and massive transfusion
Haemorrhagic disease of newborn
FFP - Dosage

• 10-20 mL/ Kg ( 3-4 bags ≈ 1 litre )

• Raise the coagulation factors by 20% immediately after infusion (Normal levels: 50-150%)

• Very judicious use in dilutional coagulopathy (can worsen dilutional coagulopathy due to its large
volumes – 1 bag ≈ 250 mL)

• Post transfusion assessment of levels of aPTT, PT and fibrinogen is done for monitoring the
effect of FFP. FFP should be thawed at 37°C in water bath prior to transfusion.

• Thawed plasma should be transfused as soon as possible, or within 24 hours, if stored at 2-6°C

• Plasma should be ABO compatible with the recipient blood, but not necessarily group specific.
The donor’s plasma should not contain ABO antibodies that might interact with A or B antigens on
the recipient’s red blood cells
Platelet preparation
Differential Centrifugation (Red cells with SAGM)
First Centrifugation

Closed System

Whole Blood Satellite Bag Satellite Bag

1. 1500 RPM (747G) X 12 min
Main Bag 1 2 At 22deg C
First
Differential Centrifugation
Second Centrifugation

1. 3300 RPM (3373G) X 25 min

Platelet-rich
RBC’s at 22deg C
Plasma
Second

Plasma
Platelet storage

• Stored at 22 -24°C for 5 days in a platelet agitator with continuous agitation

Indications for platelet transfusions

•BLEEDING due to thrombocytopenia

•Due to platelet dysfunction

•Prevention of spontaneous bleeding with counts < 20,000

 Platelet transfusion- selection of platelets

 ABO group specific as far as possible. Any group in emergency.

 Pre and post transfusion platelet count to monitor the patient

 Each unit of random donor platelets (bag volume: 50 ml) prepared from 450 ml of blood will raise the platelet
count by approximately
• Adult: 5 – 10 x 109/L in an adult of 70 kg body weight
• In child: 20 x 109/L
• In infant: 75-1000 x 109/L (75,000-100,000/µl)

 One apheresis unit (bag volume: 300ml) is equivalent to 4-6 random donor platelets and will raise the platelet
count of an adult by approximately 30,000-60,000/µl under optimal conditions.
 In small children (< 20 kg), dosing is 10–15 ml ⁄kg; in older children, an adult dose of platelets should be used
Apheresis
Indications for cryoprecipitate
Precipitated proteins of plasma, rich in FVIII, von-Willebrand factor, fibrinogen, FXIII and
fibronectin obtained on thawing FFP at 40C.

Indications:
Hemophilia A
Von Willebrand disease
Congenital or acquired fibrinogen deficiency
Acquired Factor VIII deficiency (e.g. DIC, massive transfusion)
Factor XIII deficiency
Source of Fibrin Glue used as topical hemostatic agent in surgical procedures and to remove
fragmented renal calculi.
Preparation of Cryo precipitate Satellite bag containing AS;
Cryo final product cryosupernatant
supernatant

2 3

Plasma Satellite bag to be filled

Cryo with plasma, final product
supernatant cryoprecipitate
Packed red cells (final
Cryoppt
product of bag – red cells
in additive solution)

1. 3373G (3300 rpm) X 20MTS at 4 C

Storage of cryoprecipitate:
Cryoprecipitate is frozen at -800C for one year.

Reconstituting cryoprecipitate (Thawing and issue of Cryoprecipitate)

Once thawed at 37 0C, cryoprecipitate should be kept at 2-6°C and administered within 4 hours.

Lab tests that need to be done prior and for monitoring the patient:

PT
APTT
Fibrinogen
FVIII assay in patients with hemophilia A who are on recombinant FVIII concentrate
Cryoprecipitate
•One bag produced from a 450 ml donation will contain 80-120 units of factor VIII in a volume of 15 -
30 ml and atleast 150 mg of fibrinogen.

Transfusion trigger:

•Fibrinogen: If fibrinogen > 100mg/dl but < 150mg/dl; use 1 unit/10kg; if fibrinogen< 100 mg/dl; 2
units/10 kg in adults.
•In infants 1 unit of cryoprecipitate increases fibrinogen by 60-100 mg/dl
Fibrinogen required
• (Desired fib level mg/dl – initial fib level) x plasma volume /100

Eg: 50mg/dl to reach 150mg /dl

Plasma volume – 2500ml

100 x 2500 / 100 = 2500mg

How many bags of cryo?

• 1 bag of cryo: 150- 250 mg of fibrinogen

• Hence 10-12 bags required

Cryosupernatant
• By product of cryoprecipitate preparation
• Contains stable coagulation factors 2,7,9 and 10.

• Indications:

 In deficiency of stable clotting factors (Coagulopathy due to warfarin)

Burns
TTP – supply ADAMTS13

Doses: similar to FFP

 Granulocyte concentrate
• Indications:
Septicaemia not responding to antibiotics – especially in chemotherapy patients.
Neutrophil count <500/cumm (0.5x 109/L), having gram negative infections not responding to
antibiotics
Fungal infections not responding to therapy

• Source:
Buffy coat
By apheresis – single donor

• Dosing and administration of granulocytes:

Adult: 1x 1010 granulocytes daily (Apheresis – 1 unit, 18-20 units of buffy coat)
 To be transfused as soon as possible. If delay to be stored at 22-24 0 C without agitation for upto 24
hours
 To be discontinued when granulocyte count exceeds 1 x 10 9/L or when patient becomes afebrile
Leucoreduced blood components
Technologies for leukoreduction:

Centrifugation – Removal of buffy coat –

70-80% of leucocytes removed
•Prevents FNHTR
•Does not prevent HLA alloimmunisation &
CMV transmission
Filtration- using filters 3 log or 4 log
reduction at prestorage, post storage or at
bedside
Washing of red cells with saline –
Removes 70-95% of leucocytes but can result
in loss of 15- 20 ml of red cells
Freezing and thawing of red cells

•In CMC: renal and cardiothoracic patients

 Preparation of LRRC

• Quadruple bags are centrifuged at

high spin of 3600rpm at 22 degree
Celsius for 10 minutes Platelet Poor Plasma
• After the high spin the bags are Buffy Coat Layer
placed in the component
extractor/separator without any Red Cell Concentrate
disturbance
• The red cells and plasma are
separated into transfer bags
Red Cell Additive
(SAG-M)
• Simultaneous extraction of
Red Cells and Plasma

• The buffy coat with platelets

bag are hung for 2 hours

• After 2 hours remove the

bags and centrifuge them at
low spin at 900rpm for 8
minutes
• We get buffy coat settled at the
bottom and platelets above it,
which are separated using
component extractor
Indications of leukoreduction
 Irradiated components
• To prevent TA-GVHD (Transfusion associated Graft versus Host disease): a phenomenon
that can occur after blood transfusion in which donor T cells proliferate and target host
organs, primarily the skin, liver, intestinal tract, and marrow.
• Affects immunocompromised individuals mainly, but can develop in immunocompetent
individuals also.
• Treating cellular blood components with 25 Gy γ-irradiation reliably
inactivates donor lymphocytes through cross-linkage of DNA, and
prevents TA-GVHD

• Does not damage platelets or granulocytes, and therefore shelf life of

irradiated platelets remains same i.e. 5 days

• Shortens shelf life of red cell components and it is recommended that

irradiated red cells be used within 28 days from the time of irradiation

• Effects of irradiation on red cells include increased plasma membrane

permeability, with potassium leakage, hemolysis, and problems with
adenosine triphosphate maintenance
Quality control of blood/blood components
• At least 1% of total components prepared are subjected to quality control at random. The
individual parameters to be assessed are as follows and should be fulfilled by at least 75% of the
components tested (Drugs and Cosmetics Act, 1940).

• Indian standards for quality control 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 lndia
NABH Accreditation Standards for Blood Banks
When to perform quality control
 For platelet products - done on expiry date (end of storage period) of the
component

 On installation and after repair of equipments (refrigerator, centrifuges,

deep freezers etc)

 Modification in procedure for components preparation

Recruitment of new personnel

Factors Affecting the Quality of Blood Components

1. Selection of donor
• Antiplatelet drug therapy, defer for 72 hours

2. Quality of blood bag and anticoagulant preservative solution used

3. Techniques of phlebotomy
• Clean venipuncture
• Minimal tissue trauma
• Flow-continuous and uninterrupted and should be completed in 8-10 min
• Frequent gentle mixing.

4.Time period: Separation into components should be done within 8 hrs of blood
collection
5. Transit temperature: 20-240C for not more than 8 hours

6. Refrigerated centrifuge calibration for maximum yield in minimum time

• Critical variables – speed, temp, duration, rotor size
• Accurate balancing – dry weight preferred
• Bag position
• Swinging cups better than fixed angle cups

7. Storage temperature
 4 ± 20C = WB, PRBC
 200C-240C = PRP-PC, BC-PC, AP-PC
 ≤ - 800C = FFP, Cryoprecipitate

8. Uninterrupted, gentle flat bedded platelet incubator/agitator

5. 60-70 cycles /min
6. 1½” inch movement on either side.
QC – Whole blood and red cell concentrate
Whole blood specific gravity: 1.05
Plasma: 1.02
Red cells: 1.097
QC of platelet concentrate (Apheresis)

Parameter Quality requirement Frequency of control

Volume 200-300 ml 4 units / month

Platelet count > 3.0-7.0 x 1011 4 units / month
pH > 6.0 4 units / month
RBC Traces to 0.5 ml 4 units / month
contamination
Residual < 5.0 x 106 4 units / month
leucocytes
 Storage period of blood components
Blood component Temperature & storage period
Whole blood 2-6 o C for 35 days
Packed cells 2-6 o C for 35 days
Red cells with additive solution 2-6 o C for 42 days
SAGM
Fresh Frozen plasma -80 0 C for 1 year
Cryoprecipitate -80 0 C for 1 year
Platelets 22–24oC platelet agitator for 5 days
 FFP/ cryoprecipitate need to be thawed at 37 0C in a water bath prior to transfusion and should be
transfused within 30 minutes. If not transfused immediately, it should be kept at 2-6 0 C and
transfused within 24 hours of thawing. Thawed products cannot be refrozen and it will be
discarded if not used.

 Transfuse platelets as soon as possible. DO NOT REFREIGERATE platelets

• Components may be suitable for reissue if temperature is maintained and returned to the BB
within 20 minutes of issue from BB.
• If temperature exceeds > 100C or the unit has been spiked, it will not be reissued and will be
discarded.
 Time Limits for Infusion
Blood/ Start infusion Complete infusion
blood product

Whole blood/ within 30 min. of within 4 hour

red cells removing pack (less in high
from ambient temperature)
refrigerator

Platelet immediately within 20 min

concentrates

FFP within 30 min within 20 min

53
References:
• Clinical Practice Guidelines from the AABB Red Blood Cell Transfusion Thresholds and Storage.
• BJH Guidelines for the use of fresh-frozen plasma, cryoprecipitate and cryosupernatant.
• British Committee for Standards in Haematology (BCSH) Guideline on the Administration of
Blood Components.
• The clinical use of Blood – World Health Organisation
• DGHS technical manual.
• CMC SOP BB
Thank you