Blood Collection & Storage

Donor Blood Collection

  • Approximately 470ml donor blood is collected via aseptic venepuncture into a sterile blood pack
  • The first 30ml is diverted into sample tubes for laboratory testing
  • 66.5ml of CPD preservative  is used per pack of whole blood to extend shelf life to 35 days
    • citrate - anticoagulant, by binding to Ca2+
    • phosphate - buffer which provides phosphate source for metabolism
    • dextrose - provides substrate for continued glycolysis and energy production
    • (adenine) - provides a substrate for ATP production. Present in the alternative preservative CPDA1

Mandatory Testing

All donated blood is tested for

  • ABO and RhD blood groups
  • Red cell antibody screening
  • Syphilis serology
  • Viral screening for
    • HIV1 and HIV2 antibodies and HIV 1 RNA
    • Hepatitis B surface antigen and HBV DNA
    • Hepatitis C antibodies and HCV RNA
    • Human T-cell lymphotropic virus (HTLV) 1 and 2 antibodies

All platelet components are screened for bacterial contamination 24hrs after collection

  • Platelet bacterial contamination is the most significant infectious risk of transfusion
  • Samples are cultured in both aerobic and anaerobic bottles

Blood Fractionation

  • Donated blood is no longer kept as whole blood, instead it is separated into components
  • This allows for
    • Storage of different components under different conditions that optimises storage life
    • Administration of individual components for a specific indication, avoiding wastage of other components
  • Whole blood is centrifuged to separate it into its component parts, within 24hrs of collection
    • Plasma (55%)
    • Buffy coat (leukocytes and platelets) (< 1%)              NB "buffy" = yellow-brown colour
    • Erythrocytes (45%)

Packed Cells

  • 105ml SAGM is used as the additive solution to extend shelf life
    • Saline
    • Adenine
    • Glucose (actually dextrose)
    • Mannitol - protects cell membrane, reduced haemolysis
  • Then filtered to leucodeplete the sample
    • ↓ risk of febrile, non-haemolytic reactions and CMV transmission
  • Resultant Packed Cell bag contains
    • Volume 260ml
    • Haematocrit 0.58
  • Stored at 2 - 6C
    • Large inhibition of cellular metabolism and energy consumption. Glycolysis is ↓ to 1/30th of rate at 37C
    • Inhibits bacterial growth
    • Lower temp → risk of freezing
    • Higher temp → faster metabolism, drop in pH and loss of 2,3 DPG
  • Shelf life 42 days
  • Criteria is: minimum acceptable survival rate of  70% of transfused red cells at 24hrs post transfusion


  • Pooled Platelets bag is derived from pool of buffy coats from four ABO identical donors and resuspended in nutrient additive solution (SSP+)
  • Leucodepleted and irradiated (to prevent graft vs host disease from any remaining leukocytes)
  • Stored at 20 - 24C with continuous gentle agitation
  • Shelf life 5 days
  • Resultant Pooled Platelets
    • Volume 334ml
    • Platelet count 269 x 109/unit

Fresh Frozen Plasma

  • Separated from whole blood and frozen with 18hrs of collection
  • Contains all coagulation factors including labile Factors VIII and V
  • Stored at -25C or lower for up to 12 months
  • Once thawed, it must be used within 5 days (guidelines changed from 48hrs in 2018)
  • Resultant FFP
    • Volume 284ml


  • Prepared by thawing FFP between 1 - 6C and recovering the precipitate
  • It is then refrozen
  • Contains most of the
    • Factor VIII
    • fibrinogen
    • Factor XIII
    • von Willebrand factor
    • fibronectin
  • Stored at -25C or lower for up to 12 months
  • Resultant cryoprecipitate bag
    • Volume 35ml

Storage Lesion of Whole Blood at 4C

  • Loss of functional platelets and granulocytes
  • Loss of some coagulation factors
  • ↓ 2,3 DPG → shift ODC to left
  • Small amount of haemolysis
  • Formation of aggregates
  • Biochemical changes
    • ↑ plasma [K+]
    • ↑ rbc [Na+]
    • ↓ pH
    • loss of Ca2+
Change Duration of Storage (days)
  0 7 14 21 28
RBC Survival (%) 100 98 85 80 75
2,3 DPG (%) 100 99 50 15 5
pH 7.2 7 6.9 6.8 6.7
Na+ (mmol/L) 168 166 163 156 154
K+ (mmol/L) 3.9 11.9 17.2 21 28
Glucose (mmol/L) 19.2 17.3 15.6 12.8 12.2
Free Hb (ug/L) 1.7 7.8 12.5 19 29

Red Cells

  • A storage time increases, some rbc become spherical due to metabolic changes → ↑ cell rigidity
  • Anaerobic metabolism → lactic acid production → ↓ pH 6.5


  • Platelet function rapidly lost at 4C
  • by 24 - 36 hrs, most platelets are non- functional

Coagulation Factors

  • Factors V and VIII are labile factors
    • FVIII ↓ to 50% by 24hrs, 6% after 21 days (at 2 - 6C)
    • Normal hameostasis requires FVIII at least 35% of normal
    • FV ↓ to 50% by 14 days
    • Normal haemostasis requires FV at least 5 - 20% of normal
  • Other factors not significantly affected until 21 days of storage

White Cells

  • Granulocytes lose their phagocytic and bacteriocidal properties within 4-6hrs of collection
  • Maintain their antigenic properties

Blood Groups & Compatibility

Blood Groups

  • Both donor blood and recipient blood must have their type determined to ensure that compatible donor blood is given to the recipient
  • Blood grouping or typing involved determining
    • ABO type
    • presence of Rhesus D antigen
    • presence of any other "atypical" antibodies



  • Major source of incompatibility reaction
  • Antigens present on surface of red cell membrane
  • Four blood groups
    • A
    • B
    • AB
    • O
  • Normal healthy individuals, in early childhood, develop IgM antibodies to A or B antigens that are not expressed on their own red cells


  • Rhesus system comprises of 61 antigens
  • Rh antigens are located on the surface  red cell membrane
  • RhD is the most immunogenic and important Rhesus antigen and is the one tested for
  • However, all Rh alloantibodies (usually IgG) are potentially capable of causing
    • Severe haemolytic transfusion reaction
    • Haemolytic disease of the foetus and newborn

Atypical Antibodies

  • Many other antigen systems may also cause haemolytic transfusion reactions, but are less common:
    • Duffy
    • Kell
    • Lewis
    • Kidd


Red Cells
  • Should give ABO compatible Packed Cells (see table below)
  • If recipient has atypical antibodies, need to do serological crossmatch
  • Should give ABO matched platelets
  • If it is necessary to provide platelets other than the patient’ s own blood group, the patient’ s age, diagnosis, therapy, component type availability (ie apheresis platelets vs pooled platelets) as well as any special circumstances (eg HLA matched) may influence the decision to give either antigen-incompatible or antibody-incompatible platelets
  • If an ABO antigen compatible/plasma incompatible platelet transfusion (eg group A patient given group O platelets) is given
    • The recipient may develop a positive direct antiglobulin test (DAT) which may result in haemolysis
    • This is of greater importance in children as they have lower levels of soluble A and B substance in their body fluids
  • If an ABO and/or RhD antigen incompatible/plasma compatible platelet transfusion (eg group O RhD negative patient given A RhD positive platelets) is given
    • The post-transfusion platelet increment and platelet survival may be lower in some patients
    • Sensitisation to foreign  red cell antigens   and in particular RhD may also occur
FFP and cryoprecipitate
  • Should be compatible with ABO group of recipient to avoid potential haemolysis caused by donor anti-A or anti-B

ABO component compatibility table

  Red cells Platelets Plasma components
Patient group unknown O# A or O (low titre anti-A/B) AB*
Patient group O
First choice O O O
Second choice   A** A or B
Third choice   AB AB
Patient group A
First choice A A A
Second choice O O (low titre anti-A/B) AB
Third choice   AB or B, O  
Patient group B
First choice B B B
Second choice O O (low titre anti-A/B) AB
Third choice   AB or A, O  
Patient group AB
First choice AB AB AB
Second choice A or B O (low titre anti-A/B) A*
Third choice O A or B, O  

# If patient is a female of child bearing potential, O RhD negative red cells should be used until patient’ s blood group is established.

* Group A plasma may be used as per local institutional policies.

** Group A platelets that have an A2 subgroup do not express significant amounts of A antigen and are, therefore, compatible with group O recipients.

Compatibility Testing

Group and Hold / Type and Screen

  • ABO and RhD   typing of recipient's red cells
  • Atypical antibody screen to detect antibodies in recipient's plasma
  • Identification of  red cell antibodies   (performed if positive antibody)  screen detected
  • Crossmatching appropriate donor  red cells


  • Crossmatching is used to confirm compatibility between the patient's blood (plasma)  and the donor red cells
  • This may be performed by either serological or electronic methods

Serological Crossmatch

  • Indirect antiglobulin technique (IAT)
    • Generally used when crossmatching for patients with clinically significant IgG antibodies.
    • Patient's plasma is incubated with donor red cells and tested.
  • Immediate spin crossmatch
    • Designed to detect ABO incompatibilities between donor red cells and recipient plasma.
    • Only used if patient currently has no clinically significant antibodies, and there is no history of clinically significant antibodies
    • Patient's plasma is tested against donor red cells

Non serological crossmatch

  • Electronic (Computer) crossmatch
    • ABO compatibility of patient and donor  red cells is checked by the laboratory information system (LIS) with no serological crossmatch tests needing to be performed
    • Only used if patient currently has no clinically significant antibodies, and there is no history of clinically significant antibodies
    • Allow red cells to be issued with minimal delay once blood group and antibody screen completed
    • Specific criteria must be met in order to utilise electronic crossmatching (refer to  ANZSBT guidelines )
    • Many hospitals now use electronic crossmatch unless atypical antibodies are detected, meaning that there is no need to ask for a crossmatch, over a group and hold, unless there are atypical antibodies

The 72 hour Sample Validity Rule

  • Sample validity refers to the period  when a  pretransfusion sample may be held for testing and used to provide crossmatched blood
  • Sample validity period  depends on the patient's transfusion and obstetric history.  Red cell antibodies can rapidly appear in response to stimulation by transfused red cells or as a result of pregnancy
  • If the patient has been transfused with red cells, or is currently (or has been) pregnant in the 3 months preceding pretransfusion sample collection, the sample will have a validity of 72 hours. This is known as the 72-hour rule
  • A new sample will need to be collected once 72 hours elapses if further transfusion is required
  • Longer expiry times are often applied if the patient does not have a history of transfusion or pregnancy

Incidence of haemolytic reactions

Extent of testing Relative Safety
ABO compatible blood 99.4%
plus Rhesus compatible 99.8%
plus negative antibody screen 99.94%
plus Coombs test (IAT) 99.95%