Red Blood Cell and Platelet Preservation: Historical Perspectives and Current Trends Flashcards
First blood transfusion recorded in history
In 1492, blood was taken from three young men and given to the stricken Pope Innocent VII
The first example of blood preservation research.
In 1869, when Braxton Hicks recommended sodium phosphate.
Discovered the ABO blood groups and explained the serious reactions that occur in humans as a result of incompatible transfusions
Karl Landsteiner
Carried out vein-to-vein transfusion of blood by using multiple syringes and a special cannula for puncturing the vein through the skin.
Edward E. Lindemann
Designed his syringe-valve apparatus that transfusions from donor to patient by an unassisted physician
Lester J. Unger
Reported the use of sodium citrate as an anticoagulant solution for transfusions.
Albert Hustin
Determined the minimum amount of citrate needed for anticoagulation and demonstrated its nontoxicity in small amounts.
Richard Lewishon
Introduced a citrate-dextrose solution for the preservation of blood.
Frances Payton Rouse & J.R. Turner
Pioneered on developing techniques in blood transfusion and blood preservation which led to the establishment of a widespread system of blood banks.
Dr. Charles Drew
Introduced the formula for the preservative acid-citrate-dextrose (ACD)
J.F. Loutit & Patrick L. Mollison
Introduced an improved preservative solution called citrate-phosphate-dextrose (CPD)
Gibson
Amount of whole blood in a unit
450 mL ± 10% of blood (1 pint)
Minimum hematocrit level required before blood unit collection
38%
Level of anticoagulant required when collecting 500ml of blood
70ml
The donor’s red blood cells are replaced within
1 to 2 months after donation
A volunteer donor can donate whole blood every
8 weeks
Components of whole blood
Packed RBCs
Buffy coat (WBCs & platelets)
Plasma
A unit of whole blood–prepared RBCs may be stored for
21 to 42 days
Process of collecting specific blood components
Apharesis
Average life span red of blood cell
120 days
Three areas of RBC biology that are crucial for normal erythrocyte survival and function:
- Normal chemical composition and structure of the RBC membrane
- Hemoglobin structure and function
- RBC metabolism
A semipermeable lipid bilayer supported by a mesh-like protein cytoskeleton structure
RBC membrane
The main lipid components of the membrane
Phospholipids
Proteins that extend from the outer surface and span the entire membrane to the inner cytoplasmic side of the RBC
Integral membrane protein
Membrane protein located and limited to the cytoplasmic surface of the membrane forming the RBC cytoskeleton.
Peripheral membrane proteins
The biochemical composition of the RBC membrane
Approximately 52% protein, 40% lipid, and 8% carbohydrate.
How does ATP levels affect RBC deformability?
The loss of ATP leads to a decrease in the phosphorylation of spectrin and, in turn, a loss of membrane deformability
How does membrane calcium levels affect RBC deformability?
An accumulation or increase in deposition of membrane calcium also results in an increased membrane rigidity and loss of pliability
Organ that functions in extravascular sequestration, and removal of aged, damaged, or less deformable RBCs or fragments of their membrane
Spleen
Cells with a reduced surface-to-volume ratio
Spherocytes
Cells with a permanent indentation in the remaining cell membrane
Bite cells
Ability of a normal RBCs to remain flexible, deformable, and permeable
Deformability
It prevent colloid hemolysis and control the volume of the RBCs.
The permeability properties of the RBC membrane and the active RBC cation transport
How does RBC volume and water homeostasis are maintained
By controlling the intracellular concentrations of sodium and potassium
The erythrocyte intracellular-to-extracellular ratios for Na+ and K+
1:12 and 25:1
Cytoplasmic calcium-binding protein, that is speculated to control energy-dependent calcium-ATPase pumps.
Calmodulin
Importance of calmodulin
Prevents excessive intracellular Ca2+ buildup
Why does RBCs mainly use anaerobic metabolic pathways to produce ATP?
Because the function of the RBC is to deliver oxygen, not to consume it
RBC metabolism may be divided into
Anaerobic glycolytic pathway and three ancillary pathways
The three ancillary pathways
Pentose phosphate pathway
Methemoglobin reductase pathway
Luebering-Rapoport shunt
It generates about 90% of the ATP needed by the RBC
Glycolysis
Provides approximately 10% of the ATP needed by the RBC
Pentose phosphate pathway
This pathway permits the accumulation of an important RBC organic phosphate, 2,3-diphosphoglycerate (2,3-DPG)
Luebering-Rapoport shunt
Significance of 2,3-DPG found within RBCs
The amount of 2,3-DPG found within RBCs has a significant effect on the affinity of hemoglobin for oxygen and therefore affects how well RBCs function post-transfusion.
Hemoglobin’s primary function
Gas transport: oxygen delivery to the tissues and carbon dioxide (CO2) excretion
One of the most important controls of hemoglobin affinity for oxygen is the RBC
2,3-DPG
Unloading of oxygen by hemoglobin is accompanied by
Widening of a space between β chains
Binding of 2,3-DPG
Formation of anionic salt bridges between the chains
Lower affinity to oxygen
The resulting conformation of the deoxyhemoglobin molecule is known as
Tense (T) form
Loading of oxygen by hemoglobin is accompanied by
Established salt bridges are broken
β chains are pulled together
Expelling 2,3-DPG
Higher affinity for oxygen
The resulting conformation of the oxyhemoglobin molecule is known as
Relaxed (R) form
The allosteric changes that occur as the hemoglobin loads and unloads oxygen are referred to as the
Respiratory movement
Sigmoid curve relationship between the dissociation and binding of oxygen by hemoglobin is known as
Hemoglobin-oxygen dissociation curve
The dissociation and binding of oxygen by hemoglobin are not directly proportional to the partial pressure of oxygen (pO2) in its environment. True or false?
True
The normal position of the oxygen dissociation curve depends on three different ligands normally found within the RBC:
H+ ions
CO2
Organic phosphates
Plays the most important physiological role in oxygen dissociation curve
2,3-DPG
In the hemoglobin-oxygen dissociation curve, how does shift to the right works?
Shift to the right of the hemoglobin-oxygen dissociation curve alleviates the tissue oxygen deficit. This rightward shift of the curve, mediated by increased levels of 2,3-DPG, decreases hemoglobin’s affinity for the oxygen molecule and increases oxygen delivery to the tissues.
In the hemoglobin-oxygen dissociation curve, how does shift to the left works?
It increases the hemoglobin-oxygen affinity and a decreases oxygen delivery to the tissues.
Multiple transfusions of 2,3-DPG–depleted stored blood can shift the oxygen dissociation curve to the:
Left
The goal of blood preservation
To provide viable and functional blood components for patients requiring blood transfusion.
It is a measure of in vivo RBC survival following transfusion
RBC viability
How to maintain optimum viability of RBC
Blood is stored in the liquid state between 1°C and 6°C for a specific number of days, as determined by the preservative solution(s) used
DPG-depleted RBCs indicate
Impaired capacity to deliver oxygen to the tissues
2,3-DPG levels in stored blood
Decreased
Amount of iron contained in one unit of RBC
Approximately 220 to 250 mg of iron
The purpose of the addition of various chemicals, along with the approved anticoagulant preservative CPD
It was incorporated in an attempt to stimulate glycolysis so that ATP levels were better maintained.
Chemical incorporated into the CPD solution (CPDA-1) that increases ADP levels
Adenine
How does CPDA-1 increases ADP levels
It drives glycolysis toward the synthesis of ATP
Composition of CPDA-1
0.25 mM of adenine plus 25% more glucose than CPD
Storage time of adenine-supplemented blood
35 days
Storage time of blood with Acid citrate-dextrose (formula A)*
21 days
Storage time of blood with Citrate-phosphate dextrose
21 days
Storage time of blood with Citrate-phosphate- double-dextrose
21 days
Anticoagulant Preservative Solutions used for apharesis components
Acid citrate-dextrose (formula A)*
The reported pathophysiological effects of the transfusion of RBCs with low 2,3-DPG levels and increased affinity for oxygen include
An increase in cardiac output, a decrease in mixed venous (pO2) tension, or a combination of these
Blood stored in all CPD preservatives becomes depleted of 2,3-DPG during:
The second week of storage
Required time to restore normal levels of 2,3-DPG after transfusion
Approximately 24 hours
Material used to store blood
Polyvinyl chloride (PVC) plastic bags
Functions of Citrate (sodium citrate/citric acid)
Chelates calcium; prevents clotting
Functions of Monobasic sodium phosphate
Maintains pH during storage; necessary for maintenance of adequate levels of 2,3-DPG.
Function of Dextrose
Substrate for ATP production (cellular energy)
Function of Adenine
Production of ATP (extends shelf-life from 21 to 35 days)
Preserving solutions that are added to the RBCs after removal of the plasma with or without platelets
Additive solutions (AS)
Reason for developing Additive solutions (AS)
Because the removal of the plasma component during the preparation of packed RBCs removed much of the nutrients needed to maintain RBCs during storage
Efficiency of additive solution in reducing hematocrit
Additive solutions reduce hematocrits from around 65% to 80% to around 55% to 65% with a volume of approximately 300 to 400 mL
Additive solutions licensed in the United States
Adsol
Nutricel
Optisol
SOLX
Benefits of RBC Additive Solutions
- Extends the shelf-life of RBCs to 42 days by adding nutrients
- Allows for the harvesting of more plasma and platelets from the unit
- Produces a packed RBC of lower viscosity that is easier to infuse
The additive solution is contained in bag called
Satellite bag
Additive solutions AS1 AS3 AS5 AS7 are approved to store pRBCS for
42 days
Composition of AS1
Saline
Adenine
Dextrose
Mannitol
Composition of AS5
Saline
Adenine
Dextrose
Mannitol
Composition of AS7
Saline
Adenine
Dextrose
Mannitol
Sodium bicarbonate
Composition of AS3
Saline
Adenine
Dextrose
Sodium citrate
Purpose of dextrose
Supports ATP generation by glycolytic pathway
Purpose of adenine
Acts as substrate for red cell ATP synthesis
Purpose of citrate
Prevents coagulation by chelating calcium, also protects red cell membrane
Purpose of sodium biphosphate
Prevents excessive decrease in pH
Purpose of mannitol
Osmotic diuretic acts as membrane stabilizer
Primarily used for autologous units and the storage of rare blood types
RBC freezing
Allows individuals to donate blood for their own use to meet their needs for blood transfusion
Autologous transfusion
Commonly used cryoprotective agent for RBC freezing
Glycerol
The usual storage temperature for frozen RBC
Below –65°C
Although storage (and freezing) temperature depends on the concentration of glycerol used.
Two concentrations of glycerol used to freeze RBCs
High-concentration glycerol (40% weight in volume [wt/vol])
Low-concentration glycerol (20% wt/vol)
Glycerol concentration that is commonly used by blood banks to freeze RBCs
High-concentration glycerol (40% weight in volume [wt/vol])
Shelf life of frozen RBCs
10 years
What to do before transfusing frozen RBCs
Transfusion of frozen cells must be preceded by a deglycerolization process. Removal of glycerol is achieved by systematically replacing the cryoprotectant with decreasing concentrations of saline. The usual protocol involves washing with 12% saline, followed by 1.6% saline, with a final wash of 0.2% dextrose in normal saline.
Unit of blood that is processed in an open system must be transfused within
24 hours
RBCs in CPD or CPDA-1 anticoagulant preservatives or additive solutions are glycerolized and frozen within
6 days of whole blood collection
System that allows the glycerolization and deglycerolization processes to be performed under sterile conditions
Closed-system
System glycerolization and deglycerolization processes that are performed in non-sterile environment
Open system
When closed-system is used, RBCs with CPDA-1 can be stored for
Up to two weeks
Deglycerolized RBCs using the open system must be transfused within
24 hours
RBC preparations (glycerolization and deglycerolization) using the closed-system may be stored for
14 days at 4°
Freezing speed when using high glycerol technique
Slow freezing
Freezing speed when using low glycerol technique
Fast freezing
RBCs using high glycerol technique is frozen at
-45C
RBCs using low glycerol technique is frozen at
-145C
Frozen RBCs using high glycerol technique is stored at
-65C for 10 years
Frozen RBCs using low glycerol technique is stored at
-120C for 10 years
Process by which ATP and 2,3- DPG levels are restored or enhanced by metabolic alterations.
Rejuvenation
What to do before transfusing rejuvenated RBCs
Rejuvenated RBCs must be washed to remove the inosine (which may be toxic) and transfused or frozen for long-term storage.
Rejuvenated RBCs may be prepared up to
3 days after expiration when stored in CPD, CPDA-1, and AS-1 storage solutions
Rejuvenation solution contains
Phosphate
Inosine
Pyruvate
Adenine
Importance of RBC rejuvenation
Invaluable for preserving selected autologous and rare units of blood for later use
Rejuvenated RBCs must be transfused within
24 hours
Only countries in which blood substitutes are approved for clinical use
South Africa
Mexico
Russia
Blood substitutes
hemoglobin-based oxygen carriers (HBOCs)
Perfluorocarbons (PFCs)
Blood substitute that was approved for clinical use in South Africa in 2001 to treat adult surgical patients who are anemic, and in Russia for acute anemia
Hemopure
Synthetic hydrocarbon structures in which all hydrogen atoms have been replaced with fluorine
Perfluorocarbons
Characteristics of Perfluorocarbons
Chemically inert
Excellent gas solvents
Carry O2 and CO2 by dissolving them
Size of Perfluorocarbons
0.2 μm in diameter
Advantage of small size of Perfluorocarbons
Because of their small size, they are able to pass through areas of vasoconstriction and deliver oxygen to tissues that are inaccessible to RBCs
Involved in the blood coagulation process and are given to treat or prevent bleeding
Platelets
Platelets are given either:
Therapeutically to stop bleeding or Prophylactically to prevent bleeding
Shelf life of platelet concentrates
Maximum of 5 days
Storage requirements for platelet concentrates
Platelets are stored at 20°C to 24°C with maintaining continuous gentle agitation for a period of 5 days
Importance of agitation when storing platelet concentrates
Agitation has been shown to facilitate oxygen transfer into the platelet bag and oxygen consumption by the platelets
When storing platelet concentrates, the positive role for oxygen has been associated with
Maintenance of platelet component pH
Key parameter for retaining platelet viability in vivo when platelets were stored at 20°C to 24°C
pH
The loss of platelet quality during storage is known as
Platelet storage lesion
How does platelet storage influence platelet storage lesion?
During storage, a varying degree of platelet activation occurs that results in release of some intracellular granules and a decline in ATP and ADP
Effect of reduced oxygen tension (pO2) in the plastic platelet storage container
Increase in the rate of glycolysis by platelets to compensate for the decrease in ATP regeneration from the oxidative (TCA) metabolism
The increase in glucose consumption by platelets causes
Increase in lactic acid that must be buffered
Increase of lactic acid in platelet concentrates causes
Fall in pH
The principal buffer for storage of platelet concentrates (PCs) in plasma
Bicarbonate
What happens when the pH of platelet concentrates fall down below 6.2?
Reduce platelet viability
Loss of membrane integrity
Irreversibly swollen
Aggregate together
Lyse and will not circulate when infused
During storage of platelet concentrates, the pH will remain stable as long as the production of lactic acid does not exceed the buffering capacity of the plasma or other storage solution. True or false?
True
Quality-control measurements required by various accreditation organizations for platelet concentrates
Platelet concentrate volume
Platelet count
pH of the unit
Residual leukocyte count (if claims of leukoreduction are made)
Visual inspection process for platelet concentrates
Platelet swirl
The absence of platelet swirling is associated with
Loss of membrane integrity during storage, resulting in the loss of discoid shape with irreversible sphering
In Vitro Platelet Assays Correlated With In Vivo Survival
pH
Shape change
Hypotonic shock response
Lactate production
pO2
Clinical Use of Platelets
Used to treat bleeding associated with thrombocytopenia, a marked decrease in platelet number, or dysfunctional platelets
How to estimate the efficacy of the platelet transfusion
Estimated from the corrected count increment (CCI) of platelets measured after transfusion
It is a calculated measure of patient response to platelet transfusion that adjusts for the number of platelets infused and the size of the recipient, based upon body surface area (BSA)
Corrected count increment (CCI)
Formula of corrected count increment (CCI)
CCI = (postcount – precount) × BSA/platelets transfused
When to compute for CCI?
10 to 60 minutes after transfusion
Platelets are prepared as concentrates from whole blood
Whole blood-derived platelet concentrates
Platelets are prepared as concentrates from apheresis
Apheresis platelets
Greater than 92% of platelet transfusions are from
Apheresed platelets
8% of platelet transfusions are from
Whole blood-derived platelets (WBD)
Primary means of treating thrombocytopenia
Platelets
One unit of whole blood-derived platelet concentrate contains
≥5.5 × 10^10 platelets suspended in 40 to 70 mL of plasma
Pooled units of platelet only have a shelf life of
4 hours
One unit of apheresis platelets contain how many platelets
≥3.0 × 10^11
One unit of apheresis platelets is equivalent to how many units of whole blood-derived platelets
4 to 6 units
Two FDA approved platelet additive solutions for the storage of apheresis platelets for 5 days
PAS-C (Intersol)
PAS-F (Isoplate)
With the addition of a PAS, residual plasma is reduced to how many percent?
35%
Advantages of platelet additive solutions (PAS)
Provides more plasma for fractionation
Improve the quality of platelets during storage
Reduce adverse effects associated with transfusion of plasma
Promote earlier detection of bacteria
Indicates the capacity of platelets to circulate after infusion without premature removal or destruction
Viability
Platelets have a life span of ___ after release from megakaryocytes
8 to 10 days
Viability of stored platelets is determined by measuring
Pretransfusion and post-transfusion platelet counts and expressing the difference based on the number of platelets transfused (CCI)
The observation of the swirling phenomenon indicates
Retention of platelet viability properties in stored units
It is defined as the ability of viable platelets to respond to vascular damage in promoting hemostasis
Platelet function
The major concern associated with storage of platelets at 20°C to 24°C
Potential for bacterial growth
Conditions that provide a good environment for bacterial proliferation
Room temperature storage and the presence of oxygen
Most common infectious complication of transfusion
Sepsis
Commercial systems approved by the FDA for screening platelets for bacterial contamination
BacT/ALERT (bioMérieux)
eBDS (Pall Corp.)
When to test platelet samples for bacterial detection
After 24 hours of storage
How does BacT/ALERT measure bacterial contamination?
Measures bacteria by detecting a change in carbon dioxide levels associated with bacterial growth
How does eBDS system measure bacterial contamination?
Measures the oxygen content of the air within the sample pouch for 18 to 30 hours following incubation. A decrease in oxygen level indicates the presence of bacteria
During the practice of screening platelets for bacterial contamination, false-negative cultures can occur when
Bacteria are present in low numbers and when the pathogen is a slow-growing organism
A single units of WBD platelets pooled and tested ≥24 hours after collection, and stored in an FDA-cleared container for extended pool storage for up to 5 days (consistent with the container package insert)
Prestorage pooled WBD platelets
The most common source of bacterial contamination
Entry of skin plugs into the collection bag
Represent stored single units of WBD platelets that are pooled within 4 hours prior to transfusion
Poststorage pooled WBD platelets
Pooled ABO-matched, leukoreduced WBD platelets that have been cultured and are ready for transfusion
Acrodose platelets
The only platelet pooling systems available in the United States that provide a clinically equivalent alternative to apheresis platelets
Acrodose systems
First rapid test to detect bacteria in platelets
Pan Genera Detection (PGD) test (Verax Biomedical)
What is PGD test?
An immunoassay that detects lipoteichoic acids on gram-positive bacteria and lipopolysaccharides on gram-negative bacteria
Amount of sample required for PGD test
500 μL
In PGD test, the optimum time for sampling is
At least 72 hours after collection
PGD rapid test has a sensitivity of _____ and a specificity of _____
99.3%; 60%
The process of treating platelet components to reduce or inactivate any residual pathogens (bacteria, viruses, parasites, and protozoa) that may be present
Pathogen inactivation (PI)
Pathogen inactivated component are referred to as being
Pathogen reduced (PR)
The only FDA approved technology for the manufacture of certain pathogen- reduced apheresis platelet and plasma products
INTERCEPT® Blood System
How does INTERCEPT system inactive pathogens
It uses amotosalen that is activated by ultraviolet A (UVA) light and binds to the nucleic acid base pairs of pathogens, preventing replication
How is it possible for INTERCEPT system to specifically target pathogen nucleic acid?
The targeting of nucleic acids is possible because platelets, like RBCs, do not contain functional nucleic acids
For 5-day apheresis platelets, the requirements to control the bacterial contamination risk are
Culturing the product at least 24 hours after collection
Pathogen-reducing it within 24 hours after collection
Initial/first-time testing of a platelet component to detect the presence of bacterial contamination
Primary testing of platelets
How to perform primary testing of platelets?
Testing is conducted early in the storage period of platelets using a culture-based device or other adequate or appropriate method found acceptable by the FDA
Any additional test of a platelet component to detect the presence of bacterial contamination in a unit that previously showed no bacterial contamination upon primary testing
Secondary testing of platelets
How to perform secondary testing of platelets?
Secondary testing of platelets is usually conducted late in the storage period and may be conducted with either a culture-based bacterial detection device, or a rapid bacterial detection device acceptable to the FDA
Secondary testing is currently mandatory with a time-consuming test. True or false?
False; optional & rapid tests
The requirement to test every product with a bacterial detection device cleared by FDA is defined as
Safety measure test
Platelets are cultured at least 24 hours after collection with an FDA-cleared device, and secondarily retested with a bacterial detection test labeled as
Safety measure
The only rapid bacterial detection device cleared as a “safety measure” test and is used to extend dating of platelets to day 7
Verax PGD test
How to extend platelet dating beyond 5 days
FDA recommends performing secondary testing on apheresis platelets previously cultured or PRT-treated, or on single units of WBD platelets previously cultured, using a device cleared by the FDA as a “safety measure.”
Each unit of whole blood collected contains approximately
450 mL of blood and 63 mL of anticoagulant or;
500 mL of blood and 70 mL of anticoagulant
How often can a blood donor can give blood
Every 8 weeks
One criterion used by the FDA for approval of new preservation solutions and storage containers is an average 24-hour post transfusion RBC survival of more than:
75%
Approved preservative solutions for storage of RBCs at 1°C to 6°C for 21 days
ACD
CPD
CP2D
Approved preservative solutions for storage of RBCs at 1°C to 6°C for 35 days
CPDA-1
Additive solutions approved in the United States for RBC storage for 42 days
Adsol
Nutricel
Optisol
SOLX
The only FDA-approved rejuvenation solution
Rejuvesol
Used primarily to salvage O type and rare RBC units that are outdated
Rejuvenation
Used with specific anticoagulant preservative solution up to 3 days past outdate
Rejuvenation
RBC substitutes
Hemoglobin-based oxygen carriers
Perfluorocarbons
What is the maximum volume of blood that can be collected from a 110-lb donor, including samples for processing?
450 mL
The majority of platelets transfused in the United States today are:
Apheresis platelets
Which anticoagulant preservatives provides a storage time of 35 days at 1°C to 6°C for units of whole blood and prepared RBCs if an additive solution is not added?
CPDA-1
What are the current storage time and storage temperature for platelet concentrates and apheresis platelet components?
5 days at 20°C to 24°C
Whole blood and RBC units are stored at what temperature?
1°C to 6°C
What is the lowest allowable pH for a platelet component at outdate?
6.2
The INTERCEPT pathogen reduction system uses
Amotosalen and UV light
