Blood banking intro

What is immunohematology?

The study of immunology as it relates to red blood cells

What do blood banks do?

Select, collect blood products, perform testing and distribute products. They are established by the AABB and regulated by the FDA

What do transfusion services do?

Similar to blood banks but do no collection prodcuts

What is the rbc membrane freely permeable to?

• Water

• anions (Cl and HCO3)

• O2

• CO2

What is the RBC membrane not freely permeable to?

• Na

• Ca

• K

Transport across the rbc membrane is controlled by?

Active transport and requires energy to perform

What does active transport result in?

• Energy (specifically ATP) depletion

What does energy depletion result in for the RBC?

• accumulation of Ca, Na, and K

• water loss

• all results in a rigid cell

What ways does the rbc get its energy?

• anaerobic glycolysis (90%)

• monophosphate shunt (10%)

• methemoglobin reductase pathway (very small amount)

What does the methemoglobin reductase pathway do?

Keeps iron in the Fe2+ state for oxygen transport

What does 2,3 DPG do?

Allows for greater O2 release at certain PO2

What are the primary goals of rbc preservation?

• maintain viability and function

• prevent detrimental physical and chemical changes

• minimize bacterial proliferation

What is meant by viability when refering to rbcs?

The measure of in vivo survival of rbcs after transfusion

What is the minimum requirement for a unit of pRBC to be viable?

75% survival of transfused rbcs after 24 hours

At what temperature are RBCS stored at?

1-6 degrees C

What is the approximate life-span of rbcs?

2 weeks (3 months from processing)

When rbcs are stored what substances start to decrease and what happens as they do?

• ATP and 2,3 DPG

• RBC O2 affinity increases resulting in a less functional rbc

What is the lesion of storage?

Loss of rbc viability with storage associated biochemical changes

What biochemical changes happen during rbc storage?

• decreased pH

• Increase in lactate

• decrease in glucose availability

• decrease in ATP

• Increase in plasma K levels

• decrease in 2,3 DPG

• left shift in oxygen curve

• increase in plasma hgb

What happens to coagulation factors in stored rbcs?

• decrease in liable coagulation factors after 24 hours

• very few functional platelets left

At day 0 of storage what are the biochemical stats of the rbcs?

• 100% viable cells

• pH 7.55

• ATP 100%

• 2,3 DPG 100%

• Plasma K 5.1 mmol/L

• plasma Na 169 mmol/L

• Plasma Hgb 78 mg/L

At 21-35 days of storage what are the biochemical stats of rbcs?

• 71% viable cells

• pH 6.71

• ATP 35-57%

• 2,3 DPG 100%

• Plasma K 79 mmol/L

• plasma Na 111 mmol/L

• plasma Hgb 65.8 mg/L

What are blood preservatives designed to do?

• prevent clotting

• maintain cell viability and function

What is the amount of pack red blood cells to anticoagulant

405-495 ml of blood to 63 ml of anticoagulant/preservative

What are some examples of rbcs additives?

• dextrose: supports atp production

• adenine: substrate for ATP production

• phosphate buffer: controls pH

• citrate: prevents clotting

What are the additives with a 21 day storage limit?

• CPD (citrate-phosphate-dextrose)

• CP2D (citrate-phosphate-dextrose-dextrose)

What is the additive with a 35 day storage?

• CPDA-1 (citrate-phosphate-dextrose)

• used by the majority of blood centers in the US

When are additives added?

When rbcs are separated from plasma

What does adding additives do for the rbcs?

• removes 40% of adenine and glucose

• results in increased viability of the rbcs units

How are additives added to rbcs?

In a closed system within 72 hours of collection, increases unit expiration up to 42 days

What are some examples of additives?

• Adenine-Saline solution (AS)

  • NaCl

  • Dextrose

  • Adenine

  • Mannitol

• AS-1 (Adsol)

• AS-3 (Nutricel)

• AS-5 (Optisol)

What must the blood storage bags be able to do?

• be permeable to CO2

• Made of Polyvinyl Chloride to allow “breathing”

What temperature are whole blood and red blood cells stored at?

1-6 degrees Celsius

What temperature is plasma (FFP) stored at?

-18 degrees Celsius or less

What temperature are platelets stored at?

20-24 degrees Celsius

What temperature are frozen red blood cells stored at?

• 65 C or less

• must be coated with protective glycerol before freezing

What are frozen red blood cells used for?

• autologous units

• rare blood types

• can be stored for 10 years

What are rejuvenation solutions used for?

• rare units

• regenerate ATP and 2,3 DPG

What is used to rejuvenate rbcs?

Rejuvesol

How is polyheme and hemopure made?

• hgb is extracting from rbc units

• hgb is then modified to form a polymer

• hgb is then added to a solution that can be transfusion

What are the advantages of blood substitutes?

• long shelf life

• very stable

• no antigenicity

• blood typing is not required

What are the disadvantages of blood substitutes?

• short intravascular half life

• possible toxicity

• increased O2 affinity

• increased Oncotic effect (causes cancer)

What are the advantages of perfluorochemicals?

• biologic inertness

• lack of immunogenicity

• easily synthesized

What are the disadvantages of perfluorchemicals?

• adverse clinical effects

• retention in tissues

• High O2 affinity

• O2 required with infusion

• deep freeze storage required

What is Inactine?

An additive that chemically inactivate pathogens in Rbc concentrates

What is Aziridino derivative of ethyleneimine (PEN110) and what can it do?

• is a small charged molecule

• binds to viral DNA and RNA

• prevents replication

• kills pathogens

• causes a 5 log reduction in titers of enveloped and non-enveloped viruses

• inactivates bacteria, lymphocytes and protozoa

What must be done to rbcs prior to transfusion that contain PEN110?

They must be washed

What is agglutination?

The clumping together of RBCs or any particulate matter resulting from an interaction of antibody and its corresponding antigen

What is an agglutinin?

An antibody that agglutinates cells (or other particulate matter)

What is an agglutinogen?

A substance that stimulates the production of an agglutinin

• acts as an antigen

What are the two stages of agglutination?

• sensitization

• Lattice formation

What happens during the sensitization phase of agglutination?

• Antibody attachment

• equilibrium constant

  • amt of Ab-Ag at equilibrium

  • the higher the constant, the higher the rate of association

  • affected by concentration of Ab and Ag

What happens during the lattice formation phase of agglutination?

Formation of bridges between sensitized cells

What factors affect the sensitization phase?

• Antibody specificity

• Antigen-antibody ratio

• pH

• Temperature

• Incubation time

• Ionic repulsion

What is specificy?

Primary determinant

What are IgM antibody usually?

Cold reactive that work best at room temp or lower

What are IgG antibodies usually?

Warm reactive, usually clinical significant antibodies

What does the antigen-antibody ratio affect?

• Speed of antibody uptake

• increased antibody equals increased sensitivity

What are the prozone and postzone?

When there is too much antibody and when there is too much antigen

What is the optimal pH for routine testing?

7.0 (6.5-7.5)

What temperature do IgM antibodies perform best at?

below 37 degrees

What temperature do IgG antibodies perform best at?

37 degrees

What range is antibody identification designed to cover?

22 to 37 degrees celcius

• this ensurees indentification of any possibliy present antibodies

What antibodies are clinically insignificant?

Antibodies that react below 37 degrees

How does incubation time affect sensitization?

• allows for antibody uptake

What are the uptake times for antibodies?

• 25% of antibodies are taken up in the first 15 minutes

• 75% of antibodies are taken up in the first hour

What factors affect lattice formation?

• zeta potential

• enzymes

• antihuman globin

• additional reagents

What creates ironic repulsion?

• Na and Cl ions

What is used to minimize the effect of ionic repulsion?

Low Ionic Strength Saline (LISS)

What is zeta potential?

Occurs due to a net negative charge of RBCs from sialic acid. Forms an ionic cloud

How can the zeta potential be altered?

By using a suspending medium that can mitigate the effect.

• albumin

• LISS

• Polybrene and Polyethylene Glycol (PEG)

How do enzymes affect lattice formation?

• Decrease the surface charge by cleaving surface proteins

• remove obstructions

What are some frequently used enzymes to decrease surface charge?

• papain - from papaya

• Ficin - from figs

• Bromelin - pineapple

What are some surface antigen binding sites eliminated by enzymes?

M, N, S, s, Fya, Fyb

What is antihuman globulin?

Antibody to human antibody/complement

What does antihuman globulin detect?

Cells coated with antibody, complement or both by cross linking sensitized cells

What regents can be used that affect lattice formation?

• Dithiothreitol (DTT)

• 2-mercapthoethanol (2 ME)

• ZZAP

What does DTT do?

Destroys IgM antibodies

What does 2 ME do?

• destroys IgM antibodies

• leave IgG antibodies intact

What does ZZAP do?

• dissociates IgG from RBC

• alters RBC surface charge

What other factors of the RBC affect lattice formation?

• RBC shape

• RBC membrane flexibility