Lecture

IMMUNOHEMATOLOGY
Module 1: Introduction to Immunohematology
Semi-permeable lipid bilayer:
RED BLOOD CELLS - phospholipids: arranged in a bilayer structure
COMPOSITION → outer leaflet: phosphatidylcholine and
A. 8% whole blood sphingomyelin
→ 55% plasma → inner leaflet: phosphatidylethanolamine and
7% proteins phosphatidylserine (exposure to outer leaflet:
54% albumin increased vascular adherence; macrophage
38% globulin recognition and phagocytosis)
7% fibrinogen - supported by:
1% others → cytoskeleton
91.5% water → globular proteins
1.5% other solutes integral: spans entire layer from the inner
electrolytes to the outer surface
nutrients - glycophorins A, B, C, D: can be
vitamins the blood group antigens
gases - anion exchange channel protein
wastes (band 3)
regulating substances peripheral: found on the cytoplasmic side
→ 45% formed elements of the membrane
4.8-5.4 million RBCs/uL - spectrin
5,000-10,000 WBCs/uL - actin (band 5)
60-70% neutrophils - ankyrin (band 2.1)
20-25% lymphocytes - band 4.1 and 4.2
3-8% eosinophils - band 6
0.5-1% basophils - adducin
150,000-400,000 platelets/uL - p55

B. 92% other fluids and tissues Deformability

- influenced by:
Volume: 4-6 L → ATP: decreased phosphorylation of spectrin
pH: 7.35-7.45 → Ca+: when increased: loss of membrane
liability
FUNCTIONS - resulting to loss of membrane
1. respiratory function → formation of spherocytes: reduced surface to
2. protective/defensive volume ratio
3. nutritional function → bite cells: permanent indentation
4. regulatory function
5. excretory function Permeability and active RBC cation transport
- prevent colloid hemolysis
BIOLOGY AND PRESERVATION - control RBC volume: controlled Na+
1. normal chemical composition and structure of (extracellular cation; intra to extracellular ratio
the RBC membrane is 1:12) and K+ (intracellular cation; intra to
2. normal hemoglobin structure and function extracellular ratio is 25:1)
3. RBC metabolism - freely permeable to water and anions
- Ca+: actively pumped out of the cell through
MEMBRANE calcium-ATPase pumps (controlled by
- 52% proteins; 40% lipid; 8% carbohydrates calmodulin)

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 Lecture
IMMUNOHEMATOLOGY
Module 1: Introduction to Immunohematology
RBCs when ATP depleted → Ca+ and Na+: increased
intracellularly; K+ and water: decreased/lost →
dehydrated, rigid cell → sequestered by the spleen

HEMOGLOBIN STRUCTURE AND FUNCTION

95% of RBC dry weight
33% RBC weight by volume

Consists of:
- globin: tetramer of 2 pairs of unlike globin
polypeptide chains
- 4 heme (ferriprotoporphyrin IX) groups
Protoporphyrin ring
Ferrous iron (Fe 2+)
A: pO2 is almost 100% concentrated with O2
Globin chain components → as RBCs travel to the tissues → B: pO2 drops by
Embryonic Hbs 40mmHg: 75% saturated with hemoglobin; 25% O2 is
- Gower I: ζ2ε2 released
- Gower II: α2ε2
- Portland: ζ2γ2 Shift to the right
Normal adult hemoglobin
- HbA: α2β2 chains (95-97%)
- HbA2: α2δ2 chains (2-3%)
- HbF: α2γ2 chains (1-2%)

Respiratory movement
Oxygen unload (deoxyhemoglobin)
1. binding of Hb with 2,3-diphosphoglycerate
2. formation of cationic salt bridges between β
chains
3. β chains are widened
4. tense (T) form
5. lower affinity for oxygen - mediated by increased levels of 2,3-DPG →
oxygen saturation of Hb → 50% of O2 (normally,
Oxygen loading (oxyhemoglobin) 25%) is released at 40mmHg; 50% Hb
1. salt bridges are broken - anemia; hypoxia; acidosis
2. β chains are pulled together
3. 2,3-DPG is expelled ↓ pH ↑ 2,3-DPG
4. relaxed (R) form ↑ temperature ↑ P50
5. higher affinity for oxygen
decreased affinity of Hb for O2 → increase in O2
delivered to the tissues

NORMAL OXYGEN DISSOCIATION CURVE

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 Lecture
IMMUNOHEMATOLOGY
Module 1: Introduction to Immunohematology
- reduced glutathione + NADPH: main line of
Shift to the left defense against oxidative injury)
- prevent globin denaturation and precipitation
- inherited defects: G6PD deficiency → heinz
bodies

if functionally deficient: glutathione becomes insufficient

to neutralize intracellular oxidants (keeps Hb in a
functional state → maintains RBC integrity by reducing
sulfhydryl groups of Hb)

3. Methemoglobin Reductase Pathway

- maintain Fe+ in the reduced functional state
- NADH methemoglobin reductase and NADPH
methemoglobin reductase

- alkalosis; ↑ HbF; multiple transfusions of absence of methemoglobin reductase + absence of

2,3-DPG depleted stored blood NADH → accumulation of methemoglobin

↑ pH ↓ 2,3-DPG no more than 1% RBC Hb should exist as a

↑ abnormal Hb (carboxyhemoglobin; methemoglobin) methemoglobin
↓ Temperature ↓ P50
4. Luebering-Rapoport Shunt
increased affinity of Hb for O2 → decrease in O2 deliver - causes extraordinary accumulation of 2,3-DPG
to the tissues - stores of 2,3-DPG: reserve for additional ATP
generation
METABOLIC PATHWAYS
- mainly anaerobic: mature red blood cell has no RED CELL ANTIGENS
mitochondria
- necessary for maintaining:
1. hemoglobin function
2. membrane integrity and deformability
3. RBC volume
4. adequate amounts of reduced pyridine
nucleotides

1. Embden-Meyerhof Glycolytic Pathway

- provides 90% of ATP
- 2 molecules of ATP
- generates NADH from NAD+

2. Hexose Monophosphate Shunt

- pentose phosphate/phosphogluconate
pathway
- produces pyridine nucleotide: NADPH from
NADP+
- 5-10% of glucose is metabolized
RED CELL PRESERVATION

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IMMUNOHEMATOLOGY
Module 1: Introduction to Immunohematology
- to provide viable and functional blood components CP2D: contains 100% more dextrose/glucose than
requiring blood transfusion CPD; 60% more glucose than CPDA-1
- loss of RBC viability (storage lesion) → 2,3-DPG is CPDA-1: contains 25% more glucose than CPD +
reduced (also depleted by two weeks of storage) adenine → increase ADP levels → driving glycolysis
- rate of restoration of 2,3-DPG depends on: toward ATP synthesis
1. acid-base status of patient
2. phosphorus metabolism Chemicals in Anticoagulant Solutions
3. degree of anemia
4. overall severity of the disorder

FDA requires an average of 24 hr post-transfusion rbc

survival of more than 25%

● PVC (polyvinyl chloride → breaks down at lower

temperatures) bags contain plasticizer
(diethylhexyl phthalate) → leeches into the lipids
of plasma medium and blood membranes during
storage → some stabilize RBC membrane and
Additive Solutions
reduce extent of hemolysis
- contained in a satellite bag and added to RBCs after
mst of the plasma has been depressed
● Polyolefin bag → do not contain diethylhexyl
- 100ml added to RBC concentrate (plasma removed)
phthalate; latex-free
prepared from a 450ml blood collection
- reduce hematocrits from 70-85% to 50-60%
Storage Lesions
Advantages:
1. extends shelf-life of RBCs to 42 days by adding
nutrients
2. allows for the harvesting of more plasma and
platelets from the units
3. produces an RBC concentrate of lower viscosity
that is easier to infuse

Anticoagulant Preservative Solutions

Mannitol: protects against storage-related hemolysis

ACD-A: apheresis

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IMMUNOHEMATOLOGY
Module 1: Introduction to Immunohematology
w/out glucose
RED CELL FREEZING
- done for autologous units; storage of rare blood types TRENDS IN PRESERVATION RESEARCH
- done on RBC <6 days old Development of:
- stored at -65C 1. improved additive solutions
- use of cryoprotectant (glycerol): deglycerolization is 2. procedures to reduce and inactivate the level of
done prior to transfusion: pathogens in RBC units
1. wash red cells with 12% saline 3. methods to produce RBCs through
2. 1.6% saline bioengineering (blood pharming)
3. final wash with 0.2% dextrose in normal saline 4. RBC substitutes
(0.85-0.90% of NaCl)
- good for 10 years storage IMPROVED ADDITIVE SOLUTIONS
- longer storage periods → improved logistics of
High glycerol vs. low glycerol technique providing RBCs for clinical use; increased benefits
associated with the use of autologous blood

PROCEDURES TO REDUCE AND INACTIVATE

PATHOGENS
1. use of alkylating agents → react with nucleic
acids of pathogens
- S-303, cerus/baxter
- inactive, vitex
2. riboflavin and UV-treated RBCs
Advantages
1. long term storage Before approval for use (US): potential toxicity;
2. maintenance of RBC viability immunogenicity; cellular function; cost
3. low residual leukocytes and platelets
4. removal of significant amounts of plasma FORMATION OF O-TYPE RBCs
proteins Can “A” and “B” be converted to “O”?
→ possible by using of enzymes to remove
Disadvantages carbohydrate moieties
1. time-consuming process
2. higher cost of equipment and materials Immunodominant sugar (Type A: GalNAc; Type B: Gal)
3. storage requirements (-65C) → when removed → formation of type O antigen
4. higher cost of product
BLOOD PHARMING
REJUVENATION - creating RBCs in the laboratory
- restoring ATP and 2,3-DPG levels - increase the amount of blood for transfusion
- done with outdated liquid RBC - “Arteriocyte”: company that develops system in
- done 3 days after outdate producing O-negative RBCs → technology: NANEX →
HSC (hematopoietic stem cell from umbilical cord:
RBC unit + 50ml of rejuvenating solution (rejuvesol) → source of type O, Rh-negative
incubate at 37C → washed then transfused within 24hrs
RBC SUBSTITUTES
- rejuvenating solution contains either: Artificial oxygen carriers
1. PIGPA (phosphate inosine glucose pyruvate Advantages:
adenine

2. PIPA
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IMMUNOHEMATOLOGY
Module 1: Introduction to Immunohematology
- metabolism: aerobic → TCA cycle/Krebs cycle
- functions:
1. platelet plug formation to arrest bleeding
2. stabilize hemostatic plug by fibrin formation
3. vascular integrity

Whole blood → light spin (2000 x gravity for 3 mins at

room temperature; about 22C) → RBC: stored at 1-6C
and plasma: platelet-rich → platelet concentrate
preparation

Hemoglobin-based oxygen carriers

- from human, bovine & recombinant hemoglobin

Advantages:
1. long shelf-life
2. very stable
3. no antigenicity (unless bovine)
4. no requirement for blood typing

Disadvantages:
1. short intravascular half-life Platelet concentrates:
2. possible toxicity - prepared from whole blood and apheresis (automated
3. increased O2 affinity process: blood from the body → separate components
4. increased oncotic effect in a closed system → platelets taken; remaining blood
→ back to the body)
Perfluorocarbons - storage: 20-24C with continuous agitation for up to 5
- synthetic hydrocarbon structures; hydrogen atoms days
have been replaced by fluorine → chemically inert; - FDA: expiration time is midnight of day 5
excellent gas solvent that can carry O2 and CO2
MEASUREMENT OF VIABILITY & FUNCTIONAL
Advantages: PROPERTIES OF STORED PLATELETS
1. biological inertness Platelet viability:
2. lack of immunogenicity - capacity to circulate after infusion
3. easily synthesized - without premature removal or destruction
- determined by:
Disadvantages: → measuring pre- and post-transfusion platelet
1. adverse clinical effects count (1hr and/or 24 hrs) → corrected count
2. high O2 affinity increment: expressing the difference based on
3. retained in tissues the number of platelets transfused
4. requirement for O2 administration when infused → disappearance rate of infused radiolabeled
5. deep-freeze storage temperatures platelets to normal individuals whose donation
provided the platelets
PLATELETS
PRESERVATION Platelet function: ability to respond to vascular damage
Platelets: in promoting hemostasis
- small, disk-shaped; 2-4um in diameter
- circulate in the blood: 9-12 days

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IMMUNOHEMATOLOGY
Module 1: Introduction to Immunohematology
SCREENING PLATELETS FOR BACTERIAL
QUALITY CONTROL CONTAMINATION
- platelet concentrate volume Possible causes: contamination at the phlebotomy site
- platelet count (alcohol then iodine solution); patient has an
- pH unrecognized bacterial infection during the donation
- residual leukocyte count (if leukoreduction is made) process; environmental contamination during
processing
before transfusion: visual inspection of platelet swirl →
swirling phenomenon: retention of viability → absence Methods:
→ loss of membrane integrity during storage → loss of 1. bacT/ALERT (bioMerieux)
discoid shape with irreversible sphering - culture-based system
- detecting changes in CO2 levels associated with
STORAGE LESION bacterial growth

2. eBDS (Pall Corp.)

- Measures the O2 content of the air within the
sample pouch following incubation for 18-30 hrs
→ when decreased: presence of bacteria

3. scansystem (Hemosystem)
- Laser-based, scanning cytometry method
CLINICAL USE OF PLATELETS
- treat bleeding (thrombocytopenia) Pan Genera Detection test → first rapid test to detect
- increase circulating platelet count bacteria in whole blood-derived platelets
- treat hereditary platelet conditions - lipoteichoic acids on gram-positive bacteria;
lipopolysaccharides on gram-negative bacteria
PLATELET CONCENTRATES - both aerobes and anaerobes are detected
1. Random-donor platelets - specificity: 99.8%; 10^3 to 10^5 CFU (colony
- prepared from whole blood forming units)/ml
- should contain at least 5.5x10^10 - can be performed just prior to release of platelet
- stored at 20-24C with continuous agitation products
- contain sufficient plasma → pH greater than or
equal to 6.2 500ml is required following pretreatment → sample is
- shelf-life: 5 days loaded to a disposable plastic cartridge with built in
controls → turn from yellow to blue-violet when test is
1 dose of platelet concentrate requires 4-6 whole blood ready to be read within 20 mins
bag donation → possibility of alloimmunization:
patient might induce formation of antibodies against (+): pink color bar in either the gram-positive or
mixed blood of the donors gram-negative window

2. Single-donor platelets AABB issued Interim Standard 5.1.5.1.1: prohibits use

- from apheresis of less-sensitive methods → microscopy; pH; glucose
- contain at least 3.0x10^11
- stored at 22-24C with agitation DISADVANTAGES OF CULTURE METHODS FOR
- at least 300ml of plasma DETECTING BACTERIAL CONTAMINATION OF
- shelf-life: 5 days PLATELETS
1. product loss due to sampling

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IMMUNOHEMATOLOGY
Module 1: Introduction to Immunohematology
- apheresis
2. delay in product release, further reducing - cryopreservative: DMSO (dimethylsulfoxide)
shelf-life - frozen at -80C
3. false-negative results - storage: up to 2 years
4. cost - platelets are thawed and centrifuges to remove DMSO
5. logistical problems of culturing WBD platelets - in vivo recovery: 33%

TRENDS IN PLATELET PRESERVATION RESEARCH indicated for those who are refractory to allogeneic
Development of: platelets (had formed antibodies against other people’s
1. methods to allow platelets to be stored for 7 components → destroy platelets → platelet count: not
days increased)
2. additive solutions (“synthetic media”
3. procedures to reduce and inactivate the level of
pathogens in platelet units
4. platelet substitutes
5. new approaches for storage of platelets at 1-6C
6. processes to cryopreserved platelets

ADVANTAGES OF USING PLATELET ADDITIVE

SOLUTIONS
1. optimizes platelet storage in vitro
2. saves plasma for other purposes
3. facilitates ABO-incompatible platelet
transfusions
4. reduces plasma-associated transfusion side
effects (febrile; allergic reactions); reduce risk of
TRALI (transfusion related acute lung injury)
5. improves effectiveness of photochemical
pathogen reduction technologies

DEVELOPMENT OF PLATELET SUBSTITUTES

1. washed platelets treated with paraformaldehyde
- with subsequent freezing in 5% albumin
and lyophilization
2. freeze-drying of trehalose-loaded platelets

Other methods:
- fibrinogen-coated albumin microcapsules and
microspheres
- fibrinogen binding: modified RBCs with procoagulant
properties
- platelet-derived microparticles
- liposome-based hemostatic products

FROZEN PLATELETS
- a research technique

- not licensed by the FDA

- used occasionally in the US (autologous transfusions)
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