Notes On Immunohematology
Notes On Immunohematology
Notes On Immunohematology
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
T.O.R.
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
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
T.O.R.
Lecture
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
ACD-A: apheresis
T.O.R.
Lecture
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
2. PIPA
T.O.R.
Lecture
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
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
T.O.R.
Lecture
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
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
T.O.R.
Lecture
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
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