1 - BASIC CONCEPTS OF IMMUNOHEMATOLOGY

BLOOD

Components:

• Fluid that provides the major transport system in the body

• Composed of solid (cellular components) and liquid (plasma or serum component

Plasma

liquid portion of blood sample collected with an anticoagulant while serum is the liquid portion of a blood sample that has clotted

Plasma

makes up approximately 55% of the blood volume and is composed of greater than 90% water

Serum

liquid component most often used in blood bank testing

Erythrocytes, leukocytes and thromobocytes

Cellular components suspended in the plasma

Main function of BLOOD

Transport of :

• Oxygen to the tissues and carbon dioxide to the lungs for expiration (this is the function of the erythrocytes)

• Nutrients, hormones, and chemical substances to the tissues

• Waste products to site of removal

Coagulation

Final important function of BLOOD

Coagulation

protects the body by preventing bleeding

White cells

involved in phagocytosis and immunity

pulmonary and systemic

heart

Blood circulation:

Blood flows through the body in 2 distinct circulations: (2), the ______ is the pump for each circulation

Pulmonary circulation

Oxygen and carbon dioxide exchange between the blood and the inspired air

Systemic circulation

Oxygenated blood is pumped by the heart throughout the body providing the oxygen required for the various metabolic processes tissues

Hemoglobin

Oxygen enters the red cells in the lungs and binds to an intracellular protein called

porphyrin ring

heme

alpha; beta

Hemoglobin consists of a ________ with a central iron atom (_____) and a globular protein made up of 2 ___ and 2 ___ globin chain (four-sub units) each of which carries one heme group

hemoglobin

Each red cell contains many __________ molecules

Oxygen-dissociated curve (normal sigmoid curve)

The relationship between oxygen concentration in the blood (pO2) and the percentage of oxygen bound to hemoglobin (sO2) form a sigmoid curve

increases

higher

LEFT

Oxygen-dissociated curve (normal sigmoid curve):

A raise in pH _________ the hemoglobin/oxygen affinity making it more difficult for hemoglobin to release oxygen; consequently, hemoglobin holds on to the oxygen molecule maintaining a ______ saturation; this results in a shift of the curve to the ______

decreases

decrease

left

normal

24

The level of 2,3 DPG progressively ________ as blood donor blood is stored; theoretically, this ________ can result in reduced oxygen delivery to the tissues and a shift of curve to the _____; most transfused patients are unaffected by this change as the 2,3 DPG in donor red cells returns to ______ within ____ hours of infusion

LEFT-SHIFTED CURVE

Implications:

• Increased oxygen affinity (R state)

• Reduced oxygen delivery to tissues

2,3 diphosphoglycerate (2,3 DPG)

substance produced by RBCs that diminish the hemoglobin/oxygen affinity and facilitates the release of oxygen from the red cells; therefore the oxygen dissociation is shifted to the right

LEFT-SHIFTED CURVE

Caused by:

• High pH (more basic)

• Low temperature

• Low 2,3-DPG

• Fetal Hb (HbF)

• Methemoglobenemia

• High O2 affinity Hb variants

RIGHT

Oxygen-dissociated curve (normal sigmoid curve):

Red cells produce 2,3 diphosphoglycerate (2,3 DPG); this substance diminishes the hemoglobin/oxygen affinity and facilitates the release of oxygen from the red cells; therefore the oxygen dissociation is shifted to the ______

RIGHT-SHIFTED CURVE

Implications:

• Reduced oxygen affinity (T state)

• Increased oxygen delivery to tissues

RIGHT-SHIFTED CURVE

Caused by:

• Low pH (more acidic)

• Increased CO2

• High temperature

• High 2,3-DPG

• Low O2 affinity Hb variants

RBC membrane

a semi-permeable lipid bi-layer (mainly phospholipids) supported by a protein meshlike cytoskeleton structure

1. integral proteins (transmembrane proteins)

2. peripheral proteins

2 kinds of proteins of RBC membrane

Integral proteins

extend from the outer surface and span the entire membrane to the inner cytoplasmic side of the RBC: glycophorin A, glycophorin B, glycophorin C, anion-exchange-channel protein

Peripheral proteins

are located and limited to the cytoplasmic surface of the membrane forming the red cell cytoskeleton: spectrin, actin (band 5), Ankyrin (band 2.1), band 4.1 and 4.2, band 6, adducin

deoformability and permeability

2 important characteristics of RBC

• Decrease in the phosphorylation of spectrin (due to decrease ATP levels)

• Accumulation/increase deposition of membrane calcium

The loss of RBC flexibility and deformability is due to? (2)

• vascular sequestration; lysis of RBC

• Spherocytosis

• bite cells

• shortened

The loss of deformability leads to:

• Easy extra _______________ and ___________ at the sinusoidal spaces of the spleen

• ____________ (spherocytes are formed due to the loss of RBC membrane and consequently a reduced surface-to-volume ratio)

• Formation of __________ (due to the removal of a portion of RBC membrane leaving a permanent indentation in the remaining cell mebrane)

• Survival of these forms is ________.

Spherocytosis

loss of RBC membrane and consequently a reduced surface-to-volume ratio

Formation of bite cells

due to the removal of a portion of RBC membrane leaving a permanent indentation in the remaining cell mebrane

RBC membrane

freely permeable to water and anions: chloride and bicarbonate can traverse the membrane in less than a second

RBC membrane

relatively impermeable to cations such as sodium anc potassium

RBC volume and water homeostasis

are maintained by controlling the intracellular components of sodium and potassium

1:12

Erythrocyte intra cellular-to-extra cellular ratios for sodium

Erythrocyte intra cellular-to-extra cellular ratios for Potassium

25:1

calcium; sodium

potassium; water

decrease

When RBC are depleted, ______ and ______ are allowed to accumulate intracellularly and _________ and ______ are lost, they become dehydrated and subsequently sequestered by the spleen, resulting in a _________ in RBC survival

anaerobic

deliver

The RBC metabolic pathways that produce ATP are mainly _________ because the function of RBC is to ________ oxygen and not to consume it

glycolysis (glucose breakdown)

Since mature RBC is non-nucleated and no mitochondrial apparatus for oxidative metabolism, energy is generated almost exclusively by?

AP-ML

1. Anaerobic Glycolytic Pathway

2. Pentose Phosphate Pathway

3. Methemoglobin Reductase Pathway

4. Luebering Rapaport Shunt

Metabolic pathways are divided into (4)

Anaerobic Glycolytic Pathway

Generates about 9o% of the ATP needed by the RBC

Pentose Phosphate Pathway

Increased following:

○ Increased oxidation of glutathione

○ Decreased activity of the anaerobic glycolytic pathway

Pentose Phosphate Pathway

Produces approximately 10% of the ATP needed by the RBC

glutathione

Heinz bodies

Heinz bodies

Pentose Phosphate Pathway:

• When the pathway is deficient, the amount of reduced _________ becomes insufficient to neutralize intracellular oxidants

• The result is denaturation and precipitation of globin as aggregates (____________) within the cell; __________ makes RBC less deformable than normal RBC

Methemoglobin Reductase Pathway

This pathway is necessary to maintain the heme iron to hemoglobin in the ferrous functional state

methemoglobin reductase

NAD

methemoglobin

Methemoglobin Reductase Pathway:

In the absence of the enzyme __________________________ and the action of ______, there is accumulation of ________________, which results from a conversion of ferrous to the ferric form

Methemoglobin

is a non-functional form of hemoglobin and a loss of oxygen transport capabilities

Luebering Rapaport Shunt

This permits the accumulation of 2,3 diphosphoglycerate (2,3 DPG)

Luebering Rapaport Shunt

The large amount of 2,3 DPG found within RBC has a significant effect on the affinity of hemoglobin for oxygen

75%

24

RBC preservation:

Successful transfusion

____ of cells that have been transfused should remain viable for ___ hours for transfusion to be considered successful

• Decrease in PAG: pH, ATP, glucose consumption

• Loss of red cell function

• Building of lactic acid

Storage of blood may lead to various biochemical changes (3)

decrease

LEFT

less

As blood is stored, 2,3 DPG levels ________

There is a shift to the ______ of the hemoglobin dissociation curve, and ______ oxygen is delivered to the tissues

Acid-citrate dextrose (ACD), CPD, CP2D

(3) are approved preservative solutions for whole blood storage at 1-6°C for 21 days

ACD

Since _____ has a lower pH, 2,3 DPG is lost early during storage

Adenine

It is incorporated to CPD (forming CPDA-1) in order to increase ADP levels, thereby driving glycolysis toward the synthesis of ATP

21 days

Storage time of ACD and CPD

CPD

It is more superior than ACD in preserving 2,3 DPG

35 days

Storage time of CPDA-1

42 days

Storage time of CPDA-2 and SAG-M

Additive solutions (added to PRBCs)

• A new blood collection system employs a primary bag containing standard anticoagulant and an accessory bag (or satellite bag) containing additive solution •

• After the plasma is removed from a unit of whole blood. the additive solution is added to red cells to provide nutrients for improved viability

Saline (S), Dextrose or Glucose (G), Adenine (A)

Composition of additive solutions for PRBCs

Hogman

Hogman (Sweden) and Lovric (Australian) additive solutions differ only in that _______ uses standard CPD anticoagulant in the primary bag with an additive solution containing the SAG

Hogman

Ths additive solution was modified with the addition of mannitol to maintain the integrity of RBC membrane

Lovric

doubled the dextrose concentration with the additive solution containing saline, adenine, glucose, tri-sodium citrate, citric acid, and sodium phosphate

Additive solutions licensed in the US

• Adsol (AS-1) Fenwal Laboratories

• Nutrical (AS-3) Medsep Corporation

• Optisol (AS-5) Terumo Corporation

ADSOL

It contains buffered adenine, glucose, mannitol (to retard hemolysis)

42 days

Storage time of ADSOL

Retard hemolysis

Purpose of mannitol in ADSOL

• 2.00

• 2.22

• 2.22

Formulation of additive solutions:

Adenine (mM)

• AS-1

• AS-2

• AS-5

• 11.oo

• 55.51

• 45.41

Formulation of additive solutions:

Glucose (mM)

• AS-1

• AS-2

• AS-5

• 41.20

• -

• 28.82

Formulation of additive solutions:

Mannitol (mM)

• AS-1

• AS-2

• AS-5

• 154.00

• 70.1

• 150.04

Formulation of additive solutions:

NaCl (mM)

• AS-1

• AS-2

• AS-5

• - -

• 23.00

• - -

Formulation of additive solutions:

Na2HPO4 (mM)

• AS-1

• AS-2

• AS-5

• CPD

• CD2D

• CPD

Formulation of additive solutions:

Primary bag anticoagulant

• AS-1

• AS-2

• AS-5

3

1 to 4

37 deg C

Rejuvenation solutions:

Generally, red cells stored in the liquid state for less than __ days after expiration date can be rejuvenated by incubation for __ to __ hours at ____ with rejuvenation solution

Rejuvesol (Cytosol Laboratories)

the only FDA-approved rejuvenation solution; consists of phosphate, inosine, pyruvate and adenine (PIPA)

Red Cell Freezing

primarily done for autologous units and storage of rare blood types; individuals may donate blood for their own future use (autologous transfusion)

Autologous transfusion

individuals may donate blood for their own future use

Red cell freezing

involves the addition of a cryoprotective agent to red cells that less than 6 days old

Glycerol

• The most commonly used for red cell freezing

• It is added to red cells slowly with mixing to enable glycerol to permeate the red cells; the red cells are rapidly frozen and stored in a freezer with:

  • High concentration glycerol 40% w/v (this is most commonly done)

  • Low concentration glycerol 20% w/v

1. Hemoglobin-based oxygen carrier

2. Perfluorochemicals (PFC)

2 categories of blood substitutes

Hemoglobin-based oxygen carriers

include stroma free hemoglobin solution (SFHS), chemically modified hemoglobin-solution-recombinant hemoglobin and encapsulated hemoglobin

Perfluorochemicals

chemically inert but excellent gas solvents; they carry O, and CO, by dissolving much as 40% to 70% oxygen per unit volume

Intravenous fluids (IV-fluids)

• Provide the normal maintenance fluid requirements of a patient in whom the oral route is unavailable

• Provide replacement fluids for abnormal losses incurred as a result of surgery, trauma or other pathology

• Correct electrolyte disturbance or hypoglycemia

• Act as vehicle for the administration of certain drugs

• Maybe maintenance fluids and replacement fluids

Maintenance fluids

Used to replace the normal physiological losses that occur in a patient through skin, lung, feces and urine

Maintenance fluids

Are mainly composed of water in the form of dextrose solution (electrolytes maybe added) since a considerable proportion of these losses is water

crystalloid

All maintenance fluids are _________ solutions, eg:

• 5% dextrose solution

• 4% dextrose in sodium chloride (0.18%)

1. Pyrexia

2. High ambient temperature or humidity when losses will increase

Volume of maintenance fluid required by the patient depends on (2)

Replacement fluids (or plasma substitutes)

used to replace abnormal losses of blood, plasma or other extra cellular fluids by increasing the volume of the vascular component

Replacement fluids (or plasma substitutes)

Used principally in the:

• Treatment of patients with established hypovolemia (e.g. hemorrhagic shock)

• Maintenance of normovolemia in patients with ongoing fluid losses (surgical blood loss)

Balanced salt solutions examples

Replacement fluids (or plasma substitutes):

• NSS (0.9% NaCl)

• Ringer's lactate

• Hartmann's solution

• All colloid solutions

Phenotype frequencies

are expressed as percentage or decimal

Balanced Salt Solutions

solution of NaCl with electrolyte composition resembling that of extracellular fluid

Phenotype frequencies

are determined testing red cells from large random population of the same race. The percentage of positive or negative reaction is determined with a given known antiserum

100% or 1.00

All possible phenotype frequencies for a given system totals to?

0.04

4

The frequency of Jk(at) persons is 77%. Consequently, Jk(a-) persons is 23%. If the patient has other antibodies such as anti-c, anti-K, and anti-Jka then the calculation is as follows:

• C = 20%

• k= 91%

• Jk(a-)=23%

Then 0.2 x 0.91 x 0.23 = _____

This means that __ compatible units is obtained from screening 100 units of blood

Lectins

are specific antibodies derived from plants; prolectins are derived from snails

Anti-A

Anti-B

Anti-H

Anti-A

LECTINS (4)