PHYSIOLOGY

LABCON: BLOOD PHYSIOLOGY 2.01

PAMANTASAN NG LUNGSOD NG MAYNILA
SEPTEMBER 27, 2016 COLLEGE OF MEDICINE 2020

BLOOD TYPING H SUBSTANCE

 Aka Paragloboside
 Serological test used in detecting the presence of antigen
 Biochemical Composition:
or antibody in the red cell membrane in order to
a) Glycolipid backbone, Type 2 chain
establish the blood group of an individual 
b) Terminal galactose on the precursor substance
 Forward typing – detects the presence of an antigen in
is attached to the B –acetylglucosamine in a
the red cell membrane as shown by the agglutination of
beta 1 -> 4 linkage (Type 2 chain) instead of
the red cells upon addition with reagent anti-sera
the 1 -> 3linkage (Type 1 chain).
 Reverse typing – detects the presence of antibodies
against red cell antigens when added with Known A or B
cells.

PROCEDURE
Materials were prepared and the slides were wiped with
clean tissue paper. The finger was disinfected with alcohol and was
allowed to dry. The finger was pricked at about 2 mm depth
against the direction of the fingerprints. 3 drops were placed
separately on the slide and was dropped with the different anti-
sera each corresponding to the A, B and D antibodies.

H Antigen
 H gene or FUT 1 gene or Zz gene
 Inheritance of at least one H gene (HH of Hh) elicits the
production of α-2-L-fucosyltransferase, which transfers
L-fucose from Guanosine – Diphosphate L – fucose (GDP-
Fuc) donor nucleotide to the terminal galactose of the
precursor chain
 Occurs in 99.99% of the general population
 H substance must be formed for the other sugars to be
attached in response to an inherited A and/or B gene
 Bombay phenotype (hh genotype) individuals lack the
normal expression of the ABH antigens because α-2-L-
fucosyltransferase is not produced. The Bombay
phenotype is devoid of antigens of the ABO system.

ABO BLOOD GROUP

 Designated by the International Society of Blood
Transfusion (ISBT) as ISBT 001
 First human blood group system discovered
 Discovered by Karl Landsteiner in 1901 when he drew
blood from himself and five associates, separated the
cells and serum and then mixed each cell sample with
each serum
 RBC membranes have glycoproteins antigens on their
external surfaces; the presence or absence is the basis
of the classification of the blood group
 The ABO gene is located on the long arm of
chromosome 9
 A unique blood group system as it is the only blood
group that has naturally-occurring antibodies against
the antigens that are lacking in the red cell membrane.
 ABO gene codes for the production of specific
glycosyltransferase that add sugars to a basic precursor
substance on the RBC.

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 2.01 – LabCon: BLOOD PHYSIOLOGY
A Antigen
 A allele codes for the production of α-3-N-
acetylgalactosaminyltransferase which transfers an N-
acetyl-D-galactosamine (GalNac) sugar from uridine
diphosphate-N-acetyl-D-galactose (UDP-GalNac) donor
nucleotide to the H substance
ABO ANTIBODIES
B Antigen
 B allele codes for the production of α-3-D-
galactosyltransferase which transfers a D-galactose
(Gal) sugar from UDP-Gal donor nucleotide to the H
substance
Rh BLOOD GROUP
Type O
 No production of a catalytically active polypeptide so
the H substance remains unmodified
 Has the highest concentration of the H antigen
Type AB
 Both A enzyme (α-3-N-acetylgalactosaminyltransferase)
and B enzyme (galactosyltransferase) produces the
immuno-dominant sugar counterparts
 B enzyme competes more efficiently for the H substance
SUMMARY
Rh Antibodies
1D Physiology: LabCon: Blood Physiology [Francia, Galima, Habana, Ibrahim]
 Mostly IgM in nature but has some IgG or IgA
 Cold-reacting antibodies
 Do not cross the placenta
 Binds complement
 Present at low concentrations at birth (undetectable in
serum); increases titer from 3 – 6 months and reaches
peak levels at 5 – 10 years of age
 Naturally – occurring
 Highly reactive antibodies; if a patient is transfused
with the wrong red cells, the donor red cells would be
destroyed almost immediately at a rate of
approximately 1 mL of red cells per minute.
 Anti – A, Anti – B, Anti – A,B
 Presence of antibodies are detected using reverse/back
typing
 Rh was named from the Rhesus monkey to which the
antibodies were isolated from and investigated by
Landsteiner and Weiner in the 1930s
 Designated as ISBT 004
 Nonglycosylated proteins
 Different types of Antigen: D, C, c, E, and e
 In blood typing. Rh positive is the presence of D antigen
 Antibodies against Rh Antigens are not naturally-
occurring, they develop from prior exposure to the
antigen through blood transfusion or pregnancy.
 Antibodies are mostly IgG which are capable of crossing
the placental barrier
 Warm reacting antibodies (react best at 37˚c)
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 2.01 – LabCon: BLOOD PHYSIOLOGY
ERYTHROBLASTOSIS FETALIS
 Aka Hemolytic Disease of the Newborn
 Occurs when an Rh negative mother and an Rh positive
father produces an offspring that is Rh positive
 During the first pregnancy, the mother (without
antibody) is sensitized through the introduction of fetal
cells.
 In a second pregnancy, producing another Rh positive
child, the mother develops and has increased titer of
anti-D which crosses the placenta and therefore
destroying the cells of the fetus (can be fatal)
 Destruction of the cells causes the release of
haemoglobin which is then converted to bilirubin
causing the jaundiced skin of the baby – corrected by
phototherapy
 Effects to the baby: hepatomegaly, splenomegaly, and
precipitation of bilirubin in the brain (Kernicterus)
 In order to prevent grave damage to the fetus, Rhogam
is injected within 72 hours after birth or miscarriage.
Rhogam is a type artificial passive immunity containing
IgG anti-Rh antibodies. It acts as an antibody against the
fetal cells so that the mother does not develop the
antibodies against the fetal cells.
1D Physiology: LabCon: Blood Physiology [Francia, Galima, Habana, Ibrahim]
CROSSMATCHING
A. Procedure_______________________________________
A. Extract 3 ml of venous blood from subject 1
(patient/recipient) and place in a test tube.
B. Allow to clot for 10 mins then centrifuge for 5 mins to
separate serum. Place serum in a separate test tube.
C. Prick subject 2 (donor) with a lancet and place a drop of
blood in a test tube with 0.9% NaCl solution (removes
any anticoagulant or plasma protein that could interfere
with the test)
D. Centrifuge until a button of red cells collect at the bottom.
E. Discard the fluid (red cell preparation).
F. Add 2 drops of serum from subject 1 and add to red cell
prep of subject 2. Centrifuge then observe.
Crossmatching_____________________________________
● Part of pre-transfusion compatibility testing (ABO Typing,
Antibody Screen and Crossmatching)
● Major crossmatching vs. minor cross matching:
Major crossmatching (PSDR)
 2 drops of Patient Serum, 1 drop Donor’s RBCs
 To ensure that donor blood does not have
antigens that will react with
patient’s/recipient’s antibodies
 2 phases: Immediate Spin then Indirect
Antiglobulin Test
Minor cross matching (DSPR)
 2 drops of Donor Serum,1 drop Patient’s RBCs
 To ensure that donor’s serum will not have any
antibodies that will react with
patient’s/recipient’s antigens on their RBCs
 Usually performed only in sensitive cases, but if
the patient is in immediate need/the hospital
does not have enough resources, it can be
discarded.
 Less important because donor serum is
markedly diluted after a transfusion reaction –
more unlikely to produce adverse reactions.
B. Blood Compatibility
Cross matching detects ABO incompatibility
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C.Agglutination Reaction____________________________
● Agglutination occurs when the blood types are incompatible
– Antibodies in plasma attack the surface antigens of
the RBCs
● Antibodies for the ABO blood group are IgM – pentameric
structure
Note that IgM is large and does not cross the placenta so
there is no issue for ABO matching in pregnancy
● Presence of agglutination: jagged or firm cell button edge
incompatible
● Absence of agglutination: smooth swirling of free cells off
the RBC button compatible
● Can be graded according to strength of the agglutination
reaction:
Procedural Considerations__________________________
● Causes of positive (for agglutination) reactions:
o ABO incompatibility
o Rouleaux formation in patient’s serum
o Presence of alloantibodies or autoantibodies not
detected in antibody screen
o Test tube contamination
1D Physiology: LabCon: Blood Physiology [Francia, Galima, Habana, Ibrahim]
● For sample collection:
o Hemolysis of samples should be avoided
o Serum or plasma may be used but serum is preferred as
plasma may exhibit fibrin clots that may introduce error
(0.9% NaCl solution is added)
● LIMITATIONS:
o can crossmatch Rh+ donor with Rh- with no reaction if
patient has never been sensitized to produce anti-D
before.
o cannot detect all recipient antibodies to donor antigens
(Kidd and Rh antibodies may be too weak to detect but
still cause a transfusion reaction)
o will not prevent alloimmunization of recipient (only
ABO and Rh antigens matched – patient can
potentially make antibodies to other antigens).
BLEEDING TIME AND CLOTTING TIME
BLEEDING TIME
 A test to assess platelet function and the body’s ability
to form a clot. It measures the time when there would
be vasospasm and platelet plug formation.
 Used to measure the primary phase of hemostasis,
which involves platelet adherence to injured capillaries
and then platelet activation and aggregation
 The normal values for bleeding time: 1 – 6 minutes
Procedure
 Fingertip was sterilized with alcohol. Timer was set and
a deep prick was made with the use of a lancet
 A drop of blood was allowed to be formed without
pressing the finger
 The drop of blood was blotted on a piece of absorbent
paper
 Blotting was done every 30 seconds until no more blood
oozed from the pricked area. The time at which the
bleeding stopped was recorded
Hemostasis
 Control of bleeding; Normal mechanism of the body to
stop bleeding
4 Steps of Hemostasis
1. Vasoconstriction
2. Platelet Plug Formation
3. Coagulation
4. Fibrin Formation
Primary Hemostasis
 Formation of the platelet plug, it involves the first two
steps of hemostasis: Vasoconstriction (Vascular Phase)
and Platelet Plug Formation (Platelet Phase)
 The platelet phase involves: Platelet adhesion, platelet
activation & degranulation , and platelet aggregation
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 2.01 – LabCon: BLOOD PHYSIOLOGY
1. Vasoconstriction
 The first response to injury of the endothelial cells. It is
brought about by:
1. Local smooth muscle spasm
2. Local autacoid factors (Thromboxane A2
from traumatized tissues and blood
platelets)
3. Nervous reflexes (via SNS pathway)
 In the neurogenic vasoconstriction, the afferent neuron
sends impulses to the brain to produce agents that will
stimulate the SNS to cause constriction.
 In the myogenic vasoconstriction, constriction of the
vessel is caused by the response to injury of the smooth
muscle itself
2. Platelet Plug Formation
 Vasoconstriction is not enough to stop the bleeding so
platelet will come to the rescue
 Platelet Adhesion
After vasoconstriction, blood platelets immediately attach to
COLLAGEN and VON WILLEBRAND FACTOR (a plasma
glycoprotein that forms bridges between the receptors on the
surface of the platelets (GPIb) and collagen fibers in connective
tissue)
 Platelet Activation and Degranulation
Upon adhesion, the platelets now become activated.
They begin to swell, and extend cell projections in an octopus-like
manner.
Contractile proteins (actin, myosin & thrombosthenin) would
contract forcefully causing the release of granules (degranulation).
The platelets release active substances stored in their vesicles,
including ADP (which causes the surface of the platelets to become
sticky), TXA2 (same prostaglandin that would cause further
vasoconstriction) and 5HT (serotonin).
1D Physiology: LabCon: Blood Physiology [Francia, Galima, Habana, Ibrahim]
 Platelet Aggregation(Primary hemostatic plug)
New platelets adhere to those already attached to the collagen
fibers (via glycoprotein receptors GPIIb and GPIIIa on platelet
surface attached by fibrinogen)
The newly-attached platelets also release aggregator granules,
and so the process is continues. The platelets continue to
aggregate until a platelet plug sealing the blood vessel is formed.
Note:
It is important that formation of the platelet plug is
restricted to the injured area. This is prevented mainly because
ADP and other substances released from activated platelets
stimulate the uninjured endothelial cells to release nitric oxide
(NO) as well as a prostaglandin called prostacyclin. NO and
prostacyclin counteract the action of thromboxane A2.
Another mechanism to restrict the aggregation of
platelets to the injured site is by the repulsion of the platelets and
the intact endothelial cells, both containing negatively charged
surfaces.
Factors Affecting Bleeding Time
1. Depth of wound
2. Degree of Hyperemia (the increase of blood flow to different
tissues in the body)
3. Number of platelets
Abnormal results may indicate:
 Primary thrombocythemia: a condition in which your
bone marrow creates too many platelets
 Thrombocytopenia: a condition that causes your body
to produce too few platelets
 Von Willebrand’s disease: deficiency of von
Willebrand factor protein that is important in platelet
adhesion; the most common hereditary condition that
affects how blood coagulates (clots)
CLOTTING TIME
 Refers to the time required for a sample of blood to
coagulate in vitro under standard conditions
 Clotting is the transformation of blood into clot or
thrombus which consists of polymers of fibrin
 The normal values for clotting time varies widely,
depending on the method used for measuring it
 For the capillary and slide method: 3 – 6 minutes
Procedure (Capillary Method)
 Fingertip was sterilized with alcohol. Timer was set and
a deep prick was made with the use of a lancet
 The capillary tube was filled with blood. The pricked
fingertip was pressed with alcohol-soaked cotton balls.
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 2.01 – LabCon: BLOOD PHYSIOLOGY
 The capillary tube was broken off into short segments EXTRINSIC PATHWAY (FACTORS III AND VII)
every 15 seconds  In extrinsic pathway, there is damage to the blood
 The time of coagulation was noted. Coagulation has vessels (or to the endothelial cells). This pathway is
occurred when two pieces are joined together by fibrin quicker than the intrinsic pathway.
thread  These damaged tissues would release tissue factor which
would affect several things
Procedure (Slide Method)
 It will activate Factor VII which, in conjunction with the
 Fingertip was sterilized with alcohol. Timer was set and
tissue factor will activate Factor X in the presence of
a deep prick was made with the use of a lancet
calcium ions.
 The finger was squeezed to form a large drop that was
 Once Factor X is activated, in the presence of calcium,
then placed on a slide
along with the tissue factor, it will form the
 At intervals of 15 seconds, a pin was drawn from the
prothrombin activator which will convert prothrombin
center of a drop of blood to the periphery until any
to thrombin.
fibrin strand goes with the pin
 Thrombin will then activate Factor V which acts as an
 The time of coagulation was noted. Coagulation has
accelerator to the formation of the prothrombin
occurred when two pieces are joined together by fibrin
activator.
thread

Secondary Hemostasis
 Involves the activation of coagulation factors and
formation of fibrin clot
4 Steps of Hemostasis
1. Vasoconstriction
2. Platelet Plug Formation
3. Coagulation (Secondary hemostatic
plug)
4. Fibrin Formation

3. Coagulation
 Platelets alone are not enough to secure the damage in
the vessel wall. A clot must form at the site of injury. The
formation of a clot depends upon several proteins called
blood-clotting factors.
 Most of these proteins are inactive forms of proteolytic
enzymes. When converted to their active forms, their
enzymatic actions cause the successive reactions known
as the clotting cascade.
 These factors are designated by roman numerals I
through XIII (a small letter “a” is added after the Roman
numeral to indicate the activated sate).
 Initiated either by damage to blood vessels (extrinsic INTRINSIC PATHWAY (FACTORS XII, XI, IX, VIII)
 The intrinsic pathway is activated by trauma inside the
vascular system, and exposed endothelium or collagen.
This pathway is slower than the extrinsic pathway
 When blood comes in contact with a negatively charged
surface such as glass, Factor XII is activated
 Factor XII along with HMW kininogen and prekalikrein
(accelerator) will activate Factor XI;
 In the presence of calcium ions, Factor XI will activate
Factor IX
 Factor IX along with the help of platelet phospholipids
and amplified by the activation of Factor VIII by
thrombin, will then activate Factor X.
 Similar with the extrinsic pathway, when Factor X is
activated(along with the presence of calcium, platelet
phospholipids and Factor V) it will form the
prothrombin activator and lead to the common
pathway) or by trauma to the blood itself or exposure to pathway
collagen (intrinsic pathway)

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 2.01 – LabCon: BLOOD PHYSIOLOGY
4. Fibrin Formation
COMMON PATHWAY (FACTORS X, V, II, I, XIII)
 Extrinsic and intrinsic pathways meet and finish the clot
production in what is known as the common pathway.
 Both pathways leads to the formation of prothrombin
activator, which then causes prothrombin conversion to
thrombin
 In the presence of sufficient amounts of Calcium.
Thrombin causes polymerization of fibrinogen and
removes 4 low mol weight polypeptides from each of its
molecule, forming a fibrin monomer
 These fibrin monomer polymerizes with other
monomers to form fibrin fibers which form the
reticulum of blood clot(held together by weak
noncovalent hydrogen bonding)
 Thrombin also activates fibrin stabilizing factor
(Factor XIII). Activated factor XIII then causes covalent
bonds between fibrin monomer molecules which greatly
strengthens the fibrin reticulum and adds tremendously
to the 3-D strength of fibrin meshwork
1D Physiology: LabCon: Blood Physiology [Francia, Galima, Habana, Ibrahim]
Other Measurements of Clotting Factors
Prothrombin Time (PT)
 Measure of the extrinsic and common pathways of
coagulation
 More specific and sensitive
 Measures factors I (fibrinogen), II (prothrombin), V
(proaccelerin), VII (proconvertin), X (Stuart-Prower)
 Normal prothrombin time is 12 to 15 seconds
Activated Partial Prothrombin Time (aPTT)
 Measure of the intrinsic and common pathways of
coagulation
 Measures factors I (fibrinogen), II (prothrombin), V
(proaccelerin), VIII (Antihemophilic Factor A), IX
(Antihemophilic Factor B), X (Stuart-Prower), XI
(Plasma Thromboplastin Antecedent), XII (Hageman)
 Normal activated partial prothrombin time is 30 to 35
seconds
Anticoagulants
 Anticoagulants have been developed for the purpose of
delaying the coagulation process as a treatment for
some cases such as thrombolic conditions (where
abnormal clot develops in blood vessels and may cause
blockage and serious damage)
Heparin
 Location: pericapillary connective tissue
 Action: inhibition of coagulation through the inhibition
of thrombin
 Clinical administration through IV
 Monitoring: Activated Partial Thromboplastin Time
(aPTT)
Coumarin/Warfarin
 Action: Vitamin K antagonist; inhibits Factors X, IX, VII,
II
 Monitoring: Prothrombin Time (PT)
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Factors Affecting Clotting Time
1.) Degree of damage to the vascular wall
2.) Anticoagulants
3.) Diseases that involve Coagulation Factors
QUESTIONS AND ANSWERS FROM PRECEPTORS
From Dr. Solidum
1. Can you proceed with blood transfusion without
cross-matching your blood specimens?
Yes, but ONLY is dire emergency situations. Give the
patient a donor blood with the same ABO blood type.
Such that, if patient is Type A, give Type A blood.
2. A patient is having allergic reactions during blood
transfusion, assuming you are the resident-on-duty,
what will you do?
Terminate the transfusion immediately. Then, repeat the
compatibility testing from blood typing to cross-
matching.
From Dr. Banal
3. After re-testing, the donor blood and patient blood
are found compatible. However, the patient is still
having allergic reactions, what will you do?
Perform a scratch test to detect other causes of the
patient’s allergy. Most probably, the patient is not
allergic to the donor blood but to some other factors. We
can continue to the blood transfusion since we have
proven that the patient is not reacting against the donor
blood.
4. Can you proceed with an operation without knowing
the bleeding time and coagulation time of a patient?
Yes, in emergency situations only. However, while the
operation is on-going you have to be able to get the
results for these tests.
5. What other tests should you consider for patients
who will undergo surgery? From Dr. Razon
Prothrombin Time (PT). PT is a test that examines the
extrinsic pathway and common pathway of coagulation
which involve Factor 3,7, 10, 5, 2, 1.
PT is more reliable than clotting and bleeding time
because we have ruled out the external factors such as
depth of pricking, period of checking the formation of
fibrin strands, etc. Instead, the results are generated
from the specificity of the machines and specimen used
which is platelet-poor plasma.
PT International Normalized Ratio: 1
From Dr. Munarriz
1. Is Type O completely a universal donor blood?
No, because Type O blood has both Anti-A and Anti-B
which if transfused to the patient who have the occurring
complement antigens, transfusion reactions will occur
which can be fatal.
2. What is the importance of bleeding time?
Bleeding time determines platelet count, platelet
function, and vascular integrity.
1D Physiology: LabCon: Blood Physiology [Francia, Galima, Habana, Ibrahim]
3. What are the other names of all the coagulation
factors?
1 Fibrinogen
2 Thrombin
3 Tissue Factor/Tissue Thromboplastin
4 Calcium
5 Proaccelerin
6 Non-existent
7 Proconvertin
8 Anti-hemophilic Factor A
9 Anti-hemophilic Factor B
10 Stuart-Prower
11 Plasma Thromboplastin Antecedent
12 Hageman Factor, Glass Factor
13 Fibrin Stabilizing Factor
4. Memorize the coagulation cascade.
Extrinsic & Common: 3→7→10→5→2→1
Intrinsic & Common: 12→11→9→8→10→5→2→1
1. Why does our immune system do not make
antibodies against the antigen that occurs in us?
Because of clonal deletion, wherein T and B cells are
deactivated to produce antibodies after having
expressed receptor for the self-antigens.
2. Differentiate hemostasis and thrombosis.
In hemostasis, vasoconstriction is the first step. In
thrombosis, platelet plug formation is the first step.
3. Can the intrinsic pathway be activated even if you do
not have injury?
Yes, such as in case of high blood pressure, where the
flow of blood causes stress to the walls of blood vessels
which will eventually lead to endothelial damage,
thereby exposing the subendothelial collagen.
1. Are the intrinsic and extrinsic pathways activated
simultaneously?
Yes. The extrinsic pathway, with its tissue factor, is the
usual way of initiating clotting in the body. Extrinsic
pathway includes Factors 3 and 7. Factor 7 leads to the
beginning of full intrinsic pathway, which normally plays
a little role.
The thrombin produced by the extrinsic pathway is only
large enough to trigger positive feedback effects on the
intrinsic pathway. After being activated by thrombin,
intrinsic pathway then will produce large quantities of
thrombin.
REFERENCES:
1. Lecture Notes, Group PPT Presentation
2. 1D 2019 Trans
3. Harmening’s Modern Blood Banking Transfusion
Practices, 6thed
4. LabCon notes of each group
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