Mulungushi University
School of Medicine and Health Sciences
MBChB program
Human Physiology MPG 222
2nd Year 2st semester
Blood cells and coagulation Tutorial
Prepared by Dr. Masenga SK., BSc., MSc., PhD, FFGH, Pg. Dip, CT
This tutorial is based on ABO/RH typing, FBC and coagulation physiology.
1. Arrange the players of hemostasis cascade in order following vascular injury
a) Stable fibrin/platelet clot
b) Coagulation cascade activated
c) Release of tissue factor/thromboplastin
d) Exposure of subendothelial collagen
e) Fibrinolysis
f) Vasoconstriction
g) Platelet adhesion, activation and aggregation
h) Vascular injury
i) Primary hemostatic plug
Solution:
Vascular injury-> Exposure of subendothelial collagen ->vasoconstriction-> g)
Platelet adhesion, activation and aggregation-> Primary hemostatic plug ->
Release of tissue factor/thromboplastin -> Coagulation cascade activated -> Stable
fibrin/platelet clot -> Fibrinolysis
2. Explain the mechanistic rationale of platelet adherence, activation and
aggregation that results in formation of a primary and secondary hemostatic
plug.
Solution:
In blood flow, RBCs predominate in the axial stream while the bi-converse platelets are
marginated along the wall to monitor endothelium integrity. Upon vessel injury the
platelets begin to adhere to ligands on the exposed subendothelial matrix via
receptors
ADHERENCE
Platelet receptors (glycoproteins) involved in collagen adhesion
• GPIa/IIa (α2ß1) - This is a receptor for collagen type I and IV
• GPVI – for platelet adhesion and activation. It plays a key role in their
procoagulant activity and subsequent thrombin and fibrin formation
• GPIb- IX-V complex – leading role for high stress injury elimination
― composed of four subunits: GPIbα, GPIbβ, GPV and GPIX
― GPIbα subunit bears the binding site for von Willebrand factor (vWF),
α-thrombin, leukocyte integrin αMβ2 and P-selectin.
― The binding of von Willebrand factor (vWF) results in conformational
changes within the GPIb-V-IX complex. In consequence, this complex
Page 1 of 16
Mulungushi University
School of Medicine and Health Sciences
MBChB program
Human Physiology MPG 222
2nd Year 2st semester
Blood cells and coagulation Tutorial
Prepared by Dr. Masenga SK., BSc., MSc., PhD, FFGH, Pg. Dip, CT
activates GPIIb / IIIa membrane glycoproteins allowing them to bind
fibrinogen. Fibrinogen molecules then interconnect the platelets serving
as the basis for platelet aggregation. In the absence of fibrinogen, the
platelets are joined by vWF due to its ability to bind the activated GPIIb /
IIIa complex.
― Adhesion to collagen and fibrinogen
Collagen ligands include vWF. Weibel-Palade bodies of the endothelium also
synthesize vWF. Platelets also synthesize vWF in α-granules for attachment to other
platelets
ACTIVATION
Attachment of platelets to the subendothelial matrix causes transduction pathways
that activates platelets and undergo conformational change and causes them to begin
secreting chemicals via exocytosis—degranulation that assist in aggregation and
recruitment of other platelets. Platelet activation consists of platelets undergoing two
specific events once they have adhered to the exposed vWF (i.e. the damaged vessel
site). First, platelets will undergo an irreversible change in shape from smooth discs to
multi-pseudopodal plugs, which greatly increases their surface area. Second, platelets
secrete their cytoplasmic granules.
• Platelet activation is mediated via thrombin
• Thrombin directly activates platelets via proteolytic cleavage by binding the
protease-activated receptor.
• Thrombin also stimulates platelet granule release which includes serotonin,
platelet activating factor, and Adenosine Diphosphate (ADP).
• ADP is an important physiological agonist which is stored specifically in the
dense granules of platelets. When ADP is released, it binds to P2Y1 and
P2Y12 receptors on platelet membranes.
Page 2 of 16
Mulungushi University
School of Medicine and Health Sciences
MBChB program
Human Physiology MPG 222
2nd Year 2st semester
Blood cells and coagulation Tutorial
Prepared by Dr. Masenga SK., BSc., MSc., PhD, FFGH, Pg. Dip, CT
•
•
•
P2Y1 induces the pseudopod shape change and aids in platelet
aggregation.
P2Y12 plays a major role in inducing the clotting cascade.
When ADP binds to its receptors, it induces Gp IIb/IIIa complex expression
at the platelet membrane surface. The Gp IIb/IIIa complex is a calciumdependent collagen receptor which is necessary for platelet-to-endothelial
adherence and platelet-to-platelet aggregation
Platelets Granules:
• Dense granules – ADP (activation, shape change, thromboxane A2 production
via Arachidonic acid pathway), Serotonin, Ca2+
• α-granules – platelet activating factor (PAF), platelet factor 4 (heparin binding
chemokine), vWF, P-selectin and CD63 (expressed on granules), thrombin (AKA
IIa) – all for aggregation
• thromboxane A2 – vasoconstrictor, activation of other platelets and aggregation
Closing statement: Adhesion, activation and aggregation leads to primary plug.
Then fibrin attachment via fibrinogen receptors ensures further attachment and
permanence leading to secondary hemostatic plug.
Fibrinogen ligand or sites recognized by GPIIb / IIIa complex include:
―
dodecapeptide located in the C-terminal of the fibrinogen γ chain (the most
important)
―
RGD sequence of the α chain → the Arginine-Glycine-Aspartate amino acid
sequence
3. Liver failure, (especially affecting synthetic function) affects which players in
the hemostatic cascade?
Solution: List the players; Platelets, vessel (endothelia, ECM), coagulation cascade
and t-PA
• Only most coagulation factors are produced by the Liver so they will be affected
resulting in bleeding to death.
• Factors produced by the liver: I, II, V, VII, IX, X,XI, XII, XIII
• Other factors not produced by Liver
― III (thromboplastin) – from platelets and subendothelial matrix
― VIII (antihemophilic) – endothelial cells
Page 3 of 16
Mulungushi University
School of Medicine and Health Sciences
MBChB program
Human Physiology MPG 222
2nd Year 2st semester
Blood cells and coagulation Tutorial
Prepared by Dr. Masenga SK., BSc., MSc., PhD, FFGH, Pg. Dip, CT
4. Give the rationale and Name the tests used to assess bleeding/clotting function
I.
activated partial thromboplastin time (aPTT) test AKA Partial thromboplastin time
(PTT) measures the overall speed at which blood clots by means of two
consecutive series of biochemical reactions known as the intrinsic pathway and
common pathway of coagulation.
― PTT measures the following coagulation factors: I (fibrinogen), II
(prothrombin), V (proaccelerin), VIII (anti-hemophilic factor), X (Stuart–
Prower factor), XI (plasma thromboplastin antecedent), and XII (Hageman
factor).
― The test is termed "partial" due to the absence of tissue factor from the
reaction mixture. Thromboplastin is Tissue factor+ phospholipids
― typical reference range is between 30 seconds and 50 s (depending on
laboratory).
II. The prothrombin time is the time it takes plasma to clot after addition of tissue
factor. It evaluates the extrinsic pathway and common pathway. PT measures
the following coagulation factors: I (fibrinogen), II (prothrombin), V (proaccelerin),
VII (proconvertin), and X (Stuart–Prower factor).
III. INR (International normalized ratio)
― The INR is the ratio of a patient's prothrombin time to a normal (control)
sample, raised to the power of the ISI (International Sensitivity Index)
value for the analytical system being used.
―
― This measures the quality of the extrinsic pathway (as well as the
common pathway) of coagulation. The speed of the extrinsic pathway is
greatly affected by levels of functional factor VII in the body. Factor VII has
a short half-life and the carboxylation of its glutamate residues requires
vitamin K. The prothrombin time can be prolonged as a result of
deficiencies in vitamin K, warfarin therapy, malabsorption, or lack of
intestinal colonization by bacteria (such as in newborns). In addition, poor
factor VII synthesis (due to liver disease) or increased consumption (in
disseminated intravascular coagulation) may prolong the PT.
― The INR is typically used to monitor patients on warfarin or related oral
anticoagulant therapy. The normal range for a healthy person not using
warfarin is 0.8–1.2, and for people on warfarin therapy an INR of 2.0–3.0
is usually targeted, although the target INR may be higher in particular
situations, such as for those with a mechanical heart valve. If the INR is
Page 4 of 16
Mulungushi University
School of Medicine and Health Sciences
MBChB program
Human Physiology MPG 222
2nd Year 2st semester
Blood cells and coagulation Tutorial
Prepared by Dr. Masenga SK., BSc., MSc., PhD, FFGH, Pg. Dip, CT
IV.
outside the target range, a high INR indicates a higher risk of bleeding,
while a low INR suggests a higher risk of developing a clot.
― The prothrombin ratio (aka international normalized ratio) is the
prothrombin time for a patient sample divided by the result for control
plasma.
Thrombin clotting time.
― Thrombin is added directly to patients plasma to directly clot fibrinogen
― Its elevated in Heparin use, DIC, Dysfibrinogenemia, low & high fibrinogen
levels, uremia et cetera
ADDITIONAL MATERIAL
PT and PTT method:
Prothrombin time is typically analyzed by a laboratory technologist on an automated
instrument at 37 °C (as a nominal approximation of normal human body temperature).
1. Blood is drawn into a test tube (blue top) containing liquid sodium citrate, which
acts as an anticoagulant by binding the calcium in a sample.
2. The blood is mixed, then centrifuged to separate blood cells from plasma (as
prothrombin time is most commonly measured using blood plasma). In
newborns, a capillary whole blood specimen is used
3. A sample of the plasma is extracted from the test tube and placed into a
measuring test tube (Note: for an accurate measurement, the ratio of blood to
citrate needs to be fixed and should be labeled on the side of the measuring test
tube by the manufacturing company; many laboratories will not perform the
assay if the tube is underfilled and contains a relatively high concentration of
citrate—the standardized dilution of 1 part anticoagulant to 9 parts whole blood is
no longer valid).
4. Next an excess of calcium (in a phospholipid suspension) is added to the test
tube, thereby reversing the effects of citrate and enabling the blood to clot again.
5. PT Method only: Finally, in order to activate the extrinsic / tissue factor clotting
cascade pathway, tissue factor (also known as factor III) is added and the time
the sample takes to clot is measured optically.
6. PTT method only: Finally, in order to activate the intrinsic pathway of
coagulation, an activator (such as silica, celite, kaolin, ellagic acid) is added, and
the time the sample takes to clot is measured optically.
Note: Some laboratories use a mechanical measurement, which eliminates
interferences from lipemic and icteric samples.
PTT Method
Page 5 of 16
Mulungushi University
School of Medicine and Health Sciences
MBChB program
Human Physiology MPG 222
2nd Year 2st semester
Blood cells and coagulation Tutorial
Prepared by Dr. Masenga SK., BSc., MSc., PhD, FFGH, Pg. Dip, CT
•
Blood is drawn into a test tube containing oxalate or citrate, molecules which act
as an anticoagulant by binding the calcium in a sample. The blood is mixed, then
centrifuged to separate blood cells from plasma (as partial thromboplastin time is
most commonly measured using blood plasma).
A sample of the plasma is extracted from the test tube and placed into a measuring test
tube.
Next, an excess of calcium (in a phospholipid suspension) is mixed into the plasma
sample (to reverse the anticoagulant effect of the oxalate enabling the blood to clot
again).
Some laboratories use a mechanical measurement, which eliminates interferences from
lipemic and icteric samples.
5. Does temperature affect ABO typing result?
Solution:
― ABO antibodies are of the IgM class and react preferentially at 22 oC (RT) or
below. Incubation at warm temperatures may cause a false negative reaction.
Enhancement of weak reactions may be obtained by RT incubation or incubation
at 4 oC.
6. Explain the procedure and purpose and list the three (3) types of tests which
must be performed in order to determine an individual’s ABO/D type:
― ABO forward typing - used to detect the presence or absence of A and/or B
antigens on an individual's red blood cells with reagent anti-A and anti-B sera.
Agglutination of the individual's red cells by the appropriate antisera signifies the
presence of the antigen on the red cell while no agglutination with the antisera
signifies its absence.
― ABO reverse typing -used to detect ABO antibodies in an individual's serum, and
is used to confirm the ABO Forward Typing. The patient's serum is mixed with
reagent group A1 cells. Agglutination indicates the presence of Anti-A in the
patient's serum. Mixing the patient's serum with reagent group B cells similarly
allows for the detection of anti-B in the patient's serum.
~ Group A individuals lack the B antigen and their serum will agglutinate the
reagent B cells due to their naturally occurring anti-B. Their serum will not
agglutinate the reagent A cells since this antigen is present on their own
cells.
~ Group B individuals lack the A antigen and their serum will agglutinate A
cells with their naturally occurring anti-A. Their serum will not agglutinate
the reagent B cells.
Page 6 of 16
Mulungushi University
School of Medicine and Health Sciences
MBChB program
Human Physiology MPG 222
2nd Year 2st semester
Blood cells and coagulation Tutorial
Prepared by Dr. Masenga SK., BSc., MSc., PhD, FFGH, Pg. Dip, CT
~ Group O individuals lack both A and B antigens and their serum will
agglutinate both the A and B reagent red cells. Group O individuals have 3
naturally occurring antibodies in their serum: anti-A, anti-B and anti-A,B.
~ Group AB individuals have both A and B antigens on their red cells, and
their serum will not agglutinate the A or B reagent red cells.
― D typing- Detecting the D antigen consists of testing the individual's red blood
cells with anti-D. Agglutination indicates presence of D antigen and no
agglutination indicates absence.
7. Briefly show with simple note, the fibrinolytic system and its regulation (see solution
illustration to Q.13)
8. Know the meaning of full blood count parameters, their significance and
interpretation. We will review several case reports during tutorials on Monday and
Tuesday
Solution: CBC or FBC
WBC, Platelets and RBCs :
• counted per unit volume of whole blood.
• Unit volume: per cubic millimeter (mm3) which is the same as μL
• WBC: 4.0-10.0 x 103/mm3
SI units per liter: 4.0-10.0 x 109/L
• Platelets 150-450 x 103/mm3
SI units per liter: 150-450 x 109/L
• RBC 4.5-5.9 x 106/mm3
SI units per liter: 4.5-5.9 x 1012/L
•
Hemoglobin and Red cell indices: Primary purpose is differentiation of anemias and for
quality control checks
1. Hemoglobin - Carries oxygen within the RBC; Heme = contains O2 and iron (red
pigment); Globin = protein
2. Hematocrit (35-45%)- the percentage of blood that is represented by the packed
red cells
―
― Used to assess extent of patient’s blood loss
3. Red cell count
4. Mean cell (or corpuscular) volume (MCV),
5. Mean cell hemoglobin (MCH),
Page 7 of 16
Mulungushi University
School of Medicine and Health Sciences
MBChB program
Human Physiology MPG 222
2nd Year 2st semester
Blood cells and coagulation Tutorial
Prepared by Dr. Masenga SK., BSc., MSc., PhD, FFGH, Pg. Dip, CT
6. Mean cell hemoglobin concentration (MCHC)
7. Red cell distribution width (RDW) - The coefficient of variation of the red cell
volume - distribution histogram
―
― Reference range: 12% - 15%
― High RDW indicates more variation in size (anisocytosis)
― Measure of anisocytosis → condition in which RBCs are unequal in size
― Distinguishes hereditary RBC defect from acquired
8. Erythrocyte sedimentation rate
― Rate of sedimentation is determined by plasma proteins. ESR increases
with acute phase response
― This is an indirect determination of inflammation
― Used to follow rheumatoid arthritis, SLE, vasculitis and many inflammatory
conditions
― VERY LOW SPECIFICITY
― Westergren Method: 200 mm tube
― Wintrobe Method: 100 mm tube
MCV refers to the average size of the RBCs constituting the sample.
• red cell volume in femtoliters or 10-15 liter
• Reference interval for adults is typically 78 - 100 fL (femtoliters). One femtoliter is
10-15 L
•
•
Low MCV – Microcytic
•
Normal - Nomocytic
indicates Iron deficiency anemia or Thalassemia
Page 8 of 16
Mulungushi University
School of Medicine and Health Sciences
MBChB program
Human Physiology MPG 222
2nd Year 2st semester
Blood cells and coagulation Tutorial
Prepared by Dr. Masenga SK., BSc., MSc., PhD, FFGH, Pg. Dip, CT
•
High MCV – Macrocytic
Alcoholism
indicates Vit. B12 or folic acid deficiencies or
•
MCH refers to the average weight of hemoglobin in the RBCs in the sample or reflects
the Hb CONTENT (in picograms) of each red cell
• reference interval for adults is typically 26 - 32 pg (picogram) (per red cell). One
picogram is 10 -12 grams
•
•
Correlates with MCV result
o Smaller the cell → less Hgb → lower MCH
MCHC refers to the average concentration of hemoglobin in the RBCs contained within
the sample or Hemoglobin concentration of the packed red cells (minus plasma)
• Reference interval for adults is typically 32 - 36 g/dL (of erythrocytes).
•
•
•
Low MCHC: Hypochromic – less Hb gives a pale color
High MCHC Hyperchromic – the more the Hb the deeper the red
Decreased Hb, hematocrit and RBCs → Anemia
― Chronic blood loss
― Acute hemorrhage
― Hemolysis
― Bone marrow suppression
― Nutrient deficiency (B12, folic acid, iron)
Increased Hb, hematocrit and RBCs → Polycythemia
― Hypoxia
― High altitude
― Smoking
Page 9 of 16
Mulungushi University
School of Medicine and Health Sciences
MBChB program
Human Physiology MPG 222
2nd Year 2st semester
Blood cells and coagulation Tutorial
Prepared by Dr. Masenga SK., BSc., MSc., PhD, FFGH, Pg. Dip, CT
― Cardiovascular disease
― Chronic lung disease
― Congenital heart defects
Rule of three: RBC X 3 = Hgb;
Examples:
Anemias
Normocytic normochromic
Hgb X 3 = Hct
Note: ± 3
Microcytic/hypochromic anemia
Page 10 of 16
Mulungushi University
School of Medicine and Health Sciences
MBChB program
Human Physiology MPG 222
2nd Year 2st semester
Blood cells and coagulation Tutorial
Prepared by Dr. Masenga SK., BSc., MSc., PhD, FFGH, Pg. Dip, CT
White blood cells and differential
― Leukocytosis
o Increased WBCs
o Full term newborns
o Leukemic condition
o Bacterial infection
o Tissue damage
o Inflammation
― Leukopenia
o Decreased WBCs
o Viral infection
o Chemotherapy
o Severe infection
o Diurnal variation
Differential: 5 part
1. Neutrophils – bacterial infection
2. Lymphocytes- Viral infection; Acute lymphoid leukemia (ALL), Chronic lymphoid
leukemia (CLL) predominant in peadiatrics,
3. Monocytes – Chronic inflammation,malignancies
4. Basophils – least numerous of all; inflammatory response→IgE; release heparin
and histamine into blood stream
5. Eosinophils – higher levels in newborns; Allergic reactions; parasitic infections;
chronic myeloid leaukemia
― Expressed as absolute and percentage (of total WBCs); Absolute counts reflect
true increase or decrease of each specific WBC
― Absolute mon = %mon X total WBCs
Total WBCs
Neutrophils
Lymphocytes
Monocytes
Eosinophils
Basophils
Percentage
Absolute #/µL
40-80
25-45
2-10
0-5
0-2
4,500 – 11, 000
1,800 – 8,800
1,125 – 4, 950
90 – 1.100
0 – 550
0 -220
Page 11 of 16
Mulungushi University
School of Medicine and Health Sciences
MBChB program
Human Physiology MPG 222
2nd Year 2st semester
Blood cells and coagulation Tutorial
Prepared by Dr. Masenga SK., BSc., MSc., PhD, FFGH, Pg. Dip, CT
Platelets
― Thrombocytes
― Role in cessation of bleeding
― 1-2-week lifespan
― Thrombocytopenia – risk of bleeding
― Thrombocytosis – risk of inappropriate clotting
― Mean platelet volume (MPV)
o RR: ~8-12 fL
o Average volume of circulating PLTs
o Analogous to MCV
o Increased MPV – PLT destruction
o Decreased MPV – impaired PLT production
9. what is the percentage cellularity difference between a 20year old and 60year
old male? Why is this difference so?
Solution:
% cellularity of 20-year-old = 100 - 20 = 80%
% cellularity of 60-year-old = 100 - 60 = 20%
The difference is 60%±10.
― The older you grow, the fewer the bones that produce cells (bone marrow).
Cellularity decreases with age.
― The older one grows the less requirements for growth, immunity due to ageing.
10. what stimulates the bone marrow to make cells of various lineage and give
examples of some of these stimulants and the cell lineage that results?
Solution:
― Hormones: Erythropoietin (produced by kidney) acts on erythroid progenitor
cells in bone marrow
― Granulocyte-macrophage colony stimulating factor (GM-CSF) – stimulates
hematopoietic stem cells and all committed cells (progenitor cells) except
basophil and lymphoid progenitor
― SCF, stem cell factor – stimulates hematopoietic stem cells
― IL-5 stimulates Eosinophil progenitor
― IL-4 & IL-3 stimulates basophil progenitor
Page 12 of 16
Mulungushi University
School of Medicine and Health Sciences
MBChB program
Human Physiology MPG 222
2nd Year 2st semester
Blood cells and coagulation Tutorial
Prepared by Dr. Masenga SK., BSc., MSc., PhD, FFGH, Pg. Dip, CT
― IL-1, IL-6, IL-3 stimulates hematopoietic stem cells
11. Illustrate the making of Blood group A, B and O antigens on red blood cell
surface
Solution:
Page 13 of 16
Mulungushi University
School of Medicine and Health Sciences
MBChB program
Human Physiology MPG 222
2nd Year 2st semester
Blood cells and coagulation Tutorial
Prepared by Dr. Masenga SK., BSc., MSc., PhD, FFGH, Pg. Dip, CT
12. Does a patient with Hb 7g/dl require a transfusion? Give reasons for your
answer? If you were to conduct a transfusion, what pretransfusion tests would
you do?
― Generally, 7g/dl is used as transfusion criteria
― Pretransfusion tests – compatibility test
~ ABO/Rh typing
~ Direct coombs test ―detects antibodies that have coated the patient's
RBCs
~ Indirect combs test - to screen for unexpected anti-RBC antibodies in
patient serum
13. In the coagulation cascade below, show inhibitors and cofactors required. Also list
coagulation factors requiring vitamin K. Also list some disorders of primary and
secondary hemostasis
Page 14 of 16
Mulungushi University
School of Medicine and Health Sciences
MBChB program
Human Physiology MPG 222
2nd Year 2st semester
Blood cells and coagulation Tutorial
Prepared by Dr. Masenga SK., BSc., MSc., PhD, FFGH, Pg. Dip, CT
Page 15 of 16
Mulungushi University
School of Medicine and Health Sciences
MBChB program
Human Physiology MPG 222
2nd Year 2st semester
Blood cells and coagulation Tutorial
Prepared by Dr. Masenga SK., BSc., MSc., PhD, FFGH, Pg. Dip, CT
Vitamin K dependent factors :
― Coagulation factors: II (prothrombin), VII, IX and X
― Anticoagulation: protein c and S
Disorders of Primary Hemostasis: vWF, Platelet defects, or Receptor Interference
• Von Willebrand Factor disease
• Bernard-Soulier disease
• Glanzmann thrombasthenia
• Medication-induced
Primary hemostasis defects typically present with small bleeds in the skin or mucosal
membranes. This includes petechiae and/or purpura.
Disorders of Secondary Hemostasis: Clotting Factor Defects
• Factor V Leiden
• Vitamin K deficiency
• Hemophilia
• Anti-phospholipid antibody syndrome
• Disseminated intravascular coagulation
• Liver disease
• Medication-induced
Secondary hemostasis defects typically present with bleeds into soft tissue (muscle) or joints
(hemarthrosis).
Page 16 of 16