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VSP-UG-1 (Blood)
VSP-UG-1 (Blood)
VSP-UG-1 (Blood)
Nalgirkar
Professor (Physiology)
D.Y. Patil University, School of Medicine, Nerul, Navi Mumbai
BLOOD
• Introduction
• SECTION 1: Plasma proteins: Function, Clinical significance
• SECTION 2: Erythrocytes: Introduction, Erythropoiesis: Steps in erythropoiesis, factors
influencing erythropoiesis; Anemia
• SECTION 3: White blood cells (WBCs): Classification, Functions, Immunity
• SECTION 4: HEMOSTASIS - Platelets, Coagulation, anticoagulants, bleeding and clotting
disorders
• SECTION 5: Blood groups: Blood group systems (ABO & Rh), hemolytic disease of
newborn, blood transfusion
-1-
Introduction
-2-
SECTION 1
Plasma proteins
-3-
plasma. Ceruloplasmin transports copper; transferrin transports
iron in plasma.
(4) Defence mechanisms –
- -globulins are immunoglobulins or antibodies. These antibodies
are formed by B-lymphocytes. When an antigen (or invading
microorganism) enters the body, antibodies react with it. It is an
important part of our defence reactions.
(5) Blood coagulation –
- When a blood vessel is ruptured, some reactions or sequential
steps at that site form “prothrombin activator”. It converts
prothrombin into thrombin. Thrombin then acts on fibrinogen
present in plasma, and forms fibrin. This results in clot formation
or coagulation of blood. It allows for healing of the vessel.
~ Clinical significance:
- Edema in Liver and kidney diseases: Plasma protein levels
decrease in diseases of kidney and liver. Since liver synthesizes
these proteins, in severe liver diseases (cirrhosis, liver failure)
plasma proteins levels decrease. Kidneys normally do not allow
proteins to be filtered and excreted in urine. Certain diseases of
kidney (e.g. nephrotic syndrome) result in loss of proteins in urine.
- Decreased plasma protreins → decrease in plasma colloid osmotic
pressure → fluid from interstitium will not be pulled into the
capillaries in normal amount → excess accumulation of fluid in the
interstitium – it is called “edema”.
Video link: Plasma proteins in liver and kidney diseases; plasma colloid oncotic
pressure and edema.
-4-
Section 2
ERYTHROCYTES
(Red blood cells or red blood corpuscles – RBCs)
Learning objectives:
- Anemia is a very common clinical condition that results from a
decreased RBC count and/or reduced Hb. It is important to learn
the formation, life-span, and destruction of RBCs, in order the
understand the pathophysiology of various types of anemia.
Introduction
• Structure and important features of RBCs:
- RBCs are biconcave shaped cells.
- Mean RBC diameter = 7.2 to 8
- They are non-motile and non-nucleated cells.
- RBCs have no mitochondria or ribosomes.
- RBCs contain haemoglobin. Hemoglobin (Hb) performs the
function of oxygen transport.
- Normal RBC count:
a. In males ~ 5 to 6 million/mm3.
b. In females ~ 4.5 to 5.5 million/mm3.
Erythropoiesis:
It is the process of formation of RBCs.
Sites of erythropoiesis –
{NOTE: All the blood cells are formed at the sites mentioned below. The
process is also referred to as hemopoiesis or hematopoiesis.}
▪ As the embryo grows, during 1st trimester of pregnancy – RBCs are
formed in mesoderm of the yolk sac. It is “mesoblastic stage” of
erythropoiesis. (first 3 months of pregnancy)
▪ 2nd trimester of pregnancy (3 to 6 months) – RBCs are formed in the fetal
liver and spleen. It is “hepatic stage” of erythropoiesis.
▪ 3rd trimester of pregnancy (6 months onward) – bone marrow within
fetal bones starts producing RBCs. It is called “myeloid stage”.
o At birth, all bones of the child have “red bone marrow”. That is, this
bone marrow is capable of producing cells. Slowly, fat starts infiltrating
-5-
the bone marrow and it turns into “yellow bone marrow”. A yellow
bone marrow does not produce blood cells.
o By the age of 18 yrs, only a few sites in the body are still left with red
bone marrow. Then on, blood cells will be formed at these sites only.
The sites that still have red marrow are: proximal ends of long bones,
sternum and some flat bones, vertebral bodies, anterior superior iliac
spine.
Clinical significance:
Bone marrow tapping: In the conditions such as leukemia, cellularity of the
bone marrow has to be investigated. In case of adults, aspiration of the bone
marrow contents is done from the sites which have a red bone marrow. Thus,
it is important to know the loci of red bone marrow. Preferred sites for this
procedure ~ sternum, anterior superior iliac spine.
Steps in erythropoiesis:
-6-
Pluripotent hematopoietic
stem cell
Colony forming
CFUmega CFUGM {granulocyte
units – erythrocyte
{megakaryocyte} & monocyte}
[CFUe]
Early normoblast
Intermediate normoblast
Late normoblast
Reticulocyte
ERYTHROCYTE
-7-
Proerythroblast (1) Diameter – 18
(2) Nucleus – large, central
(3) Cytoplasm – thin rim, basophilic
(1) Diameter – 7 to 10
Late normoblast
(2) Nucleus – eccentric, “ink spot”
nucleus or “pyknotic” nucleus
(3) Haemoglobin increases
[Fig: It shows the steps in erythropoiesis. Note the cell size, nucleus, and
cytoplasm in each step.]
• The salient features of erythropoiesis:
1. Cell size:
Note that pluripotent stem cell, the first precursor of RBC, is 24 in
diameter, whereas the last stage (fully matured RBC) has a diameter
of 7-8 Why?
Cell division and cell maturation are occurring simultaneously in the
bone marrow. As erythropoiesis occurs in a stepwise fashion, cell
division is occurring rapidly. As cell division occurs very rapidly, the
daughter cells have little time to grow to the parent cell size. Before
they could enlarge to the parent cell size, they are further divided,
-8-
and this goes on through subsequent steps. Hence, the cell size goes
decreasing.
2. Nucleus:
Note that the pluripotent stem cell is a nucleated cell, whereas the
fully matured RBC is non-nucleated. Thus, through the subsequent
steps the nucleus decreases in size, pushed to the periphery of the
cell and eventually thrown out of the cell. {or, nucleus undergoes
“pyknosis”; it is broken down into pieces and finally turns into dust.}
3. Cytoplasm:
Initially, the cytoplasm is basophilic. [It has acidic contents, hence
attracts basic stain – ‘basophilic’.]
At the intermediate normoblast stage, haemoglobin (Hb) begins to
appear in the cytoplasm. Hb being basic in nature, attracts acidic
stain (‘acidophilic’). The cytoplasm is now polychromatophilic
(attracting both types of stain).
Eventually, when reticulocyte converts into mature RBC, the
basophilic material disappears completely and the cytoplasm is fully
acidophilic.
Video link: Erythropoiesis ~ cell size, nucleus, and cytoplasmic changes through stages of
erythropoiesis
• Steps in erythropoiesis:
1. Pluripotent hematopoietic stem cell: (pluri = many)
- It is the first precursor cell. It can form all cell types (RBCs, WBCs,
platelets).
- It is a nucleated cell. Diameter ~ 24
- It actively divides and forms two types of committed stem cells.
2. Committed stem cells: (18-20 )
- Committed stem cell of the lymphoid series → this cell then
leaves the bone marrow and goes to the lymphoid structures. It
will eventually develop into lymphocytes.
-9-
- Committed stem cell of the erythroid series → remains in the
bone marrow and continues to form the remaining blood cells.
The cells actively divide and go into the next stage.
3. Colony forming units [CFU]:
- Different colony forming units are observed in the bone marrow;
they would form the respective cells –
CFUe – colony forming unit for erythrocytes.
CFUmega – for megakaryocyte; these will eventually form platelets.
CFUGM – for granulocytes & monocyte. {WBCs are of two main
types – granulocytes and agranulocytes. Agranulocytes are
lymphocyte and monocyte. Lymphocyte forming cell (committed
stem cell) has already moved out of bone marrow. The remaining
WBC types – granulocytes & monocyte - are formed from these
colony forming units.}
4. Proerythroblast:
- This is the step in erythropoiesis when the cell can now be
morphologically identified as RBC-forming cell.
5. Early normoblast:
- Cell division continues to occur; cell size is reduced further.
6. Intermediate normoblast:
- Hemoglobin begins to appear in the cell, at this stage.
- Cell division continues. Cell size is reduced; nucleus pushed to
periphery.
7. Late normoblast:
- Last stage when nucleus is still present. Cell division will stop after
this stage.
- Nucleus is ‘eccentric’ (at one end of the cell); sometimes referred
to as “ink-spot” nucleus. Pyknosis is the beginning of
disintegration of the nucleus – pyknotic nucleus.
8. Reticulocyte:
- Remnants of nuclear material are seen in the form of a reticulum
(network); hence the name reticulocyte.
- At this stage the cell leaves the bone marrow and enters
circulation. Over the next 24 hours or so, it matures into an
erythrocyte. Normal reticulocyte count in peripheral blood = 0.2
to 2%. That is, out of every 100 RBCs in peripheral blood, 2 cells
will be reticulocytes.
- Reticulocyte count is high (up to 5%) in new-born babies. Reason:
New-born babies suffer from a relative hypoxic situation. And,
since it is the job of RBCs to carry oxygen to tissues, rate of RBC
- 10 -
production increases in them. And, RBCs are poured into
peripheral blood (at reticulocyte stage) at a faster rate.
9. Erythrocyte:
- A matured RBC is biconcave shaped, non-motile, non-nucleated
cell. Diameter = 7-8
- It also lacks mitochondria and ribosomes.
- Normal RBC count –
o 5 to 6 million/mm3 of blood (males)
o 4.5 to 5.5 million/mm3 of blood (females)
- Life span of an RBC – 120 days.
{Entire process of erythropoiesis occurs in 7 to 9 days.}
- 11 -
RBCs contain hemoglobin. Hemoglobin = heme + globin. Heme
contains iron and globin is made up of amino acids.
a. Iron in diet – required for the synthesis of heme.
b. Amino acids (proteins in diet) – necessary for the synthesis of
globin.
c. Vitamin B12 and folic acid in diet –
RBC is a non-nucleated cell but its precursors are nucleated.
Vitamin B12 and folic acid are necessary for synthesis of DNA in the
RBC precursors.
In deficiency of vit. B12 or folic acid – results in failure of nuclear
maturation in RBC precursors. Deficient nucleus → cell division
and maturation is hampered. Cells will remain large and
immature. The condition is called “megaloblastic anemia”. {Mega
= large; blast = immature cell.}
d. Other factors – (role in erythropoiesis not proven)
▪ Vitamins B1, B2, and B6.
▪ Minerals – Cu++, cobalt, zinc.
4. Hormonal factors:
❖ Androgens/testosterone –
It is the male sex hormone.
It stimulates erythropoiesis possibly in following ways: (i) It
stimulates erythropoietin production from kidney. (ii) It
potentiates the action of erythropoietin on bone marrow.
(iii) It may directly stimulate the rate of erythropoiesis.
It is due to stimulation of erythropoiesis by testosterone,
that the RBC count is more in males compared to females.
❖ Estrogens –
Estrogen is a female sex hormone. It has been hypothesized
(not proven entirely) that estrogen suppresses
erythropoiesis to some extent. {Another reason for lower
RBC count in females.}
❖ Thyroxine, growth hormone, cortisol –
Thyroid hormones stimulate growth and differentiation of
various tissues of the body. They stimulate metabolic rate,
increase oxygen demand and thus may indirectly stimulate
erythropoiesis.
Deficiency of any of the hormones mentioned above is
associated with anemia.
- 12 -
Video link: RBC count in males and females ~ The hormonal influence
Clinical significance:
- Some drugs (e.g., chloramphenicol) and radiation are known to
suppress erythropoiesis. They result in anemia and pancytopenia
(formation of all blood cells suppressed).
- Persons residing at high altitudes are chronically exposed to
hypoxic environment. As hypoxia is the most important stimulus
for erythropoiesis, these people tend to have a very high RBC
count – polycythemia.
- Erythropoietin is synthesized in kidneys. Patients of chronic renal
failure lack erythropoietin, and hence have a low RBC count.
---------------------------------------------------------------------------------------------------------
- 13 -
Hemoglobin
To iron
pool of
body Heme Globin
To
Fe++ protein
Amino acids pool of
Biliverdin
body
Bilirubin
- 14 -
hepatocyte
spleen
To liver PORTAL BLOOD
Uptake
conjugation
Bilirubin
excretion bilirubin
+
albumin
Bile
Conjugated
Duodenum bilirubin in
2nd part of
duodenum
Excreted in
Intestine – some
urine as
part of bilirubin
urobilinogen
converted to Some part is
stercobilinogen absorbed in systemic
→ oxidized to circulation
stercobilin and
excreted in stools
(imparting brown
colour to stools)
- 15 -
conjugated with glucuronic acid, (iii) Excretion: Conjugated
bilirubin is then excreted by the liver cells into the bile.
- Conjugated bilirubin (in bile) is then transported, via the bile
ducts, to 2nd part of duodenum.
- Some part of this bilirubin is converted to stercobilinogen and
then to stercobilin. Stercobilin excreted into stools, imparts brown
colour to stools.
- Remaining part of bilirubin is absorbed into blood; it reaches
kidneys and is excreted into urine as urobilinogen.
Jaundice:
It is “yellowish discoloration of mucous membranes and skin” due to
increase in bilirubin levels of blood.
Jaundice becomes clinically apparent when serum bilirubin exceeds 3 mg%.
{Normal serum bilirubin:- 0.3 – 1 mg%.}
Jaundice is detected clinically by examination of the sclera. Reason: (i) It can be
easily made out when a white sclera turns yellow. (ii) Sclera contains a protein
– elastin – for which bilirubin has high affinity. [This is also the reason why
sclera continues to look yellow even after jaundice has been cured.]
Types of jaundice –
{The 3 types of jaundice are based on the course of bilirubin after its formation
from heme.}
1. Pre-hepatic jaundice:
- The abnormality lies in the process before bilirubin goes to liver.
- Excess breakdown of RBCs → excess liberation of Hb and
excessive formation of bilirubin from heme. Hence, it is also called
hemolytic jaundice.
- Free bilirubin will be increased in serum.
2. Hepatic jaundice:
- The abnormality lies in the processing of bilirubin once it has
reached liver.
- Diseases of the liver parenchyma and injury to liver cells will cause
bilirubin to enter blood directly from liver.
- Causes: Hepatitis A, hepatitis B, and other types of hepatitis,
cirrhosis, etc.
3. Post-hepatic jaundice:
- 16 -
- Once bilirubin is conjugated in the liver, it is excreted in bile. Then,
via bile ducts it reaches intestine. If there is obstruction of the bile
ducts, bile and bilirubin will not reach intestines. Bilirubin will start
entering directly into blood. This type is also called ‘obstructive
jaundice’.
- Since bilirubin is already conjugated by liver, it is the conjugated
bilirubin that increases in serum, in this type.
- Stools are chalky, pasty coloured. Reason: Since bilirubin does not
reach intestines, stercobilin cannot be formed; no brown colour to
stools.
---------------------------------------------------------------------------------------------------------
- 17 -
the ESR. Conditions in which such proteins are increased in plasma
will therefore exhibit an increase in ESR.
- ESR increases in conditions like TB, malignancy, rheumatoid
arthritis, anemia.
➢ Significance:
▪ ESR does not have diagnostic significance as such. It does not help
in diagnosis, since there are many conditions in which ESR
increases.
▪ ESR has a PROGNOSTIC significance. {Prognosis means progress
of a disease.} Once a disease is diagnosed, ESR of the patient is
recorded. And then with continued treatment, ESR is measured at
regular intervals. If ESR decreases during the course of treatment,
it means patient’s condition is improving.
- 18 -
100
Plasma
0
Layer of lysed RBCs may not be always visible
➢ Significance of PCV:
- Anemia: Since the RBC count is decreased in anemia, the PCV
value will be lower (< 38%).
- Polycythemia: Increased RBC count; PCV value will be higher (>
48%).
- Conditions that cause loss of plasma (Hemoconcentration) –
diarrhea, vomiting, etc – would have an increase in PCV value.
{Note: It is the relative proportion of cells and plasma. If plasma
decreases in a unit quantity of blood, cells will be relatively
increase in proportion.}
- Jaundice: Normal plasma is straw-colored. The plasma bilirubin
increases in jaundice. Bilirubin is a yellow-colored pigment; it will
turn plasma color into yellow in jaundice. Thus, the separated
plasma appearing yellow in the PCV tube is a pointer to jaundice.
• Blood indices:
[These are numbers or values related to RBCs, derived from
observations and calculations.]
1. Packed cell volume (PCV): (described above)
2. Mean corpuscular volume (MCV) –
It is the average volume of a single red blood corpuscle.
A blood sample is collected. RBC count is done. And, packed cell
volume (PCV) is measured.
- 19 -
If the blood sample has a certain number of RBCs and all those RBCs,
when packed together, have a certain packed cell volume (PCV), we
can calculate the average volume of one single RBC from the formula:
- 20 -
(If these many RBCs have this much total Hb, then on an average, how much is
the Hb present in a single RBC?)
5. Colour index –
It is the ratio between Hb% and RBC%.
- 21 -
Hb level (in gram%) is converted into a percentage value. 15 gm% of
Hb is considered as 100% Hb. So, if a patient has 10 gm% of Hb, it is
66% Hb.
RBC count (in million/mm3) is converted into a percentage value. 5
million/mm3 is considered 100%. So, if a patient’s RBC count is 4
million/mm3, it is 80%.
Color index = [Hb%]/[RBC%]
~ Anemia:
It is defined as “decreased O2 carrying capacity of blood.”
O2 is carried by Hb within RBCs; anemia is due to decreased RBC count or
decreased Hb level or both.
• Classification of anemia:
3 ways of classifying anemia –
1. Etiological classification – (according to the cause)
2. Morphological classification – (according to the morphology of
RBCs)
3. Clinical classification – (according to Hb levels)
1. Etiological classification:
[Based on the cause of the anemia]
A. Blood loss anemia – (due to excessive blood loss; there may be loss
of RBCs and reduced RBC count)
(i) Acute blood loss: (within a short period of time) e.g. accidents,
surgery.
(ii) Chronic blood loss: (over a long period of time) e.g. piles,
worms in intestine, menorrhagea and polymenorrhea
(excessive menstrual blood loss), etc.
- 22 -
B. Anemia due to excessive RBC destruction – (hemolytic anemias)
(i) Congenital cause: (genetic defect in RBC)
Spherocytosis (sphere shaped RBCs; destroyed readily)
Sickle cell anemia (sickle-shaped RBCs)
Thalassemia
(ii) Acquired cause:
Malaria (malarial parasite stays within RBCs during its life cycle;
there is excessive RBC destruction)
C. Anemia due to decreased RBC production -
(i) Nutritional deficiency:
a. Protein deficiency – It leads to deficient globin formation in
hemoglobin
b. Iron deficiency anemia – Iron is required for heme
formation (of Hb); its deficiency will lead to impaired Hb
formation in RBCs. This is the commonest type of anemia in
India.
c. Vitamin B12 and folic acid deficiency (megaloblastic anemia)
– Vitamin B12 and folic acid are required for nuclear
maturation in RBC precursors. Deficiency of these factors
will hamper the cell division and maturation in
erythropoiesis.
d. Aplastic anemia (bone marrow suppression) – Certain drugs,
X-rays, toxins suppress bone marrow; this results in
depressed formation of RBCs and other blood cells.
- 23 -
MEGALOBLASTIC ANEMIA:
[Note: Intrinsic factor protects vit B12 from the digestive juices of intestine so that B12
reaches the ileum without getting degraded. Vit B12 can be absorbed from ileum only, as
its receptors are located on the ileal mucosa. [most other nutrients are absorbed from
duodenum and upper jejunum.]
- 24 -
2. Morphological classification:
This classification is based on the morphology (appearance, size) of
RBCs.
Based on cell size:
➢ Microcytic anemia –
RBCs are smaller in size; MCV < 78 3.
E.g. iron deficiency anemia. (Low iron → less Hb → RBC
remains small.)
➢ Normocytic anemia –
RBC size is normal, Hb content is also normal; RBC count is
reduced. E.g. blood loss anemia.
➢ Macrocytic anemia –
RBC size is large; MCV > 94 3.
e.g. megaloblastic anemia.
- Based on the colour index:
➢ Hypochromic anemia –
When colour index < 0.8; it is due to iron deficiency.
Thus, iron deficiency anemia would be “microcytic
hypochromic” type.
➢ Normochromic anemia –
When colour index is normal. Hb/iron content would be
normal. RBC count would be less. Thus, blood loss anemia is
of “normocytic normochromic” type.
- 25 -
• Hemoglobin: (Hb)
- It is a conjugated chromoprotein; present inside the RBCs. It
contains iron (Fe++). It is due to hemoglobin that the red cells and
blood are red in colour.
- Its main function is to transport oxygen to tissues. Hb also plays
an important role in acid-base buffering in the ECF.
- Normal level: 14-18 gm% (males); 12-16 gm% (females)
- O2 carrying capacity of blood:
1 gram of Hb → carries 1.34 mL of O2. Thus, if Hb of a person is 15
gm% (i.e. 15 gms/100 mL of blood), then his blood would carry –
(15 × 1.34 =) 20 mL/100 mL of blood.
- Hemoglobin is made up of heme & globin.
- Heme moiety consists of iron (Fe++).
- There are 4 globin chains – two chains and 2 chains – in adult
hemoglobin.
- Synthesis of Hb:
I. 2 succinyl CoA + 2 glycine → pyrrole ring
II. 4 pyrrole → protoporphyrin IX
III. Protoporphyrin IX + Fe++ → heme
IV. Heme + polypeptide → hemoglobin chain ( or )
V. 2 chains + 2 chains → HbA (adult hemoglobin)
Glycosylated Hb (HbA1C):
- Also called glycated Hb; glucose in plasma gets attached to Hb
non-enzymatically and irreversibly.
- Percentage of Hb that is in the glycated form is a good indicator
of plasma glucose over a certain period.
- If plasma glucose is consistently high, more of glucose will attach
with Hb, thus giving a high value for glycosylated Hb. [Normal:
up to 6%.]
- Once glucose is attached to Hb, the Hb remains glycosylated for
the remainder of the life span of the red cell. Hence, glycosylated
Hb value indicates the average plasma glucose level over a
period of 3 months. [Life span of an RBC is 4 months.]
(There are many other biochemical aspects related to hemoglobin, such as
derivatives of Hb, etc. It is suggested that further reading on these aspects of
Hb may be done from a textbook of biochemistry.)
- 26 -
SECTION 3
White blood cells (or white blood corpuscles – WBCs/leucocytes):
Learning objectives:
- WBCs/Leucocytes are concerned with body’s defence
mechanisms against various micro-organisms. Increase in the
number of WBCs provides a clue toward an ongoing infection in
the body.
- Different types of WBCs are involved in different varieties of
infections. Thus, a differential WBC count (counting individual
types of WBCs) gives an idea about the onset/duration of
infection, nature of infection, and so on.
- WBCs form the basis of body’s immune mechanisms. Immune-
related disorders (autoimmune diseases, immunodeficiency
syndromes, hypersensitivity & allergy) can be learnt on the basis
of understanding of WBCs and their role in immunity.
Introduction:
▪ Some characteristics of WBCs –
- There are different types of WBCs with different morphology and
functions.
- WBCs are motile cells. They can move on their own, when they
have migrated out of blood vessels. (RBCs and platelets are non-
motile)
- They are nucleated cells. (RBCs and platelets are non-nucleated)
- Most WBCs have short life-span; and they remain in circulation for
short periods of time. They are produced in bone marrow and
they are required to perform their function in tissues. Thus, they
use blood just as a vehicle to reach the tissues.
• Total leucocyte count [TLC]: 4000 to 11,000/mm3.
• Increase in leucocyte count is called leucocytosis. It occurs in various
types of acute and chronic infections.
• Decrease in leucocyte count is called leucopenia. It may occur in bone
marrow suppression.
• There are different types of leucocytes in the body. Counting each type
of leucocyte and giving its number in relative percentage (or per 100
cells) is called differential leucocyte count [DLC].
- 27 -
Leucocytes (as found in
peripheral blood)
Granulocytes Agranulocytes
Lymphocyte Monocyte
Neutrophil Eosinophil Basophil
(20-30%) (2-8%)
(50-70%) (1-4%) (0-1%)
[Absolute counts:
The percentages of the WBCs are the relative numbers. These are per 100
WBCs only. For instance, there are 60 neutrophils, 25 lymphocytes, and 15
others. If in a patient, 80 neutrophils are found (in 100 WBCs), lymphocytes and
other cells will be less as it is a count of 100 cells only. This decrease in
lymphocytes and other cells may be only a relative decrease (because
neutrophils have counted more), or there may be an actual decrease in the
number of those cells. To verify these numbers, absolute counts of each type of
cell can be derived. First find out the total WBC count per mm3. Then, find out
the differential count of each cell type. The actual count (per mm3) can then be
calculated. E.g. The TLC is 20,000/mm3, neutrophil count 80% and lymphocyte
18%. The absolute neutrophil count would be (80% of 20,000 =) 16,000/mm3.
One can then conclude that WBC count has increased because neutrophils have
increased. The lymphocytes (18%) may be relatively low, but absolute count of
lymphocytes may fall within the normal range.]
- 28 -
Video link: Significance of absolute counts
~ There are two types of WBCs – (I) Granulocytes: Those having granules in
the cytoplasm, and (II) Agranulocytes: No granules in the cytoplasm.
Agranulocytes are of two types – (1) lymphocyte (20-30%), and (2)
monocyte (2-8%)
Granulocytes are classified according to the staining property of their
granules:
1. Eosinophil – The granules take up acid stain (as their contents are basic).
2. Basophil – The granules take up basic stain (as their contents are acidic).
3. Neutrophil – The granules take up both acid and basic stains.
- 29 -
Grade II – 2 lobes ≈ 10-15%, Grade III – 3 lobes ≈ 35-45%, Grade IV
– 4 lobes ≈ 10-15%, Grade V – 5 lobes ≈ 5-10%)Normally, highest
number of neutrophils will have 3 lobes in the nucleus.
- Granules: The cytoplasmic granules are of two types – (1) Primary
granules, (2) Secondary granules.
The primary or lysosomal granules contain – (a) various
proteolytic and amylolytic granules, (b) myeloperoxidase (MPO)
granules, and (c) lysozyme enzyme granules. These granules cause
bacterial destruction or digestion.
The secondary granules contain – (a) lactoferrin, which inhibits
bacterial growth, (b) alkaline phosphatase, and (c) other
substances or enzymes.
- Life-span: Once neutrophils are formed in the bone marrow, they
remain in the bone marrow pool.
Neutrophils released from bone marrow into circulation become
part of the vascular pool of neutrophils.
Neutrophil has short life-span in blood – 4 to 8 hours.
It then enters tissues and lives for another 4-5 days.
Thus, the total life span of a neutrophil (bone marrow → blood →
tissue) is about 12 to 14 days.
- Functions of the neutrophil:
A. Antibacterial role:
When pathogenic bacteria have entered the body, neutrophils
attack and kill the bacteria. The sequence of events, at the site
of infection, would be -
➢ Vasodilatation – Local blood vessels dilate.
➢ Margination and emigration – Neutrophils, normally
travelling at the centre of the blood vessel, come near the
vessel wall and get adhered to the endothelium of the
vessel (‘margination’). They then come out of the blood
vessel to enter the infected tissue (‘emigration’).
➢ Chemotaxis – The bacteria have released certain chemicals
(or chemo-attractants). They attract the neutrophils toward
them. Neutrophil movement toward the bacteria is called
chemotaxis.
➢ Phagocytosis – Neutrophils reach the bacteria, engulf and
swallow them (‘phagocytosis’).
➢ Finally, the swallowed bacteria are digested/degraded. The
enzymes present in the neutrophil granules are responsible
for the degradation or killing of the bacteria.
- 30 -
The neutrophil can produce H2O2 and superoxide (O2-). In
the presence of the myeloperoxidase (of granules), H2O2
becomes very lethal for the bacteria.
▪ Pus formation – dead cells, tissue fluid, and dead WBCs together form a
fluid called pus.
B. Role in acute infection:
➢ Neutrophils are the “first line defence” against any
acute infection. When a micro-organism enters the
body, neutrophils are the first cells to reach the site of
infection and attack the micro-organism.
o Variations in the neutrophil count –
a. Neutrophilia: Increase in the neutrophil count. It may also
cause leucocytosis. It occurs in –
➢ Acute infections (bacterial, viral, etc)
➢ Many types of fevers
➢ Infection by pyogenic (pus-producing) bacteria
b. Neutropenia: Decrease in the neutrophil count. It may occur in
typhoid fever, and factors that cause bone marrow depression.
2. Eosinophil: (1-4%)
- 10-14 diameter
- Nucleus: Bi-lobed or spectacle shaped
- Cytoplasm: contains granules that take acid stain and hence
appear reddish orange
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- Functions of eosinophil:
a. Major basic protein (MBP) is an important constituent of
eosinophil granules. It acts mainly against parasites
(hookworm, filaria, etc) that have invaded the body.
MBP can kill larva of many parasites.
b. Eosinophils play a role in allergic conditions. They
migrate to the site of allergy and assist the mast cells
during allergic inflammation.
o Variations in eosinophil count:
- Eosinophilia: Increase in eosinophil count. It occurs in – (1)
parasitic infestations (hookworm, threadworm, etc), (2) allergic
conditions (like bronchial asthma).
- Eosinopenia: Decrease in eosinophil count. Steroids (cortisol) are
known to cause eosinopenia.
3. Basophil: (0-1%)
- 10-12 diameter
- Nucleus: Irregular, often ‘S’ shaped
- Cytoplasm: Covered with coarse basophilic granules; the granules
cover even the nuclear zone, nucleus is barely visible.
- 32 -
- Mast cells have an important role to play in allergy.
~ Agranulocytes:
1. Monocyte: (2-8%) [“second line defence”]
- It is the largest WBC; 14-18 diameter.
- 33 -
b. Since monocytes are large cells, they can phagocytose
larger micro-organisms which cannot be attacked by
neutrophils. E.g. malarial parasite.
C. Formation of tissue macrophage –
Once they leave the circulation and enter the tissues,
monocytes become tissue macrophages. E.g. Kupffer cells in
liver, alveolar & splenic macrophages. They remain attached to
the tissue for many years. Their main function is to remove
various undesirable substances (old RBCs, bacteria, foreign
bodies, etc) by phagocytosis.
D. Role in lymphocyte-mediated immunity –
➢ The macrophage produces ‘monokines’ which causes
death of the invading antigen.
➢ The macrophage takes up an antigen; it presents this
antigen to the T- and B-lymphocytes for further action.
• Monocytosis: It is an increase in the number of monocytes. It occurs in
acute bacterial infections, malaria, and other such infective conditions.
[The figure shows small & large lymphocyte. Small lymphocyte is seen just as
the nucleus occupying the entire cell. Large lymphocyte is a large cell, with
nucleus occupying almost 90% of the cell volume; a thin rim of cytoplasm is
- 34 -
visible. Monocyte is shown in the diagram to compare between large
lymphocyte and monocyte.]
How to differentiate between a large lymphocyte and a monocyte?
[From the findings of the peripheral blood smear]
Large lymphocyte Monocyte
- Cell size Double the size of 2 ½ to 3 times the
surrounding RBCs size of surrounding
RBCs
- Nucleus Occupies 90% of cell Occupies 70% of cell
volume; bean- volume; horse-shoe
shaped nucleus with shaped nucleus with
shallow indentation deeper indentation
- Cytoplasm A thin rim of More cytoplasm
cytoplasm visible visible (about 30% of
(10% of cell volume) cell volume)
# Note: In the peripheral blood smear, two types of lymphocytes are seen –
small & large. No other WBC is seen as small and large. Why then only
lymphocytes show two sizes in peripheral blood?
Reason: Small & large lymphocyte are different developmental stages. A
lymphoblast enlarges (large lymphocyte) and then divides to form small
lymphocytes. Thus, small lymphocyte is an old or more matured cell. Such
developmental stages are seen in blood only with lymphocytes. The reason is –
lymphocytes have a long life span (300 days). During development, the
lymphocytes leave the bone marrow at an early stage. They go to primary
lymphoid structures where they are processed further. From here, they come
into blood again and reach the secondary lymphoid structures where they are
stationed. Whenever required, they again come into blood and migrate to
tissues. Due to long life span and extensive travel of lymphocytes, its different
developmental stages (small & large) can be seen in peripheral blood.
- 35 -
All other WBC types have short life spans in the circulation (few hours). Each
cell type is formed as a colony in bone marrow. Then, all cells of the colony are
poured together into circulation where they stay for a short period. Hence, all
cells of a particular type are at the same developmental stage in blood.
- Functions of lymphocytes:
➢ Lymphocytes are considered as “third line defence” of the
body.
➢ They play the important role in chronic infections.
➢ Lymphocytes form the basis of “specific” immunity
(neutrophils & monocytes form the non-specific immunity);
lymphocytes have the central role in the long-term
immunity of an individual. T-lymphocytes are involved in
cell-mediated immunity and B-lymphocytes are the basis of
humoral immunity. [These roles are discussed in greater
details with the topic of “immunity”.]
---------------------------------------------------------------------------------------------------------
➢ Immunity and role of lymphocytes:
- 36 -
self. Actually, some of the proteins of these substances are the actual
antigens.
• Antibody:
Antibodies are immunoglobulins (−globulins). They are formed by the
body’s immune system in response to an antigen that has entered the
body. The antibodies react with the antigen and cause the destruction
of antigen.
- 37 -
➢ T-suppressor cells
➢ Cytotoxic T-cells
➢ Delayed type hypersensitivity T-lymphocyte [DTHL]
▪ T-helper cells are the regulatory cells; they play the central role in the
body’s immune mechanisms by helping various other cells involved in
immune mechanisms. It is these cells which are attacked by the HIV
virus, making the immune system completely deficient.
▪ T-suppressor cells limit the immune reactions so that the damage done
to the host’s innocent cells is contained.
▪ Cytotoxic T-cells are direct killer cells.
• Clinical application:
- Allergy and anaphylaxis:
A person may be hypersensitive to a particular antigen. Entry of
that antigen will induce massive antibody formation. Antigen-
antibody reaction causes release of histamine and other
mediators of allergy.
- 38 -
In a severe form of allergy, there may be systemic allergic reaction
resulting in anaphylaxis.
- Autoimmune diseases:
Normally,the immune system has the ability to distinguish
between self and non-self. However, in some individuals, the
immune mechanisms get directed toward some self protein. E.g.
myasthenia gravis.
- 39 -
SECTION 4
HEMOSTASIS
(Stoppage of bleeding by platelets; clot formation, and related issues)
Learning objectives:
- Accidents, surgeries, and many other situations result in
bleeding. Loss of blood volume may result in hemodynamic
instability. It is important to understand the inherent processes
that stop the bleeding and thereby prevent consequences of
blood loss.
Introduction:
When a blood vessel is ruptured, and bleeding starts, some events occur in
that region to stop the bleeding. These events may be collectively called
“hemostasis”. (hemo = blood; stasis = stoppage)
Following events occur, in an overlapping sequence, in the process of
homeostasis:
1. Vasospasm or vasoconstriction
2. Platelet plug formation
3. Clot formation (coagulation of blood)
4. Organization, retraction, and eventual dissolution of the clot.
(These events occur in the sequence. However, they occur in an overlapping
manner; i.e., many of them start occurring simultaneously.)
➢ Vasospasm or vasoconstriction:
- When a blood vessel is ruptured, within a few seconds the vessel
undergoes spasm or constriction. The vessel lumen is narrowed so
that the amount of blood loss can be minimized.
- Contraction of the smooth muscle in the wall of the vessel is
responsible for the vasoconstriction.
- 40 -
• Platelets (Thrombocytes):
[morphology, functions, applied aspects]
- Platelets are round or oval disc shaped blood cells; 2-4 in
diameter
- Non-motile, non-nucleated
- Formed in the bone marrow, from a large precursor cell called
megakaryocyte;
- Thrombopoietin, a protein formed in the liver & kidneys,
stimulates thrombopoiesis (formation of platelets in bone
marrow)
- Life span of a platelet:- about 9-12 days (Half-life is about 4 days)
- Normal platelet count:- 1.5 to 4 lacs/mm3
▪ Critical platelet count – When the platelet count falls to about
50,000/mm3 or less, it is considered to be a “critical” platelet count.
Morphology of platelets:
(a) Platelet canalicular system: There are two types of canaliculi in platelets
– (I) Open canaliculi:- these are open on the platelet surface; the
secretory products will be discharged by the platelets, to the exterior,
when the platelets are activated. (II) Dense tubules:- (closed canaliculi)
These tubules or canaliculi are not open to the exterior. They maintain a
high concentration of Ca++ within them.
(b) Platelet granules : Platelets contain two types of granules: (1) dense
granules: contain nonprotein substances (serotonin, ADP, etc) which are
secreted in response to platelet activation, and (2) -granules: contain
secreted proteins which include clotting factors and platelet-derived
growth factor (PDGF). [Note: PDGF is also secreted by macrophages and
endothelial cells; it stimulates wound healing.]
(c) Contractile filaments – actin, thrombosthenin. These contractile
filaments are involved in clot retraction (discussed later).
Platelet membranes contain receptors for collagen, ADP, fibrinogen, and von
Willebrand factor (vWF) of the vessel wall.
Platelet activation & aggregation: “Platelet plug formation”
- When a blood vessel is ruptured, platelets come in contact with
the exposed collagen and vWF in the vessel wall. This results in
platelet activation.
- 41 -
- Platelet-activating factor (PAF), secreted by neutrophils,
monocytes, and platelets, increases the production of
thromboxane A2.
- Ca++ is released into the cytosol of these activated platelets via
the via the canalicular system.
- This increased cytosolic Ca++ will be utilized for the following
purposes:
(a) To cause degranulation: The contents of the platelet granules
will be discharged to the exterior. Two important contents in
this context are – ADP & thromboxane A2. Release of these
chemicals will attract nearby platelets to come into this area
and adhere to these activated platelets. A positive feedback
cycle will follow. The new platelets will be activated; they will
release the chemicals, further attracting more and more
platelets to come into the region and adhere to the platelets
already activated. Finally, a platelet plug will be formed; it will
seal the gap in the injured vessel and the bleeding will stop.
(b) Contraction of the platelets in the platelet plug: “Clot
retraction” – The increased cytosolic Ca++ is utilized by the
contractile proteins (actin, thrombosthenin). Once the platelet
plug is formed, a blood clot is formed on the surface of the
platelet plug. The contractile proteins within the platelet plug
will contract so that: (1) clot size is reduced, and (2) injured &
separated ends of the vessel wall are pulled toward each other,
reducing the gap size.
• Functions of platelets:
1. Platelet plug formation and arrest of bleeding: It occurs within 1-3
minutes after the vessel injury.
2. Release of platelet phospholipids and other factors that initiate
and aid in clot formation: Platelets play a role in intrinsic pathway
for clot formation.
3. Strengthening of the clot: Platelets (entrapped in the clot) release
a factor called fibrin-stabilizing factor. Clot is a meshwork of fibrin
threads. The fibrin-stabilizing factor causes covalent bonding
between the fibrin monomer molecules and adjacent fibrin
threads, thus strengthening the clot.
4. Clot retraction: Platelets entrapped in the clot are also responsible
for reducing the size of the clot. The contractile proteins within
these platelets would cause contraction of the platelet plug. The
- 42 -
clot size is reduced and the broken ends of the vessel wall are
pulled closer.
Clinical application:
(i) Bleeding time:
- The time interval between onset of bleeding and stoppage of
bleeding.
- Normal bleeding time is 1-3 minutes.
- Stoppage of bleeding is the function of platelets; with platelet
deficiency the bleeding time will prolong.
(ii) Thrombocytopenia & Purpura
- Decreased platelet count is called thrombocytopenia.
- As a result of a decrease in platelet count, the bleeding time is
prolonged. Bleeding into the skin, after minor injuries, leads to
appearance of purple colored spots in the skin. The condition is
called purpura.
(iii) Anti-platelet agents and “platelet aggregation inhibitors” in the
treatment of myocardial infarction and stroke:
- An abnormally formed (platelet plug + clot), within intact vessels,
is referred to as a thrombus. Such a thrombus threatens to
interfere with the blood supply to an organ.
- When a thrombus forms inside coronary vessels, it may result in
myocardial infarction (“heart attack”); thrombus within cerebral
vessels may result in stroke (“paralysis” or hemiplegia).
- Aspirin is known to be an anti-platelet agent; it reduces the
stickiness of platelets, preventing their coming together in a
thrombus formation. Clopidogrel is the drug that is called
‘platelet aggregation inhibitor’.
Let us recall the events that occur after a vessel is injured – (1)
Vasoconstriction, (2) Platelet plug formation.
The next event is clot formation or coagulation.
Many students tend to believe that the clot formation occurs in order to stop
the bleeding. Actually, it is the platelet plug that has already stopped the
bleeding. Why is then the clot formation necessary when bleeding has
already stopped? (Bleeding stops in 1-3 minutes; clot formation occurs in 4-9
minutes after the injury to the vessel wall.)
- 43 -
▪ Platelets are the cellular elements of blood. The platelet
plug is an important early tool for a quick limitation of blood
loss. However, this hemostatic plug may interfere with
fluidity of blood and patency of the vessels. Hence, the
cellular elements (platelets) in the plug are lysed and
replaced by fibrin.
▪ The platelet plug is a quicker but temporary response. The
platelet aggregation and adhesion properties have a limited
time-span. However, the injury in the vessel wall takes
longer to heal. Thus, even as the platelets are removed
from the hemostatic plug, the hole in the vessel is kept
sealed by the fibrin mass which too is gradually digested
and the vessel is healed.
- 44 -
fibrin monomer molecules and the adjacent fibrin threads,
resulting in strengthening of the fibrin meshwork.
- 45 -
The clotting factors in plasma:
Factor I Fibrinogen
Factor II Prothrombin
Factor III Tissue thromboplastin
or tissue factor
Factor IV Calcium
Factor V Labile factor
(proaccelerin)
Factor VII Stable factor
Factor VIII Anti-hemophilic factor
(AHF) or anti-
hemophilic globulin
Factor IX Christmas factor
Factor X Stuart-Prower factor
Factor XI Plasma thromboplastin
antecedent (PTA)
Factor XII Hagmen factor (contact
factor)
Factor XIII Fibrin-stabilizing factor
Other factors involved High-molecular-weight
in coagulation kininogen (HMWK),
Prekallikrein,
Platelet phospholipids
- 46 -
• The clotting factors are present in inactive forms in the plasma. When coagulation is
initiated, these factors are activated. The activated factor is mentioned with a
subscript ‘a’; for instance, Factor X to Xa.
• All the factors are synthesized by the liver, with the exception of factor VIII which is
synthesized by endothelial cells.
• Vitamin K-dependent factors: factors II, VII, IX, and X are called vitamin K-dependent
factors. Vitamin K is a necessary co-factor for the enzyme that catalyzes the
conversion of glutamic acid residues to -carboxyglutamic acid residues. The factors
II, VII, IX, and X require this conversion before being released into circulation.
- 47 -
- 48 -
2. Intrinsic pathway for coagulation: (Formation of prothrombin activator
is initiated with trauma to the blood itself, or exposure of the blood to
subendothelial collagen in the injured vessel wall, or exposure of the
blood to a water-wettable surface, such as, glass)
▪ The mechanism of clot formation has been described as “enzyme
cascade reaction” (previously also called as “waterfall sequence
theory”).
“Enzyme” – Once a factor is activated, it acts ‘enzymatically’ to
activate the next factor, and so on.
“Cascade” – Cascading effect is a domino effect; the initial
reaction is with a small amount of substance, and with each
subsequent step the reactions become larger so as to make a
large final product.
- 49 -
Intrinsic mechanism for formation of prothrombin activator
XI
XIa
IX IXa
Factor X
PROTHROMBIN
THROMBIN
FIBRINOGEN
- 50 -
Intrinsic pathway for clot formation:
➢ Triggered by:
▪ Trauma to blood itself, or
▪ Blood coming in contact with subendothelial collagen (in the
vessel wall), or contact of blood with a water-wettable surface,
like glass.
Activation of Factor XII [Hagemen factor or contact factor] is the
initiating event for the intrinsic mechanism of clotting. That is, it
is the factor XII that comes in contact with subendothelial
collagen or glass, and the clotting is triggered.
• Activation of factor XII; Release of platelet phospholipids:
The above mentioned triggers would convert factor XII to XIIa. This
activation is catalyzed by high-molecular-weight-kininogen (HMWK)
and kallikrein.
- 51 -
is thus formed as a complex of Xa + factor V + platelet phospholipids.
[Note: Factor VIII is activated when it is separated from von Willebrand
factor (vWF).]
Platelet phospholipids are released by the platelets aggregated in the
platelet plug.
The remaining steps would then follow: Prothrombin activator will act
on the prothrombin (present in plasma) to form thrombin. Thrombin will
then act on fibrinogen (present in plasma) to form fibrin.
• Role of Ca++:
- Calcium ions are necessary for all the steps in clotting, except for
the first two steps in the intrinsic pathway.
- Without the ionic calcium, clot formation will not be possible.
However, in vitro, blood Ca++ never falls so low that it’s
deficiency will hinder clot formation.
- 52 -
Clinical application
Abnormalities of hemostasis
▪ Thrombosis:
- A clot formed, under abnormal circumstances, inside a vessel is called
thrombus; the phenomenon is called thrombosis.
- An abnormal clot or thrombus is formed within a vessel under
following conditions – (i) Sluggish blood flow: It allows the activated
clotting factors to accumulate, (ii) Injury to the intima of a vessel: For
instance, as occurs in atherosclerosis.
- A thrombus in coronary vessel, blocking blood flow to myocardium,
results in myocardial infarction. A thrombus in cerebral circulation
leads to stroke.
▪ Liver disease and clotting abnormalities:
- Severe liver disease results in clotting defect and excessive bleeding.
- In severe liver disease, bile salt deficiency causes a deficient absorption
of fats and fat-soluble vitamins. Vitamin K is a fat-soluble vitamin. Its
deficiency leads to insufficiency of the vitamin K-dependent factors (II,
VII, IX, X, and protein C). This can lead to a severe bleeding tendency.
▪ Hemophilia: (“phil” = love or attract. Excessive bleeding occurs into tissues as if the
tissues ‘attract’ blood.) It is an inherited disorder that results from a
congenital deficiency of factor VIII or factor IX.
- Hemophilia A or classic hemophilia is caused by factor VIII deficiency.
- About 15% of cases of hemophilia are caused by deficiency of factor IX
(Christmas factor) – hemophilia B or Christmas disease.
- von Willebrand disease results from deficiency of von Willebrand factor
(vWF).
- Factors VIII & IX are transmitted genetically via the female
chromosome as a recessive trait. Hemophilia occurs almost exclusively
in males; females do not have hemophilia because at least one of their
two X chromosomes has the genes that code for these clotting factors.
- Hemophilia is manifested as excessive bleeding tendency.
- 53 -
After the 3 steps in hemostasis (vasospasm, platelet plug formation, and clot
formation), the next event is:
➢ Clot retraction; dissolution of the clot:
(a) Clot retraction; SERUM
- Within 20-60 minutes after clot formation, the clot begins to
retract; it reduces in size.
- The contractile filaments – actin, thrombosthenin – of the
platelets within the platelet plug would contract. Two things are
achieved by this: (i) The injured ends of the vessel are pulled
closer so as to reduce the gap size. (ii) Clot size is reduced, so that
it does not obstruct the blood flow.
- As the clot contracts, the plasma/fluid entrapped within the clot is
extruded out. This fluid is called serum.
Thus, serum = plasma – the clotting factors.
Eventually, the clot is invaded by fibroblasts, which will form connective tissue
throughout the clot.
Dissolution and lysis of the clot:
- Plasminogen is a plasma protein; it is activated to form plasmin.
Plasmin is a proteolytic enzyme (similar to trypsin). It digests the
fibrin threads in the clot, and the clot will be lysed.
- 54 -
▪ Thrombomodulin-thrombin complex: All endothelial cells have a
protein called thrombomodulin bound to their surfaces. [The only
exception is cerebral microcirculation.] Thrombomodulin binds
thrombin. Thrombin is a procoagulant that activates factors V and VIII.
However, when thrombin binds to thrombomodulin, thrombin becomes
an anticoagulant. The thrombin + thrombomodulin complex activates a
plasma protein called protein C.
Activated protein C (APC), along with its cofactor protein S, inactivates
factors V and VIII.
Clotting mechanism is a “positive feedback” process. How is this process
limited? How is excessive spread of the clot prevented?
▪ Antithrombin III: It is a protease inhibitor present in plasma. It
binds to and inactivates some clotting factors. Activated
factors IX, X, XI, XII are the factors that are inhibited by
antithrombin III, thus blocking them after a certain amount of
clot is formed.
Thrombin and factor V are involved in a positive feedback that
will form more and more thrombin. However, it is blocked as
thrombin combines with antithrombin III; thrombin gets
inactivated.
▪ Fibrin threads that are formed during the process of clotting:
Thrombin is adsorbed by these developing fibrin threads. This
prevents the spread of thrombin further into the nearby blood,
and excessive spread of the clot is prevented.
▪ Role of heparin:
o Heparin is a naturally occurring anticoagulant in blood.
o It is produced by – (a) basophils in blood, (b) mast cells
located in the pericapillary connective tissue.
o Heparin acts via antithrombin III. It facilitates the binding of
clotting factors to antithrombin III, thus inhibiting the active
forms the clotting factors IX, X, XI, XII.
o Heparin also removes thrombin via its facilitation of
antithrombin III.
▪ Dissolution of the clot: fibrinolytic system
- Plasminogen is a plasma protein; it is an inactive precursor that
when activated forms a substance called plasmin or fibrinolysin.
- Plasmin is a proteolytic enzyme that resembles trypsin. Plasmin
digests the fibrin threads, which will result in dissolution of the
clot.
- 55 -
Also note: Plasminogen receptors are present on intact endothelial
cells. When plasminogen binds to these receptors, it becomes
activated. Thus, intact blood vessels are provided with a mechanism
that discourages clot formation.
- 56 -
Clinical application:
- Streptokinase and urokinase are tissue plasminogen activators (t-
PA). Streptokinase is a bacterial enzyme produced by some strains
of streptococci.
- Human t-PA is produced by recombinant DNA technology.
Streptokinase thus produced is being used in the treatment of
early myocardial infarction (MI) and stroke. [A clot in a coronary
vessel results in obstruction to the blood flow to a part of the
myocardium, resulting in myocardial ischemia and infarction. A
clot in a cerebral vessel results in stroke.]
- Streptokinase is useful if given in an evolving MI. When given
within first 4 hours after the onset of MI, it can save the
myocardium by causing clot lysis and restoration of blood flow.
• Anticoagulants:
(A) In-vitro: (These anticoagulants are used outside the body to store
blood, as a sample for lab analysis or as blood pint for trasnfusion.)
- 57 -
▪ Double oxalate is a mixture of ammonium oxalate (3 parts)
+ potassium oxalate (2 parts).
▪ Mechanism of action: precipitation of Ca++ as insoluble
oxalate crystals
(B) In-vivo: (These anticoagulants are used in the body, when there is
tendency for abnormal clot formation; such clots may obstruct the
circulation and can have serious consequences.)
1. Heparin: [Heparin was thought to be synthesized by the liver parenchyma;
hence the name ‘heparin’.]
- It is a naturally occurring anticoagulant.
- It facilitates the action of antithrombin III.
- Synthetically made heparin is used in the clinical conditions that
are associated with abnormal tendency for clot formation; e.g.
Myocardial infarction and stroke.
- 58 -
Section 5
Blood groups
Learning objectives:
- Certain situations and clinical conditions (accidents, surgeries,
etc) result in loss of blood volume. Many of them require blood
transfusion, so as to restore the lost volume. Matching of blood
groups of donor and recipient is necessary. Transfusion of
incompatible blood results in hemolysis in the recipients.
- Incompatible blood groups of mother and fetus may result in
hemolysis in the newborn.
- It is important to understand the presence or absence of blood
group antigens and their corresponding antibodies, in order to
learn the blood grouping, and consequences of mixing of
mismatched blood.
Introduction –
The population is divided into various groups, based on presence of
certain antigens on the RBC membrane.
Carl Landsteiner discovered the first blood group system (ABO system) in the
year 1900. Since then, about 20 blood group systems have been devised. (ABO,
Rh, Kell, Duffy, Lewis, M and N system, etc.)
Each blood group system has some specific significance. For instance, M and N
system is mostly used in paternity disputes.
The two systems that are clinically most important are - ABO and Rh systems.
• ABO blood group system:
(A) Antigens:
- ABO blood group system is based on two antigens – A and B.
- The blood group antigens are present on the surface of RBC.
These antigens present on red cell membrane are called
agglutinogens. [They react with agglutinins or antibodies; the
reaction is “agglutination reaction”.]
- Based on the presence or absence of the antigens, there are 4
blood groups in ABO system – A, B, AB, and O.
If a person has only A antigen on his red cells, his blood group is A.
If only B antigen is present – blood group B.
Both A & B antigens present – blood group AB.
- 59 -
Neither A nor B present – blood group O.
(B) Antibodies: (agglutinins)
- There are antibodies against antigens A and B. Antibody against A
is called “anti-A” or alpha (); antibody against B is called “anti-B”
or beta ().
- The antibodies and are present in the plasma.
[Example: Consider any blood group, say A. The person has A antigen on his red cells, he will not have
anti-A (or ) antibody in the plasma. He does not have B antigen on the red cells; so he will have anti-B
(or ) antibody in the plasma.]
• Explanation:
[The body’s immune system has got the ability to recognize and
distinguish between “self” and “non-self” (foreign). “Self” means those
proteins or substances which are present in the body since inception. It is
the body’s own substance. So, immune system does not form antibodies
against it. And, the substances or proteins which are “non-self” or
foreign, the immune system will form antibodies against them.]
Consider Landsteiner’s law now.
1. If a particular antigen is present on the red cells since the beginning,
this antigen will be considered as “self”. Antibodies will not be
formed against it. Thus, A blood group person will not have anti-A in
plasma; B blood group will not have anti-B in the plasma, and so on.
2. Second sentence of the Landsteiner law should be understood in
detail. If a particular antigen is not present on the red cells, the
antibody to that absent antigen is present in plasma of that
individual. From where this antibody has come?
It should be noted that: A & B antigens are NOT ONLY present on the
RBCs. They are present elsewhere also. They are also present outside
the body, in the nature. When a baby is born, it gradually gets
exposed to these A and B antigens (through food, milk, bacteria).
- 60 -
Now, if a person’s group is A, and he gets exposed to both A and B
antigens after birth – A antigen entering the body will be considered
as “self”, and antibodies will not be formed against it. And, B antigen
entering the body will be considered “non-self” (since B antigen is not
present in this person), and antibodies will be produced against it.
Thus, antigen and antibody put together, blood groups can be written as
follows: A, B, AB, O
Blood group Antigen (on RBC) Antibody (in plasma)
A A Anti-B (or )
B B Anti-A (or )
AB A and B -
O - Anti-A and anti-B ( &
)
- 61 -
‘AB’ blood group does not have any antibodies in plasma. So even if any other
blood group’s blood, with antigens (A or B), enters recipient’s blood, there is
no possibility of reaction. Hence, AB was regarded as “universal recipient”.
This was the explanation given for those terms. However, many new antigens
and their corresponding antibodies have been discovered since then. Hence,
the concepts of “universal” donor and recipient are not considered to be
entirely appropriate now.
• Bombay blood group: {It was discovered by the Bombay Hospital pathologists – Dr.
Bhende et al; and is found in Bombay population, hence the name.}
On chromosome 9, there are two genes – A & B. A and B antigens are formed according
to the dominant A or B gene in a person. E.g. in a person with A gene dominant, A
antigen will be formed on red cells and his blood group will be A. If B gene dominant –
blood group B; both genes dominant – blood group AB. If both A & B genes are recessive
– the blood group will be ‘O’.
There is an independent gene “H” on the same chromosome. This H gene is always
dominant, and it forms H-substance. This H-substance is necessary for the formation of A
and B antigens on red cells.
In Bombay blood group persons, the H-gene is recessive (‘h’). H-substance is not formed
in them. Consequently, even A and B antigens are not formed in them. Their blood group
is “O”. But this “O” is not because A and B genes are recessive, but because H-gene is
recessive (‘h’). Thus, Bombay blood group is “O”, and it may be written as O“h”. These
individuals have 3 antibodies – anti-A, anti-B, and anti-H – in their plasma. They should
receive blood from another Bombay blood group only.
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• Rh system:
- Rh blood group system is based on the “Rh” antigen. [Rh ~ Rhesus;
the Rh antigen was found to be present in the Rhesus species of
monkeys; hence the term “Rh”.]
- If Rh antigen is present on the red cells, the person is called “Rh-
positive”; if it is not present, the person is said to be “Rh-
negative”.
- 85-90% population is Rh-positive; 10-15% is Rh-negative.
- While noting the blood group of an individual, antigens of both
ABO system and Rh system are mentioned. E.g. B +ve means B
antigen and Rh antigen present.
- Rh antigen is of 6 types, structurally. The 6 types are:
C, c, D, d, E, e.
- In majority individuals (almost 95%) – D type is found. Hence, Rh
antigen is sometimes called D-antigen also.
• Anti-Rh antibody:
It is IgG type of immunoglobulin.
It is not present naturally in any individual (unlike the anti-A or anti-B
antibodies). For antibody formation, a person should be exposed to an
antigen which is considered by the body as “non-self”.
- In Rh +ve individuals: Anti-Rh antibody will not be formed as Rh-
antigen is “self” for them.
- In Rh –ve individuals: (by the 2nd part of Landsteiner’s law, anti-Rh
antibody should have been present since the Rh antigen is not
present) – Rh negative individuals have never been exposed to Rh-
antigen. Hence, they too would not have formed anti-Rh antibody.
Note:
Rh antigen is present ONLY on the RBCs, in blood. Compare this
with A & B antigens, which are present in blood as well as outside
the body. And, A & B antigenic substances enter the body
naturally after birth. Hence, anti-A or anti-B antibodies would
form naturally.
In the case of Rh group, there is only one possibility of formation
of anti-Rh antibody - if an Rh-negative person gets exposed to
Rh-positive blood. Only then, this Rh-negative person would
form anti-Rh antibodies.
[Anti-A and anti-B antibodies are called “natural” agglutinins; anti-Rh antibody
is called “induced” agglutinin.]
• Erythroblastosis fetalis: [Hemolytic disease of newborn]
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It is the disease occurring in fetus/newborn baby.
It occurs due to “Rh incompatibility between mother and fetus”. (That
is, Rh blood group of mother and fetus not compatible)
- Consider a woman of Rh-negative group. Let us also assume that
she has never been exposed to Rh-positive blood.
1. First pregnancy: When this woman conceives for the first time,
and if the fetus is Rh-positive (Rh-antigen inherited from the
father), the mother is likely to be exposed to the Rh-antigen
from the fetus in her uterus.
Throughout the pregnancy, the mother does not get exposed
to the fetal Rh-antigen. Reason: The placenta present in her
uterus acts as a selective barrier; fetal antigen cannot cross
placenta to enter mother’s circulation.
2. First delivery: However, at the time of delivery or if some
obstetric manipulation is applied to assist the delivery, the
placenta may be separated and may not be an effective
barrier. At this time, some fetal blood may enter maternal
circulation (“feto-maternal bleed”). Even a small quantity of
fetal blood (1 to 2 mL) will cause sufficient antigen to enter
mother’s circulation.
3. After the first delivery: The fetus will be delivered, but antigen
that has entered mother’s body for the first time will start
forming antibodies in the mother. These anti-Rh antibodies
begin to be formed within 48-72 hours of delivery, reaching
peak titre by 2-4 months.
Now, the mother has anti-Rh antibodies in her plasma.
4. Second pregnancy: If the woman conceives again, the anti-Rh
antibodies in her plasma would be a threat to the fetus if the
fetus is Rh-positive. (If fetus Rh-negative – no problem; fetal
cells do not have antigen that can react with mother’s
antibodies.)
Anti-Rh antibodies in mother’s circulation are of IgG type; they
can cross the placenta to enter fetal circulation. Those
maternal antibodies begin to enter fetal circulation now. The
antibody titre increases gradually.
5. Antigen-antibody reaction in fetus: This results in breakdown
of fetal RBCs (as antigen is present on RBCs).
Fetus is born with: (a) anemia, (b) jaundice.
Anemia is due to massive breakdown of RBCs.
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RBC breakdown liberates Hb; heme of this Hb is converted into
bilirubin. This excess bilirubin leads to jaundice.
In response to the breakdown of RBCs, fetal bone marrow produces RBCs at a
higher rate; and immature RBCs (erythroblasts) are poured in circulation –
hence the name ‘erythroblastosis fetalis’.
The condition may be life-threatening.
Treatment:
1. Preventive:
Aim is to prevent antibody formation in mother (after 1st delivery).
Mother’s immune system starts antibody formation after 48-72 hours of
antigen entry. Before this antibody formation begins, mother is given
anti-Rh serum (serum that contains pre-formed anti-Rh antibodies). The
injected antibodies will coat the Rh-antigen and destroy it. Thus, the Rh-
antigen cannot sensitize mother’s immune system and the immune
system will not form anti-Rh antibodies.
2. Curative:
If the preventive measure could not be taken after first delivery, and the
second fetus is born with anemia & jaundice – exchange transfusion (for
anemia) and phototherapy (for rapid clearance of bilirubin) is given to
the fetus.
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Blood transfusion:
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needle is inserted in the vein, blood starts flowing out into the
bottle that contains anticoagulant.
About 480 mL of blood is collected in the bottle that contains 120
mL of anticoagulant. Throughout the procedure, bottle is shaken
gently so that blood and anticoagulant mix properly.
Anticoagulant used: Acid-citrate dextrose [ACD].
Blood is then stored in the refrigeration at 0 to 40C.
Donor Recipient
Cells Cells
Major
Minor
Plasma Plasma
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~ Mixing of donor’s cells with recipient’s plasma is called major cross-
matching.
Mixing of donor’s plasma with recipient’s cells is called minor cross-matching.
Explanation:
▪ Major cross-matching: If donor’s red cells have some antigen, and
recipient’s plasma has specific antibodies against this antigen, when
donor cells are mixed with recipient’s plasma there will be agglutination
reaction seen. It means, donor cells have some antigen which is “non-
self” for the recipient and hence recipient forms antibodies against that
antigen. If in such a case, donor’s blood is given to the recipient,
recipient will form further antibodies against donor cell antigens. And,
those antibodies will react with the donor antigen (agglutination
reaction), resulting in hemolysis in the recipient. Such transfusion should
not be given. And, this cross-matching is called “major” cross-matching.
▪ Minor cross-matching: If donor’s plasma has certain antibodies which
are against an antigen present on the recipient’s cells, mixing of donor’s
plasma with recipient’s cells will also show agglutination reaction.
However, consider the transfusion scenario. Approx. 500 mL of the
donor’s blood is given to the recipient. Of this, about 260-270 mL would
be plasma. Antibodies present in this much (small) amount of plasma
will get diluted to a great extent when they enter recipient’s 5 L blood.
Hence, those antibodies will not react in a major way with the antigen
on recipient’s cells. Hence, mixing of donor’s plasma sample with
recipient’s cell sample is called “minor” cross-matching.
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o Hazards of blood transfusion:
[complications in the recipient, after receiving blood]
1. Thrombophlebitis – Inflammation of the vein through which
the blood is given. If the inserted needle is left in the vein for a
long period, it leads to this complication.
2. Congestive heart failure – it may occur in elderly patients with
cardiovascular status compromised.
3. Transmission of infection – HIV, hepatitis B (if the donor’s
blood was not screened properly); fever and chills (if donor’s
blood has pyrogens)
4. Hazards of mismatched blood transfusion –
➢ Agglutination reaction in the recipient:
Mismatched blood means ~ donor’s blood contained
antigen against which the recipient’s plasma contains
antibodies. The agglutination reaction will occur in the
recipient, resulting in massive hemolysis.
➢ Anemia:
Hemolysis (breakdown of RBCs) will lead to anemia in the
recipient.
➢ Jaundice:
RBC breakdown results in liberation of Hb; heme of the Hb
is converted to bilirubin. It will lead to development of
jaundice.
➢ Acute renal shutdown:
Free hemoglobin in plasma → combines with haptoglobin,
forming a large complex. As this complex passes through
kidney, kidneys are unable to handle it. It may result in
acute renal failure.
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