HEMATOCRIT

Blood collected in hematocrit tube is

centrifuged for 30minutes @ 3000 rpm
(revolutions per minute)—see next slide
for photo
2. the red cells settle at bottom (45%)
3. clear plasma at top (55%) and
4. in between the two a thin buffy white
layer having WBCs & platelets
Hematocrit: The Volume of Red Cells as
% of the
total blood is called Hematocrit or Packed
Figure 18.1a
 Blood Plasma
• 92% water
• 7% proteins
– synthesized mainly by Liver
– albumin 54% -maintain blood osmotic
pressure
– globulins 38%-forms antibodies or
immunoglobulins
– fibrinogen 7%-blood clotting
• 1% solutes other than proteins
– Carbohydrates, fats, electrolytes,
amino acids, enzymes, NPN(non
protein nitrogen like NH3, Creatinine,
Urea, Creatine & Xanthine), gases
 Plasma Proteins
Normal values:
Total Plasma proteins-7.3gm%
Serum albumin-4.7gm%
Serum Globulin-2.3gm%
Fibrinogen-0.3gm%
Albumin-Globulin ratio- It is an
important indicator of some kidney or
Liver diseases. Normal A/G ratio-2:1
 Separation of Plasma Proteins
1. Precipitation Method- )Used to
separate albumin from globulin) on
adding 22% sodium sulphate solution to
plasma, globulin becomes a precipitate
while albumin remains in solution.
2. Salting Out Method (used to
separate globulin into its fractions
euglobulin & pseudoglobulin):
c. Euglobulinis salted out with sodium
chloride & is insoluble in water while
d. Pseudo-globulin is salted out with
 Separation of Plasma Proteins
(contd)
3. Electrophoresis:
a. Basis: Different plasma proteins have
different electrical charge on them so have
different rates of migration.
b. Technique- dome on Tiselius apparatus
by paper or cellulose or starch block.
c. By this proteins are separated into
albumin (55%), alpha globulin (13%), Beta
globulin (14%), gamma globulin (11%) &
Fibrinogen (7%). See nxt slide photo
Plasma proteins
 Separation of Plasma Proteins
(contd)
4. Ultracentrifugation Method- Different
plasma proteins have diff densities is the
basis of this technique. This also
determines molecular weight of plasma
proteins.
Molecular Weight
Albumin- 69,000
Globulin-1,56,000
Fibrinogen-4,00,000
5. Immuno electrophoresis:
Electrophoretic pattern formed by antigen-
 Osmotic Pressure-
Starling’Hypothesis
Osmotic Pressure exerted by plasma
proteins plays an important role in
exchange of various substances between
blood & tissues via cappilary membranes.
According to Starling Hypothesis – the Net
Filtration through capillary membrane
is proportional to the hydrostatic
pressure difference across the
membrane minus the oncotic
pressure difference.
 Functions of plasma proteins
1. Coagulation of blood (fibrinogen)
2. Defence of body (immunoglobulins)
3. Transport- egiron is transported by plasma
protein transferin
4. Maintain osmotic pressure of blood.
5. Regulate acid base balance-as albumin is a
good buffer
6. Role in ESR- Globulin & fibrinogen
accelerate the rouleux formation in RBCs
which is responsible for ESR (erythrocyte
sedimantation rate)
7. During circulation because of globulin &
 Plasmapharesis
Def: it is used as a blood purification
procedure for treating autoimmune
disorders. Also called therapeutic plasma
exchange.
Basis- In some autoimmune disorders
antibodies produced in body for defense in
fact kill own body cells. So if these
antibodies can be removed, autoimmune
damage to cells can be prevented.
Procedure: venous blood of patient is
passed through cell separator machine.
After this machine separates cells from
 Plasmapharesis (contd)
Uses:
1. idioptahic thrombocytopenic purpura
(ITP)Antibodies produced in body for defense kill
own leading to bleeding tendency. Plasmaparesis is
used to remove these antibodies
2. Gullian barre Syndrome in which paralysis of
legs occurs due to auto-antibodies directed against
own nervous system. It is fatal disease &
PLASMAPHARESIS IS LIFE SAVING PROCEDURE .
3.Myasthenia gravis-autoimmune muscle
weakness
4. Chronic demyeliniating polyneuropathy-
autoimmune damage to myelin sheath leading to
 RBCs-Important questions
1. Cytoskeleton of RBcs- actin & spectrin adhered
to membrane protein of RBC by another protein called
ankyrin.
Hereditary Spherocytosis- is due to deficiency of
spectrin. In this cells are spherocytic and fragile and
cause hemoysis.
2. Rouleux Formation: When blood is taken out RBCs
become spiles one above each other- called rouleux
formation.
3. Pathological Polycythemia: RBC count > 7
million/cumm. Two types:
e. primary polycythemia (or polycythemia vera)-
both RBC & WBC count extremely high, occurs in
mailignancy of bone marrow.
f. Secondary Polycythemia- occurs due to lack of
 Factors Necessary for
A. General-Erythropoeisis
1.EPO (erythopoetin) EPO- A
glycoprotein secreted by
Peritubularcappilaries of kidney. its release
is
stimulated by Hypoxia. On release in 4-5
days it
increases nucleated RBCs in circulation by
following
actions:
g. Production of pro-erythroblasts from
 Factors Necessary for Erythropoeisis
(contd)
2. Thyroxine : reqd for normal body
metabolism
3. Hempoetic Growth factors –Growth
inducers like interleukin- proliferation of
pleuri-potent stem cells.
IL-3 , IL-6,IL-11 are associated with
erythropoeisis.
4. Vitamins:
e. B12 (extrinsic factor) reqd. for
maturation of RBCs-deficiency causes
Pernicious anemia, a type of
megaloblasticanemia. B12 reqdf for DNA
 Factors Necessary for
Erythropoeisis (contd)
5. Factors reqd for Hb formation- iron,
proteins, copper, cobalt & nickel, Vit C,B2,
B5,B6.
 ESR
When blood mixed with an anticoagulant and
allowed to stand still vertically in a tube, the RBCs
settle down due to gravity and a clear supernatant
plasma layer foms on top. The rate at which the
RBCs settle down is called ESR.
2 methods to Determine ESR:
1.Wintrobe: short tube 110 mm open at one end
only, 1ml blood mixed with EDTA (anticoagulant) is
put in it up to’0’ mark & kept for one hour in
wintrobe stand vertically. It is read after 1 hr. Also
used for PCV estimation.
2. Westergreen-long tube 300mm open on both
ends. 1.6 ml blood mixed with 0.4 ml 3.8% sodium
Significance of ESR:
Easy, cheap. non secific test increased in
any infection or inflammation any where in
body.
But on treatment of infection its value
comes down showing improvement so it
can be used to assess response of
treatment.
Eg in TB cases, in Joint pains due to
Rheumatoid arthritis or acute rheumatic
fever.
 Blood Indices
1. Mean Corpuscular Volume- (MCV): it
is average volume of of a single RBC in
cu.mm.
Normal -90 cumm (78-90)
Macrocyte- Increase in RBC volume.eg in
pernicious &
megalobasltic anemia
Microcyte: Decrease in RBC volume.eg
Iron Deficiency
anemia
2.Mean Corpuscular hemoglobin:
(MCH): It is the quantity of Hbpresent in
one RBC, expressed in mico micro (or
picograms).
Normal MCH: 30 pg (27-32): called
Normochromic
Normal or decreased in Pernicious or
megalobalstuc anemia (so RBCs in this
anemia are macrocytic & normochromic
or hypochromic)
Decreased in Iron deficiency anemia
3. Mean Corpuscular Hemoglobin
Concentration (MCHC) :It is the conc of
Hb in one RBC ie amount of Hb in relation
to volume of RBC & its Unit is %. It is the
most important absolute value in the
diagnosis of anemia.
Normal- 30 % (30-38%)
Decreased-Iron def anemia.
4.Color index- Ratio between the % of Hb
& the % of RBcs in blood. Normal-1.0 (0.8-
1.2). High in pernicious anemia, low in iron
 Calculation of Blood Indices
Suppose RBC count-4milion/cumm, Hb-
8gm%, PCV-30%
2. Color Index: Hb%/RBC%
Hb%= Hb in person/Normal Hb expected X
100
Hb%= 8/15 X 100= 53.3%
RBC%= RBC count in person/RBC count
expected X 100
RBC%= 4/5 X 100= 80%
So C.I.= 53.3/80=0.67
 Suppose RBC count-4milion/cumm, Hb-
8gm%, PCV-30%

2.Calculate MCV
MCV= Volume of Packed cells in mlper
1000ml of blood / RBC count in milions per
cummof blood
Volume of Packed cells in mlper
1000ml of blood= 30 X 10= 300
MCV= 300/4= 75 cumm
 Suppose RBC count-4milion/cumm, Hb-
8gm%, PCV-30%

3. Calculate MCH-
MCH= Hb in gm per 1000mlof blood/ RBC
count in million per cumm
Hb in gm per 1000mlof blood- 8 X 10 = 80
gm
MCH= 80/4=20 pg
4. Calculate MCHC-
MCHC= Hb%/PCVX 100 = 8/30 X
100=26.67%
 Fragility of RBCs
Hemolysis means destruction of RBCs
It can be either due to
c. Osmotic fragility- ie RBC breakdown in
hypotonic saline
d. Mechanical fragility- RBC breakdown
due to mechanical trauma
Fragility Tests:
 Fragility Tests
Done by using sodium chloride from 1.2 to 0.2 %
CONCENTRATION. Solutions of diferent strength are
made in Cohn’s tubes.
Then one drop of blood to be tested is put in each tube,
mixed well and left for sometime.
a. Direct Observation of tube: If no hemolysis-
fluid is turbid, if hemolysis starts-turbidity decreases,
if hemolysis complete-turbidity disappears &
fluid is clear.
b. Do centrifuge & see- If no hemolysis- cells
settle at bottom with clear fluid above, if
hemolysis starts- cells sediments less and fluid slightly
reddish, if hemolysis complete-the fluid is more
reddish and no sediments.