THE BLOOD

BLOOD

One of the largest organs of the body.

An average 70 kg man has almost 5L
blood (5.5 kg).
Blood circulates throughout the body
and supports the functions of all other
body tissues.
 Blood integrates
tissues and organs
and provide
a special means of
communications.
 THE FUNCTIONS OF BLOOD
Respiration: transport of O2 and CO2.
Transport: hormone, nutrients, metabolic waste.
Excretion of metabolic wastes to the kidney, lungs
and skin.
Regulation of body temperature by distribution of
body heat.
Defense against infections (WBCs, antibodies).
Maintenance of acid-base balance.
Nutrition: transport of absorbed food material.
 PHYSICAL PROPERTIES OF BLOOD

Specific gravity:
- Whole blood: 1.055 - 1.065
- Plasma: 1.024 - 1.028
Viscosity: 5-6 times that of water.
Mass: 6-8% of the body weight.
Blood volume: ~ 8% of body weight.
~ 86% ml/kg body weight.
5-6L in adults
[Infants have a larger blood volume in proportion to
body weight than adults].
Osmotic pressure: 7-8 atmosphere at body temperature.
 Composition of Blood

Formed Elements (45%),

i- Red blood cells (erythrocytes).

ii- White blood cells (leukocytes).
iii- Platelets (thrombocytes)

Fluid medium i.e. the plasma (55%).

 Gross composition of Plasma
and Blood Cells

Constituents Plasma Red blood cells

Water 91-95% 65%

Solid 8-9 % 35%

Protein 6-8 gm % 31-33%

Specific gravity 1.026

 Haematocrit or Packed Cell Volume (PCV)

Anti-coagulant
Anti-coagulant
Whole
WholeBlood
Blood No
Noclotting
clotting
(EDTA,
(EDTA,Oxalate,
Oxalate, Centrifugation
Centrifugation

Citrate,
Citrate,heparin)
heparin)

Plasma
Plasma

PCV
PCV
PCV
PCV==0.45
0.45L/L
L/L
==0.41
0.41L/L
L/L

PCV
PCV isisanaemia
anaemia
isispolycythaemia
polycythaemia
 SERUM

No anticoagulant
Whole blood Clot formation

Centrifuge
Clot +
Clear yellowish
fluid (serum)
 ERYTHROCYTE SEDIMENTATION RATE
(ESR)

Rate of Sedimentation of Erythrocytes.

ESR at 20 3 C (Westergress Method)
Male = 0-5 mm
Female =0-7 mm
- ESR - Non-specific indicator of infection.
- ESR - For monitoring status of chronic
inflammatory diseases.
 Haemopoisis

The process of formation of blood.

Erythropoisis
Formation of Leucopoisis
Erythrocytes Formation of
Leucocytes

Thrombopoisis
Formation of
Thrombocytes
Process Product
Erythropoesis RBC

Leucopoiesis WBC

Granulopoiesis Granulocytes

Lymphopoiesis Lymphocytes

Megakaryocytes Platelets
 Site of Haemopoisis

Fetal Life
1-2 m Yolk Sac
2-6 m Spleen
1-9 m Liver
from 4 m Bone marrow
At Birth Bone marrow
Adult life Bone marrow
 ErythropoisisA two stage
differentiation system

Stage 1: From Pluripotent stem cells

to committed cells

Stage 2: From committed cells to the

recognisable precursors
 Erythropoisis
Stem cell (pluripotent)
Committed stem cells( CFU)
Pronormoblast
Basophil normoblast
Polychromatic normoblast I and II

Orthochromatoblast
Reticulocytes
Erythrocytes
Control of Erythrocyte Synthesis
Cellular hypoxia Oxygen sensor

E-Releasing factor Serum

Erythropoietin (E) Kidney

Erythropoietin (E) Serum

Stem cell Bone Marrow

 Erythropoisis

Stem cell

Pronormoblast

Basophilic normoblast

Polychromatophilic normoblast

Orthochromatic normoblast

Reticulocytes

Mature red cell

 Current model of the feedback circuit which regulate the
rate of RBC synthesis to the need for O2 in the peripheral tissues.
(Surface receptors and intracellular secondary messenger):

Bone marrow
stem cell Erythroid tissue
DNA, mRNA

Erythropoitein Red cell mass

Atm.O2
Cardiopulmonary
function
Blood volume
Hb concentration
Renal vacc O2 affinity
Renal O2
Consumption Erythropoietin O2Sensor
Produce Epo

Kidney
(Liver, Macrophages)
 Erythropoiesis
The proliferation and differentiation of cells
from pleuripotent noncommitted stem cells
of the bone marrow.
Two main compartments:
 Erythropoiesis compartments

Erythropoiesis compartments

The erythroid progenitor cell compartment The erythroid precursor cell (erythron)
1.The erythroid progenitor cells
The earliest recognizable committed
progenitor for erythroid cells is the CFU-
GEMM.
The next are the BFU-E.
The final progenitor cells are the CFU-E.
 2. The erythroid precursor cell
It is the morphologically recognizable
erythroid cell within the normal bone
marrow.
 Cells involved in erythropoiesis
1.Pluripotent stem cell:
Most primitive
haemopoietic cell.
Extensive capacity
to proliferate.
Mature into other cell types:
Multipotent myeloid stem cell.
Lymphoid stem cell.
 Cells involved in erythropoiesis
2. Pronormoblast:
earliest recognized cell in erythron.
A large cell.
Basophilic cytoplasm.
Has a large nucleus.
High conc. Of m.RNA.
1% of protein is Hb.
1000 receptor/cell for Epo.
 Cells involved in erythropoiesis
3. Basophilic normoblast:
Nucleolus is lost.
The golgi apparatus remains
prominent.
 Cells involved in erythropoiesis
4. Polychromatophilic
normoblast:
Hb production.
smaller nuclear:cytoplasmic
ratio.
Chromatin is more clumped and
condensed.
 Cells involved in erythropoiesis
5. Orthochromatic normoblast:
cytoplasm is more eosinophilic.
Final nucleated stage.
> 300 receptors/cell for Epo.
 Cells involved in erythropoiesis
6. Reticulocyte:
No nucleus.
Reticular networkes of polyribosomes.
It enters blood stream and circulate for 1-2
days they become mature RBC.
Rounder, faintly polychromatic, larger
diameter than RBC.
95% of cell protein is Hb.
No receptors for Epo.
 Maturation
Maturation of the proerythroblast within the bone marrow
to the end of the basophilic erythroblast stages takes about
60 hrs.
Maturation of the polychromatophilic erythroblast takes
about 30 hrs, the late erythrocyte stage about 50 hrs and
the reticulocyte in the steady state remains in the bone
marrow for 40-48 hrs.
Transcription factors
for differentiation and maturation
Random process.
Epo prevent programmed cell death.
Commitment of haemopoietic cells to the erythroid lineage
involves action of several transcription factors including:
TAL1, LMO2, and GATA-2.
Genes for - and - chain of Hb are activated and
controlled by cis-acting DNA sequence.
Other specific proteins: glycophorin A and EpoR.
Transferrin and genes of haem synthesis are also activated by cis-
acting mechanism.
Transcription factors
for differentiation and maturation
Cis-acting control DNA is activated by trans-acting factor.
Trans-acting NF-E2 binds to specific sequence in locus
control region for -globulin, probably also to -globulin.
Other cis-acting promoter regions are involved in the
regulation of genes coding for enzymes of haem synthesis
including: porphobilinogen deaminase, ferrochetalase and
-amino laevulinic acid synthetase.
The m.RNA for these factors disappear after
proerythroblast stage.
Transcription factors
for differentiation and maturation
Upon binding Epo, cell surface EpoR dimerizes and
activates specific intracellular kinases including: Janus
family tyrosine protein kinase-2, phosphoinositol-3 kinase,
and mitogen-activated protein kinase, and the RAS
pathway.
Other trans-acting DNA binding proteins are the erythroid
Kruppel factor and the human stem cell leukemia genes.
Intrautreine erythropoiesis
and postnatal changes
Erythropoiesis occurs in two distinct waves
during embryogenesis:
The primitive wave in the extra-embryonic sac
of the 14-19 day human embryo.
The definitive wave in the fetal liver and spleen
are the main sites of erythropoiesis in the 2nd
trimester of pregnancy and the fetal bone
marrow in the 3rd trimester.
Intrautreine erythropoiesis
and postnatal changes
The placenta activates fetal erythropoiesis by producing
factors that stimulate erythropoiesis.
The major site of Epo gene expression in the fetus is in the
kidney.
After birth, during the 1st 4yrs of life, nearly all the
marrow cavities contain red haemopoietic marrow with
very few fat cells. By the age of 25yrs the no. of fat cells
increase.
 Erythropoiesis in adults
Erythropoiesis occurs within the
haemopoietic marrow.
Pregnancy is characterized by increased
erythropoiesis within the maternal and fetal
compartments.
 Blood

Cells Plasma
Blood Cells
 BLOOD CELLS
Erythrocytes (Red blood cells)
Leukocytes (White blood cells)
Granulocytes
Neutrophils
Basophils
Eosinophils
Monocytes
Lymphocytes
T
B
Megacaryocyte (Platelets)
 NORMAL RANGES
RBC
Men 4.6 6.2 x 1012/l.
Women 4.2 5.4 x 1012/l
Total number of red cells in circulation = 2.5x1013
WBC
Men and women 5-7 x 109/l.
Platelets
Men and women 250 x 109/l
Hb
Men 14 16 g/l
Women 12 16 g/l
PCV (Haematocrit)
Men 0.42 0.52 l/l
Women 0.37 0.47 l/l
 Red blood cells
(Erythrocytes)

Biconcave
Biconcavedisks:
disks: Highly
Highlyspecialized
specialized
-- Diameter
Diameter 66--99m
m
-- Thickness:
Thickness: 11--22m
m
-- Volume;
Volume; ~~88
88fl.
fl.

Deformable
Deformable-- i.e.
i.e.can
canchange
changeshape
shapeto
totransverse
transversesmallest
smallest
blood
blood vessels.
vessels.
Contain
Containhaemoglobin
haemoglobin(~ (~33%).
33%).
No
Nonucleus
nucleusor
ormitochondria.
mitochondria.
Function:
Function: Transport
TransportofofOO22and
andCOCO22..
Normal
NormalRange:
Range:
5.51.0
-- 5.5 12
1.0xx10
1012/L/L
4.8 1.0
-- 4.8 12
1012/L
1.0xx10 /L
 Red blood cells
(Erythrocytes)
Deliver oxygen to tissues and CO2 from
tissues to lungs.
Synthesis is increased by erythropoietin
Red cell life span is 120 days.
Senescent red cells are destroyed by spleen
and replaced by juvenile cells released by
bone marrow.
An average 70 Kg adult male produces
2.3x106 red cell/sec.
 ERYTHROPOIETIN
A polypeptide hormone.
Glycoprotein of 166 a.a. (mol. li 34 Kdl).
Major regular of human erythropoiesis.
Synthesized mainly in kidney, released in response to
hypoxia and arts on bone marrow.
Interacts with progenitor of red cells (BFU E) via
specific receptors causing proliferation and
differentiation.
Also interacts with late progenitor cell (CFU E) to
cause proliferation and differentiation.
Requires cooperation of other factors e.g. interleukin-3
and insulin like growth factor.
 ERYTHROCYTE STRUCTURE
Biconcave shape. Spherical.
Simple structure:
Membrane surrounding cytoplasm.
Almost 95% of solutes in cytosal is
haemoglobin.
No intracellular organnels
Non-nucleated
Has a cytoskeleton, which plays an
important role in determining shape.
Has deformability due to special structure
of cytoskeleton
Erythrocytes
Erythrocytes Composition:
Composition:

-- Major
Majorcation:
cation: KK++

-- Other + ++ ++
Othercation:
cation: Na
Na+,,Ca
Ca++,,Mg
Mg++

-- Major
Majoranion:
anion: --Cl
Cl
--HCO
HCO33
--Hb
Hb
--Inorganic
Inorganicphosphate
phosphate
--2,3
2,3diphosphoglycerate
diphosphoglycerate
HAEMOGLOBIN
 TYPES OF HAEMOGLOBINS
In Adults
Hb : ~97% 2 2
HbF : <1% 2 2
Hb A2 : 2.5 3.5% 2 2
At Birth
HbF : 2 2
Hb A : 2 2
During Embryonic life
Hb Gower 1
Hb Gower 2
Hb Portland
 HAEMOGLOBIN IN THE RED CELLS

Haemoglobin
Major solute in red cells.
Globular protein
Conjugated protein: globin + haem.
Made of 4 subunits (Quarternary structure)
4 globins + 4 haems haemoglobin.
Binds O2 to haem group to form
oxyhaemoglobin
Hb + 4 O2 Hb (O2)4.
Contd..
GLOBIN CHAINS OF HAEMOGLOBIN

Amino acids in globin chains

Globin 141 a.a.
-like globin chains: 146 a.a.
Structure of globin chains
Globular, compact structure
~75% -helices
Have a hydrophobic cavity for binding
heme.
 Haemoglobin
Globin Chains

Heam Group
 HEME GROUP

Protoporphyrin IX
Has tetra pyrolle rings linked together by
methylene bridges.
Fe++ coordinates with 4 N of the 4 pyrolle rings:
Bind with coordinate covalent bond to Histidine
F8.
Binds to O2 between Fe++ and His E7.
If Fe is oxidized to ferric (Fe+++) the Hb is
known as met Hb, which cannot binds O2.
STRUCTURE
OF HEME
GROUP
 HAEMOGLOBIN IN THE RED CELLS

Haemoglobin..Contd
Allosteric protein: has 4 O2 binding sites
O2 binding curve of Hb is sigmoidal.
Shows cooperative effect: i.e. binding of
some O2 molecules makes it easy for
other O2 molecules to bind.
O2 affinity of Hb is affected by pO2,
pCO2,, H+, 2,3 DPG.
 HAEMOGLOBIN IN THE RED CELLS

Haemoglobin..Contd
Affinity for O2 depends on partial pressure of
O2, CO2, and H+, 2,3 DPG level.
Binds CO2 to N-terminal of -globin chain to
form carbamino Hb.
Carboxy Hb.
Hb + 4CO Hb (CO)4.
Has high affinity for CO
 THE BOHR EFFECT

In Lungs:
High PO2, H+, CO2 high affinity of Hb for
O2 (O2 dissociation curve shifts to left).

In Tissues
Low P O2, H+, CO2, 2,3 DPG Low
affinity of Hb for O2 (O2 dissociation curve shifts
to right)
 THE BOHR EFFECT
Lungs CO2 Exhaled

2CO2 + 2H2O

2H2CO3
Carbonic Peripheral
Anhydrase Tissues
HCO3 - + 2H+ Hb + 4O2
4O2

4O2 Hb + 2H+ 2H+ + 2HCO3-

Carbonic
2H2CO3- Anhydrase

2CO2 + 2H2O
Generated by TCA Cycle
O2 Dissociation curve of Haemoglobin
Saturation%

100

50

26 pO2(Torrs)
 BINDING OF 2,3 DIPHOSPHOGLYCERATE

1 molecule of 2,3 DPG /Hb molecules

2,3 DPG binds between 2 -chains of HbA.
It is formed from 1,3 DPG (a glycolytic
intermediate).
In peripheral tissues level of 2,3 DPG is high. It
binds Hb and decreases affinity for O2.
HbF cannot bind 2,3 DPG and has higher
affinity for O2.
O2 can be transported from mother to fetal
blood
Red Cell Metabolism
 SUMMARY OF RED CELL
METABOLISM
Highly dependant on glucose as energy source.
Glucose is metabolized by:
Glycolysis (~ 95%)
Pentose phosphate pathway (~ 5%)
Glycolysis produces lactate + ATP
2,3 DPG regulates O2 affinity of Hb.
PPP produces NADPH, necessary for keeping red
cells in reduced state.
No synthesis of glycogen, fatty acids, proteins or
nucleic acids in red cells
Contd..
 SUMMARY OF RED CELL
METABOLISM
Contd
Reduced glutathione is important as it keeps the:
red cells and other proteins in reduced state.
reduces oxidizing radicals (peroxides) generated
in red cells
2GSH G-S-S-G

H2O2 2H2O

Iron of Hb is kept in reduced state (Ferrous, Fe++)

by NADH-dependant methaemoglobin reductase.
 GLUCOSE TRANSPORTERS IN RED CELL
MEMBRANE

Glucose uptake by red cells is by

facilitated diffusion.
Proteins involved in facilitated diffusion of
++ +
glucose are glucose transporters (~ 2%
to membrane protein of RBC).
Almost 7 different glucose transporters
have been identified in different tissue.
Glucose transporters in red cells
membrane are insulin-independent.
Glucose Metabolism in Erythrocytes

95% Oxidised by glycolysis

Glucose

5% Oxidized by pentose-
phosphate pathways
 The role of glycolysis in the functional requirements of
mature red cells:

Function EMP PPP

- Maintenance of shape ATP

- Membrane structure and

Function GSH

- Regulation of O2 transport 2,3-DPG

ATP

- Reducing potential NADP GSH

NADPH
 PRODUCTION OF POWERFUL OXIDANT IN
RED CELLS DURING METABOLISM
During metabolism, there is production of:
Superoxides (O2): O2 + e O2
Hydrogen peroxide (H2O2)
O2 + O2 + 2H H2O2 + O2
Peroxyl radicals (ROO)
Hydroxyl radicals (OH*)
These oxidizing radicals are highly reactive
molecules and can react with proteins, nucleic
acids, lipids and other mol. to alter their structure
and produce tissue damage.
Red cell need several reducing reactions to keep it
in reduced state and protect it from damage by
oxidizing radicals.
 PROTECTION OF RED CELLS
FROM HAEMOLYSIS
By:
Super oxide dismutase
O2- + O2- + 2H+ H2O2 + O2
Catalase:
H2O2 + 2H + 2H2O
Glutathione
2GSH + RO OH GSSG + H2O + ROH
Glutathione Oxidised Glutathione
 Glucose-6-Phosphate Dehydrogenase
(G-6-PD)

- G-6-PD is the first enzyme of the Pentose Phosphate Pathway.

- Catalyses the following reaction:
G-6-P + NADP + 6-Phosphogluconolactone+
NADPH+ H+
- NADPH is necessary for the red cell integrity and stability.
- Co-enzyme for glutathione reductase which converts oxidised
glutathione to reduced glutathione. This reduces oxidising radicles
and protects red cells from damage.
-Deficiency of G-6-PD leads to hemolytic anaemia under oxidative
stress(e.g. antimalarial drugs, fava beans, infections, diabetic
acidosis)
Other Blood Cells
 PLATELETS (Thrombocytes)
Discoid, anucleated cells with agranular
cytoplasm.
- Diameter = 3 m
- Thickness = 1 m
- Volume = 7 fl.
250x109 platelets/litre.
Synthesis increased by thrombopoietin.
Synthesised from megakaryocytes.
Contd..
 PLATELETS (Thrombocytes)
Contd...
Survival in circulation 10-12 days.
Primary role:
- in haemostasis: stick to the edges
of wounds and form a plug to arrest
blood loss.
Platelets also involved in development of
atherosclerosis and hence can lead to
thrombosis.
 White Blood Cells (Leucocytes)

Two Main Groups:

i. The Phagocytes ----------Play a role in protecting the
a- Granulocytes: the body against infection by
- Neutrophils phagocytosis.
- Eosinophils
- Basophils
b- Monocytes
ii. The Lymphocytes (immunocytes)--Function in protecting.
a- B-Lymphocytes-----------------Provide humoral immunity.
b- T- Lymphocytes----------------Provide cellular immunity.
Total leucocytes: 4.00-11.0x 106/l
 GRANULOCYTES

Have numerous lysosomes and granules

(secretory vesicles).
Also known as polymorphonuclear leukocytes
(PMN) as they have multilobular nuclei
Types of granulocytes:
Neutrophils,
basophils and
eosinophils
are distinguished by their morphology and staining
properties of their granules.
FUNCTIONS OF GRANULOCYTES

Neutrophils: Phagocytose bacteria and play

a major role in accurate
information.
Basophils: Resemble mast cells and
contain histamine and heparin
play a major role in immunologic
hypersensitivity reaction.
Eosinophils: Involved in certain allergic
reactions and parasitic infection.
Granulocytes

Neutrophils

Eosinophils

Basophils
 NEUTROPHILS

Responsible for acute inflammatory response

Increase Cause entry of Cause Spontaneous

vascular activated Activation subsidence
permeability neutrophils of (resolution) of
into tissues Platelets invading organism
that have been
dealt with
successfully
By:
Platelet, activating factor (PAF)
Eicosanoids (various prostaglandins
and leukotriens)
 FUNCTIONS OF MONOCYTES

Monocytes are precursors of

macrophages, which are actively
involved in phagocytosis.
 FUNCTIONS OF LYMPHOCYTES

B-Lymphocytes: Synthesize and secrete

antibodies (humoral immunity)
T-Lymphocytes: Involved in cellular immune
mechanism e.g
- killing virally infected cells
and some cancer cells.
- activate B cells to make
antibodies.

Lymphocytes
 PLATELETS

Involved in coagulation
of blood

PLATELETS
Haemolysis of Erythrocyte
 Haemolysis
Haemolysed red cells

>120 days

Free Haemoglobin
In Reticuloendothelial cells
 Haemolysis of Erythocytes

After a life span of 120 days, erythrocytes are haemolysed

In: - Spleen
- Bone marrow
- Other REC

Signal for haemolysis:

- Loss or alteration of:
- Cytoskeleton structure
- Active ion pump
- Membrane lipids
- Membrane glycoproteins

Most intracellular components are reutilized.

 Fate of Haemoglobin

Haem

Haemoglobin

Globins
 Haemolyzed RBC
RE System
Haemoglobin

Met haemoglobin

Globin + Heme + Fe++

CO BLOOD

Amino Biliverdin
acids Transferrin - Fe++
Bilirubin
Blood
Bilirubin - Albumin Apotransferrin Bone
complex marrow
Liver Albumin
Fe++ reutrilization
Bilirubin
 In R.E.S.

Heme Oxygenase

Heme Biliverdin + CO + Fe++

2H+

NADPH - Cytochrome
Reductase

3O2 + 3NADPH + H+ 3 NADP+ NADPH-

Bilirubin
Reductase

BILIRUBIN
 In Liver
Glucuronyl
Bilirubin + UDP - Transferase Bilirubin
Glucuronic acid Monoglucuronide
+ UDP

UDP-
Glucuronyl Transferase glucuronic acid

Bilirubin Diglucuronide

To the Bile
 Daily excretion of Bile Pigments

250-350 ng Bile Pigment excreted in Feces/day

1-2 mg Bile Pigment excreted in urine/day

Plasma Level of Bilirubin

Total Bilirubin = < 17 mol/L

Direct Bilirubin = < 2 mol/L

 Disorder of Bile Pigment Metabolism

Causes:

1. An increase load of bilirubin arriving at the liver:

- due to increased red cell destruction.
- Absorption of large haematoma.
2. Defective uptake and transport by the liver cells:
- Gilberts disease.
3. Disturbance of conjugation:
- Liver cell destruction.
- Reduced glucuronyl transferase activity.
- Neonatal jaundice.
- Crigler-Najjar Syndrome
- Gilberts disease.
4. Disturbance of excretion of conjugated bilirubin:
- Liver cell destruction.
- Intra and extrahepatic cholestasis.
- Dubin-Johnson Syndrome
 Jaundice

Elevation of bile pigments in blood.

Bile pigments escape into tissues - yellow

colouration.

Due to:

- Production of bile pigments.

- Failure of liver to conjugate and excrete
bile pigments.
- Decreased excretion of bile pigment
due to obstructive of bile duct.
 Types of Jaundice

Hemolytic or prehepatic.

Hepatic.

Obstructive as posthepatic.

Congenital non-hemolytic.
 Hemolytic Jaundice

Increase destruction of erythrocytes.

Formation of bilirubin

Elevation of serum bilirubin.

e.g. - In hemolytic anemia

- Infection
- G-6-PD deficiency
 Hepatic Jaundice

Caused by liver dysfunction.

Results from damage to parenchymal cells.

Decreased conjugation of bilirubin.

e.g. - Liver poisons (chloroform

phosphorus, CCl 4)
- Toxins.
- Hepatitis virus.
- Engorgement by hepatic vessels in
cardiac failure
- Cirrhoses.
 Obstructive Jaundice

Results from blockage of the hepatic or

common bile duct.

Passage of blood into liver cell is normal.

Conjugation of bilirubin in liver is normal.

Failure of conjugated bilirubin to be excreted

by bile capillaries.

Bilirubin reabsorbed by hepatic veins and

lymphatics.
Congenital Neonatal Hyperbilirubinaemia

Activity of glucuronyl transferase in liver.

Conjugation and excretion of bilirubin.

Unconjugated level in blood.

Often occurs in neonatal period.

Treated by phototherapy.
 Congenital Hyperbilirubinaemia

Gilberts Disease:
- Defective bilirubin transport into liver cells.
- Occassionally reduced glucuronyl transferase activity.
- Elevated plasma unconjugated bilirubin (20-35 mol/L)
- Harmless.

Crigler-Najjar Syndrome:
- Deficiency in glucuronyl transferase.
- Significantly elevated plasma unconjugated bilirubin
(350 mol/L)
- Hyperbilirubinaemia in first few days of life.
- Kernicterus in newborn.
Contd..
 Congenital Hyperbilirubinaemia
Contd.

Dubin-Johnsons Syndrome

- Defective excretion of conjugated bilirubin.

- Mildly raised conjugated bilirubin.
- Bilirubin in urine.
- Harmless.
Types of Bilirubin present in different Jaundice

Defect Types of Bilirubin

- Increased production Unconjugated bilirubin

- Haemolytic disease

- Reduced liver uptake of bilirubin Unconjugated bilirubin

- Drug competition

- Reduced conjugation of bilirubin Unconjugated bilirubin

- Developmental defect
- Drug competition
- Inherited enzyme defects
(Gilberts disease,
Criglar Najjar disease)
Contd.
Types of Bilirubin present in different Jaundice
Contd.

Defect Types of Bilirubin

- Decrease secretion of conjugated Mainly conjugated

bilirubin bilirubin
- Drug competition
- Inherited defects.

- Obstruction of biliary tree Mainly conjugated

(cholestasis) bilirubin
- Within the liver
(liver cirrhosis, drugs
side-effects)
- Outside the liver
(gallstone, neoplasm)
Anaemia
 Anaemia

Decrease in the level of:

- Haemoglobin or/and

- RBC count or/and

- PCV (Hematocrit)
 Classification of Anaemia

Classified mainly in two ways:

1. According to the morphology of the

average red cells.

2. According to the pathophysiologic

mechanism of the red cell production.
 Classification of Anaemia

By Red Cell Morphology:

An anaemic state with altered or normal red cell morphology
i.e. MCV and MCH:

MCV: denotes the mean corpuscular volume of

red cells (Normal = 85-100 f/l)

MCH: denotes mean corpuscular hemoglobin

(Normal = 27-32 pg).
 Mean Corpuscular Haemoglobin (MCH)

MCH expresses the amount of haemoglobin

in red blood cell in picogram (pg).

Hb (g/dl blood) = MCH

RBC Count X 1012/l

Normal range 27-32 pg

 Mean Corpuscular Haemoglobin
Concentration (MCHC)

MCH expresses the amount of haemoglobin in

RBC. It is expressed in gm/deciliter.

Hb (g/dl) = MCHC g/dl

PCV (l/l)

Normal Range 30-35 g/dl

 Classification of Anaemia Contd.

(a) Normocytic Normochromic Anaemia

(MCV = 85-100 fl) (MCH = 31-35 pg)

1. Acute bleeding.

2. Haemolytic anaemia:
(a) Extracorpuscular defects immune and non-immune.
(b) Intracorpuscular defects, membrane,and metabolic
defects and hemoglobinopathies.
(c) Combined defects.

3. Marrow failure associated with hypoproliferation of

hematopoietic cells:
(a) Aplastic anaemia.
(b) Pure red cell aplasia.
(c) Anaemia of chronic renal failure.
(d) Anaemia of endocrine disease.
(e) Toxic depression of bone marrow.
 Classification of Anaemia Contd.

(b) Microcytic - Hypochromic Anaemias

(MCV = < 87 fl) (MCH = < 30 pg)

1. Iron deficiency.
2. Thalassaemias.
3. Sideroblastic anaemia:
(a) Reflactory.
(b) Reversible.
(c) Pyridoxine - responsive.

(c) Macrocytic - Normochromic Anaemias:

(MCV = > 103 fl) (MCH = 31-35 pg)

1. Megaloblastic anaemia:
(a) Vit. B12 deficiency.
(b) folic acid deficiency.
(c) Others.
2. Non-Megaloblastic Macrocytic Anaemia.
 Classification of Anaemias
(Contd)

2. According to the pathophysiologic mechanism:

(A) Increased loss of RBCs

- Acute bleeding

(B) Increased destruction of RBCs:

- Haemolytic anaemia

(C) Decreased production of RBCs:

1. Marrow failure associated with hypo-proliferation

of hematopoietic cells.
2. Marrow failure associated with ineffective
erythropoiesis.
 Causes of Anaemias

1. Dyshaemopoietic anaemias: Due to insufficient blood

production.

2. Haemolytic anaemias: Due to excessive

intra-vascular destruction.

3. Haemorrhagic anaemias: Due to extravascular

blood loss.

4. Anaemias of unknown causes.

 Dyshaemotopoietic Anaemias

Deficiency of Factors Essential for Erythropoiesis.

- Iron deficiency.
- Trace metal (copper) deficiency.
- Haemopoietic principle deficiency
- Extrinsic factor (Vit. B12)
- Intrinsic factor (in gastric juice)
- Other vitamin deficiencies:
- Folic acid deficiency.
- Pyridoxine deficiency.
- Riboflavin deficiency.
- Nicotinic acid deficiency.
- Internal secretion deficiency:
- Thyroid hormone deficiency
- Pituitary hormone deficiency
- First class proteins deficiency:
- Milk and milk product.
- Eggs.
- Meat proteins.
 Causes of Deficiency in Factors Essential for Erythropoiesis

- Food Intake Defect - (Nutritional Anaemias):

- Deficiency of:
- Proteins
- Iron and other metals.
- Vitamin C
- Vitamin B12
- Folic acid

- Defect of Digestion - due to impaired gastric function:

- Achlorhydria
- Deficiency of intrinsic factor
- Presence of autoantibodies

- Defects of absorption and transport:

- Fatty diarrhea, sprue, coeliac disease, diarrhea
- Transferrin deficiency, ceruloplasmin deficiency.
 Causes of Deficiency in the Factors (Contd.)

- Defects of storage:
- Liver damage.
- Failure to utilize the factors essential for haemopoiesis:
- Failure of iron utilization.
- Sepsis.
- Chronic infection (TB, Syphilis)
- Nephritis.
- Cachexia of malignant disease.
- Leukaemia.
- Liver cirrhosis.

- Toxic and aplastic conditions:

- Idiopathic aplastic anaemia.
- Damage by Benzol, X-rays, Radium.
 Haemolytic Anaemias

Causes:
- Infections:
- Sepsis and septicaemia:
- Streptococcus, Clostridium, Welchi
(qas gangrene)
- Typhoid fever
- Viral infection
- Poisons:
- Chronic lead poisoning
- Acute lead poisoning
- Chemicals (phenylhydrazine, saponins)
- Snake venoms
- Allergic haemolytic anaemia:
- Pollens or vegetables
 Haemolytic Anaemias (Contd.)

Causes:
- Paroxysmal haemoglobinuria.
- Intravascular haemorrhage due to cold exertion.
- Hereditary Intracorpuscular Defects:
- Abnormal haemoglobins (Hb S, Hb C).
- Thalassaemias ( and )
- Enzyme deficiency (G-6-PD and PK deficiency)
- Hereditary abnormalities in corpuscular shape:
- Congenital haemolytic icterus.
(Hereditary spherocytosis)
- Hereditary Elliptocytosis.
- Hereditary Defects of Unknown cause:
- Familial non-spherocytic haemolytic anaemia
 Haemorrhagic Anaemias

- Acute haemorrhage:
- Accidents
- Surgery
- Chronic haemorrhage:
- Epistaxis, Menorrhagia
- Haemorrhoids
- Bleeding duodenal ulcer
- Haemorrhagic disease:
- Congenital coagulation defects:
- Haemophilia (Def. of factor VIII)
- Christmas disease (Def. of factor IX)
- Acquired coagulation defects:
- Vitamin K deficiency
- Liver disease
- Congenital platelet defects:
- Familial thrombocytopenia
- Acquired platelet defects:
- Irradiation
- Drugs (cytotoxic drugs)
 Anaemias of
Unknown Causes

Refractory anaemias.

Anaemia secondary to other

diseases.

Anaemia due to exertion.

 Pernicious Anaemia
(Addisonial Megaloblastic Anaemia)

Most common megaloblastic anaemia.

Due to the absence of intrinsic factor in the gastric juice

(atrophy of gastric mucosa).

Intrinsic factor is needed for absorption of Vit. B12.

Vit. B12 necessary for Haematopoises.

Intrinsic factor Vit. B12 absorption

Ineffective erythropoiesis Megaloblastic red cells

 The
Blood
Plasma
 The Blood Plasma

-Contains 91-95% water.

- Solutes in plasma range from 5-9%
- Proteins are the major solute in the
plasma and their level ranges from
6-8 gm %.
Principal Inorganic Constituents of Human Blood Plasma

Anions Concentration Cations Concentration

meq/Liter meq/Liter

Total 142-150 Total 142-158

Bicarbonate 24-30 Calcium 4.5-5.6
Chloride 100-110 Magnesium 1.6-2.2
Phosphate 1.6-2.7 Potassium 3.8-5.4
Sulfate 0.7-1.5 Sodium 132-150
Iodine,total 8-15* Iron 50-180*
Protein bound 6-8* Copper 80-160*

*These concentrations are in micrograms per 100 ml.

Principal
PrincipalNon-Protein
Non-ProteinOrganic
Organic Constituents
Constituents
of
of Human
HumanBlood
BloodPlasma
Plasma

Constituents
Constituents Normal
NormalRange
Range

Non-Protein
Non-ProteinNN:: 25
25--4040
Amino
Aminoacid
acidNN 44--88
Amino
Aminoacids
acids 36
36--6565
Bilirubin
Bilirubin 0.2
0.2--1.4
1.4
Creatine
Creatine 0.2
0.2--0.9
0.9
Creatinine
Creatinine 11--22
Uric
Uricacid
acid 22--66

Carbohydrates:
Carbohydrates:
Glucose
Glucose 65
65--9090
Fructose
Fructose 66--88
Contd...
Contd...
Principal
PrincipalNon-Protein
Non-ProteinOrganic
Organic Constituents
Constituents
of
of Human
HumanBlood
BloodPlasma
Plasma

Constituents
Constituents Normal
NormalRange
Range

Organic
Organicacids:
acids:
Citric
Citricacid
acid 1.4
1.4--3.0
3.0
-ketoglutaric
-ketoglutaricacid
acid 0.2
0.2--1.0
1.0
Lactic
Lacticacid
acid 88--17
17
Lipids:
Lipids:
Total
Totallipids
lipids 285
285--675
675
Neutral
Neutralfatfat 80
80--240
240
Cholesterol,
Cholesterol,total
total 130
130--260
260

Phosphoglyceride:
Phosphoglyceride:
Total
Total 150
150--250
250
Plasma Proteins
 TOTAL PLASMA PROTEINS
The normal serum protein level is 63-83 g/L.
The type of proteins in serum include:
a. Albumin
b. Globulins
globulin: 1 & 2globulins
globulin: 1 & 2 globulins
globulins
c. Fibrinogen
Under different pathological conditions the protein
levels depart from the normal range.
 Functions of Plasma proteins
Transport: e.g.
- Transferrin transports iron.
- Ceruloplasmin transports copper.
- Albumin transports fatty acids, bilirubin
calcium, many drugs etc.
- Transcortin transports cortisol and corticosterone
- Retinol binding protein transports retinol.
- Lipoproteins transport lipids.
- Haptoglobin transports free haemoglobin.
- Thyroxin binding globulin transports thyroxin.
 Functions of Plasma proteins
(contd)
Osmotic regulation:
- Plasma proteins are colloidal and non-diffusable and
exert a colloidal osmotic pressure which helps to
maintain a normal blood volume and a normal water
content in the interstitial fluid and the tissues.
- Albumin content is most important in regulation of
colloidal osmotic or oncotic pressure.
- Decrease in albumin level results in loss of water from
blood and its entry into interstitial fluids causing edema.
Catalytic function (enzymes):
- e.g lipases for removal of lipids from the blood.
 Functions of Plasma proteins
(contd)
Protective function:
- Immunoglobulins combine with foreign antigens and
remove them.
-Complement system removes cellular antigens.
- Enzyme inhibitors remove enzymes by forming
complexes with them. e.g. 1 antitrypsin combines
with elastase, trypsin and protects the hydrolytic
damage of tissues such as lungs.
- Some proteins increase during acute phase and protect
the body. E.g. 1 antitrypsin, 2 macroglobulins
 Functions of Plasma proteins
(contd)
Blood clotting:
- Many factors are involved in clotting mechanism and
prevent loss of excessive amount of blood. e.g. clotting
factors IX, VIII, thrombin, fibrinogen etc.
- An excess of deficiency leads to a disease. e.g
hemophilia, thrombus formation.
Anticoagulant activity (thrombolysis):
- Plasmin breaks down thrombin and dissolves the clot
Buffering capacity:
- Proteins in plasma help to maintain acid-base balance.
Specific Functions of some proteins

PROTEIN
PLASMA FUNCTION
CONC. (g/L)
Pre-albumin
0.3 Binds T3 & T4
Albumin
40.0 Transport,
1- globulin : 1- antitrypsin colloid oncotic pressure
3.0 Anti proteinase
2- globulins
0.4 Copper transport
ceruloplasmin
1.2 Binds haemoglobin
haptoglobulin
3.0 Transport, anti-proteinase
2-macroglobulin
Contd...........
Specific Functions of some proteins

PROTEIN PLASMA FUNCTION

CONC. (g/L)

- Globulins
Transferrin 2.5 - Iron - transport
Hemopexin 1.0 - Binds haem
Plasminogen 0.7 - Fibrinolysis
Fibrinogen 4.0 - Haemostasis
- Globulin
IgA 0.9-4.5 -Ig in external secretions
IgM 0.7-2.8 - First Ab synthesised
IgG 8-18.0 -Main classes of antibody
IgE - Involved in allergy
IgD
MEASUREMENT OF PROTEIN
FRACTIONS

The protein fraction in plasma can be

separated and estimated using the following
methods:
Zone electrophoresis
Immunochemical methods
Chemical methods
Ultracentrifugation
 CHARACTERIZATION, MEASUREMENT AND
ISOLATION OF PLASMA PROTEINS

Physical Techniques
1. Ultracentrifugation (analytical or Sedimentation
velocity ultracentrifuge) at 60,000 per.min. (Refractive index
the boundary between the solvent and the protein is
visualized by an optical system - called Sehlieren System).

Advantage
Most useful for the determination of the mol. wt of proteins

Disadvantage
High cost of each analysis and poor resolving capacity (when applied
to whole serum or plasma)
 CHARACTERIZATION, MEASUREMENT AND
ISOLATION OF PLASMA PROTEINS

Electrophoresis
Protein in aqueous solution are charged groups (e.g.
carboxylic (Asp. Glu), amino groups (Lys, Arg), they can
be separated under an electric field using various stabilizing
media.
N.B. Amino groups undergo ionic dissociation at alkaline
pH and carboxylic undergo dissociation at acid pH. Most
proteins are -ve at pH 8.6. The pH at which +ve charges
equal to -ve charges is characteristic for a protein and is
called isoelectric point PI).
Boundary electrophoresis: Separation in free liquid media
Zone electrophoresis - Separation in stabilizing media
(e.g. Pager, Cellulose acetate, Starch, Polyacrylamide, Agarose)
 Electrophoresis
- Separates proteins on the basis of their charge.
- Types:
- Free boundary: separation under an electric
field in a fluid media. Separates plasma proteins
five bands: albumin(54-58%), 1 globulins
(6-7%), 2 globulins( 8-9%), globulins (13-14%),
globulins (11-12%).
- Zone electrophoresis: Separation under an electric
field in a solid media e.g. paper, starch, cellulose,
Acrylamide etc. Separates plasma proteins into:
Albumin, 1 globulins, 2 globulins, globulins,
globulins and fibrinogen.
 NORMALHUMAN
NORMAL HUMANSERUM
SERUM
PROTEINELECTROPHORESIS
PROTEIN ELECTROPHORESIS
SERUM PROTEIN DEFECTS

Normal serum protein levels:

Total serum protein level: 63-83 g/dL.
Hyperproteinaemia:
Total serum protein level: > 90 g/dL.
Hypoproteinaemia:
Total serum protein level: < 63 g/dL.
 INDIVIDUAL PROTEIN
INDIVIDUAL PROTEIN
FRACTION
FRACTION
 ALBUMIN

A low molecular weight protein (M.Wt= 65,000).

Functions include:
Transport
Osmotic pressure regulation
Synthesized in the liver.
Deficiency: in liver disease and kidney disease.
 GLOBULINS
Heterogenous group
Can be separated into different fractions on the basis of their
electrophoretic mobility and sedimentation coefficient:
1-Globulin - 1- Fetoprotein
1- Antitrypsin
2- Globulin - 2- Fetoprotein
Haptoglobin
- Globulin - Transferrin
Ceruloplasmin
- Globulin - Antibodies (immunoglobulins)
 FIBRINOGEN
A globulin of very high mol. wt.
Can be precipitated easily.
Can be converted to fibrin which
causes the blood clot formation.
Synthesized exclusively in the liver.
 BIOCHEMICAL ABNORMALITIES
OF PROTEINS

Total protein abnormalities.

Abnormalities of individual protein fraction:
Serum albumin.
Carrier proteins.
Protease inhibitors.
Immunoglobulins.
Embryonic and fetal protein abnormalities.
associated with human neoplasia.
TOTAL SERUM PROTEIN
ABNORMALITIES
Hypoproteinaemia may result from:
1. Water access caused as a result of:
a. Overhydration.
b. Artifactual cause - blood taken from the drip arm.
2. Excessive loss of protein (mainly albumin):
a. Through the kidney in nephrotic syndrome
b. From the skin after burns
c. Through the skin in protein losing enteropathy.
3. Decreased synthesis of proteins
a*. Severe dietary protein deficiency e.g. in Kwashiokar
b*. Severe liver disease (mainly albumin).
c. Severe malabsorption.
* There may be no fall in total protein if -globulin is raised
 HYPOLBUMINAEMIA

Normal albumin level = 32-52 g/L.

Hypoalbuminaemia: the level of albumin <32 g/L.
Frequently encountered.
Consequence:
Oedema
Hypocalcaemia
Alteration in the levels of protein-bound
substance due to loss of carrier protein.
 CAUSES OF
HYPOALBUMINAEMIA
Decrease albumin synthesis:
a. Liver disease (specially chronic diseases).
b. Malnutrition.
c. Alcoholism
Increased albumin loss:
a. Renal disease (nephrotic syndrome).
- Loss of albumin in urine (proteinuria).
b. Extensive burns:
- Loss of albumin through skin - transdution.
 CAUSES OF
HYPOALBUMINAEMIA .......Contd
Defective intake:
a. Malabsorption due to gastro-intestinal disease
Protein-losing enteropathy (rare)
Excessive loss of protein from the body into the gut.
Occurs in a variety of conditions such as :
a. Ulceration of the bowel.
b. Lymphatic obstruction.
c. Intestinal lymphaangiectasis.
 CAUSES OF
HYPOALBUMINAEMIA .......Contd
Haemodilution
a. Over hydration.
b. Late stage of pregnancy.
Artefactual
a. Blood drawn from drip arm.
Non-specific causes (common)
In many acute conditions including minor illnesses such as colds and boils.
Often in hospitalized patients.
Upright position when drawing blood.
Newborn babies.
Increased degradation of albumin. In:
Idiopathic
Familial idiopathic hypercatabolic hypoproteinemia.
Wislcott-Aldrich syndrome
ABNORMALITIES OF
ABNORMALITIES OF
CARRIER PROTEINS
CARRIER PROTEINS
 1-globulin
The normal serum level of 1-globulin is
1-3g/L.
1-lipoprotein transport cholesterol.
In a rare genetic disorder, 1-lipoprotein deficiency
(Tangiers disease), its level is reduced causing the
accumulation of cholesterol esters in tissues resulting
in:
Tonsillar enlargement.
Hepatomegaly.
Lymphadenopathy
 1-FETOPROTEIN (AFP)
AFP is synthesized in fetus at 14-40 weeks of
gestation.
AFP levels decline rapidly after 2 weeks of age.
In adults it is found primarily in:
association with hepatocellular cancer of liver and
embryonic tumor of the ovary and testes.
Cases of gastric and prostatic carcinoma.
Viral hepatitis.
Cirrhosis.
AFP detection is very useful in diagnosis of primary
liver cancer.
 2-GLOBULIN

The normal 2globulin level is 6-10 g/L of serum.

2-Macroglobulin make up most of 2-globulin fraction.
It is a large molecule
In nephrotic syndrome, it is retained in serum and
levels are found to increase.
Haptoglobin : binds free haemoglobin. Low levels are
found in hemolytic conditions since the haptoglobin/
haemoglobin complex is catabolised better than free
haptoglobin.
 -GLOBULIN
Normal level of -globulin in serum is 7-11 g/L.
-lipoprotein transport cholesterol in serum.
Abetalipoproteinaemia is the complete absence of -
lipoprotein, pre -lipoprotein and chylomicron. This
causes:
. Inability to transport lipid from intestine or the liver.
. Plasma cholesterol deficiency.
It is clinically characterized by intestinal malabsorption under
steatorrhea, progressive atasia, retinitis pigmentation and
crenation of erythrocytes.
High levels of -globulin are found in pregnancy, biliary
obstruction and nephrotic syndrome.
 TRANSFERRIN

Transferrin is a -globulin.
It binds free iron in serum.
Normally it is about one third saturated with iron.
Transferrin levels are decreased in:
Liver disease (e.g. cirrhosis).
Chronic infections.
Nephrosis.
Congenital atransferrinaemia.
Increased serum transferrin levels occur during
increased transferrin synthesis caused as a result of
iron deficiency anaemia.
 ALTERATION OF PLASMA PROTEIN
CONCENTRATION
PROTEIN INCREASED IN DECREASED IN
Albumin Dehydration - Acute and chronic liver
disease.
- Malnutrition
- Malabsorption
- Cirrhosis of liver
- Burns
- Severe trauma
- Nephrotic syndrome

Transferrin - Iron deficiency - Protein losing conditions

- In woman taking - Infection; and
oral contraceptives. - Neoplastic disease
Contd................
 ALTERATION OF PLASMA PROTEIN
CONCENTRATION ..........Contd

PROTEIN INCREASED IN DECREASED IN

Ceruloplasmin - Chronic liver disease Wilson disease
- Some infections.
Haptoglobulin Haemolytic anaemia

1-Antitrypsin Pulmonary emphysema.

2-Macroglobulin Nephrotic syndrome Liver disease in children
collagen disorder leading to cirrhosis.

-Fetoprotein Hepatocellular carcinoma

Fibrinogen - Congenital fibrinogen def.
- Shock.
- Complication of pregnancy.
- Major surgery
- Snake bites.
- Disseminated carcinoma
 INFLAMMATORY RESPONSE

Assessment of the presence and

degree of inflammation can be
obtained from the levels of acute
phase protein
Positive acute phase proteins:
Increase during inflammation.
Negative acute phase proteins:
decrease during inflammation.
 IgG
Cryoglobulins - Pure monoclonal IgM
IgA
- Mixed.
- Consist of complexes of
immunoglobulins or altered
immunoglobulins.
- Insoluble at 4oC. Aggregate at
30oC
ACUTE PHASE PROTEINS
Indicators of inflammatory disease with:
ESR
Leukocytosis
Fever
Indicate active state of inflammation.
Constitute: 1-antitrypsin
Carrier proteins:
Haptoglobin.
Ceruloplasmin.
Fibrinogen.
C-reactive proteins
1-acid glycoprotein
CLINICAL INDICATIONS FOR ASSESSMENT
OF ACUTE PHASE PROTEINS

Presence of inflammatory disease.

Differential diagnosis of inflammatory
disease.
Estimation of the endpoint of therapy.
Monitoring therapeutic effectiveness.
Postsurgical follow-up in patients at risk
of postoperative infections.
Follow-up of patient with malignancy.
POSITIVE ACUTE PHASE PROTEINS

1-antitrypsin.
1-antichymotrypsin.
1-acid glycoprotein.
Ceruloplasmin.
Haptoglobin.
Complement component C3 and C4.
Antithrombin III.
SPECIFIC INDICATIONS FOR QUATIFICATION
OF SOME ACUTE PHASE PROTEINS

PROTEIN DISEASE

1-antitrypsin - Chronic obstructive

pulmonary disease.
- Neonatal hepatitis syndrome
cytogenic cirrhosis.

Ceruloplasmin Hepatitis or cirrhosis (unexplained)

Haptoglobin In-vivo haemolysis.

Ineffective erythropoiesis
 EMBRYONIC AND FETAL PROTEIN
ASSOCIATED WITH HUMAN NEOPLASIA

Several fetal proteins and synthesized in human tumors.

They are released in biological fluid.
Useful in - diagnosis of malignancy
- monitoring of therapy for cancer
- evaluation of prognosis:
The protein often found associated with tumors are:
1-fetoproteins
2-H fetoprotein
2-S fetoprotein
regain alkaline phosphatase
fetal sulphoglycoprotein antigen
-fetoproteins
Carcinoembryonic antigen of the gastrointestinal tract.
 INHERITED ABNORMALITIES OF THE
Plasma Proteins
DEFICIENCY ASSOCIATED ABNORMALITY

1-Antitrypsin Obstructive pulmonary disease (Chronic or

emphysema) liver disease.

Anti-thrombin Thrombosis
Pulmonary embolism

Immunoglobulin Severe recurrent or chronic infection

Complement Severe, recurrent infection.

C1 esterase inhibitor Recurrent non-pruritic swelling of skin and

mucus membrane (hereditary angioneurotic
edema)
 PLASMA PROTEIN CHANGES IN
LIVER DISEASES

Liver disease HPT A1b C3 LDL IgG IgM IgA TRF Pre- 1-
Alb AT
Pure Biliary
Obstruction
Advanced Hepatic ()
Cirrhosis
Acute Viral () () ()
Hepatitis
Infection () ()
Mononucleosis

= Decrease () = May be decreased

= Increase () = May be increased
NEGATIVE ACUTE PHASE
PROTEINS

Albumin.
Transferrin.
Pre-albumin.
 BIOCHEMICAL
BIOCHEMICAL
INVESTIGATIONS IN
INVESTIGATIONS IN
THEDIAGNOSIS
THE DIAGNOSISOF
OF
DISEASESTATES
DISEASE STATES
TYPES OF BIOCHEMICAL TESTS

1. Discretionary tests.
2. Profile and screening
investigations.
a. On patients.
b. On apparently healthy
individuals.
 COMMONLY REQUESTED
DISCRETIONARY BIOCHEMICAL TESTS
TEST SUSPECTED DISEASE
- Bilirubin Liver disorders.
- Glucose Diabetes Mellitus
- Iron and Total Iron Anaemias
Binding capacity
- Urea Renal function
- Creatinine Renal function
- Uric acid Gout
- Electrolytes Water and electrolyte balance
- Plasma enzymes Liver, cardiac, muscle, etc.
- Cholesterol/Lipids Cardiac diseases
- Blood gases Acid-Base balance
 EXAMPLES OF ORGAN-SPECIFIC
PROFILES
TESTS
- Electrolyte profile Na+, K+, CI-, HCO-3
- Liver function tests Bilirubin, alkaline phosphatase,
alanine transaminase (SGPT), Plasma
albumin.
- Bone Profile Cax2, alkaline phosphatase, phosphate.
- Kidney function tests Creatinine, urea.
- Acid-Base balance pH, PCO2, HCO-3
- Cardiac profile Lactate dehydrogenase (LDH), Creatinine
phosphokinase (CPK), Aspartate
transaminase (SGOT)
- Endocrine profile T3, T4, TSH, and Thyroid function
 METHODS USED IN IDENTIFICATION
AND QUANTITATION OF NORMAL AND
ABNORMAL BLOOD PROTEINS

a. Plasma Proteins
b. Haemoglobin
 METHODS FOR PLASMA PROTEIN
ESTIMATION
Quantitation:
Total Protein
Albumin
Globulin
Manually - Biuret method. Colour development with Cu+2
reagent.
Autoanalyser - SMAC
- American monitor
Method for specific protein:
Immunodiffusion e.g. transferrin, immunoglobulins
Nephelometric method e.g. Albumin, 1-antitrypsin,
immunoglobulins.
RIA method e.g. Ferritin, Immunoglobulin, Protein Hormones.
 IDENTIFICATION
Electrophoresis:
Widely used method.
Simple.
Proteins are separated on the basis
of the charges under an electric field.
Useful investigation of disease states
e.g. liver, renal diseases, infections.
 IMMUNODIFFUSION

Used for specific protein

identification.
Simple procedure.
Proteins are identified on the basis
of precipitation reaction with
respective antibodies.
IMMUNOELECTROPHORESIS

Complex procedure.
Accurate.
Proteins are identified on the
basis of their change and
precipitation reaction with
respective antibody.
 METHODS USED FOR
ESTIMATION OF HAEMOGLOBIN
Estimation of total haemoglobin:
a. Manually: Cyanomethaemoglobin method
- Not used commonly.
- Not very accurate.
b. Autoanalyzer: Coulter Counter with
haemoglobinometer attachment:
- Widely used.
- Very accurate.
- Simple.
- Estimates total RBC, WBC, MCV, MCH,
MCHC.
 INHERITED ABNORMALITIES
OF PLASMA PROTEINS
DEFICIENCY ASSOCIATED ABNORMALITY

1-Antitrypsin Obstructive pulmonary disease (Chronic

bronchitus or emphysema), liver disease.

Anti-thrombin Thrombosis.
Pulmonary embolism.

Immunoglobulin Severe recurrent or chronic infection

Complement Severe, recurrent infection.

C1 esterase inhibitor Recurrent non-pruritic swelling of skin and

mucus membrane (Hereditary
angioneurotic edema).
 EMBRYONIC AND FETAL PROTEIN
ASSOCIATED WITH HUMAN NEOPLASMA

Several fetal proteins are synthesized in human tumors.

They are released in biological fluid.
Useful in: - diagnosis of malignancy.
- monitoring of therapy for cancer.
- evaluation of prognosis.
The protein often found associated with tumors are:
-feto proteins
2ferroprotein.
- -fetoprotein.
- Alkaline phosphatase.
- Fetal sulphoglycoprotein antigen.
- -fetoproteins.
- Carcinoembryonic antigen of the gastrointestinal tract.
 IDENTIFICATION OF
HAEMOGLOBIN TYPES

a. Electrophoresis at alkaline acid pH

- Simple procedure
- Accurate
- Useful for identification of several Hb
variants (not all).
- Proteins can be quantitated by using
a densitometer.
 IDENTIFICATION OF
HAEMOGLOBIN TYPES
b.Isoelectric Focussing:
Separation on the basis of
isoelectric pH of haemoglobin
variants.
Simple method.
Does not separate all variants.
LABORATORY INVESTIGATIONS
OF ANAEMIA

Haemoglobin, RBC and PCV.

Red cell indices.
Red cell morphology.
Iron and TIBC estimation.
Hb A2 and F estimation.
Haemoglobin electrophoresis at
acid and alkaline pH.
METHODS USED FOR INVESTIGATION
OF HAEMOGLOBINOPATHIES

Detection of haemoglobinopathies and

thalassaemias - Haematological Tests.
Hb
RBC count
PCV
MCH
MCHC
Red cell morphology
Contd...........
METHODS USED FOR INVESTIGATION
OF HAEMOGLOBINOPATHIES ...Contd

Differentiation and confirmation of

haemoglobinopathies and thalassaemias:
Electrophoresis.
Hb A2 quantitation.
Hb F quantitation.
Hb stability test.
Determination of /non- globin chain
ratio.
Studies at gene level.
 BIOCHEMICAL TEST IN THE
INVESTIGATION OF
G-6-PD PD DEFICIENCY

1. Estimation of red cell G-6-PD activity

Spot tests
Spectrophotometric method.
2. Phenotyping by electrophoresis.
 ESTIMATION OF G-6-PD
ACTIVITY REACTION

Glucose-6-Phosphate

NADP+ G-6-PD
NADPH*
6-phospho-gluconolactone
* NADPH is estimated by measuring
a. Fluorescence (under UV Lamp)
b. Absorbance at 340 nm
PATHOLOGICAL CHANGES IN
LIVER DISEASE
a. Liver cell damage
(acute hepatitis, toxins, chronic hepatitis, prolonged
biliary obstruction, cirrhosis, hepatic congestion).
b. Cholestasis
Intrahepatic cholestasis:
(Viral hepatitis, biliary cirrhosis, infiltration of the
liver).
Extra hepatic cholestasis
(Gallstone in the common bile duct, fibrosis of the
bile duct, carcinoma of head of pancreas, external
presence of tumour).
 CONSEQUENCES OF LIVER
DISEASES

Synthesis of plasma proteins.

Release of hepatic proteins and
enzymes.
Excretion of some metabolites.
 LIVER FUNCTION TESTS

The principal function of the liver include:

Conjugation and excretion of bilirubin.
Metabolism of carbohydrates, proteins and
lipids.
Detoxication of drugs, metabolite and
hormone.
Excretion of various natural and foreign
substances into the biliary tract.
Storage.
LIVER FUNCTION TESTS

Total bilirubin.
Transaminase (SGPT & SGOT)
Alkaline phosphatase.
Albumin
Total protein
 TESTS PERFORMED IN SUSPECTED CASES
OF DIFFERENT LIVER DISEASES
Tests Acute Chronic Cirrhosis Choles- Hepatic Hepato-
Hepatitis Hepatitis tosis Infiltration cellular
Carcinoma
Plasma Bilirubin
SGOT
SGPT
Urinary Bilirubin
Urobilirubin

Hepatitis associated
antigen

Plasma protein
electrophoresis

Alkaline phosphatase

5 Nucleotidase

-Fetoprotein

-Glutamyl Transfe-rase
 TYPES OF BILE PIGMENTS PRESENT IN PLASMA, URINE
AND FEACEA IN DIFFERENT TYPES OF JAUNDICE

Plasma Uric Feaces

Disease Total Excess Conju- Uro Bilirubin Bilirubin Urobilirogen
Bilirubin gated Bilirubin
(Direct Van der
Brough Reaction)
Normal Present - Present Absent Present
Haemolytic + - Increased Absent G
Jaundice
Hepatic (Infective G + Variable + Low
hepatitis)
Post Hepatic III G Absent G Absent
FUNCTIONS OF THE KIDNEY

To excrete water and ions from

the body.
To maintain the composition of
plasma normal by excreting or
reabsorbing substances.
To maintain acid-base balance.
To excrete metabolic end
products, hormones, drugs.
To control blood pressure.
 CHOICE OF RENAL FUNCTION TESTS

TESTS CONDITION

- Examination of urine Suspected renal damage.

- The water deprivation or Most useful single test to confirm renal

vasopressin test tubular impairment.

- Creatinine clearance Quantitative test for glomerular impairment.

- Estimation of plasma urea Guide to progress and prognosis if there

and creatinine is severe renal damage or obstruction.
PLASMA PROTEIN CHANGES
IN RENAL DISEASE
Albumin

2-globulins

-globulins (often)

C3 and C4 in acute glomerulonephritis

C3, normal C4 in membrane proliferative

glomerulonephritis.
 URINARY PROTEIN CHANGES IN
RENAL DISEASES
Glomerulal Overflow Revert Tubular Nephrogenic
Proteinura* Proteinura Disease Proteinuria** Proteinuria
Albumin Bench flow 2-Globular IgG, IgM
protein
Transferrin Myoglobin ad -Globular IgE

Acid Glycoprotein Haemoglobin Slightly albumin

and transferrin
1-Antitrypsin Acid glyco- 2-Microglobulin
protein
IgG 1-Antitrypsin Lysozone

* Ratio of albumin to low mol-wt. protein 20:1

** Ratio of albumin to low mol-wt. protein 1:1
BLOOD TESTS FOR BONE
DISEASE

Blood Ca+2 level.

Blood phosphate level.
Alkaline phosphatase level.
Parathyroid hormone level.
 BLOOD TESTS FOR DIAGNOSIS
OF DIABETES MELLITUS

Random blood glucose estimation.

Fasting blood glucose estimation.
Testing for glucose in urine.
Two-hour post-glucose blood glucose
level.
Glucose Tolerance Test (GTT).
 MUSCLE DISEASE TESTS

Creatine phosphokinase (CPK).

Creatine and Creatinine in Serum.
Calcium.
Na+ in Serum.
K+ in Serum.
Mg+2 in Serum