Transcript Blood Biochemistry BCH 577

Blood Biochemistry BCH 577
Prof. Omar S. Al-Attas
Professor of Biochemistry
Biochemistry Department
King Saud University
Chapter 1
Introduction To Hematology
• Blood is the only tissue that flows throughout your body.
• This red liquid carries oxygen and nutrients to all parts of the
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body and waste products back to your lungs, kidneys and liver
for disposal.
It is also an essential part of your immune system, crucial to fluid
and temperature balance, a hydraulic fluid for certain functions
and a highway for hormonal messages.
It represent about 8% of total body weight
Average volume of 5 liters in women
Average volume 5.6 liters in men
99% of blood cells are erythrocytes
Plasma accounts for the remaining of the blood volume.
Consist of three types of cellular elements (Erythrocytes,
Leukocytes and Platelets)
The cellular elements are suspended in plasma
Hematopoiesis
• The cells normally found in the circulatory blood are of three main types:
• The erythrocytes ( RBC) are largely concerned with oxygen transport.
• The leukocytes (White blood cell) play various roles in defense against infections and
tissue injury.
• The thrombocytes (platelets) are immediately involved in maintaining the integrity of
blood vessels and in the prevention of blood clots
Hematopoiesis (poiesis= formation)
• Is the term used to describe the formation and development of blood cells.
• Cellular proliferation, differentiation and maturation take place in the hematopoietic
tissue, which consists primarily of the bone marrow. Only mature cells are released to
the peripheral blood.
• Hematopoietic begins in the nineteenth day after fertilization in the yolk sac of human
embryo. Then the fetal liver becomes the chief site of blood cell production( at about
this time the yolk sac discontinues its role in the hematopoiesis. Also at this time
hematopoiesis also begins to lesser degree in the spleen, kidney, thymus and lymph
nodes.
• At about 4-5 months of gestation hematopoiesis commences in the bone marrow where
it is fully active by the seventh or eight month and at birth partially the whole bony
skeleton contain active marrows.
• During childhood and adolescence there is a marked recession of marrow activity in the
long bones so that in the adult activity is limited to the trunkcal skeleton and skull.
Site of Hematopoiesis
Cont…
• In the yolk sac, most of
hematopoiesis activity at this
site is confined to
erythropoiesis (erythrocytes
formation). Cell production
at this site time is called
Primitive erythropoiesis
because this erythroblast and
Hb are not typical of that seen
in later developing
erythroblasts
• Hematopoiesis in the bone
marrow is called medullary
hematopoiesis.
Hematopoietic Tissues
Spleen, is located in the upper left
quadrant of the diaphragm (muscle
near the stomach)
It is enclosed by capsule of connective
tissue which contains the largest
collection of lymphocytes and
mononuclear phagocytes in the body.
Splenic function:
• Immune defense
• Culling(The removal of aging or
abnormal red blood cells)
• Pitting (The ability of the spleen to
clear inclusions while maintaining
the integrity of the red cell)
1.
Culling
• The discriminatory filtering and destruction of senescent or damaged
red cells by the spleen
• ATP is important in cation pump of erythrocytes and because of
entering the spleen through the slow transit become concentrated in the
hypoglycemic due to low concentration of glucose and slow circulation
in the splenic cords, the supply of glucose in the damaged or senescent
erythrocytes is rapidly diminished.
• This decrease the availability of ATP and contributes to the demise of
these red cells. Slow passage through a macrophage-rich route before
allows the phagocytic cells to remove these old or damaged erythrocyte
before or during their squeeze through 3µm pores to cords and
sinuses.
• Normal RBC withstand this adverse environment and eventually reenter
the circulation.
Cont…
Pitting
• The spleens ability to “pluck out” particles from intact
erythrocytes.
• The pinched off cell membrane can reseal itself, but
the cell cannot synthesize lipids and proteins for new
membrane because of its lack of cellular organelles.
Therefore extensive pitting causes reduced surfacearea to volume ratio resulting in the formation of
spherocytes.
For Example:
• Howell Jolly Bodies: Small, round or oval structure pinkish or bluish in
color, observed in erythrocytes in various anemias and leukemias and
after splenectomy, they also represent unphysiological red cell nuclear
remnant.
• Pappenheimer Bodies: Basophilic containing iron granules observed in
various types of erythrocytes
• Heinz bodies: Resulting from oxidative injury to unprecipitation of Hb
(abnormal Hb) and also erythrocytes with enzyme deficiency. Blood cells
coated with Ab are also susceptible to pitting by macrophages. The
macrophage removes the antigen-antibody complex and the attached
membranes.
The presence of spherocytes on a blood film is evidence that the red blood cells
has undergone membrane assault in the spleen.
Inclusion Bodies
Pappenheimer bodies
Cont…
Defense
• Spleen is rich supply of lymphocytes and phagocytic cells as well as its
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unique circulation
Reservoir for platelets
Massive splenomegaly may result in pooling of 80-90% of the platelets
producing peripheral blood thrombocytopenia.
Condition associated with an enlargement of the spleen are also frequently
accompanied by leukopenia and anemia, the result of splenic pooling and
sequestration
Removal of the spleen results in transient thrombocytosis, with the platelet
count returning to normal in about 10 days.
Splenectomy does produce characteristic erythrocyte abnormalities that are
easily noted on blood smears by experienced technologist.
After splenectomy, the red cells contain granular inclusion such as Howelljolly bodies, pappenheimer bodies. Target cells, cells with excess membrane
to volume ratio, have several mechanism of formation
The splenectomized patients, these cells are probably formed as a result of
excess lipid on the membrane since the missing spleen would normally have
groomed the excess lipid from reticulocytes in their maturation process.
Hypersplenism
• Under certain conditions the spleen may become enlarged;
consequently exaggeration of its normal filtering and
phagocytizing. So anemia leukopenia, thrombocytopenia, then
plasma volume increases consequently cytopenias occurs.
A diagnosis of hypersplenism is made when four conditions
are met:
1. Anemia, leukopenia or thrombocytopenia in the blood.
2. 2. Cellular or hyperplastic bone marrow ( increase in normal
cells of the tissue)corresponding to the peripheral blood
cytopenias.
3. The occurrence of the splenomegaly
4. 4. The correction of cytopenia as following splenectomy.
Hypersplenism
Primary
Occurs when no underlying
cause identified. The spleen
behaves normal but causes
disease.
The most common cause is
congestive splenomegaly
associated with liver
cirrhosis and portal
hypertension.
Thrombosis of the splenic or
portal veins may contribute
Secondary
Caused by a disorder.
The causes: Inflammatory
and infectious diseases
increase the defense
function of the spleen.
Gaucher’s diseasemacrophages
accumulates large
quantities of
undigestable substance
causes splenomegaly
Lymphnodes
The lymphatic system is composed of lymph
node (bean-shaped)
2. Lymph Vessels:
• Node are composed of lymphocytes,
macrophages and reticular meshwork.
• The morphology: lymph nodes contain the
following: an inner area called medulla,
outer area called cortex.
• The medulla: surround the efferent
lymphatics and contains the B lymphocytes
• The cortex contains the T-lymphocytes
• The lymph nodes act as filter removing
foreign particles from the lymph by
phagocytic cells. Antigens pass through the
nodes, they contact and stimulate immune
complex lymphocytes to proliferate and
differentiate into effector cells
Thymus
3. Thymus
• The thymus is a well developed
organ at birth and continues to
increase in size until puberty, and
eventually would begin to atrophy
( hardening).
• It is bilobular organ packed with
small lymphocytes and a few
macrophages.
• Thymus is to serve for maturation
of T-lymphocytes. The thymic
hormone, thymosin, is important
in the maturation of virgin
lymphocytes into
immunocompetent T-cells.
Bone Marrow
4. Bone Marrow
• In adult the marrow normally
consist of islands of cellular active
marrow separated and supported
by fat (Yellow fat).
• Red marrow contains both
erythroid and myeloid precursors
with ration of (M:E) of 1.5:1 to 4:1
• For the first four years of life
nearly all marrow cavities are
composed of red hematopoietic
marrow. After 4 years of age, the
red marrow in shafts of long bones
is gradually replaced by yellow fat
tissues.
The Assessment of Marrow
Activities
• In general if a patient has a normal peripheral blood count
and a bone marrow aspirate contains what appears to be
adequate number of cells, present in normal proportions it
is reasonable to assume normal activity
• Cellular or even hypercellular marrow may be associated
markedly defective output of cells to the peripheral
circulation:
e.g. megaloblast anemia is featured by excessive cellular
destruction in the marrow itself.
• Other cases a defective peripheral cell count may be
associated with hypercellular marrows, there being
excessive destruction of cells in the circulation or
sequestration in an enlarged spleen.
Chapter 2
Derivation of Blood Cells
• Replacement of effete peripheral hematopoietic cells
is the function of more primitive elements in the bone
marrow called stem cells.
Stem cells are characterize by:
 Ability to differentiate into distinct cells lines with
specialize functions.
Ability to regenerate themselves in order to maintain
the stem cell compartment.
Cont…
• Myeloblasts and erythroblasts (pronormoblasts) because of the
high mitotic index, were believed to responsible for maintaining
normal numbers of mature blood cells.
• But these two types of cells have limited ability to proliferate into
the billions of blood cells replaced daily.
• So two different theories on stem cells have been proposed to
verify the existence of stem cell different from myeloblasts and
erythroblasts.
1. Monophyletic theory: Pluripotential stem cell under unknown
hormonal factors may give rise to each of the principle blood
cell lines.
• These pluripotential cells have the capability to self-renewal,
proliferation and differentiation into all hematopoietic cells lines.
2. Polyphylitic theory. Each blood cell type come from a separate
stem cell. So each monopotential stem cell differentiates into
only one type of blood cell.
Cont…
• (Erythrocytic, myelocytic or megakaryocytic) It has
been suggested that nodules appearing at 7th days
were formed from more mature unipotent committed
stem cells
• In the day 14 the nodules showed mixed cell
populations. The cell that formed colony termedColony-forming unit spleen (CFU-S)
• Suggesting the nodules at this time were derived from
more primitive multipotential stem cell.
• Stem cells number one per 1000 nucleated cells in the
marrows
Erythropoiesis
0 The earliest recognizable erythroid cells in the bone marrow is the
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pronormoblast which is a long cell measuring 15-20 µm in diameter. With
dark blue cytoplasmin, a central rounded nucleus with nucleoli and slightly
clumped chromatin. The deep color of the more immature cells due to the
presence of large amount of RNS which is associated with active protein
synthesis.
They also contain Hb (which stain pink) in the cytoplasm; the cytoplasm
stain pale blue as it does its RNS and protein synthetic apparatus while the
nuclear chromatin becomes more condensed.
Any remaining nuclear material is removed by a process of pitting during
passage through the splenic sinus walls.
The successive cytoplasmic charge from blue to pink earning them name
basophilic, polychromatic and orthochromatic
After extruding the nucleus from the normoblast the new stage of cells is
called reticulocytes which still contain some ribosomal RNA and still able to
synthesis Hb. This cell spends 1-2 days in the bone marrow and also
circulates in the peripheral blood for 1-2 days before maturing mainly in the
spleen when RNS is completely lost and completely pink-staining mature
erythrocytes (red cell).
Kinetics of Erythropoiesis
• In man, time required for erythropoiesis to proceed from the undifferentiated stem
cell to reticulocyte is about 7 days and the final maturation of these cells in the
peripheral blood and spleen takes about 24 hours
• This means that some 210 thousand million erythrocytes must be produced by the
marrow each day about 9 thousand million per hour. This requires the synthesis of
6.5g Hb and involves the turnover of about 22mg of iron.
To maintain regular erythropoiesis
1. Erythropoietin- the principle site of erythropoietin production in the kidney.
Erythropoiesis is regulated by hormone erythropoietin which:
• Acts primarily on more mature committed erythroid cells of erythroid colonyforming units.
• Increasing Hb synthesis in red cells precursors.
• Decreasing maturation time of red cell precursors.
• Releasing marrow reticulocytes into peripheral blood at an earlier stage than normal.
Cont…
For normal erythropoiesis to be established there must be
adequate supply of stem cells in a satisfactory environment
containing all the essential materials for their normal growth and
differentiation.
The materials include beside those require by all cells certain specific
factors:
• Vit B12, folate, Vit C, Vit E, Vit B6, pyroxidine, thiamine, riboflavin.
• Metal: Iron, manganese, cobalt
• Amino acids
• Hormone: erythropoeitin, thyroxine and androgens
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Erythrocytes
The mature erythrocytes of the
peripheral blood of a man are:
 Non-nucleated and contain organelles
 Contain enzyme of both anearobic glycolytic
pathway of Embden-Myerhoff and aerobic
pentose P pathway
• It stains pink to orange because of the
large amount intracellular acidophilic
protein called Hb.
• The 7 μm RBC must be flexible
corpuscle to squeeze through the tiny 3
μm fenestrations of the capillaries of
the spleen.
Erythrocytes Membrane
• The cells flexibility is a property of both the erythrocyte
membrane and the fluidity of the cell’s content which is
main hemoglobin.
It is a biphospholipid protein composed of the following:
 49 % protein (composed of contractile protein, enzyme,
surface antigens)
 43% lipid (95% of lipid is equal amounts of unspecified
cholesterol and phospholipids. The remaining are
glycolipids. The polar lipids on the external and internal
surfaces and non polar at the center of the membrane.
 8% CHO ( occurs on the external surface)
Cont…
Cholesterol responsible for the passive cation permeability if the
membrane. It appears that membrane cholesterol exist in free equilibrium
with plasma cholesterol.
Increase cholesterol in plasma (such as occur in lecithin-cholesterol
acyltransferase LCAT deficiency) results in accumulation of cholesterol on
membrane.
These cholesterol laden erythrocytes appears distorted with formation of
target cells and spicules (tiny spike-like structure)
Increase in cholesterol: Phospholipids increases the microviscosity and
the degree of order of the membrane.
The phospholipids are:
 Phosphatidylethanolamine (Cephalin)
 Phosphatidylcholin (Lecithin)
 Sphengomyelin
 Phosphatidylserine
Cont…
• Glycolipids is in the form of glycosphingolipids (cerebrosides and gangliosides) are
responsible for some antigenic properties of the membrane in particular those coresponding
to the A,B, H and lewis blood groups.
• Glycoprotien have similar antigenic properties.
Proteins in the membrane
Two types (both synthesized during cell development)
Integral protein consist of two types: Glycophorin A and band 3.
 Glycophorin A serves as receptors for certain viruses and lectins.
 Band 3 (the name is from its migration with erythrocyte proteins on SDS polyacrylamide gel
electrophoresis. It is responsible for an ion transport across the membrane.
Peripheral proteins lack CHO moieties and are to the cytoplasmic side of the lipid bilipid layer
These proteins include:
• Enzyme glycealdehyde-3-p dehydrogenase.
• Skeletal proteins e.g. spectrin actin viscoelastic properties and contribute to cell shape
deformability and membrane stability.
• Calcium is a membrane component. 80% of intracellular calcium is found in membrane.
 It is maintained at an extremely low intracellular concentration by the activity of an ATP pump.
 The accumulation of calcium cation induces irreversible cross-linking and alteration of
cytoskeletal proteins.
Erythrocyte Metabolism
• Although the binding, transport and release of oxygen and carbon
dioxide is a passive process not requiring energy, a variety of energydependent metabolic processes occur that are essential to cell viability.
• The metabolism of the red cell is limited because of the absence of a
nucleus, mitochondria and other subcellular organelles.
• The most important metabolic pathway in the mature erythrocytes
require glucose as substrate.
Metabolic Pathways
These pathways include:
a. Embden-Meyerhof pathway
b. Hexose-monophosphate (HMP) shunt
c. Methemoglobin reductase pathway
d. Rapoport-leubering pathway
These pathways contribute:
 The first pathway for providing energy for maintaining high intracelllur
K+ , low intracellular Na + and very low Ca2+ (cation pump)
 The second pathway provides reducing power to protect Hb in reducing
state.
 The third pathway regulates oxygen affinity of Hb
 Maintains Hb in reduced state
Embden-Meyerhof Pathway
• 90-95% of the red cell’s glucose consumption is utilized by this pathway. Normal
red cells have glycogen deposits. They depend entirely on environmental glucose
for glycolysis.
• Glucose enters the cell by the facilitated diffusion an energy-free process.
ATP’s
• Are necessary to maintain red cell shape and flexibility.
• Membrane integrity through regulation of intracellular cation concentration:
• Na+ & Ca2+ are more concentrated in the plasma.
• K+ is more concentrated within the cell.
Erythrocyte osmotic equilibrium is maintained by:
• The selective permeability of the membrane
• By the cation pumps located in the cell membrane
Cont…
• The Na+ & K+ pump hydrolysis
ADP + Pi
One ATP
 In the expulsion of 3 Na+ and the uptake of 2K+ .
 Ca2+ is maintained in low concentration by the action of a similar but
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separate cation pump that utilized ATP for fuel.
Excess leakage of Ca2+ into the cell or failure of the pump causes
rigid shrunken cells with protrusions (echinocytes)
An increase in calcium is associated with excess K+ leakage from cell.
Magnesium is another major intracellular cation. It reacts with ATP
to form the substrate complex, Mg-ATP for Ca2+ - MgCT- ATPase
(calcium cation pump).
Upon the exhaustion of glucose, the fuel for the cation pumps is no
longer available. Cells cannot maintain normal intracellular cation
concentrations and this leads to cell death
Hexose Monophosphate Shunt (HMP shunt)
• 5% of cellular glucose enters the oxidative HMP shunt, an ancillary aerobic energy
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system.
In the pathway glucose-6- P is converted to 6-phosphogluconate and so to ribulose-5P.
NADPH is generated and is linked with GSH which maintains sulfhydryl (-SH)
groups intact in the cell including those in Hb and RBC membrane.
Reduced glutathione (GSH) protects the cell from permanent oxidant injury (H2O2).
Oxidants, within the cell will oxidize Hb-SH groups, unless they are reduced by
glutathione.
This reduction oxidizes glutathione (GSSG), which in turn is reduced by adequate
levels of NADPH. The red cell normally maintains a large ratio of NADPH to NADP+.
Failure to maintain reducing power through levels of GSH or NADPH leads to
oxidation of Hb - SH groups, followed by denaturation and precipitation of Hb in the
form of Heinz bodies.
Heinz bodies with a portion of the membrane are then plucked out by the
macrophages of the spleen.
Reduced GSH is also responsible for maintaining reduced-SH groups at the
membrane level. Decrease of GSH lead to injury of membrane sulfhydryl groups
resulting in leak of cell membrane.
Methemoglobin Reductase Pathway
• The methemoglobin reductase pathway, an offshoot of the
Embden-Meyerhof pathway, is essential to maintain heme
iron in the reduced state, Fe++
• Hemoglobin with iron in the ferric state Fe++ is known as
methoglobin. This form of Hb cannot combine with O2
• Methemoglobin reductase together with NADPH produced
by the Embden-Meyerhof pathway protect the heme iron
from oxidation.
• The absence of this system, the 2% of heme methemoglobin
formed daily will eventually raise to 20-40% surely limiting
the oxygen-carrying capacity of the blood.
Rapoport-Luebering Pathway
• The Rapoport-Luebering Pathway is a shunt of Embden-
Meyerhof pathway.
• DPG is present in the erythrocytes in a conclusion of 1,ol
DPG/ 1 mol Hb, and it binds exclusively to deoxyHb.
• As more DPG binds to deoxy Hb, glycolysis is stimulated to
produce more DPG and ATP.
• Increase in DPG concentration facilitate the release of O2 to
the tissues by causing a decrease in Hb affinity for oxygen.
Thus, the red cell has built-in mechanism for regulation of
O2 delivery to the tissues
Hexose monophophate shunt
pathway
Chapter 3
Lifespan and Faith of RBC
0 Normally, the average life span
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of red blood cells is 120 days.
This cells live in blood
circulation.
In in typical adult produces 200
billion RBC per day.
At the end of their lifespan, they
become senescent, and are
removed from circulation.
In many chronic diseases, the
lifespan of the erythrocytes is
markedly reduced (e.g. patients
requiring haemodialysis).
The destruction of RBC is about
2-3 million per second on
average.
Causes of reduction in the life
span of RBC.
1. Defects in RBC
(curpuscular defects)
e.g. Hereditary spherocytosis
Sickle cell anemia,
Thalassemias
2. Deficiency of red
cell enzyme such as: G6PD,
Pyruvate kinase def.,
Autoimmune disorder,
Hypersplenism
Cont…
The fate of the RBC
0 After 120 days , the RBC becomes
more fragile due to decrease NADPH
activity.
0 Younger RBC RBCs can easily pass
through the capillaries which have the
diameter smaller than the mature
RBC’c
0 With a fragile membrane, the mature
RBC are destroyed while trying to
squeeze through capillaries.
0 The destruction most occurs in the
capillaries of the spleen. This is why
spleen is called the “grave yard of the
RBC”.
0 The hemoglobin is released and taken
up the macropharges
Pathway of RBC destruction
Cont…
Bilirubin
0 Is the yellow breakdown product of normal heme catabolism.
0 Bilirubin is excreted in bile and urine, and elevated levels may
indicate certain diseases.
0 It is a toxic waste product in the body
0 It is extracted and biotransformed mainly in the liver and
excreted in bile and liver.
0 Elevation in serum and urine bilirubin is associated with
juandice
Bilirubin Excretion
Bilirubin diglucuronide
2 glucuronate
liver
Bacterial enzyme
Intrahepatic
urobilinogen cycle
Bilirubin
8H
Bacterial enzyme
kidneys
intestines
Urobilinogen
kidneys
Urobilin
urine
Stercobilin
feces
Bacterial enzymes
Stercobilinogen
Cont…
•The globin is recycled into amino acids,
which in turn are recycled or catabolized as
required.
•Heme is oxidized with the heme porphyrin
ring being oped by the endoplasmic
reticulum enzyme, heme oxigenase.
•The oxidation occurs on the specific carbon
producing equimolar amount of the
biliverdin, iron, and carbon monoxide (CO).
This is the only reaction in the body that is
known to produce CO.
•Most of the CO is excreted through the lungs,
with the result that the CO content of expired
air is a direct measure of the activity of heme
oxygenase in an individual.
Cont…
0 In the first reaction, a
bridging methylene
group is cleaved by
heme oxygenase to form
linear Bilivirdin from
cyclic heme molecule.
0 Fe 2+ is release from the
ring in this process
Cont….
•In the next reaction, a second
bridging methylene (between rings
III and IV is reduced by the
biliverdin reductase producing
bilirubin
In Blood
0 The bilirubin synthesized in
spleen, liver and bone
marrow is unconjugated
bilirubin
0 It is hydrophobic in nature so
it is transported to the liver as
complex with the plasma
protein, albumin
Unconjugated bilirubin(Free
Bilirubin)
0 Lipid soluble
0 1gm albumin binds 8.5 mg of
bilirubin
0 Fatty acids and drugs can
displace bilirubin
0 Indirect positive reaction in
van den Berg test
Cont…
Bilirubin is conjugated in a
two step process to form
bilirubin mono and diglucuronide
0 Unconjugated (indirect)
0 Conjugated (Direct) bilirubin is
released into the bile by the liver
and stored in the gallbladder, or
transferred directly to the small
intestines.
0 Bilirubin is further broken down
by bacteria in the intestines, and
those breakdown products
contribute to the color of the feces.
0 A small percentage of these
breakdown compounds are taken
in again by the body, and
eventually appear in the urine
Erythrocytes generated in
the bone marrow are disposed of
in the spleen when they get old
or damaged.
0 This releases hemoglobin, which
is broken down to heme as the
globin parts are turned into
amino acids.
0 The heme is then turned into
unconjugated bilirubin in the
reticuloendothelial cells of the
spleen.
0 This unconjugated bilirubin is
not soluble in water, due to
intramolecular hydrogen
bonding. It is then bound to
albumin and sent to the liver.
Jaundice
Treatment for Neonatal Jaundice
Bilirubin Lab values
Bilirubin form
Normal value
Total (elderly, adult, child)
(newborn)
Critical value (a(newborn)
0.1 to 1.0 mg/dL
1.0 to 12.0 mg/dL
>12 mg/dL
>15 mg/dL
Pre-hepatic, unconjugated, indirect
0.0 to 0.8 mg/dL
Post-hepatic, conjugated, direct
0.0 to 0.25 mg/dL
Fecal urobilinogen
40 to 280 mg/day
Urine
0.0 to 0.02 mg/dL
Conjugated bilirubin - water soluble direct reaction with dyes
Unconjugated bilirubin - water insoluble alcohol is needed for dye (indirect) reaction
Observe the color changes associated with heme degradation by
watching the progress of a bruise (dark red to green to yellow).
Chapter 4
Anemia
Diagnosis of Anemia:
• History- Information solicited
by the physician which should
include:
 Dietary habits
 Medications taken
 Possible exposure to chemical
or toxins
The most complaint is tiredness.
Muscle weakness and fatigue
when there is not enough
oxygen available to burn fuel for
production of energy. When no
oxygen to the brain, headache
vertigo and syncope may occur.
Physical Examination :
General Signs
• Organomegaly of the spleen and
liver may develop since they are
important in hematopoietic system
production and destruction.
• Heart abnormalities may occur as a
result of increased cardiac
workload associated with the
physiologic adaptations to anemia.
Specific Signs
Jaundice in hemolytic anemias
Red Cell Indices
MCH, MCV, MCHC , RDW- These indices are used for
classifying anemias.
MCH- Mean Cell Hemoglobin
-is derived from the Hb devided by RBC
Hb (g/dl) x 10 ÷ RBC (10¹²/l)
MCV-Mean Cell Volume
-is calculated by deviding the PCV by RBC
PCV (l/l) x 1000 ÷ RBC (10¹²/l)
MCHC-Mean Cell Hemoglobin Concentration
-is derived from the Hb, MCV, and RBC
Hb (g/dl) ÷ PCV (l/l)
RDW- red Cell distribution Width
is derived from pulse height analysis can be expressed either as standard
deviation or as the coefficient variable (CV) (%)
Classification of anemia
Morphologic
0 Normocytic: MCV= 80-100fL
0 Macrocytic: MCV > 100 fL
0 Microcytic : MCV < 80 fL
Pathogenic (underlying mechanism)
0 Blood loss (bleeding)
0 Decreased RBC production
0 Increased RBC destruction/pooling
Other causes: Inadequate production of mature
red cells
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Deficiency of essential substances like iron, folic
acid, vit B12, protein and other elements like
copper, cobalt, etc.
Deficiency of erythroblast.. i.e.Aplastic anemia,
Pure red cell aplasia
Infiltration of the bone marrow i.e. leukemia,
lymphoma, carcinoma, myelofibrosis
Endocrine abnormalities i.e. myxedema, addisons
disease, pituitary insuficiency
Chronic renal disease
Chronic inflammatory disease
Cirrhosis of liver
Hypochromic Microcytic Anemia
The etiologic possibilities are
 Iron deficiency
 Thalassemia
 Sideroblastic anemia
 Anemias of chronic disease.
Severe microcytic anemia (MCV
<70 fL) is
caused mainly by iron deficiency
or thalassemia.
Normochromic Normocytic Anemia
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Chronic inflammatory
disease—
(1)infection (2)collagen
vascular disease
(3)inflammatory bowel disease
Recent blood loss
Malignancy/Marrow infiltration
Chronic renal failure
Transient erythroblastopenia of
chidhood
Marrow aplasia/hypoplasia
HIV infection
Hemophagocytic syndrome
Macrocytic Anemia
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Megaloblastic anemias
• Vit.B12 def. - (1) pernicious anemia (2)
malabsorption
• Folate def. - (1) malnutrition (2)
malabsorption
(3) chronic hemolysis (4)drugs phenytoin, sulfa
Hemolysis
Myelodysplastic syndrome
Marrow failure - Aplastic anemia
Chronic liver disease
Hypothyroidism
Macrocytic anemia may be the result of
megaloblastic (folate or vitamin B12
deficiency) or nonmegaloblastic causes.
Folate deficiency can in turn be due to
either reduced intake or diminished
absorption. Severe macrocytic anemia
(MCV >125 fL) is almost always
megaloblastic.
Normocytic Anemias
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These may be classified as follows:
underproduction of erythrocytes due to
(1) the anemia of chronic disease
(2) marrow failure
(3) renal failure (decreased erythropoietin)
loss or destruction of erythrocytes due
to
(1) hemolysis
(2) acute blood loss
The reticulocyte count is useful in
drawing this distinction, being elevated in
(b) and reduced in (a).
The causes of normocytic anemias include
aplastic anemia, bone-marrow
replacement, pure red-cell aplasia,
anemias of chronic disease, hemolytic
anemia, and recent blood loss. A number of
anemias have a genetic etiology. Examples
of such inherited disorders include
hereditary spherocytosis, sickle-cell (SC)
anemia, and thalassemia
Iron Deficiency Anemia
0 It is a condition when supply of iron in the body to bone marrow
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falls short of that required for the production of red blood cells. It
is the commonest cause of anemia throughout the world.
The incidence of anemia in the general population is about 1.5%.
Iron deficiency related to inadequate replacement of lost iron is
the most frequent cause of asymptomatic anemia and has a
variety of causes.
Iron deficiency is common among women of childbearing age;
10% to 20% of menstruating women have abnormally low
concentrations of hemoglobin (usually <12 g per 100 mL).
Between 20% and 60% of pregnant women have hemoglobin
levels <11 g per 100 mL. Anemia was found in 6% of white
women and 17% of black women during the first trimester and
in 25% of white women and 46% of black women during the
third trimester.
Iron
Function as electron transporter;
Vital for life
Must be in ferrous (Fe++) state of
activity.
0 In anaerobic condition, easy to
maintain ferrous state
0 Iron readily donates electrons to
oxygen, superoxide radicals, H2O2
OH Radicals
0 Ferric (Fe+++) ions cannot
transport electrons or O2.
0 Organisms able to limit exposure
to iron had major survival
advantage
Cont…
Iron absorption
0 Duodenum
0 Proximal jejunum
(influenced by rate of
erythropoiesis)
Factor Affecting Iron Absorption
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Form of iron
Acids
Amount of iron
Rate erythropoiesis
Sickle-cell anemia is a molecular disease
of Hb
Comparison of normal and sickle-shaped
erythrocytes
Normal and sickle-cell hemoglobin
Mutation of Sickle Cell Gene
Characterization of HbS
 HbS has between 2 & 4 more net + charges per
molecule than net HbA
A
S
pI of Oxy Hb
6.87
7.09
= 0.22
pI of deoxy Hb
6.88
6.91
=0.23
 Non-polar residue on the outside of HbS (due to Val)
causing low solubility
 Sticky patch on the outside of its β chains & are present on
both deoxy HbS & oxy HbS but not on HbA
Biochemical basis for sickling
0 The substitution of nonpolar
Valine for a charge glutamate
forms a protrusion on the betaglobin that fits in to a
complementary site on the
alpha-chain of another
hemoglobin molecule in the cell
0 At low oxygen tension, HbS
polymerizes inside the RBC
then subsequently assembling
in to a net work of fibrous
polymers that stiffen and
distort the cell, producing rigid
misshapen erythrocytes-sickle
shaped erythrocytes.
Signs and Symptoms of Sickle cell
Episodes of pain
Pain develops when sickle-shaped red
blood cells block blood flow through tiny
blood vessels to your chest, abdomen and
joints. Pain can also occur in your bones.
Frequent infections
Sickle cells can damage your spleen, an
organ that fights infection. This may make
you more vulnerable to infections.
Vision problems
Tiny blood vessels that supply your eyes
may become plugged with sickle cells. This
can damage the retina — the portion of the
eye that processes visual images.
Prevention of Sickle Cell Disease
0 Screen for Hb S at birth.
This method of finding
allows institution of early
treatment and control
0 Prenatal diagnosis.
Prenatal testing is sensitive
and rapid and must be
accompanied with genetic
testing and psychological
counseling
Thalasemia
0 Diverse group of disorders which manifest as anemia of varying
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degrees.
Result of defective production of globin portion of hemoglobin
molecule.
Distribution is worldwide.
May be either homozygous defect or heterozygous defect.
Defect results from abnormal rate of synthesis in one of the globin
chains.
Globin chains structurally normal (is how differentiated from
hemoglobinopathy), but have imbalance in production of two
different types of chains.
Results in overall decrease in amount of hemoglobin produced and
may induce hemolysis.
Two major types of thalassemia:
 Alpha (α) - Caused by defect in rate of synthesis of alpha chains.
 Beta (β) - Caused by defect in rate of synthesis in beta chains.
May contribute protection against malaria.
Cont…
0 Also called Alpha Thalassemia
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Minor.
Caused by two missing alpha
genes. May be homozygous (a/-a) or heterozygous (--/aa).
Exhibits mild microcytic,
hypochromic anemia.
MCV between 70-75 fL.
May be confused with iron
deficiency anemia.
Although some Bart's
hemoglobin (γ4) present at
birth, no Bart's hemoglobin
present in adults.
Alpha thalassemia
0 Has wide range clinical expressions.
0 Is difficult to classify alpha thalassemias due to wide variety of
possible genetic combinations.
0 Absence of alpha chains will result in increase of gamma chains
during fetal life and excess beta chains later in life; Causes
molecules like Bart's Hemoglobin (γ4) or Hemoglobin H (β4),
which are stable molecules but physiologically useless.
0 Predominant cause of alpha thalassemias is large
number of gene deletions in the alpha-globin gene.
0 Are four clinical syndromes present in alpha
thalassemia:
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Silent Carrier State
Alpha Thalassemia Trait (Alpha Thalassemia Minor)
Hemoglobin H Disease
Bart's Hydrops Fetalis Syndrome
Alpha thalassemia
Alpha thalasemia
Cont…
ALPHA THALASSEMIA
Silent Carrier state
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Deletion of one alpha gene, leaving
three functional alpha genes.
Alpha/Beta chain ratio nearly normal.
No hematologic abnormalities present.
No reliable way to diagnose silent
carriers by hematologic
methods; Must be done by genetic
mapping.
May see borderline low MCV (7880fL).
Beta thalassemia
Beta thalassemia
Chapter 5
Collecting and Handling of
Blood
THE VASCULAR SYSTEM
VEINS
•have thinner walls because blood in
them is under less pressure
•Collapse more easily
•Dark bluish red (oxygen poor)
ARTERIES
•Have thick walls to withstand the
pressure of ventricular contraction, that
creates a pulse
•Normal systemic arterial blood is bright
red.
Capillary
• only one cell
•Can easily be punctured to provide
blood specimen
VASCULAR ANATOMY (phlebotomy related)
2 basic patterns of the veins
VASCULAR ANATOMY (phlebotomy related)
H PATTERN
M Pattern
VASCULAR ANATOMY (phlebotomy related)
OTHER VEINS:
•Veins on the back of the hand
or at the ankle may be used,
although these are less
desirable and should be
avoided in diabetics and other
individuals with poor
circulation.
•Leg, ankle and foot veins are
sometimes used but not
without permission of the
patient’s physician due to
potential medical
complications
SOURCE AND COMPOSITION OF BLOOD SPECIMENS
ARTERIAL BLOOD
Primarily reserved for blood gas evaluation and certain emergency situations
VENOUS BLOOD
•affected by metabolic activity of the tissue it drains and varies by collection site
chloride, glucose, pH, CO2, lactic acid and ammonia levels differ may from
arterial blood
CAPILLARY BLOOD
•Contains arterial and venous blood plus tissue fluid
•Calcium, potassium and total protein are normally lower
TYPES OF BLOOD SPECIMENS
SERUM- Serum is that part of blood
which is similar in composition with
plasma but exclude clotting factors of
blood.
PLASMA- Plasma is considered as the
medium of blood in which RBCs (Red
Blood Cells), WBC (White Blood Cells)
and other components of blood are
suspended
WHOLE BLOOD
VENIPUNCTURE EQUIPMENT
Venipuncture
can
be
performed by 3 basic
methods
Evacuated tube system
(ETS) – most preferred because
blood is collected directly from the
vein in the tube, minimizing the
risk of specimen contamination
and exposure to the blood
Needle and syringe
– used
on small, fragile and damaged veins
Winged
infusion
set
(butterfly) – can be used with
the ETS and syringe
•Used to draw blood from infants
and children, hand veins and other
difficult to draw situations
VENIPUNCTURE EQUIPMENT
1. Tourniquet
•Applied to a patient’s arm during venipuncture
•Distends the veins, making them larger and easier to find, stretches the
wall so they are thinner and easier to find
•Must not be left on longer than 1 minute because specimen quality
may be affected
2. Needles
3. Evacuated Tube System (Vacutainer)
VENIPUNCTURE EQUIPMENT
•The bevel is the slanted opening at the end of
the needle.
•bevel of the needle must face upward when
the needle is inserted into the vein.
VENIPUNCTURE EQUIPMENT
Tube Additives
A.Anticoagulants
•Prevent blood from clotting and include EDTA, citrates, heparin and oxalates
B. Antiglycolitic agents
•Prevent glycolysis which can decrease glucose concentration by upto 10 mg/dl
per hour
•Sodium fluoride : most common antiglycolitic agent
Preserves glucose for upto 3 days, and inhibits bacterial growth
C. Clot activators
•Are coagulation factors like thrombin
•Glass particles (silica)
•Inert clays ex. Diatomite (celite)
Enhance clotting by providing more surface for platelet activation
ORDER OF DRAW AND ADDITIVE CARRY OVER
COMMON TESTS AFFECTED BY ADDITIVE CONTAMINATION
Citrate – ALP, Ca,Phosporus
EDTA - ALP, Ca, CK,PTT,K,PT,Serum Iron, Na
Heparin – Activated CT, ACP, Ca, PT, PTT Na, Li
Oxalates- ACP, ALP, Amylase,Ca, LDH, PT, PTT, K, Red cell
Silica (clot activator) – PTT, PT
Sodium fluoride – Na, BUN
PROCEDURAL ERROS RISKS
1.Hematoma formation
- rapid swelling near the venipuncture site due to blood leaking into
the tissues
Situations that can trigger hematoma formation?
Capillary Specimen Collection
Collection sites
1.Fingers – adults and children over the age of 2
2.Heels - infants
Neonatal Bilirubin Collection – must be protected from
light
•Neonatal Screening
screens for
phenylketonuria, a disorder
which could be managed by
dietary adjustment if
diagnosed early.
End