Lab diagnosis of anaemia/ oral surgery courses

INDIAN DENTAL ACADEMY
Leader in continuing Dental Education
www.indiandentalacademy.com

 S M Kawthalkar- Essentials of haematology
 K P Sembulingum- medical physiology
 Textbook of Pathologic basis of diseases:
Robbins and Cotran 7th edition
 Textbook of pathology-Harshmohan 5th
edition.
 Erythropoeisis – Jr. of pathol;1996

 Introduction
 Anaemia
 Establishing its presence & severity
 Hb concentration
 Peripheral blood smear
 Reticulocyte count
 Red cell indices
 Types of anaemias
 Other disorders

 Erythrocytes
 Erythropoiesis
 Haemoglobin
 Variants of haemoglobin
 Iron kinetics
 Heme breakdown

 Erythrocytes are also known as red blood cells,
erythrocytes, or red corpuscles.
 The term erythron refers to all the erythrocytes
and their precursors in the blood, bone marrow or
at extra-medullary sites.

 Characteristics of individual Erythrocytes
 Diameter = 6 to 8 microns
 Thickness = 1.5-1.8 microns (a biconcave disk)
 Production rate = 2.5 million/sec. or 200
billion/day
 Life span = approximately 120 days

 Sites of development
 Erythropoietic tissue originates in the yolk sac
then moves to the liver and spleen during fetal
life.
 Eventually erythropoiesis settles in the medullary
cavity of the skeleton.
 By about 18 years of age the axial skeleton and
proximal ends of the long bones are the site of
erythrocyte production.

 Stages of
Erythropoiesis: -
 Erythropoiesis starts
with the pluripotent stem
cell, as does all
hematopoiesis.
 In the presence of the
proper growth factors
the pluripotent stem cell
differentiates into the
CFU-GEMM.
 Partially under the
influence of EPO the
CFU-GEMM
differentiates into a
BFU-E. www.indiandentalacademy.com

 The BFU-E is considered the earliest cell in the
erythrocyte series.
 The BFU-E then differentiates into the CFU-E.
 The CFU-E is heavily under the influence of
erythropoietin and will differentiate into the
identifiable cells in the erythrocyte cell line.

 Erythropoiesis –
 General overview of cellular changes
 Cell volume decreases as the cell matures. On the
average the size goes from erythroblast measuring
15-20 microns to a mature erythrocyte measuring 6-8
microns in diameter. Chromatin condenses.

 The earliest morphologically identifiable
erythroid cell in the bone marrow is the
proerythroblast (pro-normoblast) a large cell
(15-20) microns has very fine uniform
chromatin pattern, one or more nucleoli, and
dark blue cytoplasm.

 The next cell in the maturation process is the
basophilic (early) normoblast .
 This cell is smaller in size (12-16 um) and has a
coarser nuclear chromatin with barely visible
nucleoli.
 The cytoplasm is deeply basophilic.

 RNA activity decreases in cytoplasm resulting in
lighter blue cytoplasm as the cell matures.
 The early normoblast cytoplasm is deep blue; the
reticulocyte cytoplasm is pinkish blue, the pink
coming from the beginning of hemoglobin
production.

 The more differentiated erythroid cell is the
polychromatic (intermediate) normoblast (12- 15 um).
 The nuclear size is smaller and the chromatin becomes
clumped.
 Polychromasia of cytoplasm results from admixture
of blue ribonucleic acid and pink haemoglobin.
 This is the last erythroid precursor
capable of mitotic division.

 The orthochromatic (late) normoblast is 8 to 12
um, nucleus is small, dense and pyknotic and
commonly eccentrically located.
 The cytoplasm stains mostly pink due to
haemoglobinization.
 It is called orthochromatic because
cytoplasmic staining is largely similar
to that of erythrocytes.

 The nucleus is ultimately expelled from the
orthochromatic normoblast with the formation
of reticulocyte.
 The reticulocyte still has remnants of the
ribosomal RNA in it.

 After 1 -2 days in the bone marrow and 1-2 days in
peripheral blood, reticulocytes lose RNA and become
mature pink-staining erythrocytes.
 Blue color of cytoplasm due to presence of RNA indicating
protein synthesis. Pink color of cytoplasm due to presence
of hemoglobin production. Perinuclear halo indicates
mitochondria and Golgi apparatus surrounding nucleus.
 14-16 erythrocytes are produced from one
proerythroblast.

 Pronormoblast usually divide within 12 hours of
stimulation to make 2 daughter cells (early
normoblast)
 Early normoblast require about 20 hours to develop.
 Intermediate normoblast stage lasts about 30 hours.
 Late normoblast last about 48 hours.
 Reticulocytes are released from bone marrow after 2
to 3 days and circulate for an additional 1 or 2 days
before maturing into an erythrocyte.
 A mature erythrocyte lives about 120 days.

 Composition of Bone Marrow
 Pronormoblast = 1 % or less
 Early normoblast = 1 to 4%
 Intermediate normoblast = 10 to 20%
 Late normoblast = 5 to 10%

 Reticulocytes: - About 1% of the circulating
erythrocyte mass is generated by the marrow
each day so that on a given day there should be
1% reticulocytes in the peripheral blood.
 These replace the 1% of the erythrocyte mass
that dies of old age each day.

 The reticulocyte is an immature erythrocyte.
 It lacks a nucleus but contains various organelles
(mitochondria and ribosomes) and still doesn't
have its full compliment of hemoglobin.

 If stained with a
supravital stain such as
Methylene Blue, it stains
to form a mesh-like
pattern precipitates the
remaining RNA in the
reticulocyte.
 With Wright's stain the
reticulocytes may appear
polychromatic (blue/pink
cytoplasm), depending on
the amount of remaining
RNA.

 The rate of reticulocyte release from the marrow
into the peripheral circulation is governed
primarily by the rate at which O2 is being
supplied to the tissues.
 A decrease in 02 (hypoxia) is recognized by the
kidney which is stimulated to release
erythropoietin.

 Hemoglobin: - Hemoglobin is a conjugated globular
protein that constitutes 33% of the
erythrocyte's weight by volume.
 Its function is to carry O2 from the lungs to the
tissues and carry CO2 from the tissues to the
lungs.

 Hemoglobin has a molecular weight of 68,000
making it a large protein.
 Each normal erythrocyte contains 200-300 million
molecules of hemoglobin, 65% of hemoglobin
synthesis occurs during the nucleated stages of
maturation and 35% during the reticulocyte stage.

 Adult males- 13-17 gm/dl
 Adult females (nonpregnant) – 12-15 gm/dl
 Adult females (pregnant) – 11-14 gm/dl
 Children, 6-12 years – 11.5-15.5 gm/dl
 Children, 6 months-6 years- 11-14 gm/dl
 Infants, 2-6 months- 9.5- 14 gm/dl
 Newborns-13.6-19.6 gm/dl

 Composition of
hemoglobin: - Each
hemoglobin molecule
consists of 4 heme
groups and 1 globin
molecule.
 Each heme group
contains a
protoporphyrin ring
plus an iron molecule.
 Each globin consists
of 4 polypeptide
chains (2 pairs).

 Globin formation: - The polypeptide chains of
globin are produced on the ribosomes.
 The four most common chain types are alpha,
beta, gamma and delta chains.
 Each of these chains differs from the others in
their amino acid sequence.
 Each globin molecule is made of 2 pairs of chains
and each chain is made of 141-146 amino acids.

 Heme formation: Heme formation takes place in the
mitochondria then the cytoplasm of erythroid
precursors through the reticulocyte stage.
 It begins with production of a protoporphyrin ring,
iron then incorporates with protoporphyrin to form
heme.
 Porphyrias are a group of disorders
that have specific enzymatic
problems in the chemical pathway
leading to heme production.

 Configuration :
 The polypeptide chains of adult hemoglobin are
organized into 2 alpha chains 2 beta chains; each
chain has an attached heme group.
 In this configuration hemoglobin carries its
maximum amount of O2.

 A complete hemoglobin molecule is spherical, has
4 heme groups attached to 4 polypeptide chains
so it may carry up to 4 molecules of O2. Heme is
suspended between portions of a polypeptide
chain.

 Embryonic hemoglobin is found in developing fetus
until approximately 12 weeks of age. It has a
particular chain composition and is very primitive
hemoglobin.

 Fetal hemoglobin (hemoglobin F) is present in
fetal blood during the 5th week, through the 9th
month of gestation and after birth. The switch to
adult hemoglobin in not complete for 3 to 6
months. Hemoglobin F functions very well in the
low O2 environment in the uterus because its
affinity for O2 is higher than normal adult
hemoglobin.

 Adult hemoglobin (hemoglobin A) is made of 2
alpha and 2 beta chains. It makes up 95 to 97%
of adult hemoglobin.
 Adult erythrocytes also contain hemoglobin A2 (2
alpha and 2 delta chains) and hemoglobin F.

 Glycosylated hemoglobin is a sub fraction of
hemoglobin A. It's of interest for monitoring
glucose metabolism in diabetics because the
formation of glycosylated hemoglobin depends on
the glucose concentration in the body.

1) Hemoglobins with the oxygen-carrying capacity
altered.
In these hemoglobin different molecule replaces
the 02 molecule.

 Carboxyhemoglobin –
 O2 has been replaced by CO (carbon monoxide).
CO preferentially binds to hemoglobin over 02 by
an affinity 210 times greater than O2. The blood
is cherry red and the patient has a "healthy
flush."

 Sulfhemoglobln –
 Sulfur has replaced the oxygen. This is also a
stable form of hemoglobin. It may result in
denatured hemoglobin, which precipitates out as
Heinz, bodies. It is associated with administration
of oxidizing drugs (certain antibiotics),
Clostridium infection, and severe constipation.
Levels seldom reach 10% and this is usually not
life threatening.

 Methemoglobin is the oxidized state of
hemoglobin with iron in the ferric state.
 Hemoglobin is unable to bind to O2 in this state.
This is an inherited or acquired state. In cases of
acquired methemoglobinemia if exposure to
causative drugs or oxidant chemicals are removed,
the methemoglobin will revert to normal
hemoglobin.

2) Abnormal hemoglobins from genetic alteration of
the amino acid sequence.
This condition is called a hemoglobinopathy and
results in structural rearrangements of the
hemoglobin molecule. Alteration has occurred
during synthesis of one of the globin chains, e.g. in
sickle cell disease there is a substitution of one
amino acid on the beta chain for another amino
acid. The sickled cells are not capable of normal
oxygen transport.

 Abnormal hemoglobins from the altered rate of
synthesis of one chain.
 These conditions are called thalassemias. A
normal sequenced chain is produced but the
decreased rate of synthesis results in disease,
which in turn results in a decreased amount of .
hemoglobin produced. Any excess chains will form
inclusions in the erythrocyte cytoplasm, which
mark the cells for destruction by the
macrophages.

 ❖ Protein synthesis and production of hemoglobin
in the various stages of erythropoiesis:
 Pronormoblast- Most of the Iron to be used in
hemoglobin synthesis is taken into the cell at this
stage. The very blue cytoplasm means protein (for
hemoglobin) is being produced.

 Early Normoblast - Hemoglobin appears for the
first time, if any asynchrony between
development of the nucleus and the cytoplasm
occurs, it's first detected at this stage.
 Late normoblast - The nucleus is extruded in later
period of this stage. After this stage cell no
longer can undergo mitosis.

 Reticulocyte - Part of this stage occurs in the
bone marrow, part in peripheral blood. At this
stage the cell has approximately 2/3 of its total
hemoglobin content. The cytoplasmic color at this
stage is called polychromatic or
polychromatophilic when viewed with Wright's
stain. If stained with methylene blue, the
remaining RNA will precipitate resulting in a
reticular appearance.

 Mature erythrocyte - All the hemoglobin is now
produced so there is no need for continued RNA
and protein synthesis. These cells are pliable and
deformable, making them capable of unusual
changes in shape for passage through the
microcirculation.

 The source of iron used in heme production is
ingestion. After being absorbed into the
bloodstream, the iron is transported by
transferrin, a carrier protein, to the bone marrow
where it is incorporated into heme molecules.
 Excess iron is stored in erythrocytes, liver,
spleen, and the mononuclear phagocyte system
(macrophages). The storage forms of iron are
ferritin and hemosiderin.

 Tests for evaluation of iron
 1) Serum Ferritin - soluble storage form of iron
that is proportional to iron stored in tissues
 2) Serum Iron - iron available in the serum for
erythrocyte production. This test isn't as closely
related to body stores, as is ferritin,

 Total iron binding capacity (TIBC) - a measure of
transferrin, which is not bound to iron. The TIBC is
inversely related to iron level. The percent saturation
of transferrin is calculated by dividing the serum iron
divided by the TIBC. This is a more accurate test
than serum iron but not as accurate as ferritin.
 Serum Transferrin measured by EIA (enzyme
immunoassay). Serum transferrin correlate well with
TIBC values.

 During its 120 day life span the erythrocyte has
traveled 200-300 miles. The process of aging is
called senescence. Enzyme activity decreases
(esp. glycolytic enzyme which helps in breakdown
of glucose, the source of erythrocyte energy),
and the cell looses its deformability.

 MCHC -(mean corpuscular hemoglobin
concentration) increases, the cell becomes
rounder, and the MCV (mean corpuscular volume)
decreases.
 90% of destruction of senescent erythrocytes
occurs by extra vascular hemolysis.

 Macrophages of the mononuclear phagocyte
system remove them from circulation.
 Macrophages of the spleen are especially active in
removing aging, dead and abnormal erythrocytes
(e.g. cells containing Heinz bodies or Howell-Jolly
bodies, siderocytes, target cells, schistocytes,
tear drop cells and antibody-coated
erythrocytes.)

 Some essential components of heme (iron plus
amino acids from globin of hemoglobin) are
recovered from the macrophages.
 Iron is transported via transferrin to the red
bone marrow. Amino acids from globin are
returned to the liver where they are used to build
more proteins. The protoporphyrin ring of heme is
disassembled and form the body.

 Its alpha carbon is exhaled in the form of CO2.
The opened tetrapyrrole, biliverdin, is converted
to bilirubin which is then carried to the liver by
the plasma protein, albumin.
 In the liver bilirubin is conjugated to glucuronide
to make it water soluble and excreted along with
bile into the intestines.

 In the intestines it is converted by bacteria into
stercobilinogen and excreted in the stool; some is
eliminated as urobilinogen in the urine.
 Stercobilinogen and urobilinogen are what give
feces and urine their color.

 When there is excess erythrocyte breakdown,
jaundice (icterus) results; seen as yellowness of
the skin and sclera due to increased serum
bilirubin and deposition of bile pigments.
 Unconjugated bilirubin (prehepatic) and
conjugated bilirubin (posthepatic) are measured
serum as indirect (unconjugated) and direct
(conjugated) bilirubin; used to monitor amount of
hemolysis.

 About 10% of normal erythrocyte destruction
occurs by intravascular hemolysis While in
circulation the red cell is subjected to metabolic
and mechanical stresses: turbulence, endothelial
damage and fibrin deposition resulting in red cell
fragmentation (schisiocytes) and/or intravascular
hemolysis

 When the erythrocyte ruptures, hemoglobin is
released into the blood. The hemoglobin
dissociates into alpha-beta dimers and is picked
up by haptoglobin, a protein carrier, to prevent
renal excretion of hemoglobin.
 Haptoglobin carries the hemoglobin to the liver
for further catabolism where the process
proceeds as with extra vascular hemolysis.

 As haptoglobin is depleted, unbound hemoglobin
dimers appear in the plasma (hemoglobinemia) and
are reabsorbed by the kidney up to a certain level
and convened to hemosiderin; beyond this level
hemoglobin shows up in the urine (hemoglobinuria).
 Intravascular hemolysis results in pink, red or
brown plasma (hemoglobinemia). Urine may also
show red color (hemoglobinuria).

 TO BE
CONTINUED……..

 PART I:
 Erythrocytes
 Erythropoiesis
 Haemoglobin
 Variants of haemoglobin
 Iron kinetics
 Heme breakdown
 PART II:
 Hb concentration
 PCV
 Peripheral smear

 Definition: -ANAEMIA is defined as a
reduction in the concentration of circulating
hemoglobin or oxygen carrying capacity of blood
below the level that is expected for healthy
persons of same age and sex in the same
environment.

 Various methods are available for estimation of
haemoglobin.
 Out of these, Cyanmethaemoglobin method is the
most accurate and is recommended by the
International Committee for Standardization in
Haemalotogy.

 * Methods for estimation of haemoglobin:
 1) Colorimetric method: Colour comparison is
made between the known standard and the test
sample, either visually or by photoelectric
colorimeter.

 In this method a specified amount of blood is
mixed with a solution containing potassium
ferricyanide and potassium cyanide (Drabkin’s
solution); potassium ferricyanide converts
haemoglobin to methaemoglobin while
methaemoglobin combines with potassium cyanide
to form cyanmethaemoglobin.

 Most forms of haemoglobins present in blood (e.g.
oxyhaemoglobin, carboxyheamoglobin,
methaemoglobin etc.) except sulfhaemoglobins are
completely converted to a single compound,
cyanmethaemoglobin.

 After, completion of the reaction, absorbance of
the solution is measured in a spectrophotometer
at 540 nm.
 To obtain the haemoglobin concentration of
unknown sample, its absorbance is compared with
that of the standard cyanmethhaemoglobin
solution concentration of which is known.

 Gasometric method: Oxygen-carrying capacity
of blood is measured in van Slyke apparatus;
not suitable for routine use.
 Chemical method: - Iron content of blood is
measured and value of haemoglobin is
calculated indirectly; tedious and time
consuming method

 Specific gravity method: Simple, rapid and
inexpensive method in which a rough estimate
of haemoglobin is obtained from specific
gravity of blood; used for mass screening like
selection of blood donors.

 Mild : Haemoglobin from lower limit of normal to
10.0 gm/dl
 Moderate : 10.0- 7.0 gm/dl
 Severe : < 7.0 gm/dl

 PCV is the volume of packed red cells obtained
after centrifugation of a sample of anti-
coagulated venous or capillary blood.
 It is expressed either as a percentage of volume
of whole blood or as a decimal fraction.

 Uses of PCV are:
 (I) Detection of anaemia and polycythaemia; PCV
is normally about three times the haemoglobin
concentration when the latter is expressed in
gm/dl
 (II) Calculation of red cell indices such as mean
cell volume (MCV) and mean cell haemoglobin
concentration (MCHC)
 (III) Checking the accuracy of haemoglobin value.

 Red cell count in millions / cmm x 3 =
Haemoglobin in gm/dl
 Hb gm/dl x 3 = PCV in %
 Rule of 3 applies mainly to normocytic
normochromic specimens

 Anticoagulated whole blood is centrifuged in a
wintrobes tube at 2300 rpm for 30 min to pack
the red cells.
 The level of the column of red cells is directly
read from the tube.
 Wintrobe tube is 110 mm in length with 3mm
internal bore, is marked at every 1mm up to 100
and has a capacity for about 1 ml of blood.

 After centrifugation, three layers can be
distinguished-
 A column of straw coloured plasma at the top
 A thin greyish layer of white cells and platelets in
the middle (buffy layer)
 And a column of red cells at the bottom

 This method is simple, rapid and needs only a small
quantity of blood.
 It requires microhematocrit centrifuge (or table top
centrifuge with microhaematocrit head) and capillary
haematocrit tubes (75 mm long with a 1 mm bore)
 Two types of capillary hematocrit tubes are available
anticoagulated (coated with heparin so that capillary
blood can be directly collected) and plain (without
anticoagulant so that anticoagulated blood is needed)

 A) Anaemias due to impaired red cell production
 1) Anaemias due to deficiency of nutrients
 Iron deficiency anaemia
 Megaloblastic Anaemia due to deficiency of folate or
vitamin B12)
 2) Anaemia of chronic disease
 3) Sideroblastic anaemia
 4) Aplastic anaemia and related disorders
 5) Anaemia of chronic renal disease
 6) Anaemia of liver disease
 7) Anaemia in endocrine disorders
 8) Myelophthisic anaemia (due to replacement of marrow
by metastatic carcinoma, leukemia, lymphoma, infections,
storage disorders, etc.)
 9) Congenital dyserythropoietic anaemiawww.indiandentalacademy.com

 B) Anaemias due to excessive red cell
destruction (Haemolytic anaemias)
 Abnormality Intrinsic to red cells
 1) Defects in red cell membrane
 Hereditary spherocytosis
 Hereditary elliptocytosis
 2) Defects in haemoglobin
 Quantitative: Thalassemias
 Qualitative: Sickle cell disease
 Haemoglobin D, E, or C disease
 3) Defects in enzymes
 Glucosc-6-phosphate dehydrogenase deficiency
 Pyruvate kinase deficiency

 Abnormality Extrinsic to red cells
 1) Immune haemolytic anaemias
 Autoimmune
 Alloimmune
 Drug-induced
 2) Mechanical hemolytic anaemia
 Microangiopathic
 Cardiac
 March hemoglobinuria
 3)Direct action of physical, chemical, or
 4) Hypersplenism
 C) Anemia due to excess blood loss

 Peripheral blood smear or film provides an
important information regarding the underlying
cause of anaemia.
 Peripheral blood smear is prepared by spreading a
drop of capillary or venous blood across a glass
slide and staining it with a Romanowsky stain.

 A well-made blood film should show three zones-
thick area or the 'head’, ’body’ and the thin
portion or the ‘tail' of the smear.
 The smear should be smooth and uniform in
appearance with gradual transition from thick to
thin portion.
 It should not cover the entire area of the slide.

 The blood film should be examined in an orderly
manner under low and high powers and oil
immersion lens for cell morphology , presence of
nucleated red cells, approximate number of
WBCs, differential leukocyte count, abnormal
WBCs, parasites and adequacy of platelets.
 Valuable information regarding the cause of
anemia can be obtained by observing the red cell
morphology.

 Normocytic normochromic: normal size and colour
7-8 um; pink with small area of central pallor.

 Anisocytosis : significant variation in size of red
cells
 Poikilocytosis : significant variation in shape of
red cells
Any kind of
anaemia

 Macrocytic : larger than normal (round or oval)
Larger than normal > 7.7 um,
well filled with Hb
Young RBC
DNA synthesis -impaired
B12 or folate deficiency
Accelerated erythropoeisis

 Sickle cells ; elongated and narrow cells with one
or both ends curved and pointed
Form assumed under
hypoxia
Molecular aggregation of
Hbs
Sickle cell anaemia

 Target cells : cells with accumulation of Hb in the
center and periphery with clear intervening area
producing a bull’s eye or target- like appearance.
Splenectomy decreases rate
and extent of loss of lipids
from reticulocytes
Accumulation of both
cholesterol and phospholipid on
RBC
Congenital
Post splenectomy
In liver disease especially
obstructive jaundice

 Schistocytes : irregular fragmented cells
appearing as helmet- shaped and triangular
RBCs lose fragments after
impact with fibrin stands, walls
of diseased vessels and
artificial surfaces in circulation
Microangiopathic hemolytic
anaemia
Hemolytic anaemia due to
physical agents
Also in uremia, malignant
hypertension

 Tear drop red cells : cells with a tapering drop
like shape
Usually microcytic, often
hypochromic
Distorted or fragmented RBC
Especially in myelosclerosis
Thalassemia

 Polychromatic red cells : slightly larger cells with
faint blue-gray tint due to presence of ribosomal
RNA.
Elliptical in shape, not
hypochromic
Hereditary elliptocytosis
Megaloblastic anaemia

 Basophilic stippling (punctate basophilia) :
presence of fine or coarse purple-blue granules
(representing ribosomal aggregates)
Lead posioning
Thalassemia

 Dimorphic red cells: presence of two different
populations of cells e.g. macrocytic and
hypochromic, normocytic and hypochromic.
Sideroblastic anaemia,
Partially treated
anaemia,
Myelodysplasia and
post blood tranfusion

 Rouleaux : arrangement like a stack of coins
Hyper gammaglobunemia
Multiple myeloma

 Howell jolly bodies : round purple nuclear
remnants
Thalassemia
Post splenectomy
Nucleated red cells

To be continued…www.indiandentalacademy.com

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