Microcytic

Hypochromic
Anaemia
and Revision
 Causes
Decreased production

Blood loss

Haemolysis
Microcytic Hypochromic Anemia: Diagnosis

Mild (MCV > 70 fl)

Iron deficiency
Thalassemia
Lead toxicity
Sideroblastic anaemia
Anaemia of chronic disease

Severe (MCV < 70 fl)

Iron deficiency
Thalassemia
Normal
Hypochromia
Microcytic Hypochromic
Normal but too thick
Anisocytosis
+++

Polychromasia
Poikilocytosis
++
Polychromasia
Teardrops
Dimorphic

Post Bleed, Blood

transfusion, Iron therapy
 Target Cells

Nucleated Red blood

cell
NRBC
Polychromatic

Schistocytes
Fragmented

Microangiopathic
Haemolytic Anaemia
(MAHA)
Spherocytes
Stomatocytes
Folded Red cells
Elliptocytes
Agglutination
Rouleaux
Crenated Red Cells
Howell Jolly
Bodies
Basophilic Stippling
 Pappenheimer Bodies

Howell Jolly
Malaria
Water Artefact
 Haematology
Revision
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The differential count must add up to 100%
The absolute values must equal the Total leukocyte count.
MCV (mean cell volume) is the average size of the red cells.
Below NRR in microcytosis and above NRR in macrocytosis.
MCH (mean cell haemoglobin) is the average amount of
haemoglobin In the red cells.
Below NRR in hypochromia.
MCHC (mean cell haemoglobin concentration) is the average
concentration in the red cells. It is increased in spherocytosis.
Anisocytosis: Variation in size of the red cells.
Poikilocytosis: Variation in shape of the red cells.
Round macrocytes: Are seen in Liver disease along with
target cells.
Oval macrocytes: Are seen in Megaloblastic anaemia (Vit
B12/ folate deficiency) along with hypersegmentation of the
neutrophils. (more than 5 lobes).
Microcytes: Seen in Iron deficiency anaemia along with
Hypochromic red cells.
Polychromasia: Seen in Bone marrow regeneration after a
bleed or iron therapy.
Reticulocytes: Are polychromatic cells stained with a
supravital stain. (New Methylene Blue)
 Red cell classification
1. Normocytic Normochromic
2. Microcytic Hypochromic
3. Macrocytic
 Platelet comment:
If a count has been done:
Above NRR: Thrombocytosis
Below NRR: Thrombocytopaenia

If a count had NOT been done:

Platelets appear above or below NRR
Comment on large or giant platelets
Large platelets are greater than one third the size of a red cell
Giant platelets are greater that the size of a red cell
 What is the name for platelets which are within the Normal
Reference Range (NRR) but are non functional?

Thromboasthenia
Which immature White Cell had no granules in the
cytoplasm?

Myeloblast
What is the difference between Acute & Chronic
Leukaemia?
Acute has 30 to 90% blast cells.
Chronic has more mature cell present and only a few
blast cells.
Name the white cells in order of maturity
Myeloblast
Promyelocyte
Myelocyte
Metamyelocyte
Band Neutrophil
Segmented Neutrophil
Extravascular Pathway for RBC Destruction
ferrous (Fe+2) is
more readily
absorbed in the
small intestine
than the ferric (Fe Phagocytosis & Lysis
+3) form, which is
not very soluble.
Hemoglobin

Bilirubin Haem Globin

Fe2+
Amino Acids

Excreted
Recycled
John Santangelo
Bone Marrow
The peripheral blood is the mature blood
that circulates in the Arteries and Veins,
i.e. outside the Bone Marrow

The normal cells below, appear in the peripheral blood


The nucleolus is the site of assembly
of ribosomal proteïns, which are
located in various particles dispersed
over the cytoplasm.
Mitochondria are present but have a
rather small size.
Myeloblast
Myeloblasts are rather small cells
with a diameter between 12 and
20µm, from which the major part
is occupied by a large oval
nucleus.
Their nucleus is composed of
very fine nonaggregated
chromatin and possesses 3 or
more nucleoli. (arrows)
The cytoplasm has basophilic
character and has no granules,
which is a major difference with
its successor, the promyelocyte.
Black Arrows Myeloblast
Nucleoli 14um to 19um Diam
Cytoplasm has
3 or more nucleoli
no Granules

Auer Rods

Condition?
Acute Myeloid Leukaemia (AML)
A myeloblast is a large cell, 12-20 microns in diameter, with a nucleus
that takes up most of the cell.

Nuclear chromatin is fine, with little condensation and fairly

homogenous in appearance.
Nucleoli may vary in number and are tend to be large.
The scanty cytoplasm is blue without granules.

Blue arrows:
Nucleated Red Cells

White arrows:
Nucleoli
Myeloblast
ACUTE Chronic Myeloid Leukaemia
Note the lack of Nucleoli
lio
cle
Nu

The granules are coarse and
range in colour from azurophilic
to dark blue.
The nuclear chromatin is coarse
but evenly dispersed and one or
more nucleoli are usually seen.
Promyelocyte
It is the largest cell of the various
stages of granulocyte maturation,
and is distinguished from a blast
by the appearance of variable
numbers of primary granules in
the cytoplasm and the
development of the Golgi
apparatus.
Primary granules may appear
before the Golgi apparatus is
seen.
 A promyelocyte may be the same
size as the myeloblast or even larger.

A promyelocyte may be the same

size as the myeloblast or even
larger. Unlike the myeloblast, the
promyelocyte's cytoplasm contains
large black or purple granules.

Nucleoli may be present.

Promyelocyte
Toxic Myelocyte Myelocytes.

The chromatin structure is denser

than that of promyelocytes.

The granules do not lie over the

nucleus (as can be seen by turning
the fine focus adjustment of the
microscope to and fro).

Normal Myelocyte

Orange granules start to appear

in the cytoplasm of the
myelocyte
Myelocytes (10-18um) are slightly smaller than promyelocytes and
have eccentric round-oval nuclei, often flattened along one side.
The chromatin is fine, but shows evidence of condensation.
Nucleoli may be seen in early stages
but not in the late myelocyte.

Primary azurophilic granules are still

present, but secondary granules
predominate.
Secondary granules (neut, eos, or
baso) first appear adjacent to the
nucleus.
In neutrophils this is the "dawn" of
neutrophilia.
Myelocyte
 Metamyelocyte
When the nucleus of a
mature myelocyte
starts to indent, it is
referred to as a
metamyelocyte until
the indentation is
greater than 50% of
the diameter of the
nucleus, at which time
the stage is referred to
as a neutrophilic band.

The cytoplasm is all pink (acidophilic) with only secondary,

neutrophilic granules and the nuclear chromatin is clumped.
The cell is only slightly smaller than a mature myelocyte.
Metamyelocytes (10-18um) are slightly smaller than myelocytes.

They have kidney shaped indented nuclei and relatively dense

chromatin, especially along the nuclear membrane.
The cytoplasm is faintly pink
with almost no blue
background.
Numerous secondary
Metamyelocyte granules (neutro, eos, or
baso) clearly outnumber
primary granules.
Zero to one percent of the
peripheral blood white
cells may be
metamyelocytes
(juveniles).
 Band Neutrophils

10-15 microns
Band shaped nucleus with coarse, clumped chromatin structure.
The narrowest part is at least 1/3 of the widest.
No nucleoles
Pale pink to colourless cytoplasm
Plentiful very fine violet granules
 Segmented Neutrophil
When the first segmentation of
the nucleus occurs, the cell is
Lobe
referred to as a segmented
neutrophil.
Segment Further segmentation usually
occurs with the number of
segments varying between 2
and 5.

The lobes are connected by chromatin filaments of varying

lengths.
The filaments may not be visible when the lobes are partially
superimposed but distinct lobes are usually identifiable.
 Transition between
a Metamyelocyte
and a Band Cell.

Band Cells Metamyelocyte

 Green arrows:
Nucleated Red Cells
 Orthochromatic normoblasts
• The E5 orthochromatic normoblast is smaller than
the E4 stage.
• In the E5 orthochromatic normoblast hemoglobin is
abundant while ribosomes decrease in number.
• The nuclear chromatin is pyknotic and ready to be
extruded.
 Polychromasia
• Polychromatophilic red cells are larger than mature
erythrocytes and still contain ribosomes and
mitochondria, giving the cell a blue tint.
• When polychromatic RBCs are stained with a
supravital dye (new methylene blue or cresyl violet)
ribosomes clump to form a blue stained granulars
called reticulum. These are called reticulocytes.
 Mature red cells
• Normal mature erythrocytes are biconcave discs
(approximately 7 um in diameter; 2.5 um thick at the
periphery; 1.0 um thick at the center).
• This results in an area of central pallor (about 1/3
the dia.) seen in mature RBCs stained with Wright's-
Giemsa.

Erythrocytes
 Megakaryocytes
• Megakaryocytes undergo endomitosis - a process
whereby DNA is duplicated without cell division.
• Megakaryocytes are the largest normally occurring
cells in the marrow (35-160um)

Platelet
Precursors
 Thrombopoietin

• Produced in kidney and liver

• Cause megakaryocyte maturation

• Bound to platelets- feedback

 Functions of platelets

1.Hemostasis

2. Coagulation of blood

3. Clot retraction

4. Phagocytosis

5. Storage and transport- serotonin &

histamine
 Thrombocytopenia:
• Bone marrow depression

• Hypersplenism

• Viral infections
Unknown cause

Adrenalin • Idiopathic

• Epinephrine administration
 Thromboasthenia
Platelet function disorder
Tests:
Old Tests:
Bleeding & Clotting time
New Test:
PFA 100
Platelet Function Analysis 100
Auer Rods
Acute Myeloid leukaemia with Auer Rods

Auer Rods
Giant Platelets
 Howell Jolly Bodies
Blue Arrows
Howell Jolly Bodies

Red Circle
Pappenheimer Bodies
Pappenheimer Bodies
Siderocytes
 Dohle Bodies
Toxic Lymphocyte EBV (Ebstein Barr Virus)
Toxic Granulation
Toxic Granulation
Toxic Granulation
Basophilic Stippling
Basophilic Stippling
Dimorphic
Dimorphic Blood picture
Nucleated Red
Blood Cells
NRBC
Nucleated Red Cells
Supravital Staining of
Reticulocytes
Reticulocytes (Polychromatic)
Polychromasia
Macrocytes
Macrocytes
Round
Macrocyte
Oval
Pelger Huet anomaly
Pelger Huet
Platelet Satellitism
Blasts, AML
Mast Cell

Nucleated Red Cell

Mature Basophils
Metamyelocyte
Microcytic Hypochromic
Microcytic Hypochromic
Myelocyte
Promyelocyte in
Mitotic Division
Hampden Bridge
Kangaroo Valley
Platelet Clumping
Rouleaux
Sickle Cell
Spherocyte
Spherocytosis
Stomatocyte
 Target Cells

Not a Kmart Cell

Haemoglobin SC Disease
Autoagglutination
Neutrophilia
Fragmented cells
 Pelger Huet Anomaly
Pelger-Huët anomaly (PHA) is a benign dominantly
inherited defect of terminal neutrophil differentiation
secondary to mutations in the lamin B receptor (LBR) gene.
Characteristics observed on blood smears include
leukocytes with dumbbell-shaped bilobed nuclei;
a reduced number of nuclear segments;
coarse clumping of the nuclear chromatin in neutrophils,
lymphocytes, and monocytes.
Pelger-Huët cells survive normally in circulation. They have
normal leukocytic function and can phagocytize and kill
micro-organisms.
Examination of a peripheral blood smear in an individual
heterozygous for Pelger-Huët anomaly (PHA) is remarkable
for neutrophils with a predominance of bilobed, spectacle-
shaped nuclei.

Cells that contain twin, joined, and plump nuclei resembling

dumbbells are predominant.
A thin bridge, which is thinner than that observed in a normal
band neutrophil, joins the 2 lobes.
About 69-93% of the neutrophils show nuclear segmentation
that is arrested at the bilobe level.
A small population of neutrophils that possess a nonlobulated
oblong or peanut-shaped nucleus is often present.
 Iron Exists in several oxidation states.

• There are 2 forms of iron:

Heme; animal
Non-heme; plants

• In the human body and food, iron exists in

Ferric (Fe+3) and ferrous (Fe+2) forms.

• Heme contains the iron bound to the porphyrin ring,

called a metalloporphyrin.
 Iron in human body

Total iron in human body ~3.8 g (men),

~2.3 g (women);

Iron is localized mostly in cells;

1-2 mg are taken up from food (10 % absorption);

1-2 mg are released with skin and gut tissues.
􀃆 Fe metabolism is conservative;

Transferrin is the major Fe binder in blood plasma.

Most ferritin is found in hypatocytes (liver), spleen,
bone marrow;
 Storage and Excretion

Storage
Iron is delivered by transferrin to the liver, bone marrow and spleen.
Primary storage form of iron is Ferritin in cells and tissues.

Secondary storage form of iron is Hemosiderin (protein).

Stored more in the liver, then bone marrow and spleen.

Excretion
Not needed iron as ferritin it is excreted with the mucosal cells that are
shed off into the lumen of GI tract every 2-3 days.
Most iron is lost from the GI Tract,, then skin, kidney.
Small amount lost in urine.
During menstrual cycle lost iron accounts for 17.5mg per cycle.

Every mL of Blood contains 0.5 mg iron.

 Common diseases

Iron deficiency
Iron overload
Anaemia of chronic disease
Vegetarian groups

Iron Functions

Oxygen carriers
Haemoglobin

Oxygen storage
Myoglobin
 Ferritin

A routine blood test – reflects iron stores

Low serum levels

Indicate Iron deficiency (high specificity)

High serum levels

Iron overload
Other - Ferritin may be increased in serum by:
Tissue release (hepatitis, leukaemia, lymphoma)
Acute phase response (tissue damage, infection, cancer)
Interpretation
Low levels always indicate Fe deficiency.
 Iron Loss
Physiological
Cell loss: gut, desquamation
Menstruation (1mg/day)
Pregnancy, lactation

Pathological
Bleeding
Gut, menorrhagia, surgery, gross haematuria
Iron Absorption Regulation

Increased
Low dietary iron
Low body iron stores
Increased red cell production
Low haemoglobin
Low blood oxygen content

Decreased
Systemic inflammation
 Iron Deficiency

Laboratory changes:
Low iron (poor specificity)
Low ferritin (excellent specificity)
Elevated Transferrin (TIBC)
Low transferrin saturation
Hypochromic, microcytic Anaemia
 Causes of Iron deficiency

Increased demand for iron

Infancy and adolescence
Pregnancy and lactation

Iron loss
Bleeding

Decreased iron intake or absorption

Hepciden
In physiologic conditions
Menstruation

In pathologic conditions
Surgery,
Haemoglobinuria,
haemoptysis
Gastrointestinal tract pathology

In therapeutic procedures
Phlebotomy

In blood donation
 Iron Deficiency
Clinical Manifestations

Fatigue
Decreased exercise tolerance
Tachycardia
Dermatologic manifestations
Decreased intellectual performance
Dysphagia
Depression, increased incidence of infections
Restless legs syndrome
 Iron Deficiency Anemia (IDA)

• Most common cause of anemia

• Microcytic hypochromic anemia
• MCV, MCH, MCHC are reduced
• blood film : small red cells (microcytic)
: pale red cells (hypochromic)
Microcytic Hypochromic
 Megaloblastic Anaemia
(Pernicious Anaemia)
Hypersegmented Neutrophils
and
Oval Macrocytes
Laboratory findings (2)

• Iron metabolism tests

– serum iron concentration 

– total iron-binding capacity (TIBC) 
– saturation of transferrin 
– serum ferritin levels 
– sideroblasts 
 Hemochromatosis

Also known as accumulating disease.

This is a genetic disorder in which the
intestine is not able regulate iron absorption.

Result: iron absorption increases = build up

of excess iron = organ damage.

Most often seen in Caucasian males around

age 20
 Accumulation

Iron accumulation in tissues

causes damage/ failure to:
Liver
Heart
Pancreas
Skin
May generate free radicals which
can damage normal cells.
 Too much Iron
Haemochromotosis
Uncontrolled Iron absorption leads to Iron overload

Iron accumulates in: And causes:

1. Liver
1. Cirrhosis
2. Heart
2. Cardiomyopathy
3. Pancreas
3. Diabetes
4. Skin
4. Bronzing
5. Joints
5. Arthritis

It is Autosomal recessive
 Iron
Iron study
study

1. Serum iron

2.Total iron binding capacity (TIBC)

3. %Transferrin saturation (% sat)

4. Ferritin
Macrocytic Anaemia's
The Mean Cell Volume (MCV) is
typically greater than 100 fL

Presence of hypersegmented neutrophils.

Oval Macrocytes
 Megaloblastic
Non production of HCL and Intrinsic Factor
Due to damage to the Parietal cells of the
Stomach
Or
Parietal cell antibodies
And
Intrinsic Factor antibodies
Therefore B12 folate cannot be taken orally
The term 'pernicious anaemia' is sometimes
also incorrectly used to indicate megaloblastic
anaemia due to any cause of vitamin B12
deficiency.
Its proper usage refers to that caused by
atrophic gastritis, parietal cell loss, and lack of
intrinsic factor only.
Peripheral nerve can result from Vitamin B12
deficiency.
Tests:
Old Tests:
1. Fractional Test meal.
2. Schillings Test.
Latest Tests:
Parietal Cell antibodies and Intrinsic factor
antibodies.
Serum B12 and folate + Red cell folate.
 Types of macrocytic anaemias
Common causes of macrocytic anaemias.
1. Megaloblastic anemias (DNA replication disorders)
The cells are larger because they cannot produce DNA quickly
enough to divide at the right time as they grow, and thus grow
too large before division.
Causes for the DNA synthetic problem
Lack of certain vitamins needed to produce DNA (notably
folate and B12).
Poisons or inhibitors of DNA replication, such as some
kinds of antiviral drugs and chemotherapeutic agents.
 Oval Macrocytes

Hypersegmentation
of the Neutrophil
More than 5 lobes
 A history of alcohol abuse may be an important clue as to the cause of the
increased MCV.
A thorough examination of the patient's medication regimen is also crucial in
the workup of macrocytosis.
Dyspnoea is a consequence of anemia. In acute or severe anaemia, the
volume of haemoglobin in the blood is inadequate to provide appropriate
oxygenation of the tissues.
Fatigue may be attributed to underlying disease, if present, or to inadequate
blood volume.
Headache is a symptom of anaemia due to decreased oxygenation of the
tissues.
A sore tongue may be glossitis or atrophy of the tongue, which are common
findings in folate and vitamin B-12 deficiencies.
Diarrhoea or other gastrointestinal symptoms may be present in patients with
tropical or celiac sprue. Sprue may cause folate or vitamin B-12 deficiencies.
Long-term use of alcohol may have a direct toxic effect on the bone marrow,
causing macrocytosis.
A variety of medications may have an effect on the MCV.
Paresthesia or gait disturbances suggest vitamin B-12 deficiency.
Recent acute blood loss may signal that reticulocytosis is causing the MCV
increase.
Causes
Vitamin B-12 deficiency is a cause of macrocytosis.
Because DNA synthesis requires cyanocobalamin (vitamin B-12) as a
cofactor, a deficiency of the vitamin leads to decreased DNA synthesis in
the erythrocyte, thus leading to macrocytosis.
A dietary deficiency of vitamin B-12 is rare, although deficiency can result
from the following:
Lack of intrinsic factor in patients with postgastrectomy status or who have
pernicious anaemia
Malabsorption of vitamin B-12 secondary to small bowel overgrowth,
tapeworm, familial factors, drugs, ileal bypass, ileal enteritis, or sprue
Folate also is needed as a cofactor in the synthesis of DNA. Folate
deficiency may be caused by any of the following:
Dietary deficiency
Increased requirements of pregnancy
Congenital deficiency
Drug-induced macrocytosis is the most common cause in non-
alcoholic patients.
Usually, no associated anaemia is present.

The following categories of drugs are known to cause

macrocytosis:

Folate antagonists (methotrexate)

Purine antagonists (6-mercaptopurine)
Pyrimidine antagonist (ara-C)
Alkylating agents (cyclophosphamide)
Azidothymidine (AZT)
Trimethoprim
Oral contraceptive pills (OCPs)
Dilantin
Arsenic
Lab Studies
Complete FBC with platelet count
The haemoglobin/hematocrit level may provide a guide to
diagnosis, and it determines presence and severity of
anaemia.
WBC and platelet count may be decreased in primary marrow
disturbances.
The MCV is a calculated average volume of the erythrocytes.
An MCV greater than 100 fL is, by definition, macrocytosis.
Because evaluation of the RBC size is key to the diagnosis of
an acnemia, the MCV is considered to be the most important
of the RBC indices.
Reticulocyte count
This study helps to determine if hemolysis is present;
it also indicates malfunctioning bone marrow.
Expect marked reticulocytosis (>4) in haemolytic
anaemia's.
Reticulocyte count less than 1% indicates inadequate
marrow production.
Reticulocyte count must be corrected for degree of
anaemia.
 The Reticulocyte Count:
Count 1000 Red cells noting the number of reticulocytes.

Formula: Number of reticulocytes

---------------------------------- X 100
1000

= Reticulocytes%

e.g. If 15 reticulocytes were counted in the 1000 red cell that were counted, then,
15 divided by 1000 multiplied by 100 = 1.5%
Normal Retic Count = 0.5 – 2.5%

Reticulocyte Count Correction for Anaemia

Since the Reticulocyte count is expressed as a relative percentage, a change

in this percentage might be due to an overall change in the number of
circulating erythrocytes, and must be corrected to an absolute percentage in
cases of anaemia. This correction is accomplished as follows:

Patient's HCT
Corrected Retic Count = Retic % X -------------------
Normal HCT (45
 Follow up tests

1. Serum B12/folate
2. Red cell folate
3. Intrinsic factor antibodies
4. Parietal cell antibodies
 Anaemia of chronic disease, increasingly referred to as
"anaemia of inflammation”
75% clinical causes are secondary to:

Infection.

Inflammation - including connective tissue disorders.

Neoplasia. (New growth)

Anaemia is also commonly associated with chronic renal

failure.
The anaemia of chronic disease is still poorly understood:
New discoveries suggest that the syndrome is likely largely the
result of the body's production of hepcidin.
Hepcidin ia a master regulator of human iron metabolism.
Inflammatory conditions produce cytokines.
In response to cytokines, the liver produces increased amounts
of hepcidin.
Hepcidin in turn stops ferroportin from releasing iron stores.
Ferroportin is a transmembrane protein that transports iron
from the inside of a cell to the outside of it.
Inflammatory cytokines also decrease the ability of the bone
marrow to respond to erythropoietin.
Cytokines also promote the production of white blood cells.
Therefore, the increased production of white blood cells
causes fewer stem cells to differentiate into red blood cells.
Diagnosing Anaemia of chronic disease

A full blood count can be very useful and is required for

diagnosis as it gives the levels of haemoglobin in the blood.

It will also give information about the size of the red blood cells,
as well as how much haemoglobin each cell contains.

Usually an anaemia of chronic disease will normocytic and

normochromic.

Another useful blood test is an 'iron study' as this will show how
much iron there is in the blood as well as showing various other
factors such as how much iron the blood has the potential to
bind.
Investigations

Anaemia of chronic disease typically occurs despite adequate

reticuloendothelial iron stores.

Reduced serum iron, transferrin and total iron binding capacity.

Measuring serum ferritin is essential in investigating

unexplained anaemia:

Serum ferritin concentration is directly related to

reticuloendothelial iron stores, and normally 1 μg/l serum ferritin
roughly corresponds to about 8 mg of storage iron.

Reduced serum ferritin provides unequivocal evidence of

diminished iron stores and occurs in no other condition.
 In anaemia of chronic disease the Ferritin is
Normal or above the NRR

In iron deficiency disease the Ferritin is below

the NRR
Platelet satellitism