1
SCOPE
 To cover the morphological appearance of blood cells
that can be seen on a blood film, which will aid in
diagnosis and management of hematological and non
hematological disorders.
2
OBJECTIVES
 To present preparation of blood films and
morphological abnormalities that can occur due to
preparation.
 To describe normal morphology and morphological
changes in
 Red cells
 White cells
 Platelets
3
PREPARATION OF BLOOD FILMS
• A blood film may be made from:
– Fresh blood
– EDTA - anticoagulated blood.
• Spreading:
– Manual
The cover glass smear.
 The wedge smear .
 The spun smear.

– Automated
4
PREPARATION OF BLOOD FILMS
• The are two additional types of blood smear used
for specific purposes
• Buffy coat smear for WBCs < 1.0×109/L
• Thick blood smears for blood parasites.
• Labeling of blood films
• By pencil
• Bar-coding
5
CHARACTERISTICS OF A GOOD SMEAR
 Thick at one end, thinning out to a smooth
rounded feather edge.
 Should occupy 2/3 of the total slide area.
 Should not touch any edge of the slide.
 Should be margin free, except for point of
application.
6
Tail
Body
Head
7
FIXATION
 Thin films are fixed in absolute methanol for 10–20
minutes after air drying.
 Poor fixation and characteristic artifactual changes occur
if there is more than a few per cent of water in the
methanol.
 This renders interpretation of morphology, particularly
red cell morphology, impossible giving a mistaken
impression of hypochromia.
8
STAINING - ROMANOWSKY STAINS
 Romanowsky stains are universally employed
for staining blood films and are generally very
satisfactory.
 There are a number of different combinations
of these dyes, which vary, in their staining
characteristics.
9
STAINING - ROMANOWSKY STAINS
1. Giemsa stain
1. methylene azure and eosin
2. May-Grünwald–Giemsa (MGG) stain.
1. Giemsa and May-Grünwald
3. Wright’s stain
methylene blue and eosin
4. Jenner’s stain
5. Leishman stain
6. Field’s stain
1.
10
STAINING CHARACTERISTICS OF A CORRECTLY
STAINED NORMAL FILM:
 Nuclei
Purple
 Cytoplasm

Erythrocytes
 Neutrophils
 Lymphocytes
 Monocytes
 Basophils
Deep pink
Orange-pink
Blue
Grey-blue
Blue
 Granules

Neutrophils
 Eosinophils
 Basophils
 Monocytes
 Platelets
Fine purple
Red-orange
Purple-black
Fine reddish (azurophil)
Purple
11
STAINING
• Staining must be performed at the correct pH.
• low pH
– basophilic components do not stain well. Leucocytes will appear pale
• High pH
– Uptake of the basic dye may be excessive leading to general
overstaining,
– Makes it difficult to distinguish between normal and polychromatic red
cells,
– Granules of normal neutrophils become heavily stained, simulating
toxic granulation.
• Stain solutions may need to be filtered shortly before use, to
avoid stain deposit on the blood film, which can be confused
with red cell inclusion.
12
STAINING
TOO ACIDIC SUITABLE
TOO BASIC
13
ARTEFACTUAL CHANGES PRODUCED BY 5% WATER IN THE
METHANOL USED FOR FIXATION
14
STAINING FOR MALARIAL PARASITES
 The detection and identification of malarial parasites
done on blood films stained with a Giemsa (or
Leishman) stain at pH 7.2.
 At this pH, cells that have been parasitized by either
Plasmodium vivax or Plasmodium ovale have different
tinctorial qualities from non-parasitized cells and are
easily identified.
15
EXAMINING BLOOD FILMS
• The blood film acts as a quality control for the cell
counters.
• Patient identification
– Age, sex and number should match that on the FBC
• Macroscopic examination
– confirm adequate spreading
– look for any unusual spreading or staining characteristics.
• Blue coloration-commonest macroscopic abnormality
16
MACROSCOPY
Bluish coloration
• Hypergammaglobulinia
due to:
– Paraprotein
– Reactive increase in
immunoglobulinscirrhosis or rheumatoid
arthritis
• Old smears, excessive
staining times
• Aged buffers
17
MACROSCOPIC ABNORMALITIES
Caused by :
 Precipitation of cryoglobulin,
 Gross red cell agglutination,
 Platelet clumping
 Presence of clumps of tumour cells
 Leucocytosis
 High MCV
18
MICROSCOPIC EXAMINATION OF
BLOOD FILMS
 Find an optimal area for the detailed examination and





enumerations of cells.
The RBCs should not touch each other.
There should be no area containing large amounts of
broken cells or Precipitated stain.
The RBCs should have a graduated central pallor.
Nuclei and cytoplasm of WBCs should be the proper
color.
Platelets should be clearly visible.
19
MICROSCOPIC EXAMINATION OF BLOOD FILMS
• Low power(x 10)
– Assess quality of smear
– Assess whether red cell agglutination, excessive rouleaux
formation or platelet aggregation is present
– Assess number, distribution and staining of leucocytes.
– Allows rapid scanning of the film to detect scanty abnormal
cells.
 At x 40:
 Appreciate variation of size ,shape, presence of toxic
granulation
 White cell differential counts
 At x 100 oil immersion:
 Cellular inclusions and cytoplasmic granules, malaria parasites 20
STORAGE INDUCED ARTEFACTS
• Prolonged storage of EDTA-anticoagulated blood
causes:
– Loss of central pallor simulating spherocytosis
– Crenation or echinocytic changes in red cells.
– Degeneration of neutrophils
– Lobulation of some lymphocyte nuclei
• Excess EDTA may cause crenation of red cells and
accelerates the development of storage change
21
Smear from a blood specimen
transported in a hot motor vehicle,
showing red cell budding and
fragmentation.
Blood film showing storage
Artefact -crenation (echinocytosis),
a disintegrated cell and a neutrophil
with a rounded pyknotic nucleus.
22
RED BLOOD CELLS
Examine for :
1. Size and shape.
2. Relative hemoglobin content.
3. Polychromatophilia.
4. Inclusions.
5. Rouleaux formation or agglutination
Note :The percentage of pyknocytes and schistocytes
in normal blood does not exceed 0.1% in adults,0.31.9% in full term infants and 5.6% in preterm
23
RBC MORPHOLOGY
 7-9 m with 1/3 central
pallor
 About the size of nucleus
of normal lymphocyte
24
ANISOCYTOSIS
 Is an increase in the variability of erythrocyte size
beyond that which is observed in a normal healthy
subject.
 Is a common, non-specific abnormality in
haematological disorders.
 Increase in the red cell distribution width (RDW) is
indicative of anisocytosis.
25
MICROCYTOSIS
 Decrease in the size of the erythrocytes.
 The nucleus of a small lymphocyte, is a useful guide
to the size of a red cell.
 Microcytes result from a defect in haemoglobin
formation.
 Causes
 Inherited-thalassemia
 Acquired-iron deficiency anaemia
26
HYPOCHROMIC MICROCYTIC RED BLOOD CELLS
27
MACROCYTOSIS
 Increase in the size of erythrocytes.
 The erythrocytes of neonates and pregnant
women show a considerable degree of
macrocytosis .
28
CAUSES OF MACROCYTOSIS
• Associated with reticulocytosis
– Haemolytic anaemia
– Haemorrhage
• Associated with megaloblastic erythropoiesis
– Vitamin B12 deficiency and inactivation of vitamin B12 by chronic exposure to
nitric oxide
– Folic acid deficiency, antifolate drugs
– Scurvy
– Drugs interfering with DNA synthesis
• Associated with macronormoblastic erythropoiesis
– Myelodysplastic syndromes including primary acquired sideroblastic anaemia
–
–
–
–
Multiple myeloma
Ethanol intake
Liver disease
Phenytoin therapy
• Uncertain mechanism
– Cigarette smoking
– Chronic obstructive airways disease
29
HYPOCHROMIA
 Reduction of the staining of the red cell ; there is an
increase in central pallor, which occupies more than
the normal approximate one-third of the red cell
diameter.
 Any of the conditions leading to microcytosis may
also cause hypochromia.
30
ANISOCHROMASIA
 Increased variability in the degree of staining or
haemoglobinization of the red cell.
 There is a spectrum of staining from hypochromic to
normochromic.
 Commonly indicates a changing situation, such as iron
deficiency developing or responding to treatment
DIMORPHISM
 Indicates the presence of two distinct populations of red
cells (hypochromic,microcytic cells and another
population of normochromic cells)
31
CAUSES OF A DIMORPHIC FILM
• Iron deficiency anaemia (following administration of
iron or blood transfusion)
• Macrocytic anaemia, post transfusion,
• Double deficiency of iron and either vitamin B12 or folic
acid, unmasking of iron deficiency
• Following treatment of megaloblastic anaemia
• Delayed transfusion reactions.
32
POLYCHROMASIA
• Red cells that are pinkish-blue as a consequence of uptake
both of eosin (by haemoglobin) and of basic dyes (by residual
ribosomal RNA).
• They are readily recognized, in MGG-stained films, by their,
– Greater diameter,
– Lack of central pallor
– Polychromatic qualities
 Increase –
 Increasing altitude (physiological response)
 Hypoxic stimulus
 Normal response to anaemia when there are no factors limiting
erythropoiesis.
 In myelofibrosis
 Metastatic carcinoma of the bone marrow.
33
SHAPE
• The normal shape and flexibility of a red cell are
dependent on the integrity of the cytoskeleton to which
the lipid membrane is bound.
• An abnormal shape can be caused by:
– Primary defect of the cytoskeleton or membrane:
– Secondary to red cell fragmentation or to polymerization,
crystallization or precipitation of haemoglobin
34
POIKILOCYTOSIS
• State in which there is an increased proportion of cells of
abnormal shape.
• May result from the production of abnormal cells by the
bone marrow or from damage to normal cells after
release into the blood-stream.
• If poikilocytosis is very marked, diagnostic possibilities
include myelofibrosis, congenital and acquired
dyserythropoietic anaemias
• The presence of poikilocytes of certain specific shapes,
e.g. spherocytes or elliptocytes, may have a particular
significance
35
SPHEROCYTOSIS
• Spherocytes are cells that, are spherical or near-spherical
in shape.
• They are cells that have lost membrane without
equivalent loss of cytosol, as a consequence of an
inherited or acquired abnormality of the red cell
cytoskeleton and membrane.
• In a stained blood film, spherocytes lack the normal
central pallor.
36
Spherocyte
 Absent central pallor
 Look smaller
 Causes
 Hereditary
spherocytosis
 Immune hemolytic
anemia
37
ELLIPTOCYTOSIS
 Increased numbers of elliptocytes
38
STOMATOCYTE
 Mouth like
 Membrane defect
 Smear artifact
 Hereditary
stomatocytosis
 Liver disease
39
TEARDROP CELLS (DACROCYTES)
 Occur when there is bone
marrow fibrosis or severe
dyserythropoiesis and also in
some haemolytic anaemias.
 They are particularly
characteristic of megaloblastic
anaemia, thalassaemia major and
myelofibrosis
40
TARGET CELLS
 Target cells have an area of increased
staining which appears in the middle of
the area of central pallor
 Formed as a consequence of there being
redundant membrane in relation to the
volume of the cytoplasm
 Target cells may also be an artefact, as a
result of using dirty slides
 Hemoglobinopathies, Thalassemia and
Liver disease
41
SICKLE CELLS
 Molecular aggregation of
Hb-S
 SS, SC, S-thal
 Rarely S-trait
42
BASOPHILIC STIPPLING
 Precipitated RNA
 Lead or heavy metal
poisoning
 Alcohol abuse
 Hemolytic anemia
43
BURR CELLS
 Altered lipid in cell
membrane
 Artifact
 Uremia
 Renal failure
 Gastric Ca
 Transfused old blood
44
SCHISTOCYTES
 Fragmented
RBC(mechanical or
phagocytosis)
 DIC
 TTP
 HUS
 Vasculitis
 Prosthetic heart valve
 Severe burns
45
INCLUSIONS IN ERYTHROCYTES
• Howell–Jolly bodies are medium sized, round,
cytoplasmic red cell inclusions that have the same
staining characteristics as a nucleus and can be
demonstrated to be composed of DNA.
• Howell–Jolly body is a fragment of nuclear material.
• It can arise by karyorrhexis (the breaking up of a
nucleus) or by incomplete nuclear expulsion, or can
represent a chromosome that has separated from the
mitotic spindle during abnormal mitosis.
• Hemolytic anemia and absent or hypofunctioning spleen
46
HOWELL JOLLY BODY
47
CIRCULATING NUCLEATED RED
BLOOD CELLS
• Are found in erythrocytes within the bone marrow in
haematologically normal subjects but, since they are
removed by the spleen, they are not seen in the
peripheral blood.
• They appear in the blood following splenectomy and are
present in other hyposplenic states,
• They can be a normal finding in neonates (in whom the
spleen is functionally immature)
48
CIRCULATING NUCLEATED RED
BLOOD CELLS
 Except in the neonatal
period and occasionally in
pregnancy, the presence of
NRBC in the peripheral
blood is abnormal,
generally indicating
hyperplastic
erythropoiesis or bone
marrow infiltration.
49
WBC CORRECTION FOR THE PRESENCE OF
NUCLEATED RED BLOOD CELLS
 If five or more nucleated red blood cells (nRBC) are seen
per 100 WBC’s when performing a differential, the total
white count MUST be corrected for the presence of these
nucleated red cells.
 Corrected WBC Count = WBC x 100/( nRBC + 100)
50
RED CELL AGGLUTINATION
 Red cell agglutinates are
irregular clumps of cells
 Mature red cells
agglutinate when they
are antibody-coated.
51
ROULEAUX FORMATION
• Are stacks of erythrocytes resembling a pile of coins.
• Increased when there is an increased plasma concentration of
proteins of high molecular weight.
• The most common causes
– Are pregnancy (in which fibrinogen concentration is increased),
– Inflammatory conditions (in which polyclonal immunoglobulins, α2
macroglobulin and fibrinogen are increased)
– Plasma cell dyscrasias such as multiple myeloma (in which increased
immunoglobulin concentration is caused by the presence of a
monoclonal paraprotein).
– HIV
• May be artefactually increased if a drop of blood is left standing
for too long on a microscope slide before the blood film is spread.
52
ROULEAUX FORMATION
53
WHITE BLOOD CELLS
Examine,

Even distribution and estimate the number
present (also, look for any gross abnormalities
present on the smear).

Perform the differential count.

Morphologic abnormalities.
54
NORMAL PBF
55
MANUAL DIFFERENTIAL COUNTS
 These counts are done in the same area as WBC and
platelet estimates with the red cells barely touching.
 This takes place under × 100 (oil) using the zigzag
method.
 Count 100 WBCs
 Reporting results
 Absolute number of cells/µl = % of cell type in
differential x white cell count.
56
Increased neutrophils count (neutrophilia)




Acute bacterial infection.
Many inflammatory processes.
During physical stress.
Granulocytic leukemia.
Decreased neutrophil count (neutropenia)

Typhoid fever

Brucellosis

Viral diseases, including hepatitis, influenza, rubella, and
mumps.

A great infection can also deplete the bone marrow

of neutrophils.

Many drugs used to treat cancer produce bone marrow
depression.
`
57
NEUTROPHILS
 Left shift
 Causes: pregnancy,
infection, hypoxia,
shock
58
HYPRESEGMENTATION
 Megaloblastic
erythropoiesis
 Iron deficiency
 Uraemia
 Infection
 Hereditary neutrophil
hypersegmentation
 Myelodysplastic
syndrome
59
HYPOSEGMENTATION
 Pelger–Huët anomaly
 Bilobed neutrophils
with reduced specific
granules (lactoferrin
deficiency)
 Acquired or pseudoPelger–Huët anomaly
60
NUCLEAR ABNORMALITIES
Dense chromatin clumping
• Myelodysplastic
syndromes
Detached nuclear fragments
• Dysplastic granulopoiesis
due to HIV infection or
administration of drugs
interfering with DNA
synthesis , including
chlorambucil,
patient on combination chemotherapy for
lymphoma showing a neutrophil with a
61
detached nuclear fragment
NUCLEAR ABNORMALITIES
Ring nuclei
• Chronic granulocytic
leukaemia
• Acute myeloid leukaemia
• Chronic neutrophilic
leukaemia
• Megaloblastic anaemia
Botryoid nucleus
• Heat stroke
• Hyperthermia
• Burns
Severe burns showing a neutrophil with a
botryoid nucleus
62
REDUCED GRANULATION
Reduced granulation
 Myelodysplastic
syndromes and acute
myeloid leukaemia
 Congenital lactoferrin
(specific granule)
deficiency
blood film of a patient with AML showing
three blasts and a hypogranular neutrophil.
63
INCREASED GRANULATION
Increased granulation
• ‘Toxic’ granulation
• pregnancy, infection,
inflammation, G-CSF
and GM-CSF therapy
• Aplastic anaemia
• Hypereosinophilic
syndromes
• Chronic neutrophilic
leukaemia]
64
ABNORMAL GRANULATION
Abnormal granulation
• Chédiak–Higashi syndrome
and related anomalies
• Alder–Reilly anomaly
• Acute myeloid leukaemia and
myelodysplastic syndromes
Vacuolation
• Infection, G-CSF therapy,
GM-CSF therapy
• Acute alcohol poisoning
• Carnitine deficiency
• Kwashiorkor
65
DOHLE BODIES
• Light blue-gray, oval,
basophilic, leukocyte
inclusions located in the
peripheral cytoplasm of
neutrophils.
• Infection, inflammation,
burns, pregnancy, G-CSF
therapy
• Myelodysplastic syndromes
and acute myeloid leukaemia
• Good indicator of neonatal
sepsis
66
MAY HEGGLIN ANOMALY
Triad
consisting of
thrombocyto
penia, giant
platelets, and
leukocyte
inclusions
giant platelet with poorly defined granulation.
A normal-sized platelet is also present. The
trilobed neutrophil contains a large, welldefined, basophilic, peripherally placed
cytoplasmic inclusion body (resembling a
Döhle body).
67
LYMPHOCYTES
Lymphocytes increase (lymphocytosis)
•
Many viral infections
•
Tuberculosis.
•
Typhoid fever
•
Lymphocytic leukemia.
A decreased lymphocyte (lymphopenia)
count of less than 500 places a patient at
very high risk of infection, particularly
viral infections.
68
LYMPHOCYTE INCLUSIONS
 Inclusions may be found
in lymphocytes in the
Chédiak–Higashi
syndrome and Alder–
Reilly anomaly
69
LYMPHOCYTE VACUOLATION
 Lymphocyte vacuolation
occurs in many inherited
metabolic disorders
including the following:
I-cell disease
(mucolipidosis type II)
70
REACTIVE CHANGES IN LYMPHOCYTES
• Plasmacytoid lymphocytes
or Türk cells.
• Plasmacytoid lymphocytes
may contain abundant
globular inclusions
composed of
immunoglobulin (‘Mott
cells’, ‘morular cells’ or
‘grape cells’)
71
AUER BODIES(AUER ROD)
72
PLATELETS
 Normal – 1-3μm (few – 5μm), irregular outlines
with fine red granules (scattered or
centralised).
 Abnormalities in size
 Larger – increased production,
hyposplenism, myeloproliferative
disorder(with ↑counts).
 Increased counts
 Infammation, bleeeding (not > 1000 x 109)
 Myeloproliferative disorder.
73
PLATELETS
• Disorders with charecteristic morphological disorders
• Bernard-soulier syndrome – giant platelets with defective
ristocetin response.
• Grey platelet syndrome – no granules, ghost like appearance.
• Platelet satelitism
• Occurring particularly but not only in EDTA -anticoagulated
blood.
• Is induced by a plasma factor, usually either Ig G or Ig M, which
causes platelets to adhere to cd16 on neutrophils .
• Platelets adhere to and encircle neutrophils and some may be
phagocytosed
74
Large granular lymphocyte
Agranular
platelet
Normal
platelet
Abnormalities in platelets
Large platelet or
macrothrombocyte
Ref 2
75
SUMMARY
 Proper preparation of blood film important .
 Blood film is important in diagnosis and
monitoring of haematological disorders.
76