Peripheral blood smear [autosaved]

PERIPHERAL BLOOD SMEAR
DR. AKANSHA ANU
GUIDE DR. VAISHALI KOTASTHANE

INTRODUCTION
•Peripheral smear is the most important investigation in haematology.
•It provides information about red cells –their number, shape, size and variations in morphology.
•It helps in diagnosis of different types of anaemias and other haematological disorders.
•DLC is very important diagnosis of various haematologic and non haematologic diseases.
•Assessment of platelet number, their aggregates and morphology is helpful in diagnosis of
various bleeding disorders.
•Smear evaluation is a check on the value obtained from automated cell counters.
•For evaluation of PS it is important to have a nicely made and stained PS.

Role of peripheral blood examination.
•Evaluation of anaemia.
•Evaluation of thrombocytopenia/ thrombocytosis.
•Identification of abnormal cells.
•Inclusion like basophilic stippling, Howell-jolly bodies.
•Infection like malaria, microfilaria etc.

Collection of blood
Advantages
Many smears can be done in just a single draw.
Immediate preparation of smear is not necessary.
Disadvantages
Platelets satellitosis causes pseudothromocytopenia and pseudoeukocytosis.
Causes :platelet specific auto antibodies that react best at room temperature.

Anticoagulants
EDTA – Most commonly used anticoagulants (CBC, Hb, TLC, DLC, PLATELET COUNT, RBC COUNT)
Sodium citrate – ESR (WESTERGREN METHOD)
Double oxalate- used in coagulation studies
Heparin – used for coagulation, for red cell enzyme studies like G6PD and pk deficiency
Sodium fluoride- estimation of blood glucose

EDTA
Collected in lavender (purple) topped tubes
Contain disodium or trisodium ethylenediaminetetraacetic (EDTA) anticouglants the blood by
chelating the calcium that is essential for coagulation
High quality of blood films can be made within 2-3 hrs of drawing
Blood films from EDTA tubes that remain at room temperature for more than 5 hrs often have
unacceptable blood cell artifacts
Echinocyte red blood cells
Spherocytes
Degenerated leukocytes
Vacuolated neutrophils

Characteristic of good smear
Good smear is tongue shaped with a smooth tail.
Does not cover the entire area of the slide.
Has both thick and thin areas with gradual transition.
Does not contain any lines or holes.

Preparation of smear
There are three types of blood smear
•The wedge smear
•The cover glass smear
•The spun smear
There are two additional types of blood smear used for specific purposes
•Buffy coat smear
•Thick blood smeras for blood parasites

Wedge technique
•Easiest to make
•Most convienient and most commonoly used technique
•EQUIPMENT
I. Spreaders
II. Clean slides
III. Blood capillary tube or micropipette 10ul

Place a drop of blood, about 2-3 mm in
diameter approximately 1cm from one end
of slides.

Place the slide on a flat surface, and hold the other end between your left thumb and forefinger.
With your right hand, place the smooth clean edge of second (spreaders) slide on specimen
slide, just in front of blood drop.
Hold the spreaders slide at a 30 degree – 45 degree angle, and draw it back against the drop of
blood.
PRECAUTIONS
Too large drop =too thick smear
Too small drop=too thin smear

Allow the blood to spread almost to the edges of the slide
PRECAUTIONS.
•Ensure that the whole drop of blood is picked up and spread
ANGLE CORRECTION:
•High Hct: angle should be lowered
•Low Hct: angle should be raised

Cover slip technique
Rarely used
ADVANTAGE- excellent leucocyte distribution.
DISADVANTAGES-labelling, transport, staning and storage is a problem.
 TECHNIQUE-
A drop of blood is placed on top of 1 coverslip.
Another coverslip is placed over the other allowing the blood to spread.
One is pulled over the other to create 1 thin smears.
Mounted on a 3x1 inch glass slide.

Automated slide making and staning.
•Perfoms a CBC for specimen.
•Dependent on the hemocrait reading, the system adjusts.
Size of the drop of blood used and
Angle and spread of the spreaders slide in making a wedge preparation.
After each blood flim is prepared, the spreadrers slide is automatically cleaned.

Automated slide making and stanning.
Films be produced approximately every 30 seconds.
Name, number, and date for the specimen is printed on the slide.
The slide is dried, loaded into a cassette,and moved to the stanning position, where a stain and
then buffer and rinse are added designated times.
When stanning is complete, the slide is moved to a dry position, then to a collection area where
it can be picked up for microscopic evaluation.

Stains for blood smear
Romanowsky stains are universally employed for stanning of blood smears
It combines of methylene blue and eosin
Basic dye
Has affinity for acidic component of the cell i.e. nucleus and eosin has affinity for basic component I;e basic
Various stains for Romanowsky are;
1.Leishman’s stain
2. giemsa stain
3 wright stain
4 field stain
5 jenner stain
6 JSB stain

Staning of thin blood smear
Leishman’s stain
Preparation
Dissolve 0.2 g of powdered LS dye in 100ml of acetone free methyl alcohol
Warm it to 50 degree c for half n hr with occasional shaking
Cool it and filter it
Procedure
Pour LS dropwise on slide and wait for 2 mins (allows fixation)
Add double the quality of buffered water over the slide
Wash in water for 1 -2 mins
Dry in air and examine under oil immersion

Giemsa stain
Prepartion
Mix 0.15 g of giemsa powder in 12.5 ml of glycerine and 12.5 ml of methyl alcohol
Before use dissolve one volume of stock solution in nine volumes of buffered water (dilution
1:9)
Procedure
pour diluted stain over slide or immense blood smear in staning trough
Wait for 15-60 mins
Wash in water
Dry it and examine under oil immersion

AUTOMATED SLIDE STAINERS
It takes about 5-10 mins to stain a batch of smears.
Slides are just automatically dipped in the stain in the buffer and a series of rinses.
DISADVANTAGES
Stanning process has begun, no stat slides can be added in the batch.
Aqueous solutions of stains are stable only for 3-6 hours.

Rapid stanning method-field’s stain
•Advantage
fast, convenient and takes about 1 minute.
Cost effective.
•Components.
Methanol
Solution b contains eosin
Solution a contains methylene blue

CAUSES AND CORRECTIONS
Too acidic stain.
Insufficient staining time.
Prolonged buffering or wasting
Old stain
Correction
Lengthen staning time
Check stain and buffer ph
Shorten buffering or wash time

Too alkaline stain
Thick blood sugar
Prolonged standing
Insufficient washing
Alkaline ph. of stain components
Corrections
Check ph.
Shorten stain time
Prolonged buffering time

Features of a well-stained PBS
Microscopically- color should be pink to purple.
Microscopically- RBC orange to salmon pink
WBS- nuclei is purple to blue
Cytoplasm is purple to pinl
Granules is iliac to violet
Eosinophil- granules orange
Basophill- granules dark blue to black.

MORPHOLOGIC CHANGE DUE TO AREA
OF SMEAR
Thin area- spherocytes which are really spheroidocytes or flayttened red cells.
True spherocytes will be found in other good areas of smear.
Thick area-rouleaux which is normal in such areas.
Confirm by examining thin areas.
If true rouleaux's two three RBCs will stick together in a stack of coins fashion.

10x objective
Assess overall quality of the smear i.e feathery edge, quality of color, distribution of cells and
the lateral edges can be checked for WBC distribution.
Snow plow effect ; more than 4x cells per field on the feathery edge; reject
Fibrin stands; reject

TOTAL LEUCOCYTE COUNT
40 X OBJECTIVE
Use dry without oil
Choose a portion of the peripheral smear where there is only slight overlapping of RBCs
Count 10 fields take the total number of white cells and divide by 10.
To do a wbc estimate by taking the average number of white blood cells and multiplying by 2000
Normal leucocyte count ranges from 4000 to 11000/ul

Observe one field and record the number of
WBC according to the different type then turn
to another field in snake like direction.

Manual differential counts
These counts are done in same area as WBCs and platelet estimates with the red cells barely
touching
This takes place under x 100 (oil) using the zigzag method
Count 100 WBCs
Expressed as percentage
Absolute number of cells/ul = %of cell type in differential x white cell count

NUCLEATED RED BLOOD CELLS
If 10 or more nucleated RBCs are seen correct the TLC
Corrected WBC count = WBC x 100 / (nRBC +100)
EXAMPLE
If WBC =5000 and 10 nRBCs have been counted
Then 5000 x 100/110 =4545
Then corrected white count is 4545

Do not count
Disintegrating cells
Eosinophil with no cytoplasmic membrane and with scattered granules
Smudge cells
Pyknotic cells

RBC MORPHOLOGY
Scan under using x 100 (Oil immersion)
Observe 10 fields
Red cells are observed for – size, shape, haemoglobin content, inclusions

RBC
RBCs are circular, homogenous disc nearly of
uniform size (7-8 um)
Deep pink cytoplasm with central pallor
<1/3rd)

HYPOCHROMIA
Decrease in haemoglobin content of RBC
Increase in central pallor(1/3)
Decrease in MCH and MCHC
Seen in various anaemias

Dimorphic anaemia
Presence of anisocytosis and anis chromia in
same film
Seen in- coexistence of iron deficiency and
megaloblastic anaemia.
Sideroblastic anaemia
Some weeks after iron therapy for iron
deficiency anaemia.
Hypochromic anaemia after transfusion with
normal cells.

MICROCYTES
Size of RBCs are reduced (<80fl)
Seen in – iron deficiency anaemia
thalassemia
anaemia of chronic disease
sideroblastic anaemia

Macrocytes
When MCH of RBC is increased
(>100fl)
Seen in – vit b12 and folate deficiency
alcoholism
hepatic disease
haemolytic states
hypothyroidism

Shape
Variations in shape is called poikilocytoses
Types
Elliptocytes
Spherocytes
Target cells
Schistocytes
Acanthocytes
Karyocytes
Echinocytes

ELLIPTOCYTES
Elipitical in shapes
Most abundant in hereditary elliptocytes
Seen in- iron deficiency anemia
megaloblastic anemia

Acanthocytes
Thorny projections on red cell membrane
Few irregular non uniform
Seen in – hypothyroidism
liver disease
McLeod phenotype

Echinocytes (Burr cells)
Numerous short regular projection
Commonly occur as an artifact during
preparation
Renal disease
Liver disease
hyperlipidaemia

LEPTOCYTES
Thin red cells with large unstained central area
Also known as pessary cells
Seen in – iron deficiency anaemia
thalassemia

Somatotypes
Red cells with central biconcave area appears
slit like in dried film
Seen in- liver disease
hereditary
alcoholism
myelodysplastic syndromes

Sickle cell
Cells are sickle (crescent) shape
Present in film of patient with homozygosity
for HbS

Tear drop cells
Also called dacrocytosis
Seen in- beta thalassemia
post splenectomy
severe iron deficiency

Spherocytes
Nearly spherical
Diameter is smaller than normal
Lack central pale area or have smaller,
eccentric pale area
Seen in- hereditary spherocytes
autoimmune haemolytic anaemia
physical or chemical injury

Target cells
Cells in which central round stained area and
peripheral rim of cytoplasm
Seen in – sickle cell anemia
thalassemia major
hemoytic anemias
postsplenectomy

HOWELL- JOLLY BODIES
Smooth single large round inclusion which are
remnant of nuclear chromatin.
Seen in- megaloblastic anaemia
haemolytic anaemia
postsplectomy
abnormal erythropoiesis

BASOPHILIC STIPPLING
Presence of irregular basophilic granules with
in RBCs which are variable in size
Fine stippling seen
Coarse stippling- lead and heavy metal
poisoning
disturbed erythropoiesis
megaloblastic anaemia
thalassaemia
infection
liver disease

Pappenheimer bodies
Smaller than Howell- jolly bodies
Composed of haemosiderin
Seen in - hyposplenesim
myodysplastic syndrome

Heinz bodies
Purple blue large single or multiple inclusion
attach to inner surface of red blood cells
Seen in – post splenectomy
oxidative stress
drugs
toxins
glutathione synthetase deficiency

Cabot ring
These are ring shaped figure of eight or loop
shaped
Observed in – megaloblastic anemia
pernicious anemia
lead poisoning

Rouleaux formation
Alignment of red cells one upon another so
that resemble stack of coins
Occurs in – multiple myeloma
chronic inflammatory disease

Agglutination
It is more irregular and round clumping than
liner Rolex
Seen in – anti RBC antibody
autoimmune haemolytic anaemia
macroglobulinemia

WBCs in PBS
GRANULOCYTES
Neutrophils
Eosinophils
Basophils
Agranulocytes
Lymphocytes
monocytes

Polymorphonuclear neutrophills
The terminal stage of development measuring
12- 14 um in diameter
Characterised by a lobulated nucleus
Two to five lobes of clumped chromatin
The cytoplasm contains fine azurophilic
granules

Hypersegmented neutrophils
Presence of even a single neutrophils with six
or more lobes
Seen in – megaloblastic anaemia
uraemia

Eosinophils
They are slightly larger than a segmented
neutrophil measuring 12-15 um
Two nuclear lobes are spectacle in shape
The cytoplasm has pale hue and has
numerous dense orange red colour

Monocytes
Monocytes are 10-11um
The nucleus is large and oval
The nuclear chromatin are delicate
The cytoplasm is abundant, is grey or light
blue grey in colour
The granules resemble fine dust and give
bluish cytoplasm a ground glass appearance

Monocities
Chronic infections and inflammatory
conditions such as :
Malaria
Typhoid
Kala-azar
Bacterial endocarditis
Crohn’s disease
Infectious mononucleosis
tuberculosis
Haematolymphoid malignancies
Acute myelomonocytic leukaemia(AML M4)
acute monocytic leukaemia (AML M5)
Myeloproliferative neoplasm
Myelodysplastic syndrome
Chronic myelomonocytic leukaemia

Lymphocytes
Small lymphocytes
Measuring 9-12um
Smaller than granulocytes
Cytoplasm is in the from of thin rim around
the nucleus
Round and slightly intended nucleus

Large lymphocytes
Measuring 12 -15um
Round in outline
Nucleus is round and slightly indented
With clumped chromatin
Cytoplasm is more abundant than
lymphocytes and pale blue in colour

Granules
Seen in
Bacterial infections
Burn
Administration of G-CSF GM –CSF

Dohle bodies
Small round or oval pale blue grey structure
Found at periphery of neutrophils
Contains ribosomes and endoplasmic
reticulum
Seen in
Bacterial infections
Inflammation
Pregnancy
Administration of G-CSF

Vacuoles in neutrophils
In fresh blood smear vacuoles seen in severe
sepsis
Indicative of phagocytosis

Alder-reilly anomaly
Commonly seen in hurler’s and hunter’s
syndrome
Granules are large discrete stain deep red
Neutophils function is normal

May hegglin anomaly
Autosomal dominant inheritance
Triad of thrombocytopaenia giant platelets
and dohle’s bodies
MYH -9 gene

Chediak-higashi syndrome
Rare autosomal recessive disease
Immune deficiency
Poor resistance to bacterial infections
Bleeding tendencies
Multiple neurological abnormalities

Pelger-huet cells
Benign inherited condition
Neutrophil nuclei fail to segment properly

Platelets
size 1-3 um
Normal count 1.5 to 4.5 lac/cmm
Non nucleated derived from cytoplasmic
fragments of megakaryocytes
Have an irregular outline and fine purple red
granules

Thrombocytosis
Essential thrombocytopenia
CML
Reactive thrombocytosis – post infection
iron deficiency
inflammation
collagen vascular disease

Platelet morphology – giant platelets
Platelets seem to be size of RBCs
Seen in
Alport syndrome
Storage disorders
Bernard syndromes

Hemoparasites
Malaria
Microscopic examination of peripheral blood
film is the gold standard for diagnosis
Number of parasitized RBCs seen in 10,000
RBCs (in 100x objective) is calculated
Approximate number of parasites is roughly
assessed assuming 1ul of blood contains
5x10’6 RBCs
Blood film evaluation
Thin film examination
Thick blood film evaluation
Malaria antigen detection test
Molecular method
Serology
Loop mediated isothermal amplification test

Plasmodium falciparum
Infected RBCs are of normal size with one or
multiple rings
Gametocytes have characteristic banana
shape
Few Maurer's clefts may be seen

Plasmodium vivax
Infected RBCs are enlarged and deform
Parasites infect the reticulocytes which demonstrate ring form, schizonts with dots and
gametocytes
Gametocytes are large and round to oval with eccentrically placed chromatin

Plasmodium ovale
Infected erythrocytes
Moderately enlarged
Oval in shape
Show red granules like schuffenr’s dots
Merozoites have daisy head diatribution
Gametocytes are small ½ to 2/3rd of red cells

Plasmodium malariae
Infected RBCs –size normal to decreased
Ameboid forms are seen as a band across the
red cells
The gametocytes are small and round
Occupies 1/3rd to 2/3rd of red cells
Hemozoin is present in liver, spleen, brain

Filariasis
Causes elephantiasis
Many caused by wuchereria bancrofti and brugia malayi
Pathology- due to adult worm obstructing lymphactics
Includes eosinophilia and elephantiasis of legs and scrotum
Diagnosis- PBS
membrane filter method
immunochromatographic test
DNA probe using PCR

filariasis
Detection of microfilariae in blood in early
stages of disese
Blood film, knott’s method (concentration of 1
ml of blood)
Best 10 pm to 2 am (nocturnal periodicity)

trypanosomiasis
Transmitted by tse tse fly
Causes African sleeping sickness and
splenomegaly

Babesiosis
It is a tick borne disease caused by protozoan
caused by parasite babesia
Vector is tick
Cause malaria like sickness
In blood organism recognised as tiny multiple
rings in red cells

Toxoplasmosis
Caused by toxoplasma gondii
Infection from cats
Immunodeficient cases- involvement of brain,
eyes, muscle, heart, lungs
Rarely trophozoites are seen in peripheral
blood.

Peripheral blood smear [autosaved]

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