2. 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.
3. 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.
4. 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.
5.
6. 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
8. 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
9. 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.
10.
11.
12. 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
13. 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
14. Place a drop of blood, about 2-3 mm in
diameter approximately 1cm from one end
of slides.
15. 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
16. 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
17.
18.
19.
20.
21. 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.
22.
23. 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.
24. 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.
25. 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
26. 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
27. 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
28. 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.
29.
30. 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
35. 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
36. Too alkaline stain
Thick blood sugar
Prolonged standing
Insufficient washing
Alkaline ph. of stain components
Corrections
Check ph.
Shorten stain time
Prolonged buffering time
37. 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.
38. 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.
39. 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
40. 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
41. Observe one field and record the number of
WBC according to the different type then turn
to another field in snake like direction.
42. 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
43. 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
44. Do not count
Disintegrating cells
Eosinophil with no cytoplasmic membrane and with scattered granules
Smudge cells
Pyknotic cells
45. RBC MORPHOLOGY
Scan under using x 100 (Oil immersion)
Observe 10 fields
Red cells are observed for – size, shape, haemoglobin content, inclusions
46. RBC
RBCs are circular, homogenous disc nearly of
uniform size (7-8 um)
Deep pink cytoplasm with central pallor
<1/3rd)
48. 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.
50. MICROCYTES
Size of RBCs are reduced (<80fl)
Seen in – iron deficiency anaemia
thalassemia
anaemia of chronic disease
sideroblastic anaemia
51. Macrocytes
When MCH of RBC is increased
(>100fl)
Seen in – vit b12 and folate deficiency
alcoholism
hepatic disease
haemolytic states
hypothyroidism
52. Shape
Variations in shape is called poikilocytoses
Types
Elliptocytes
Spherocytes
Target cells
Schistocytes
Acanthocytes
Karyocytes
Echinocytes
55. Echinocytes (Burr cells)
Numerous short regular projection
Commonly occur as an artifact during
preparation
Renal disease
Liver disease
hyperlipidaemia
56. LEPTOCYTES
Thin red cells with large unstained central area
Also known as pessary cells
Seen in – iron deficiency anaemia
thalassemia
57. Somatotypes
Red cells with central biconcave area appears
slit like in dried film
Seen in- liver disease
hereditary
alcoholism
myelodysplastic syndromes
58. Sickle cell
Cells are sickle (crescent) shape
Present in film of patient with homozygosity
for HbS
59. Tear drop cells
Also called dacrocytosis
Seen in- beta thalassemia
post splenectomy
severe iron deficiency
60. 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
61. Target cells
Cells in which central round stained area and
peripheral rim of cytoplasm
Seen in – sickle cell anemia
thalassemia major
hemoytic anemias
postsplenectomy
63. HOWELL- JOLLY BODIES
Smooth single large round inclusion which are
remnant of nuclear chromatin.
Seen in- megaloblastic anaemia
haemolytic anaemia
postsplectomy
abnormal erythropoiesis
64. 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
66. 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
67. Cabot ring
These are ring shaped figure of eight or loop
shaped
Observed in – megaloblastic anemia
pernicious anemia
lead poisoning
68. Rouleaux formation
Alignment of red cells one upon another so
that resemble stack of coins
Occurs in – multiple myeloma
chronic inflammatory disease
69. Agglutination
It is more irregular and round clumping than
liner Rolex
Seen in – anti RBC antibody
autoimmune haemolytic anaemia
macroglobulinemia
71. 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
73. 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
74. 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
77. 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
79. 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
85. 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
88. Platelet morphology – giant platelets
Platelets seem to be size of RBCs
Seen in
Alport syndrome
Storage disorders
Bernard syndromes
89. 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
90. 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
91. 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
92.
93. 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
94. 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
95. 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
96.
97. 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)
99. 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
100. Toxoplasmosis
Caused by toxoplasma gondii
Infection from cats
Immunodeficient cases- involvement of brain,
eyes, muscle, heart, lungs
Rarely trophozoites are seen in peripheral
blood.