Peripheral blood smear examination
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Peripheral blood smear examination (slide preparation and reporting)
Dr. Bahoran SinghModerator- Dr. Namrata Shetty
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Role of peripheral blood examination
• Evaluation of anemia• Evaluation of thrombocytopenia/ thrombocytosis• Identification of abnormal cells (blasts/abnormal promyelocytes/atypical lymphoid)• Infections like malaria, microfilaria• Inclusions like basophilic stippling, Howell-Jolly bodies, Cabot ring
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• Objective:
1. Peripheral Smear Preparation
2. Staining of Peripheral Blood Smear
3. Peripheral Smear Examination
Peripheral Blood Smear
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Peripheral Smear Preparation
• Wedge technique• Coverslip technique• Automated Slide Making and Staining
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WEDGE BLOOD SMEAR
• Specimen: Peripheral blood smear made from EDTA-
anticoagulated blood. Smears should be made within 1 hour of blood
collection from EDTA specimens stored at room temperature to avoid distortion of cell morphology
Blood smears can also be made from finger prick blood directly onto slide.
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• Equipment•Spreaders• Clean slides• Blood capillary tube or micropipette 10
µL
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SMEAR PREPARATION
1. Place a drop of blood, about 2-3 mm in diameter approximately 1 cm from one end of slide.
2. Place the slide on a flat surface, and hold the other end between your left thumb and forefinger.
3. With your right hand, place the smooth clean edge of a second (spreader) slide on the specimen slide, just in front of the blood drop.
4. Hold the spreader slide at a 30°- 45 angle, and draw it back against the drop of blood
6. Allow the blood to spread almost to the edges of the slide.
7. Push the spread forward with one light, smooth moderate speed. A thin film of blood in the shape of tongue.
8. Label one edge with patient name, lab id and date.
9. The slides should be rapidly air dried by waving the slides or using an electrical fan.
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The shape of blood film
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Characteristics of A Good Smear
1. Good smear is tongue shaped with a smooth tail.
2. Does not cover the entire area of the slide.3. Has both thick and thin areas with gradual
transition.4. Does not contain any lines or holes.
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The thickness of the smear
Is determined by:1. The angle of the spreader slide. (the
greater the angle, the thicker and shorter the smear).
2. Size of the blood drop.3. Speed of spreading
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The thickness of the spread
1. If the hematocrit is increased, the angle of the spreader slide should be decreased.
2. If the hematocrit is decreased, the angle of the spreader slide should be increased
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Common causes of a poor blood smear
1. Drop of blood too large or too small.2. Spreader slide pushed across the slide in a
jerky manner.3. Failure to keep the entire edge of the
spreader slide against the slide while making the smear.
4. Failure to keep the spreader slide at a 30° angle with the slide
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5. Failure to push the spreader slide completely across the slide.
6. Irregular spread with ridges and long tail: Edge of spreader dirty or chipped; dusty slide
7. Holes in film: Slide contaminated with fat or grease and air bubbles.
8. Cellular degenerative changes: Delay in fixing, inadequate fixing time or methanol contaminated with water.
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Blood films
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Slide fixation and staining
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Romanowsky stainingIt includes:• May-Grunwald –Geimsa stain,• Jenner’s stain,• Wright’s stain,• Leishman’s stain and,• Field’s stain.
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Components of Giemsa stain
It mainly composed of• Eosin Y and,• Azure B- Methylene Blue
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• Eosin-It is acidic component of stain and stain basic component of cells like hemoglobin.
• Azure B – it is basic component and stains the acidic component of cells like DNA and RNA (nucleus of WBC)
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Materials
• Slide holder or rack• Stain reagent• Methanol : fix the cells on the slide
• Buffer:• Used to maintain an adequate pH.• 0.05M Na2PO4 (pH 6.4)• Distill water kept in glass bottle for at least 24hours (pH
6.4-6.8)
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Methodology
• Put the smear into methanol jar and fix it for 1 -2 minute.• Remove excess methanol from the smear• Insert the smear into Wright’s stain jar and leave the
stain for 2 minutes.• Insert smear into a buffer jar and allow to stand for 4-8
minutes• Rinse thoroughly with a steam of distilled water• Allow to air dry• Note: time varies from manufacturers, thus ensure to
follow the exact time in the kit manual of each procedure
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Colour responses of blood cells to Romanowsky staining
• Cellular component Colour• Nuclei Chromatin Purple• Nucleoli Light blue• Cytoplasm• Erythroblast Dark blue• Erythrocyte Dark pink• Reticulocyte Grey–blue
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Cytoplasm colour
• Lymphocyte Blue• Metamyelocyte Pink• Monocyte Grey–blue• Myelocyte Pink• Neutrophil Pink/orange• Promyelocyte Blue• Basophil Blue
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Granules
• Promyelocyte(primary granules) Red or purple• Basophil Purple black• Eosinophil Red–orange• Neutrophil Purple• Toxic granules Dark blue• Platelet Purple
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Other inclusions
• Auer body Purple• Cabot ring Purple• Howell-Jolly body Purple• Döhle body Light blue
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Factors influence smear staining method
• Blood smear may be under or over stained based on the following
• Concentration of the stain used– Low concentration: pale coloured cells (under staining)– High concentration: dark stained smear (over stained)
• Time of exposure the stain and the buffer– Too long: overstaining,– Too short: understaining
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Factors giving rise to faulty staining• Appearances Causes
Too blue Eosin concentration too lowIncorrect preparation of stock
stock stain exposed to bright daylight
Batch of stain solution overused• Impure dyes• Staining time too short• Staining solution too acid• Smear too thick• Inadequate time in buffer solution
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– Too pink Incorrect proportion of azureB-eosin Y• Impure dyes• Buffer pH too low• Excessive washing in buffer
solution
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• Pale staining Old staining solution• Overused staining solution• Incorrect preparation of
stock• Impure dyes, especially
azure A and/or C• High ambient temperature
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• Neutrophil granules Insufficient azure B not stained
• Neutrophil granules Dark Excess azure BBlue/black (pseudo-toxic)
• Other stain anomalies Various contaminating
dyes and metal salts
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• Stain deposit Stain solution left in on film uncovered jar
Stain solution not filtered
• Blue background Inadequate fixation or prolonged storage
before fixation• Blood collected into heparin
as anticoagulant
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Peripheral blood smear examination• Macroscopic view : quality of the smear • Any abnormal particles present• The Microscopic analysis • begins on lower power (10x), • to assess quality of the preparation• assess whether red cell agglutination, excessive rouleaux formation or platelet aggregation is present; • Assess the number, distribution and stainingof the leucocytes; and
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• find an area where the red cells are evenly distributed and are not distorted.
• On high power(40x)-• to obtain a WBC estimate• All of the detailed analysis of the cellular
elements using high power or oil immersion(100x).
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Normal peripheral smear
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Area too thin to examine the slide
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Too thick area
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Evaluation of PBS • 1. RBC
• Size• Shape • Color • Arrangement • Inclusions
• 2. WBC• Total counts• Differential counts• Abnormal WBC
• 3. Platelets • Counts
• Abnormality •4. Parasites
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RBC
• In the blood from healthy person RBCs are – Circular , Homogenous disc nearly of uniform
size(6–8 µm)– deep pink cytoplasm with Central pallor <1/3rd
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Various changes in RBCs
1. COLOUR: It is determined by hemoglobin content of RBC.
1. Normochromic- Normal intensity of staining.2. Hypochromic-3. Hyperchromic-
• Normal Hb conc. Male-15020 g/l• Female- 13515 g/l
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Hypochromic
• Dcrease in Hemoglobin content of RBC
• increase in central pallor(>1/3rd)
• Decrease in MCH and MCHC
• Seen in Iron Deficiency anemia
• thalassaemia
• hypochromia
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Hyperchromia
• Red cells stain deeply
• Have less central pallor,
• Increase in MCH• Seen in
Megaloblastic anemia
• Hereditary spherocytosis(MCH is normal but MCHC is increased)
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• Anisochromia – presence of hypochromic cells and normochromic cells in the same film. Also called dimorphic anemia.
• Seen in – Sideroblastic anemia– Some weeks after iron therapy for iron deficiency anemia– hypochromic anemia after transfusion with normal cells.
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Dimorphic blood picture
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Polychromatophillia
Blue grey tint of red cellsDue to Hb and RNA(Residual) in young cells.• Larger than normal and
may lack central pallor.• Implies Reticulocytosis • Seen in
• Hemolysis• Acute blood loss
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Variation In Size
• Anisocytosis- Variation in size of the red blood cells
• Normal MCV is -80-100 fl• Microcytes ( MCV <80 fl)• Macrocytes (MCV >100fl)• Anisocytosis is a feature of most anemias.
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Microcytes
• Size of RBC is reduced(<80fl)
• Seen when hemoglobin synthesis is defective1. Iron deficiency anemia2. Thalassemia3. Anemia of chronic
disease4. Sideroblastic anemia
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Macrocytes• When MCV of RBC is
Increased(>100fl)• Macro- ovalocytes are
seen in Megaloblastic anemia
• Myledysplastic syndrome.
• Round Macrocytes seen in Alcoholism, Liver disease.
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Shape• Variation in shape is called Poikilocytosis.• It is of following types-
• Elliptocytes• Spherocytes• Target cells• Schistocytes• Acanthocytes• Keratocytes• Echinocytes
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Elliptocytes
• Elipitical in shapes• Most abundant in
hereditary elliptocytosis• Seen in –1. Iron deficiency anemia2. Myelofirosis with myeloid metaplasia3. Megaloblastic anemia4. Sickle cell anemia
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Spherocytes
• Nearly spherical • Diameter is smaller than
normal• Lack central pale area or
have a smaller , eccentric, pale area
• Seen in – hereditary spherocytosis– Some cases of
autoimmune hemolytic anemia
– direct physical or chemical injury
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Target cells
• Cells in which central round stained area and peripheral rim of cytoplasm
• Seen in Thalassaemia• Chronic liver disease• Hereditary hypo-
betalipoproteinemia• Iron deficiency anemia• Hemoglobinopathies (Hb C,
Hb H, Sickel cell anemia• Postsplenectomy
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Schistocytes • These are fragmaented
erythrocytes.• Smaller than normal red cells
and of varying shape• Seen in• Genetic disorder
– Thalassaemia– congential dyserythropoietic
anemia.
• Acquired disorder of RBC formation– Megaloblastic– Dyserythropoietic
• Mechanical stress MAHA• Direct thermal injury
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Acanthocytes
Red cells with small no of spicules of inconstant length, thickness and shape , irregularly disposed over the surface.• Seen in Abnormal
phospholipid metabolism• Abetalipoproteinemia• Inherited abnormalities of
red cell membrane protein• Splenectomy
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Echinocytes • Also called crenated cells• Numerous, short, regular
projection• Commonly occur as an artifact
during preparation of film• Hyperosmolarity• discocyte–echinocyte
transformation• Overnight stored blood at 20 C
before films are made.• Premature infant after
exchange transfusion• water contaminating the
Wright’s stain (or absolute methanol)
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Keratocytes
• Have pairs of spicules either one or two pairs.
• Sometimes termed as Bite cell or helmet cell
• Seen in– Mechanical damage– Removal of Heinz body
by pitting action of spleen.
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Leptocytes
• Thin red cells with large unstained central area.
• Seen in– Severe iron deficiency
anemia– Thaleasaemia
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Stomatocytes • Red cells with central
biconcave area appears slit like in dried film.
• Wet film it appears as cup-shaped.
• Seen in – Artifact– south-east Asian
ovalocytosis– liver disease,– alcoholism,– myelodysplastic syndromes.
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Sickle cell
• Cells are sickle (boat shape) or crescent shape
• Present in film of patient with homozygosity for Hb S.
• Usually absent in neonates and rare in patients with high Hb F percentage
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Tear drop cells• One side of cells is tapered
and other is blunt.• Seen in
– Myelofibrosis– thalassemia– Hemoglobin E heterozygous
+ homozygous●HbH disease●HbC trait ●Hb Lepore heterozygous + homozygous●HbO Arab disease●HbD disease●Iron defciency●Hb Lepore trait
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Structure
• Basophilic stippling (Punctate basophilia)• Howell – jolly Bodies• Cabot Rings• Malarial Stippling• Rouleaux formation
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Red blood cell inclusions
• Name of Inclusion Content• Howell-Jolly body DNA• Basophilic stippling RNA• Pappenheimer body Iron• Heinz body(supravital only) Denatured hemoglobin• Crystals Hemoglobin-C• Cabot rings Mitotic spindle remnants• Nucleus DNA
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Basophilic Stippling• Presence of irregular basophilic
granules with in Rbc which are variable in size .
• Stain deep blue with Wright’s stain• Fine stippling seen with
– Increased polychromatophilia– Increased production of red cells.
• Coarse stippling– Lead and heavy metal poisoning– Disturbed erythropoiesis
• Megaloblastic anemia • Thalassaemia• infection• liver disease
– Unstable Hb– Pyrimidine-5’-nucleotidase def.
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Howell-Jolly Bodies
• Smooth single large round inclusions which are remnant of nuclear chromatin.
• Seen in • Single –
– Megaloblastic anemia– Hemolytic anemia– Postsplenectomy
• MULTIPLE –– Megaloblastic anemia– Abnormal erythropoiesis
• Howell-Jolly Bodies
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Pappenheimer Bodies
• These are small single or multiple peripherally sited angular basophilic (almost black) erythrocyte inclusions.
• Smaller than Howell–Jolly bodies.
• composed of haemosiderin.• Their nature can be confirmed
by Perls’ stain.• Seen in
– Sideroblastic erythropoiesis– Hypospenism– Myelodysplastic syndrome– Hemolytic anemia
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Heinz bodies• Seen on supravital stains• Not seen on Romanowsky stain.• Purple, blue, large, single or multiple
inclusions attached to the inner surface of the red blood cell.
• Represent precipitated normal or unstable hemoglobins.
• seen – Postsplenectomy• Oxidative stress
– Glucose-6-phosphate dehydrogenase deficiency,
– Glutathione synthetase deficiency– Drugs– Toxins– Unstable hemoglobins
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Cabot Rings
• These are Ring shaped ,figure of eight or loop shaped
• Red or Reddish purple with Wright’s stain and have no internal structure
• Observed rarely in– Pernicious anemia,– Lead poisoning,
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Malrial stippling
• Fine granules of plasmodium vivax
• On wright stain these are fine , purplish red
• Red cells are larger than normal
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Rouleaux Formation
• Alignment of red cells one upon another so that they resemblestacks of coins.
• Occurs in• Paraproteinemia
( monoclonal gammopathy)• Elevated plasma fibrinogen
or globulin level
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Agglutination
• It is more irregular and round clumping than linear rouleaux
• Seen with cold agglutinin
• Anti RBC antibody• Autoimmune hemolytic
anemia• Macroglobulinemia
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WBC MORPHOLOGY
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Before evaluating leucocyte following must be seen-Film is well madeDistribution of cells is uniformStaining is satisfactory While scanning estimate the total leucocyte count Differential count is done at oil immersion
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• Ten microscopic fields are examined in a vertical direction from bottom to top or top to bottom
• Slide is horizontally moved to the next field• Ten microscopic fields are counted vertically.• Procedure is repeated until 100 WBCS have been
counted (zig zag motion)
Scanning technique for WBC differential count and morphologic evaluation
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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 including all cell lines from immature to mature.
Reporting results• Absolute number of cells/µl = % of cell type in
differential x white cell count
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• If 10 or more nucleated RBC's (NRBC) are seen, correct the White Count using this formula:Corrected WBC Count = WBC x 100/( NRBC + 100)Example : If WBC = 5000 and 10 NRBCs have been countedThen 5,000× 100/110 = 4545.50The corrected white count is 4545.50
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Leukocytes Normally Present in Blood
• GRANULOCYESNeutrophils ( polymorphonuclear leucocytes)Eosinophils Basophils
• Agranulocytes Lymphocytes Monocytes
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POLYMORPHONUCLEARNEUTROPHILS• 40 to 80 percent of total WBC
count(2.0–7.0 ×109/l )• Diameter - 13 µm• segmented nucleus and
pink/orange cytoplasm with fine granulation(0.2-0.3µm) stain tan to pink with Wright’s
• Lobes -2-5 • Neutrophils usually have
trilobed nucleus. • small percent has four lobes
and occasionally five lobes.
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Band forms
• neutrophils has either a strand of nuclear material thicker than a filament connecting the lobes, or a U-shaped nucleus of uniform thickness.
• Up to 8% of circulating neutrophils are unsegmented orpartly segmented (‘band’ forms)
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• Left-shift: non-segmented neutrophil > 5%– Increased
bands Means acute infection, usually bacterial
77
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Granules
• Toxic granulation- increase in staining density and number of granules
• Seen with Bacterial infections and other inflammation
• Administration of G-CSF• Anaplastic anemia
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• Hypogranular and agranular neutophils poorly stained
• seen in Myelodysplastic syndrome
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Alder–Reilly anomaly
• Granules are large,– discrete,– stain deep red – may obscure the
nucleus– Neutrophil function
is Normal
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Chédiak-Higashi Syndrome
• Granules are– also seen in other
leukocytes like lymphocytes
– Giant – Scanty azurophilic– functional defect occur
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Dohle Bodies• Small, round or oval, pale
blue-grey structure• Found at periphery of
neutrophil.• Contains Ribosomes and
Endoplasmic reticulum• Seen in – Bacterial infection
– inflammation – administration of G-CSF– during pregnancy
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May–Hegglin anomaly
• inclusions occur in alltypes of leucocytes except lymphocytes.
• contain small basophilic cytoplasmic granules
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Vacuoles in neutrophils
• In Fresh blood smear • vacuoles seen in
– severe sepsis– as an artifact with
prolonged standing
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Nuclei• Hypersegmentated
neutrophil• def.-presence of
neutrophils with six or more lobes or the presence of morethan 3% of neutrophils with at least five lobes.
• seen in Megaloblastic anemia– uraemia– iron deficiency. – Drugs-cytotoxic treatment
with Methotrexate– hydroxycarbamide
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Pelger–Huët Cells
• Pelger–Huët anomaly• Benign inherited
condition.• Neutrophil nuclei fail to
segment properly.• Majority of circulating
neutrophils have only two discrete equal-sized lobes connected by a thin chromatin bridge.
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Pseudo-Pelger cells
• Pseudo-Pelger cells or the acquired Pelger–Huët anomaly
• Acquired condition• Morphologically similar
to Pelger–Huët anomaly• seen in Myelodysplastic
syndromes,• Acute myeloid
leukaemia with dysplastic maturation,
• Occasionally inchronic myelogenous leukaemia
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Pyknotic Neutrophils (Apoptosis)
• Small numbers of dead or dying cells may normally be found in the blood
• seen in infections– invitro after standing for
12-18 hrs– Nuclei-round dense,
featureless– Cytoplasm-dark pink
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EOSINOPHILS
• Normally 1-6( 0.02–0.5 × 109/l)
• Size- 12–17 µm• Nucleus- Bilobed (spectacle
shaped)• Cytoplasm- Pale blue• Granules - Coarse spherical
gold/orange
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Eosinopenia- seen with prolonged steroid administration.• Eosinophilia- allergic conditions hay fever,
asthama• severe eosinophilia- parasitic infection
– reactive eosinophilia– Eosinophilic leukaemia– Idiopathic hypereosinophilic syndrome– T-cell lymphoma, B-cell lymphoma
and acute lymphoblastic leukaemia.
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BASOPHILS• Rarest <1• Nucleus segments fold up on
each other resulting compact irregular dense nucleus(closed lotus flower like)
• Granules-large, variable size dark blue or purple often obscure the nucleus
• Granules are rich in histamine, serotonin and heparin
• Increase in myeloproliferative disorder-CML
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MONOCYTES• 2-10% of total wbc count• Size- largest circulating leucocyte,
15–18µm in diameter• Cytoplasm- grey blue• Nucleus- large , curved , horse
shoe shape• No segmentation occur• Chromatin- fine evenly
distributed• Increase in chronic infections and
inflammatory conditions such as– Tuberculosis and Crohn’s
disease,– Chronic myeloid leukaemias– Acute leukaemias with a
monocytic component– Infectious mononucleosis
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LYMPHOCYTES• 20-40% of total wbc count• two types1. Small lymphocyte(6-10µm)2. Large lymphocyte(12-15µm)• Nucleus-single, sharply
defined, stain dark blue on Wright’s stain
• Cytoplasm- Pale blue• Large lymphocytes less
densely stain nuclei & abundant cytoplasm
• Few round purple(azure) granules are present
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Turk cells
• Türk’ cell (immunoblasts)- Transformed lymphocyte seen in bacterial and viral infection
• Size 10-15 µm• Nucleus- Round,• Large nucleolus, and
abundant, deeply basophilic cytoplasm
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Reactive lymphocytes
• Have slightly larger nuclei with moreopen chromatin
• Abundant cytoplasm that may beirregular.
• Seen in -infectious mononucleosis– viral infections
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Malignant lymphoid cells
• Commonest malignancy is Chronic lymphocytic leukemia- composed almost exclusively of small lymphocytes.
• Some times few larger nucleolated cells
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• Lymphocytes predominate in the blood films of infants and young children.
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Platelates• Size -1-3µm • Normal count - 280 130×109/µl• Non nucleated cells derived from cytoplasmic
fragments of Megakaryocytes• Has purple red granules.• Liliac color
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Thrombocytopenia
• Decreased production – Aplastic anemia – Acute leukemia – Viral infections *Parvovirus *CMV −Amegakaryocytic thrombocytopenia (AMT)
• Increased destruction – Immune thrombocytopenia – Idiopathic thrombocytopenic purpura (ITP)– Neonatal alloimmune thrombocytopenia (NAITP) – Disseminated intravascular coagulation (DIC)– Hypersplenism
• Pseudothrombocytopenia- due to clumpping of pltelates in EDTA bulb
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Thrombocytosis
• Reactive thrombocytosisPost infectionInflammationJuvenile rheumatoid arthritisCollagen vascular disease• Essential thrombocythemia
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Platelet morphology: Giant platelets
• Platelates seems to be size of rbcs.
• Seen in– May –Hegglin anomly– Bernard Soulier
syndrome– Alport syndrome– Storage pool syndrome
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Peripheral Blood Cells
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changes on prolonged storage of sample
• EFFECT ON COUNT-• Less marked in blood in ACD, CPD or Alsever’s solution than in
EDTA.• At room temperature blood is stable up to 8 h.• RBC
– Swell up the PCV and MCV increases– Osmotic fragility increases– Erythrocyte sedimentation rate decreases– At 4_ C up to 24 h– Reticulocyte count- Unchanged upto 24 h at 4 C– Hemoglobin Unchanged upto 2-3 days
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Changes in Morphology
• At 3 h changes start occure• By 12–18 h these become striking• RBC- progressive crenation and sphering• Netrophils- nuclei stain more homogeneously
– Nuclear lobes may become separated– Cytoplasmic margin may appear ragged– Vacuoles appear in the cytoplasm– Lymphocytes and monocytes undergo similar
changes
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Changes in netrophils
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Changes in monocytes
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changes in lymphocytes
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Apoptotic changes
• Cell shrinkage• Cytoplasmic condensation
around the nuclear membrane
• Indentations in the nucleus• Followed by nuclear
fragmentation.• Cell remnants form dense
basophilic masses (the apoptotic bodies)
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Disadvantages of the Peripheral Blood Smear
• Experience is required to make technically adequate
smears.
• Non-uniform distribution of white blood cells
• Larger leukocytes concentrated near edges and
lymphocytes scattered throughout.
• Non-uniform distribution of RBCs
– Small crowded red blood cells at the thick edge
– Large flat red blood cells without central pallor at the
feathered edge
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EXAMINATION OF BLOOD FILMSFOR PARASITES
• thick film- when parasites are scanty• thin film – identification of species• STAINING OF FILM
– by Leishman’s stain at pH 7.2
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Plasmodium falciparumErythrocytes throughout this series are not enlarged or distorted.• Early trophozoites• Accole form
• Crescent (‘banana-shaped’) gametocyte
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double chromatin dot
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Schizonts are commonly seen in P. vivax infection and appear as large bodies containing 12 to 24 nuclei and a loose pigmented body. This photograph shows an early schizont of P. vivax on the left and mature schizonts
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Ealy trophozoit in the form of thick ring with large chromatin dot
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Leishmaniasis (Leishman–Donovan bodies)
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African trypanosomiasis (Trypanosoma brucei gambiense)
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American trypanosomiasis ( T. cruzi);
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microfilaria
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Thank you