Peripheral blood smear examination (slide preparation and reporting)

Dr. Bahoran SinghModerator- Dr. Namrata Shetty

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

• Objective:

1. Peripheral Smear Preparation

2. Staining of Peripheral Blood Smear

3. Peripheral Smear Examination

Peripheral Blood Smear

Peripheral Smear Preparation

• Wedge technique• Coverslip technique• Automated Slide Making and Staining

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.

• Equipment•Spreaders• Clean slides• Blood capillary tube or micropipette 10

µL

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.

The shape of blood film

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.

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

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

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

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.

Blood films

Slide fixation and staining

Romanowsky stainingIt includes:• May-Grunwald –Geimsa stain,• Jenner’s stain,• Wright’s stain,• Leishman’s stain and,• Field’s stain.

Components of Giemsa stain

It mainly composed of• Eosin Y and,• Azure B- Methylene Blue

• 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)

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)

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

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

Cytoplasm colour

• Lymphocyte Blue• Metamyelocyte Pink• Monocyte Grey–blue• Myelocyte Pink• Neutrophil Pink/orange• Promyelocyte Blue• Basophil Blue

Granules

• Promyelocyte(primary granules) Red or purple• Basophil Purple black• Eosinophil Red–orange• Neutrophil Purple• Toxic granules Dark blue• Platelet Purple

Other inclusions

• Auer body Purple• Cabot ring Purple• Howell-Jolly body Purple• Döhle body Light blue

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

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

– Too pink Incorrect proportion of azureB-eosin Y• Impure dyes• Buffer pH too low• Excessive washing in buffer

solution

• Pale staining Old staining solution• Overused staining solution• Incorrect preparation of

stock• Impure dyes, especially

azure A and/or C• High ambient temperature

• 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

• 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

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

• 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).

Normal peripheral smear

Area too thin to examine the slide

Too thick area

Evaluation of PBS • 1. RBC

• Size• Shape • Color • Arrangement • Inclusions

• 2. WBC• Total counts• Differential counts• Abnormal WBC

• 3. Platelets • Counts

• Abnormality •4. Parasites

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

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

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

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)

• 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.

Dimorphic blood picture

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

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.

Microcytes

• Size of RBC is reduced(<80fl)

• Seen when hemoglobin synthesis is defective1. Iron deficiency anemia2. Thalassemia3. Anemia of chronic

disease4. Sideroblastic anemia

Macrocytes• When MCV of RBC is

Increased(>100fl)• Macro- ovalocytes are

seen in Megaloblastic anemia

• Myledysplastic syndrome.

• Round Macrocytes seen in Alcoholism, Liver disease.

Shape• Variation in shape is called Poikilocytosis.• It is of following types-

• Elliptocytes• Spherocytes• Target cells• Schistocytes• Acanthocytes• Keratocytes• Echinocytes

Elliptocytes

• Elipitical in shapes• Most abundant in

hereditary elliptocytosis• Seen in –1. Iron deficiency anemia2. Myelofirosis with myeloid metaplasia3. Megaloblastic anemia4. Sickle cell anemia

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

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

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

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

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)

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.

Leptocytes

• Thin red cells with large unstained central area.

• Seen in– Severe iron deficiency

anemia– Thaleasaemia

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.

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

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

Structure

• Basophilic stippling (Punctate basophilia)• Howell – jolly Bodies• Cabot Rings• Malarial Stippling• Rouleaux formation

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

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.

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

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

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

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,

Malrial stippling

• Fine granules of plasmodium vivax

• On wright stain these are fine , purplish red

• Red cells are larger than normal

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

Agglutination

• It is more irregular and round clumping than linear rouleaux

• Seen with cold agglutinin

• Anti RBC antibody• Autoimmune hemolytic

anemia• Macroglobulinemia

WBC MORPHOLOGY

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

• 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

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

• 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

Leukocytes Normally Present in Blood

• GRANULOCYESNeutrophils ( polymorphonuclear leucocytes)Eosinophils Basophils

• Agranulocytes Lymphocytes Monocytes

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.

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)

• Left-shift: non-segmented neutrophil > 5%– Increased

bands Means acute infection, usually bacterial

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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

• Hypogranular and agranular neutophils poorly stained

• seen in Myelodysplastic syndrome

Alder–Reilly anomaly

• Granules are large,– discrete,– stain deep red – may obscure the

nucleus– Neutrophil function

is Normal

Chédiak-Higashi Syndrome

• Granules are– also seen in other

leukocytes like lymphocytes

– Giant – Scanty azurophilic– functional defect occur

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

May–Hegglin anomaly

• inclusions occur in alltypes of leucocytes except lymphocytes.

• contain small basophilic cytoplasmic granules

Vacuoles in neutrophils

• In Fresh blood smear • vacuoles seen in

– severe sepsis– as an artifact with

prolonged standing

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

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.

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

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

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

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.

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

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

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

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

Reactive lymphocytes

• Have slightly larger nuclei with moreopen chromatin

• Abundant cytoplasm that may beirregular.

• Seen in -infectious mononucleosis– viral infections

Malignant lymphoid cells

• Commonest malignancy is Chronic lymphocytic leukemia- composed almost exclusively of small lymphocytes.

• Some times few larger nucleolated cells

• Lymphocytes predominate in the blood films of infants and young children.

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

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

Thrombocytosis

• Reactive thrombocytosisPost infectionInflammationJuvenile rheumatoid arthritisCollagen vascular disease• Essential thrombocythemia

Platelet morphology: Giant platelets

• Platelates seems to be size of rbcs.

• Seen in– May –Hegglin anomly– Bernard Soulier

syndrome– Alport syndrome– Storage pool syndrome

Peripheral Blood Cells

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

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

Changes in netrophils

Changes in monocytes

changes in lymphocytes

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)

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

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

Plasmodium falciparumErythrocytes throughout this series are not enlarged or distorted.• Early trophozoites• Accole form

• Crescent (‘banana-shaped’) gametocyte

double chromatin dot

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

Ealy trophozoit in the form of thick ring with large chromatin dot

Leishmaniasis (Leishman–Donovan bodies)

African trypanosomiasis (Trypanosoma brucei gambiense)

American trypanosomiasis ( T. cruzi);

microfilaria

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