RBC and WBC morphology

Parts of a peripheral blood smear

Head, body and tail

1-Head

2-Body

3-Tail

Examples of unacceptable smears

A: Blood film with jagged tail made from a spreader with chipped end.

B: Film which is too thick

C: Film which is too long, too wide, uneven thickness and made on a greasy slide.

D: A well-made blood film.

THE CORRECT SELECTION OF THE AREA

Too thin Too thick Good area

The "feather edge”or “tail”of the slide, should be scanned at low power to assess •white cells •any large abnormal cells or• platelet clumps

Always scan the feather edge

Good Habits

Principle White Blood Cells.1. Check for even distribution and estimate

the number present (also, look for any gross abnormalities present on the smear).

2. Perform the differential count.

Principle

Red Blood Cells, Examine for: 1.Size and shape. 2.Relative hemoglobin content. 3.Polychromatophilia. 4. Inclusions. 5.  Rouleaux formation or agglutination

Platelets. 1.Estimate number present. 2. Examine for morphologic

abnormalities.

Procedures

Observations Under ×101.Check to see if there are good counting

areas available free of ragged edges and cell clumps.

2.Check the WBC distribution over the smear.

3.Check that the slide is properly stained.4.Check for the presence of large platelets,

platelet clumps, and fibrin strands.

Observations Under× 40x : WBC Estimates

Using the × 40 high dry with no oil. Choose a portion of the peripheral smear

where there is only slight overlapping of the RBCs.

Observations Under × 100: Platelet Estimates1. Use the oil immersion lens estimate the number of

platelets per field.2. Look at 5-6 fields and take an average.3. Multiply the average by 20,000.4. Note any macroplatelets. Platelets per oil immersion field (OIF)1)<8 platelets/OIF = decreased2)8 to 20 platelets/OIF = adequate3)>20 platelets/OIF = increased

RBC Morphology

Normochromic Normocytic

Abnormal RBC morphology

Inadequqate haemoglobin formation

Abnormal erythropoiesis

Regenerating marrow• Polychromasia

• Erythroblastaemia

•Anisocytosis•Poikilocytosis•Microcytosis

•Macrocytosis

•Hypochromia

Abnormal RBC morphology

•Sickle cells

•Fragmented red cells•Bite cells•Spherocytes•Tear drop cells

•Basophilic stippling•Howell Jolly bodies•Pappenheimer Bodies

Abnormal haemoglobin formation

Damage to red cells

Red cell inclusions

Abnormal RBC morphology

•Target cells•Echinocytes•Acanthocytes•Stomatocytes•Dimorphic blood picture

•Rouleaux formation•Agglutination

Miscellaneous erythrocyte abnormalities

Normal RBCs & abnormal plasma

Hypochromia

Central pallor more than 1/3 of the red cell

Associated diseases

¨ Iron deficiency

¨ Thalassaemia

¨ Anaemia of chronic disease

¨ Sideroblastic anaemia

¨ Lead poisoning

Inadequate haemoglobin formation

Abnormal erythropoiesis

Anisocytosis

Variation in size

MCV may be normal

Associated with increased RDW

Poikilocytosis

Variation in shape

Non specific feature ofAbnormal erythropoiesis

Associated diseases

Iron deficiency anaemiaThalasaemiaMegaloblastic anaemiaMyelofibrosis

Microcytes

Compare with small lymphocyte which is slightly larger than a normal RBC

Microcytosis, hypochromia, & poikilocytosis

Elliptocytes

Hereditary elliptocytosis

Iron deficiency anaemia

Megaloblastic anaemia

•Elongated red cells •Ends are rounded•Central pallor is present

Key features

Associated conditions

Round Macrocytes

•Larger than normal , round shaped more than 100 fl

•Central pallor is present

Key features:

Round Macrocytes

Round Macrocytes

Liver disease Hypothyroidism Alcoholism Chronic

obstructive pulmonary disease

• Regenerating marrow• Hemolytic anaemia• Acute blood loss• Neonates

Due to abnormal lipid composition of erythrocyte membrane

Pathophysiology

With normal reticulocyte count

Causes

With increased reticulocyte count

Reticulocytes are larger than normal RBCs

Oval Macrocytosis

Oval Macrocytosis

Pathophysiology

Defective DNA synthesis

Slow nuclear maturation

Formation of abnormally large cells

Oval macrocytosis

Nuclear cytoplasmic asynchrony

Oval Macrocytes

Megaloblastic anaemia - Vitamin B12

deficiecy - Folate deficiency Myelodysplastic anaemia Treatment with

hydroxyurea

Causes

Regenerating marrow

Polychromasia

• Any condition associated with reticulocytosis • These are in fact

reticulocytes & can be confirmed with supra- vital stains.

Larger than normal RBCsShow bluish grey shades Central pallor not present

Key features

Associated conditions

Reticulocytosis Marked erythropoiesis

Bone marrow

Supra-vital stain

Reticulocytes

Reticulocytes are immature RBCs which have shed their nucleus but still retain ribosomes, mitochondria, remnants of

golgi bodies and other cytoplasmic granules

Reticulocyte count

Regenerating marrow

Acute blood loss

Haemolytic anaemia

Deficiency anaemias receiving therapy

Hypersplenism

New born

• Aplastic anaemia

• Drug suppression

• Megaloblastic anaemia

• Myelodysplastic syndrome

• Pure red cell aplasia

Increased Decreased

Peripheral Blood Smear ofThalassemia Major

Erythroblastaemia

• Any condition associated with severe haemolysis

• Exramedulary haematopoiesis• Myelopthesic diseases.

Pyknotic eccentric nucleus Cytoplasm pink to grey

Key features

Associated conditions

Erythroblastaemia

Normoblasts in haemolytic disease of new born

Abnormal Haemoglobin formation

Sickle Cell

• Red cells with pointed ends

• Crescent shape• No central pallor• Very dense

hemoglobin

Key features

Associated diseaseHemoglobin SSHemoglobin SCHemoglobin SD S- beta thalassaemia

Damaged red cells

RBC Fragmentation

Synonym:Schistocytes

Key features•Two to three sharp angles of spines •Central pallor is not present

RBC

Fragmented RBCs (schistocytes)

Microangiopathic hemolytic anaemia -TTP -Hemolytic uremic syndrome -DIC

Malignant hypertension Artificial heart valve

Causes

Schistocytes vs Microcyte

Key 1-Schistocytes with pointed ends & loss of central pallor

2-Microcytes showing central pallor

Bite cells

Synonym:Keratocytes

Key features•Pair of spicules Central pallor is not present•Helmet like cells are also included

PathophysiologyRemoval of Heinz body pitting action of spleenAssociated conditionsG6PD deficiency,unstable hemoglobin, Drug insult,thalassaemias

Bite cell with reticulocytosis

Microspherocytes

• Artifactual• Hereditary spherocytosis• Immune hemolytic anaemia• Thermal injury• Microangiopathic hemolytic anaemia

Smaller than normal RBCs • No central pallor• Very dense hemoglobin

Key features

Associated conditions

Microspherocytes

Compare them with small lymphocyte

Microspherocytes in Microangiopathic hemolytic anaemia

Basophilic stippling

Hemolytic anemias Pyrimidine-5’nucleotidase

deficiency

Iron deficiency Thalassemias Lead poisoning

Diffuse fine or coarse blue dots in the red cell representing usually RNA residue

Howell Jolly Bodies

Howell jolly bodies seen in post splenectomy patient

Pappenheimer Bodies

• Any condition associated with iron over load

• Sideroblastic anaemia• Thalassaemia

Small peripherally sited basophilic inclusions Their nature can be confirmed by perl stain

Key features

Associated conditions

Target Cells

Causes of target cell formation

¨ Liver disease¨ Post splenectomy

¨ Iron deficiency anaemia ¨ Thalassaemia¨ Haemoglobinopathies

•Artifacts of air drying

• Haemoglobin C

•Decreased volume

•Increased surface membrane

Echinocytes

Synonyms:

-Burr cells -Crenated cells

•Spiculated red cells with 10-30 short evenly spaced projections over the entire suface

•These retain the central pallor

Acanthocytes

Abnormal lipid metabolism

Liver disease

Splenectomy

Synonym:Spur Cells Key features•Red cell with small number of Spicules with irregular thickness & shape.•Central pallor is not present

Causes

Compare 1-burr cells with 2-spur cell

ArtifactDecreased pHLiver diseaseAlcholism

Key features•Red cell in which Central pallor areais slit like or mouth like in dried Smear.

Causes

Stomatocytes

Rouleaux Formation

• Multiple myeloma• Chronic liver disease• Chronic inflammatory

conditions• Malignant lymphoma

CausesAll the diseases associated with hypergamma globulinaemia

Agglutination

AgglutinationZeta potential

25 nm

IgM antibodies are formed against red cell antigens

These antibodies are large enough to overcome the zeta potential gap

RBCs are joined together in the form ofclumps ( grape like clusters)

RBC QUIZ

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Normochromic Normocytic

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Thal Major

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Oval Macrocytosis:Megaloblastic Anaemia

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Rouleaux Formation

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Howell Jolly Bodies

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Microcytic Hypochromic

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Polychromasia

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Sickle Cells & Target Cells

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Microspherocytes

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Tear Drop cells

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macroovalocytosis, hypersegmentation, thrombocytopenia

Megaloblastic Anemia

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Burr cells (Echinocytes)

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Spherocytes

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NRBC

White Blood Cells Morphology

Five Types of Leukocytes (WBCs)

Granular Leukocytes

Neutrophil

Eosinophil

Basophil

Neutrophil

60-70% of all WBC’s Anatomy

10-12 µm diameter 2-6 nuclear lobes Fine, pale inconspicuous

granules Physiology

Respond first to bacteria damage by chemotaxis

Phagocytosis After engulfing pathogen

releases several chemicals lysozyme strong oxidants defensins

Clues to recognize Neutrophils

BUT lighter in shade i.e. light purple

•Cytoplasm has purple/basophilic granules i.e.same as the color of the nucleus.

Neutrophil granules can be fine or coarse

¨ bilobation is not seen in normal neutrophils

• Multiple lobes of the nucleus

Eosinophil 2-4% of all WBC’s Anatomy

10-12 µm diameter 2 connected nuclear

lobes red/orange large,

uniform granules, do not obscure the nucleus

Physiology exit capillaries, enter

tissue fluid combat parasites

histamine phagocytize antigen-

antibody complexes

Basophil 0.5-1% of all WBC’s Anatomy

8-10 µm diameter bilobed or irregular nucleus round, blue-black granules

may obscure the nucleus Physiology

exit capillaries to enter tissue fluid

mature into mast cells release heparin, histamine,

serotonin – stimulate inflammation

Hypersensitivity (allergic) reactions

Agranular Leukocytes

Lymphocyte

Monocyte

Lymphocytes

20-25% of all WBC’s

Anatomy 7-15µm nucleus large and

dark stained, round or indented

cytoplasm forms a pale blue rim around the nucleus

Monocytes

3-8% of all WBC’s Anatomy

14-19 µm indented or kidney-shaped

nucleus (not round) cytoplasm foamy

Physiology slower to arrive but survive

longer enlarge, differentiate into

fixed and wandering macrophages

remove microbes, cellular debris, following injury

Platelet satellitism

Platelet satellitism describes the phenomenon of adherence of platelets to white cells.

It is an in vitro phenomenon of no clinical significance. However it is important that it is detected since the platelet count will be factitiously low.

How granulocytes are formed ?

Myeloblast is the earliest recognizable cell of the granulocytic series

It is normally present in the bone marrow

Large cell

Fine reticular chromatin

Nucleoli are present

Fine azurophilic granules may be present

How granulocytes are formed ?

Appearance of primary azurophilc granules

PromyelocyteMyeloblast

Primary azurophilic granulesFine reticular chromatin

Specific (secondary)granulesappear, with primary azurophilc granulesSlightly clumpy chromatin

Promyelocyte Myelocyte

. Note; sunrise effect

Neutrophilic granules Primary Secondary

Non specific/ Azurophilic granules

Large Blue-Black Appear in

promyelocytes, however their synthesis cease at myelocyte stage

Contains myeloperoxidase

Specific granules

Small sand-like Pink-red to Pink

violet Appear in

myelocyte

Contains alkaline phosphatase

Synonyms

Size

Color

Visibility

Consistency

Ref: Text book of Heamatology, Mckenzie, second edition

Myelocyte Metamyelocyte

Slight indentation of nucleus

Metamyelocyte Band cell

Nuclear indentation < half

Nuclear indentation > half

Band cell Segmented neutrophil

Maturation of polymorphs

Myelocyte

Metamyelocyte

Band cell

Mature polymorph

Ref: CAP Color Atlas of Hematology

Eric F Glassy, M D

Maturation of polymorphs

MyelocyteBand cellMature polymorphMetamyelocyteRef: CAP Color Atlas of Hematology

Eric F Glassy, M D

Granulopoiesis- All Stages

Auer rods ?

•Auer rods are collection of azurophilic granules containing peroxidase lysosomal enzymes organized in the form of rods

Ref: Williams Hematology Fifth edition

•Seen in 2-3% of myeloblasts

•Diagnotic feature of AML

Myeloblasts

Myeloblasts

Morphological features

Medium to large Scanty to moderate

finely granular Present in 60-70% Fine reticular Two to 3

Small to medium Scanty non-granular

Not present Fine to coarse Indistinct

Myeloblasts Lymphoblasts

Ref: Atlas of tumor pathlogy AFIP

SizeCytoplasm

Auer rods

ChromatinNucleoli

Small Lymphocytes

Normal lymphocytes are slightly larger than erythrocytes, and constitute more than 95% of the peripheral blood lymphocyte pool.

They have condensed nuclear chromatin with a thin rim of cytoplasm. Nucleoli are rarely seen.

Reactive lymphocytes

Abundant cytoplasm, fine nuclear chromatin and often nucleoli.

Key Features

Causes

Viral infections.

Large Granular Lymphocytes (LGL)

The LGL has cytoplasm,which is often lighter stained than that of small lymphocytes

Azurophilic granules are seen and the nucleus is more variable in shape and often eccentric. Some LGL are natural killers, while others are T-cells.

Monocyteso Monocytes are larger

than normal lymphocytes.

o Nucleus is kidney shaped or lobulated, with fine to coarse chromatin.

o Cytoplasm is slightly basophilic or gray-blue with occasional granules or vacuoles.

Monocytes Morphologic Spectrum

REACTIVE CHANGES IN NEUTROPHIL MORPHOLOGY

Toxic Granulation in Neutrophils

Associated conditionsBacterial infections

Other inflammatory conditions

Increased number of coarse azurophlic granules

Key Features

Dohle Body in Neutrophils

Small round, or oval pale blue – grey structure

Consist of ribosome or endoplasmic reticulum

Bacterial infection

Key Features

Associated conditions

May-Heglin anomaly

Vacuolated Neutrophils

Associated conditions

Artifact of prolonged standing

Severe sepsis

EXERCISE

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Basophil

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Lymphocyte

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Promyelocyte

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Neutrophil

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Neutrophil with Toxic Granulation

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Monocyte

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Blast

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Lymphocytes

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Monocyte

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Neutrophil

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Eosinophil & Neutrophils

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Neutrophil with Vacuoles

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Monocyte

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Basophil

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Dohle Body in Neutrophil

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Eosinophil

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Lymphocyte & neutrophil

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NRBC

Lymphocytosis