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

Good Habits
Always scan the feather edge

The "feather edgeor tailof the slide, should be scanned at low power to assess white cells any large abnormal cells or platelet clumps

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
2.

number present. Examine for morphologic abnormalities.

Procedures

Observations Under 10
1. 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 Estimates

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 = decreased 2) 8 to 20 platelets/OIF = adequate 3) >20 platelets/OIF = increased
1.

RBC Morphology

Normochromic Normocytic

Abnormal RBC morphology

Inadequqate haemoglobin formation
Hypochromia

Abnormal erythropoiesis
Anisocytosis Poikilocytosis Microcytosis Macrocytosis

Regenerating marrow

Polychromasia
Erythroblastaemia

Abnormal RBC morphology

Abnormal haemoglobin formation
Sickle cells

Damage to red cells

Fragmented red cells Bite cells Spherocytes Tear drop cells

Red cell inclusions

Basophilic stippling Howell Jolly bodies Pappenheimer Bodies

Abnormal RBC morphology

Miscellaneous erythrocyte abnormalities
Target cells Echinocytes Acanthocytes Stomatocytes Dimorphic blood picture

Normal RBCs & abnormal plasma

Rouleaux formation Agglutination

Inadequate haemoglobin formation

Hypochromia
Associated diseases
Iron deficiency

Thalassaemia
Anaemia of chronic disease Sideroblastic anaemia Lead poisoning

Central pallor more than 1/3 of the red cell

Abnormal erythropoiesis

Anisocytosis
Variation in size MCV may be normal Associated with increased RDW

Poikilocytosis
Variation in shape
Non specific feature of Abnormal erythropoiesis

Associated diseases
Iron deficiency anaemia Thalasaemia Megaloblastic anaemia Myelofibrosis

Microcytes

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

Microcytosis, hypochromia, & poikilocytosis

Elliptocytes
Key features
Elongated red cells Ends are rounded

Central pallor is present

Associated conditions

Hereditary elliptocytosis Iron deficiency anaemia Megaloblastic anaemia

Round Macrocytes
Key features:
Larger than normal , round shaped more than 100 fl

Central pallor is present

Round Macrocytes

Round Macrocytes
With normal reticulocyte count Pathophysiology With increased reticulocyte count

Due to abnormal lipid composition Reticulocytes are larger than norma of erythrocyte membrane RBCs

Causes Liver disease Hypothyroidism Alcoholism Chronic obstructive pulmonary disease

Regenerating marrow Hemolytic anaemia Acute blood loss Neonates

Oval Macrocytosis

Oval Macrocytosis
Pathophysiology
Defective DNA synthesis

Slow nuclear maturation

Nuclear cytoplasmic asynchrony

Formation of abnormally large cells

Oval macrocytosis

Oval Macrocytes Causes

Megaloblastic anaemia - Vitamin B12 deficiecy - Folate deficiency Myelodysplastic anaemia Treatment with hydroxyurea

Regenerating marrow

Polychromasia
Reticulocytosis Marked erythropoiesis

Key features
Larger than normal RBCs Show bluish grey shades Central pallor not present

Supra-vital stain

Bone marrow

Associated conditions
Any condition associated with reticulocytosis These are in fact reticulocytes & can be confirmed with supravital stains.

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

Increased

Decreased Aplastic anaemia Drug suppression

Haemolytic anaemia Deficiency anaemias receiving therapy Hypersplenism New born

Megaloblastic anaemia
Myelodysplastic syndrome Pure red cell aplasia

Peripheral Blood Smear of Erythroblastaemia Thalassemia Major

Key features
Pyknotic eccentric nucleus Cytoplasm pink to grey

Associated conditions
Any condition associated with severe haemolysis Exramedulary haematopoiesis Myelopthesic diseases.

Erythroblastaemia

Normoblasts in haemolytic disease of new born

Abnormal Haemoglobin formation

Sickle Cell Key features

Red cells with pointed ends Crescent shape No central pallor Very dense hemoglobin

Associated disease

Hemoglobin SS Hemoglobin SC Hemoglobin 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

Fragmented RBCs (schistocytes)

Causes

Microangiopathic hemolytic anaemia -TTP -Hemolytic uremic syndrome -DIC Malignant hypertension Artificial heart valve

RBC

Schistocytes vs Microcyte

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

2-Microcytes showing central pallor

Bite cells
Synonym: Keratocytes Bite cell with reticulocytosis

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

Pathophysiology Removal of Heinz body pitting action of spleen Associated conditions G6PD deficiency,unstable hemoglobin, Drug insult, thalassaemias

Microspherocytes Key features

Smaller than normal RBCs No central pallor Very dense hemoglobin

Associated conditions
Artifactual Hereditary spherocytosis Immune hemolytic anaemia Thermal injury Microangiopathic hemolytic anaemia

Microspherocytes

Compare them with small lymphocyte

Microspherocytes in Microangiopathic hemolytic anaemia

Basophilic stippling

Hemolytic anemias

Pyrimidine-5nucleotidase 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
Key features Small peripherally sited basophilic inclusions Their nature can be confirmed by perl stain Associated conditions Any condition associated with iron over load Sideroblastic anaemia Thalassaemia

Target Cells

Causes of target cell formation

Artifacts of air drying
Haemoglobin C Decreased volume
Iron deficiency anaemia Thalassaemia Haemoglobinopathies

Increased surface membrane

Liver disease Post splenectomy

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
Synonym: Spur Cells

Key features
Red cell with small number of Spicules with irregular thickness & shape. Central pallor is not present

Causes
Abnormal lipid metabolism Liver disease Splenectomy

Compare 1-burr cells with 2-spur cell

Stomatocytes
Key features
Red cell in which Central pallor area is slit like or mouth like in dried Smear.

Causes
Artifact Decreased pH Liver disease Alcholism

Rouleaux Formation
Causes
All the diseases associated with hypergamma globulinaemia

Multiple myeloma Chronic liver disease Chronic inflammatory conditions Malignant lymphoma

Agglutination

Agglutination
Zeta 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 of clumps ( 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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Megaloblastic Anemia

macroovalocytosis, hypersegmentation, thrombocytopenia

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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

Eosinophil

Neutrophil
Basophil

Neutrophil
60-70% of all WBCs Anatomy

10-12 m diameter 2-6 nuclear lobes Fine, pale inconspicuous granules Respond first to bacteria damage by chemotaxis Phagocytosis After engulfing pathogen releases several chemicals

Physiology

lysozyme strong oxidants defensins

Clues to recognize Neutrophils

Multiple lobes of the nucleus bilobation is not seen in normal neutrophils Cytoplasm has purple/basophilic granules i.e.same as the color of the nucleus.
BUT lighter in shade i.e. light purple Neutrophil granules can be fine or coarse

Eosinophil

2-4% of all WBCs Anatomy 10-12 m 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 antigenantibody complexes

Basophil

0.5-1% of all WBCs Anatomy

8-10 m diameter bilobed or irregular nucleus round, blue-black granules may obscure the nucleus exit capillaries to enter tissue fluid mature into mast cells

Physiology

release heparin, histamine, serotonin stimulate inflammation

Hypersensitivity (allergic) reactions

Agranular Leukocytes

Lymphocyte

Monocyte

Lymphocytes

20-25% of all WBCs Anatomy

7-15m nucleus large and dark stained, round or indented cytoplasm forms a pale blue rim around the nucleus

Monocytes
3-8% of all WBCs Anatomy

14-19 m indented or kidney-shaped nucleus (not round) cytoplasm foamy slower to arrive but survive longer enlarge, differentiate into fixed and wandering macrophages remove microbes, cellular debris, following injury

Physiology

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 ?

Myeloblast Promyelocyte

Appearance of primary azurophilc granules

Promyelocyte

Myelocyte

Primary azurophilic granules Fine reticular chromatin

Specific (secondary)granules appear, with primary azurophilc granules . Note; sunrise effect Slightly clumpy chromatin

Neutrophilic granules
Primary
Non specific/ Azurophilic granules Size Large Color Blue-Black Visibility Appear in promyelocytes, however their synthesis cease at myelocyte stage Consistency Contains myeloperoxidase Synonyms

Secondary
Specific granules

Small sand-like Pink-red to Pink violet Appear in myelocyte

Contains alkaline phosphatase

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

Myelocyte Metamyelocyte Band cell Mature polymorph

Ref: 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 Seen in 2-3% of myeloblasts Diagnotic feature of AML

Ref: Williams Hematology Fifth edition

Myeloblasts

Myeloblasts

Morphological features
Myeloblasts Lymphoblasts

Size Cytoplasm Auer rods

Chromatin Nucleoli

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

Ref: Atlas of tumor pathlogy AFIP

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
Key Features
Abundant cytoplasm, fine nuclear chromatin and often nucleoli.

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 Tcells.

Monocytes are larger than normal lymphocytes. Nucleus is kidney shaped or lobulated, with fine to coarse chromatin. Cytoplasm is slightly basophilic or gray-blue with occasional granules or vacuoles.

Monocytes

Monocytes Morphologic Spectrum

REACTIVE CHANGES IN NEUTROPHIL MORPHOLOGY

Toxic Granulation in Neutrophils

Key Features
Increased number of coarse azurophlic granules

Associated conditions
Bacterial infections Other inflammatory conditions

Dohle Body in Neutrophils

Key Features
Small round, or oval pale blue grey structure Consist of ribosome or endoplasmic reticulum

Associated conditions
Bacterial infection 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

?????????

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