Practical of Clinical Hematology Medical Technology Department Islamic University-Gaza Practical of Clinical Hematology Collected and prepared by Mr. Mohammed O. Jaber

RBCs Abnormal morphology Peripheral Blood Morphology

Abnormal erythrocyte morphology Is found in pathological states that may be abnormalities in Red cell distribution. Size (anisocytosis). Hemoglobin content – Color Variation . Shape (poikilocytosis). The presence of inclusion bodies in erythrocyte.

Erythrocyte Distribution Abnormalities Rouleaux formation Stacking of RBCs due to increased plasma proteins coating RBCs Agglutination Antibody-mediated clumping; temperature dependent 2

Erythrocyte Distribution Abnormalities 13- Rouleaux Formation: Morphology: Stacks of RBC's resembling a stack of coins. Found in: - Hyperfibrinogenaemia - Hyperglobulinaemia

Erythrocyte Distribution Abnormalities 14- Red cell-agglutination: Morphology: Irregular clumps of red cells Found in: - Cold agglutinins - Warm autoimmune hemolysis

Rouleaux Formation

Agglutination Reaction

Schrier, S. ASH Image Bank 2002;2002:100344 Figure 2. The patient has autoimmune cold agglutinin disease and the red blood cells (RBC) have clumped when placed on the cold slide Schrier, S. ASH Image Bank 2002;2002:100344

Variation in erythrocyte size (anisocytosis) Normocyte: normal size of RBC, The average size of an RBC is 7.2 μm with a range of 6.8 to 7.5 μm. The nucleus of a small lymphocyte (± 8,µm) is a useful guide to the size of a red blood cell. Anisocytosis: Variations in size e.g. Microcyte Macrocyte

A. Report RBC size • Microcytic: smaller than the normal RBC,( <7.2 μm), and is associated with a decrease in hemoglobin synthesis Found in: - Iron deficiency anemia. - Thalassaemia. - Sideroblastic anemia. - Lead poisoning. - Anemia of chronic disease.

Variation in erythrocyte size (anisocytosis) • Macrocyte: larger than the normal RBC (<8.2 μm) and is the result of a defect in nuclear maturation or stimulated erythropoiesis. May be round or oval in shape, the diagnostic significance being different. Found in: - Folate and B12 deficiencies (oval) - Ethanol (round) - Liver disease (round) - Reticulocytosis (round)

Macrocytic Anemia: Macro-Ovalocytes

Variation in erythrocyte size (anisocytosis) Comment: Most erythrocytes presented in the picture are microcytes (compare with the small lymphocyte). The degree of hemoglobinization is sufficient. Normal platelets and single ovalocytes are present. Staining: MGG Magnification: x1000 1. microcyte   2. normocyte

Variation in erythrocyte color A normal erythrocyte has a pinkish-red color with a slightly lighter-colored center (central pallor) when stained with a blood stain, such as Wright. The color of the erythrocyte is representative of hemoglobin concentration in the cell. Under normal conditions, when the color, central pallor, and hemoglobin are proportional, the erythrocyte is referred to as normochromic.

Variation in hemoglobin content • Hypochromia: increased central pallor and decreased hemoglobin concentration, the central pallor occupies more than the normal third of the red cell diameter. Found in: Iron deficiency Thalassaemia any of the conditions leading to Microcytosis

Variation in hemoglobin content • Polychromasia: Red cells stain shades of blue-gray as a consequence of uptake of both eosin (by hemoglobin) and basic dyes (by residual ribosomal RNA). Often slightly larger than normal red cells and round in shape - round macrocytosis. Found in: Any situation with reticulocytosis - for example bleeding, hemolysis or response to heamatinic factor replacement

Reticulocyte: Polychromasia

Polychromasia

Figure 2. Polychromasia may be seen in response to anemia Maslak, P. ASH Image Bank 2004;2004:101122

Shape Abnormalities of Erythrocytes Poikilocytosis is the general term for mature erythrocytes that have a shape other than the round, biconcave disk. Poikilocytes can be seen in many shapes.(e.g. Acanthocyte, Spherocytosis,……)

Poikilocytosis

Schrier, S. ASH Image Bank 2002;2002:100513 Figure 1. This is a periperal smear of a patient with hereditary pyropoikilocytosis Schrier, S. ASH Image Bank 2002;2002:100513

Shape Abnormalities of Erythrocytes Terminology Description Condition Target cells Central hemoglobin; target-shaped Liver disease; thalassemia: Abnormal Hgb; iron deficiency Echinocyte Short spicules, equally-spaced Uremia, hypokalemia, artifact Acanthocyte Spiculated, irregular Liver disease (alcohol), Post-splenectomy Spherocyte Spherical, no central pallor HS, Immune hemolytic anemia Schistocyte Fragmented RBC, helmet cells MAHA, burns Ovalocyte Oval/elliptical shaped Hereditary elliptocytosis, Megaloblastic anemia Sickle cell bipolar spiculated shape Hgb S-containing “banana” shaped hemoglobinopathy Teardrop cell single elongated extremity Myelophthistic changes Bite cells Irregular gap in membrane G6PD deficiency 2

III- Variation of red cells shape (Poikilocytosis) Target cell: Red cell with a “target” or bull’s-eye appearance. The cell appears with a central bull’s eye that is surrounded by a clear ring and then an outer red ring. Found in: -Obstructive liver disease - Severe iron deficiency - Thalassaemia - Haemoglobinopathies (S and C) - Post splenectomy

Target Cells Diagnostic possibilities Liver disease Hemoglobinopathy Thalassemia Iron deficiency Post-splenectomy Lipid disorders

Abnormal Shape Spherocytosis: Morphology: Red cells are more spherical. Lack the central area of pallor on a stained blood film. Found in: - Hereditary spherocytosis - Immune haemolytic anemia - Zieve's syndrome - Microangiopathic haemolytic Zieve's syndrome is an acute metabolic condition that can occur during withdrawal from prolonged alcohol abuse. It is defined by hemolytic anemia, hyperlipoproteinaemia (excessive blood lipoprotein), jaundice, and abdominal pain.[1] The underlying cause is liver delipidization. This is distinct from alcoholic hepatitis, which however may be subsequent or copresent. microangiopathic hemolytic anemia (MAHA) :In all causes, the mechanism of MAHA is the formation of a fibrin mesh due to increased activation of the system of coagulation. The red blood cells are physically cut by these protein networks, and the fragments are identical to the schistocytes seen on light microscopy.

Spherocytes

Spherocytes: Autoimmune Hemolytic Anemia

Spherocytes: Hereditary Spherocytosis

III- Variation of red cells shape (Poikilocytosis) 8- Stomatocytosis: Morphology: Red cells with a central linear slit or stoma. Seen as mouth-shaped form in peripheral smear. Found in: - Alcohol excess - Alcoholic liver disease - Hereditary stomatocytosis - Hereditary spherocytosis

Stomatocyte

III- Variation of red cells shape (Poikilocytosis) Ovalocyte : an elongated oval cell. They are a result of a membrane defect. Found in: - Thalassaemia major. - Hereditary ovalocytosis. - Sickle cell anemia

III- Variation of red cells shape (Poikilocytosis) 4- Elliptocytosis: Morphology: The red cells are oval or elliptical in shape. Long axis is twice the short axis. Found in: - Hereditary elliptocytosis - Megaloblastic anemia - Iron deficiency - Thalassaemia - Myelofibrosis

Elliptocytes: Hereditary Elliptocytosis

III- Variation of red cells shape (Poikilocytosis) Schistocyte: red cell fragments that are irregular in shape and size. They are usually half the size of the normal RBC; therefore, they have a deeper red color. Found in: - Disseminated intravascular coagulation ( DIC ) - Micro angiopathic haemolytic anemia - Mechanical haemolytic anemia

Schistocytes: Microangiopathic Hemolytic Anemia

Schrier, S. ASH Image Bank 2001;2001:100249 schistocytes Figure 1. Note the fragmented schistocytes, burr cells, and helmet cells Schrier, S. ASH Image Bank 2001;2001:100249

III- Variation of red cells shape (Poikilocytosis) 7- Schistocytosis: Morphology: Fragmentation of the red cells. Found in: - DIC - Micro angiopathic haemolytic anemia - Mechanical haemolytic anemia Disseminated intravascular coagulation (DIC), also known as consumptive coagulopathy, is a pathological activation of coagulation (blood clotting) mechanisms that happens in response to a variety of diseases. DIC leads to the formation of small blood clots inside the blood vessels throughout the body.

III- Variation of red cells shape (Poikilocytosis) 6- Blister cell:prekeratocyte Morphology: Have accentric hallow area. Resemble a women's handbag and may be called pocket-book cell. Found in: Microangiopathic hemolytic anemia

Blister cell or prekratocyte

III- Variation of red cells shape (Poikilocytosis) 10- Keratocytes (horn cell): Morphology: Part of the cell fuses back leaving two or three horn-like projections. The keratocyte is a fragile cell and remains in circulation for only a few hours. Found in: - Uraemia - Severe burns - EDTA artifact - Liver disease

Keratocyte

Bite Cells

III- Variation of red cells shape (Poikilocytosis) 12- Sickle Cells: Morphology: Sickle shaped red cells Found in: Hb-S disease

Sickle Cell Anemia: Hgb SS

III- Variation of red cells shape (Poikilocytosis) 9- Burr (crenation ) cell: Morphology: Red cell with uniformly spaced, pointed projections on their surface. Found in: - hemolytic anemia - Uremia. - Megaloblastic anemia

Echinocytes (Burr Cells)

Hepatorenal Syndrome: Burr + Spur Cells

III- Variation of red cells shape (Poikilocytosis) 11- Acanthocytosis: Morphology: are red blood cells with irregularly spaced projections, these projections very in width but usually contain a rounded end Found in: - Liver disease - Post splenectomy - Anorexia nervosa and starvation

Acanthocytes (Spur Cells)

Lazarchick, J. ASH Image Bank 2002;2002:100507 Figure 2. Higher magnification of the same smear showing the spur cells ( arrows), target cells, and polychromasia Lazarchick, J. ASH Image Bank 2002;2002:100507

III- Variation of red cells shape (Poikilocytosis) Teardrop: resembles a tear and usually smaller than the normal RBC. Found in: - Bone marrow fibrosis - Megaloblastic anemia - Iron deficiency - Thalassaemia

Maslak, P. ASH Image Bank 2002;2002:100453 Figure 1. Dacryocytes (tear drop poikilocytes) are seen in the peripheral blood of a patient with idiopathic myelofibrosis Maslak, P. ASH Image Bank 2002;2002:100453

Envelope form cell Found in thalassemia Sickle cell anemia

Erythrocyte Inclusions with Wright’s Stain Inclusion Composition Appearance Condition Basophilic Precipitated Evenly dispersed Lead poisoning stippling ribosomes fine or coarse granules thalassemia other anemias Howell-Jolly DNA in origin Dense, round Post-splenectomy bodies Nuclear fragment blue granule Pappenheimer Iron-containing Small blue granules Anemias bodies granules in clusters Heinz bodies Denatured round blue precipitates G6PD hemoglobin Cabot Rings remnants of Reddish-blue threadlike Severe anemia, nuclear membrane rings Lead poisoning Organism Small blue inclusion Malaria Babesiosis

Erythrocyte inclusion bodies 1- Howell-Jolly Bodies: Morphology: Small round cytoplasmic red cell inclusion with same staining characteristics as nuclei Found in: - Post splenectomy - Megaloblastic anemia

Howell-Jolly Body

Maslak, P. ASH Image Bank 2005;2005:101350 Figure 1. Howell-Jolly bodies are small dense particles seen in the cytoplasm of this erythroid precursor Maslak, P. ASH Image Bank 2005;2005:101350

Erythrocyte inclusion bodies 2- Siderotic Granules (Pappenheimer Bodies) RBCs which contain no hemoglobin iron granules. They appear as dense blue, irregular granules which are unevenly distributed in Wright stained RBCs. Pappenheimer bodies can be increased in hemolytic anemia, infections and post-splenectomy.

Lazarchick, J. ASH Image Bank 2004;2004:101168 Figure 2. Multiple RBCs containing Pappenheimer bodies are seen in htis view Lazarchick, J. ASH Image Bank 2004;2004:101168

Erythrocyte inclusion bodies 3- Basophilic stippling: Morphology: Considerable numbers of small basophilic inclusions in red cells. Found in: - Thalassaemia - Megaloblastic anemia - Hemolytic anemia - Liver disease - Heavy metal poisoning.

Tear Drop Cells

Schrier, S. ASH Image Bank 2002;2002:100344 Figure 3. There is extensive course basophilic stippling of the red blood cells (RBC) in this patient who has had exposure to toxic levels of lead Schrier, S. ASH Image Bank 2002;2002:100344

Basophilic Stippling

Erythrocyte inclusion bodies 4- Heinz Bodies: Represent denatured hemoglobin (methemoglobin - Fe+++) within a cell. With a supravital stain like crystal violet, Heinz bodies appear as round blue precipitates. Presence of Heinz bodies indicates red cell injury and is usually associated with G6PD-deficiency.

Heinz Bodies

Figure 2. Heinz Body Preparation Figure 2. Heinz Body Preparation. RBC are incubated supravitally in new Methylene blue to identify precipitates of oxidatively denatured hemoglobin.

IV -Erythrocyte inclusion bodies 5- Cabot Rings: Reddish-blue threadlike rings in RBCs of severe anemia's. These are remnants of the nuclear membrane or remnants of microtubules and appear as a ring or figure 8 pattern. Very rare finding in patients with Megaloblastic anemia. severe anemia's. lead poisoning. Dyserythropoiesis.

Cabot rings

Erythrocyte inclusion bodies 6- Parasites of Red Cell: Two organisms are have a tendency to invade the RBCs. All 4 species of the malaria parasite will invade RBCs. We will see the Plasmodium of different species in RBCs. Bebesia microti

Malaria

RBCs Abnormal morphology Depiction of red blood cell morphologies that may appear on a peripheral smear, showing: (A) basophilic stippling, (B) Howell-Jolly bodies, (C) Cabot's ring bodies and (D) Heinz's bodies.

Normal Peripheral Smear

Reticulocyte Manual Count by Supravital Stain: Normal Count

Reticulocytes: Elevated Count

RBC Inclusions: Composite

Hypochromic Microcytic RBC

Normal Hypochromic microcytic

Severe Hypochromia: Iron Deficiency Anemia

Mixed Population: Treated Iron Deficiency Anemia

Microcytic Hypochromia: Alpha Thalassemia (a-/--)

Morphologic Changes in Liver Disease Target Cells Spur Cells