¾ Place I volume of filtered stain in small test tube
¾ Place I volume of capillary or venous blood and mix
¾ Incubate at room temperature or at 37°C for 10–30
¾ Remix the tube contents and spread 1 drop of the
stained blood on a slide making a thin film.
When dry, examine with the oil immersion lens. Count
systematically at least 500 cells, including in this the
number of reticulocytes. Reticulocytes appear larger than
mature cells and contain irregular dark purple granules
or fine threads of ribonuclear material, calculate the
Interpretation: Reticulocyte counts are low in ineffective
erythropoiesis, e.g. myelosclerosis, aplastic anemia,
megaloblastic anemia, thalassemia, erythroleukemia and
sideroblastic anemia. Reticulocytosis occurs after blood
loss or effective therapy for certain kinds of anemia, e.g.
therapy of iron deficiency or megaloblastic macrocytic
anemia. Reticulocytosis also occurs in hemolytic anemias.
Description of abnormalities of RBC color Possible causes of abnormal RBC color
Anisochromia is demonstrated by variable staining intensities indicating
unequal hemoglobin content due to multiple populations of red blood cells
Anisochromatism: Iron-deficiency anemia treated with transfused blood
Hyperchromia is demonstrated by the presence of cells having a smaller than
normal area of central pallor, causing the cells to take on excessive staining
and demonstrate higher than normal pigmentation. Increased amounts of
these cells are called hyperchromatism
Hyperchromatism: Dehydration, increased bone marrow iron
Hypochromia is demonstrated by the presence of cells having a larger than
normal area of central pallor, causing the cells to stain weakly and appear to
have less than normal pigmentation. Increased amounts of these cells are
Hypochromatism: Anemia (iron deficiency) and decreased
Polychromatophils are cells that are stainable with many types of stains, such
as stains with both an acid and base component. They are demonstrated by
a bluish-pink tinge caused by the presence of both hemoglobin stained by
acid and cytoplasmic ribonucleic acid (RNA) stained by the basic component.
Both the larger-than-normal cell size and the presence of cytoplasmic RNA
indicate that polychromatophils are reticulocytes (newly made RBCs).
Increased amounts of polychromatophils is called polychromatosis and
occurs in accelerated RBC production
Polychromatosis: Hemorrhage, hemolysis, reticulocytosis, and
therapy for iron deficiency anemia or pernicious anemia
Description of abnormalities of RBC shape Possible causes of abnormal RBC shape
Acanthocytes are cells with irregular, thorny, spiculated membrane surface
Acanthocytosis: Abetalipoproteinemia (most common cause),
alcoholic cirrhosis, hemolytic anemia (induced by pyruvate
kinase deficiency), hepatic disease, postsplenectomy, and
retinitis pigmentosa. Drugs include heparin calcium and
Crescent bodies (achromocytes) are cells with a faint quarter-moon shape
Achromocytosis: Any condition that increases the fragility of
RBCs (i.e. sickle cell anemia, reduced oxygen supply)
Drepanocytes/sickle cells are cells formed in the shape of a sickle with a point
at one end. The presence of these cells is called drepanocytosis
Drepanocytosis: Anemia (hemolytic, sickle cell) and hemoglobin SC disease
Echinocytes/burr cells/crenated: RBCs have a cell surface with 10–30
uniformly distributed, blunt spicules. Echinocytes may be commonly due
to pH changes due to faulty drying during smear preparation, but certain
physiologic conditions, including a reversible defect in the lipid content of
the RBC membrane, have been associated with their presence. The presence
of these cells is called echinocytosis
Echinocytosis: Bile acid abnormalities, blood loss (acute),
burns (extensive), carcinoma of the stomach, disseminated
intravascular coagulation (DIC), gastric ulcers (bleeding),
increased free fatty acids, microangiopathic hemolytic
anemia, pyruvate kinase deficiency, renal failure, thrombotic
thrombocytopenic purpura, and uremia. Drugs include
barbiturates, heparin calcium, heparin sodium, and salicylates
Elliptocytes/ovalocytes have a cigar shape, which distinguishes them from the
more oval shape of the ovalocytes. They are normal constituents of mature
RBCs. Higher than normal amounts of these cells are called elliptocytosis
Elliptocytosis: Anemias (iron deficiency, pernicious, sickle
cell), hereditary elliptocytosis, leukemia, megaloblastic
hematopoiesis, and thalassemia
Leptocytes/target cells have an increased ratio of surface to volume, often
due to a shape that looks like a cup, bell, or hat. They have a colorless center
and are thinner and lighter staining than normal RBCs due to abnormally
low amounts of hemoglobin. When stained, the depth of the “cup” collapses,
causing a bulls-eye appearance. The presence of leptocytes is termed
Leptocytosis: Anemia (iron deficiency, sickle cell), cellular
dehydration, cirrhosis, hemoglobin C disease, hemoglobin SC
disease, hepatitis, jaundice (obstructive), postsplenectomy
Description of abnormalities of RBC shape Possible causes of abnormal RBC shape
Poikilocytes occur in varying shapes, ranging from slightly irregular to
dumbbell-like, pear-shaped or teardrop-shaped. Defective bone marrow
production causes poikilocytosis, a general term used to describe the
presence of cells demonstrating variation from the normal shape of the RBC
Poikilocytosis: Anemia (iron deficiency, hemolytic,
megaloblastic. pernicious), myelofibrosis, myeloid metaplasia,
Schizocytosis/schistocytes are RBCs with adhesions of spiral and triangular
RBC fragments due to hemolysis, hemoglobinopathies, or erythrocytic
mechanical damage from fibrin strands. The presence of these cells is called
Schizocytosis or Schistocytosis: Anemia (acute hemolytic,
microangiopathic hemolytic), burns (severe), disseminated
intravascular coagulation (DIC), prosthetic heart valves,
Spherocytes are cells that are globe-like rather than biconcave, with an
increased mean corpuscular hemoglobin concentration) and have a smaller
surface area relative to their size. Spherocytes are caused by mechanical
fibrin strand damage to circulating RBCs. The presence of spherocytes is
Spherocytosis: ABO hemolytic disease of the newborn,
accelerated reticuloendothelial RBC destruction, anemia
(hemolytic), following blood transfusion, hereditary
spherocytosis, and thermal injury of the cell membrane
Stomatocytes are cup-shaped RBCs with an abnormal area of central
pallor that may be oval or rectangular elongated, or slit-like. These cells are
produced by antibodies or hydrocytosis. The presence of these cells is called
Stomatocytosis: Alcoholism, cirrhosis, erythrocyte sodium
pump defect, hepatic disease (obstructive), hereditary
spherocytosis, hereditary, stomatocytosis, and Rh null cells
Description of abnormalities of RBC size Possible causes of abnormalities of RBC size
Anisocytosis is a general term that describes any variation in the size of the
Anisocytosis: Anemias (iron deficiency, pernicious), folic
acid deficiency, following blood transfusion of normal cells
into an abnormal RBC population, leukemia, newborns, and
Macrocytes are large erythrocytes having a diameter > 8 µ, a mean corpuscular
volume > 96 µ, and higher than normal hemoglobin content. They are usually
increased due to stress erythropoiesis. Increased amounts of macrocytes are
Macrocytosis: Anemia (hemolytic, pernicious), folic acid
deficiency, following hemorrhagic states, hepatic disease,
hyperthyroidism, idiopathic steatorrhea, newborns,
reticulocytosis, and thalassemia
Microcytes have an RBC diameter <6 µ, a mean corpuscular volume <76 µ,
and mean corpuscular hemoglobin concentration <27%. Increased amounts
of microcytes are called microcytosis
Microcytosis: Anemia (from chronic hemorrhage, iron
deficiency), hemoglobinopathies, hereditary spherocytosis,
Agglutination: Clumping together of RBCs is an immune mechanism caused
Agglutination: Invading antigen(s)
Basophilic stippling is demonstrated by the presence of minute basophilic
granules that cause a bluish/purple color when reticulocytes are stained.
They are caused by ribosomal aggregation that occurs as smears are
prepared. Small amounts of basophilic stippling normally occur as the
smears are dried. Increased amounts occur in conditions in which RNA has
Increased basophilic stippling: Alcoholism, anemia
(megaloblastic, sickle cell), heavy metal intoxication (bismuth,
lead, mercury, and silver), hemorrhage (gastrointestinal),
Cabot’s rings are cells containing mitotic spindle remnants appearing as fine,
threadlike filaments of bluish purple color in the shape of a single ring or a
double ring (figure-eight shape)
Presence of Cabot’s rings: Anemia (severe, pernicious), lead
poisoning, myelofibrosis, and myeloid metaplasia
238 Concise Book of Medical Laboratory Technology: Methods and Interpretations Contd...
Heinz bodies are denatured particles of hemoglobin attached to the RBC
membrane that appear when stained with cresyl blue or new methylene blue.
Heinz bodies usually indicate abnormal erythrocyte stability due to hemolytic
conditions or hemoglobinopathies
Presence of’Heinz bodies: Alpha-thalassemia, anemia
(hemolytic), glucose-6-phosphate dehydrogenase
deficiency, hemoglobinopathies, methemoglobinemia, and
post-splenectomy. Drugs include analgesics, antipyretics,
chlorates, phenacetin, phenothiazines, phenylacetamide,
phenylhydrazine phenylamine, primaquine phosphate,
Howell-Jolly bodies are nuclear fragments contained in red cells that stain
purple or violet. They are normally present in immature RBCs and in mature
erythrocytes before they pass through the splenic circulation. In conditions
causing increased RBC production, erythrocytes contain higher than normal
Presence of Howell-Jolly bodies: Anemia (hemolytic,
megaloblastic), leukemia, splenic absence congenital or surgical removal), and splenic atrophy
Platelets on RBCs appear as a halo that resists staining and can be easily
confused with RBC inclusion bodies
Rouleaux formation is demonstrated by a cellular configuration in stacks or rolls.
shaped RBCs, which inhibits adherence of the cells in a stacked shape. Rouleaux
formation may also result from a delay in slide preparation
Increased rouleaux formation: Hyperfibrinogenemia,
macroglobulinemia, and multiple myeloma
Decreased rouleaux formation: Hereditary spherocytosis
Siderocytes/Pappenheimer bodies are cells with mitochondrial concentrations
of ferritin (nonhemoglobin iron) deposits. These cells stain as purple-bluish
granules only in the presence of iron stains such as Prussian-blue reactions.
Ferritin is normally absent in RBCs. During hemoglobin formation, in the
premature infant and newborn, siderocyte free-iron granules commonly
occur in developing normoblasts and reticulocytes. The presence of
siderocytes is called siderocytosis
hemochromatosis, infection, lead poisoning, postsplenectomy, and thalassemia
Red blood cell abnormalities seen on stained smear (in brief)
Descriptive term Observation Importance
Macrocytosis Cell diameter > 8 µm
Megaloblastic anemias, severe liver disease
Microcytosis Cell diameter < 6 µm
Hypochromia Increased zone of central pallor Reduced Hb content
Polychromatophilia Presence of red cells not fully hemogolobinized Reticulocytosis
Poikilocytosis Variability of cell shape Sickle cell disease
Descriptive term Observation Importance
Anisocytosis Variability in cell size Reticulocytosis
Transfusing normal blood into microcytic or
Leptocytosis Hypochromic cells with small central zone
Iron deficiency anemia, obstructive jaundice
Spherocytosis Cells without central pallor, loss of biconcave
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