Red Cell Morphology Laboratory Medicine Department Saudi German Hospital-Jeddah

Objectives Discuss the procedure for proper red blood cell examination Discuss aspects of red cell morphology related to size Discuss aspects of red cell morphology related to color Discuss different types of poikilocytosis

Introduction Red cell morphology can be defined as the appearance of the erythrocytes on a Giemsa stained smear. Careful examination of the red cells for the purpose of identifying abnormalities is part of the differential procedure.

Why differentiating normal morphology from abnormal and artificial morphology. provide valuable diagnostic information to the physician, provide a quality control mechanism to verify red cell indices values as determined by automated or manual methods.

How Assess RBC morphology by examining the smear in the feathered (thinner) edge where the RBC are randomly distributed and, for the most part, lie singly, with occasional doublets. This area is referred to as the "critical area."

How If the area is too thin, the red cells will appear flat and somewhat square (cobblestone effect) with no central pallor. If the area examined is too thick, the cells will be too close together to evaluate the morphology of individual cells.

How To begin the red cell morphology examination, use the low power (10X) objective to locate the "critical area." The oil immersion objective (100X) is used for the actual evaluation.

Normal RBC’s Round, elastic, non-nucleated, bi-concave discs Many RBCs have an area of central pallor which covers about one-third of the cell. The pallor occurs as a result of the disc-shaped cells being spread on the slide.

Normal RBC’s Average diameter of 7.2 microns with a range of 6-9 microns, almost the same size as the nucleus of a small lymphocyte,

Critical area 10x A view of the "critical area" using the low power (10X) objective is shown here.

Critical area 100x Once the correct area has been located on low power, switch to oil immersion Notice the red cells are lying singly with occasional doublets.

Too thin The area shown in this field is too thin for accurate red cell morphology evaluation. The cells have large spaces between them, show no central pallor and many are somewhat square, showing a "cobblestone effect."

Too thick These cells are in an area which is too thick, and should not be used for red cell morphology assessment. Some of the cells appear to be stacked like coins because of the large number of cells present in this section of the slide.

The morphology seen in the too thin and too thick areas of the smear is referred to as artificial morphology.

Size Variation

Size variation Red blood cells can vary in size from smaller than normal, microcytes, to larger than normal, macrocytes. When red cells of normal size, microcytes and macrocytes are present in the same field, the term anisocytosis is used.

Normal size Size of normal RBC is almost the size of the nucleus of the lymphocyte.

Microcyte Smaller than a nucleus of the lymphocyte, central pallor is greater than 1/3 of the cell

Microcyte, increased central pallor

Microcyte, normal Hb content

Microcytes

summary microcytes have a diameter of less than 7 microns and an MCV of less than 80 cubic microns. Two types of microcytes can be seen, those with increased central pallor and those with normal central pallor.

Macrocyte (megalocyte) diameter of 9-14 microns (1.5 - 2 times larger than normal red cells) MCV is 100 cubic microns or more.

Megalocytes Megalocytes are the result of decreased DNA synthesis, frequently due to vitamin B12 and/or folic acid deficiencies. Decreased DNA synthesis causes the nucleus in the developing red cells to mature at a slower than normal rate. Since hemoglobin production is not affected, the mature red cell is larger than normal

Macrocytes

Pseudomacrocytes appears larger than the lymphocyte but in contrast to megalocytes has an area of central pallor. size is the result of exaggerated flattening and thus the presence of the central pallor. in patients with cirrhosis of the liver, obstructive jaundice, post splenectomy.

Psudomacrocytes

Summary two types of macrocytes:- True macrocytes (megalocytes). Increased MCV, MCH Pseudomacrocytes. Normal MCV, MCH

Anisocytosis Increased variation in size of the red cell population present on a blood smear. Normal, small and large cells can be seen in one field. Normal MCV, high RDW As the severity of the anemia increases, the amount of significant anisocytosis present also increases.

Anisocytosis

Anisocytosis

RBC’s

RDW RDW is an expression of the homogeneity of the RBC population size. A large RDW says there's a wide variation in the RBC diameters within the test pool.  It doesn't say the cells are large or small, rather that the population is not homogenous.  Younger cells are larger (reticulocytes). Older, and generally beat up, RBCs are smaller.

RDW

Microcytic Anemia

Macrocytic Anemia

Macrocyte

Microcytes

Anisocytosis

Pseudo-macrocyte

Normal

Anisocytosis

RBC Color

RBC Color Erythrocytes, when spread on a glass slide, show varying degrees of central pallor This central pallor is related to the hemoglobin concentration present in the red cells.

RBC Color the central area (1/3 of the cell) is white, while buff-colored hemoglobin is visible in the outer 2/3 of the cell. The MCHC (32-36 gm/dl) is the index value which is used to verify the presence of adequate hemoglobin concentration in the cells visible on the peripheral smear.

RBC Color A decreased amount of hemoglobin is referred to as hypochromasia or hypochromia. MCHC values of 30% or less reflect this condition. Hyperchromasia and hyperchromia, refer to a hypothetical situation rather than an actual occurrence.

RBC Color Cells located in the "too thin" portion of the smear often appear to be "hyperchromic". Megalocytes (macrocytes) are normochromic.

Normochromic cells

Hypochromic cells

Hyperchromia

Hypochromia

Hyperchromasia

Polychromasia

Poikilocytosis

Poikilocytosis Variations in shape.

Terminology Uniform terminology based on Greek roots

Grading system 1+ = 2 – 4 /OIF 2+ = 5 - 7 3+ = 8 - 10 4+ = >10 The terms few, moderate, many, and marked may be substituted for the 1+ - 4+ grading system.

Acanthocytes 3-12 thorn-like projections irregularly spaced around the cell. Smaller than normal and have little or no central pallor. Acanthocytes have an excess of cholesterol Large numbers of these cells on a smear can be of diagnostic significance.

Acanthocytes Abetalipoproteinemia Hereditary acanthocytosis,50 – 100% of blood cells. Alcoholic cirrhosis lipid disorders splenectomy

Acanthocyte

Codocyte Target cells are thin-walled cells showing a darkly-stained centre area of hemoglobin which has been separated from the peripheral ring of hemoglobin.

Codocyte Codocytes appear in conditions which cause the surface of the red cell to increase disproportionately to its volume. This may result from a decrease in hemoglobin, as in iron deficiency anemia, or an increase in cell membrane.

Codocyte Thalassemias, Hb C disease, post splenectomy, obstructive jaundice.

Dacrocyte Dacryocytes are pear-shaped or teardrop shaped cells. myelofibrosis/myeloid metaplasia,

Drepanocytes Drepanocytes or sickle cells are formed as a result of the presence of hemoglobin S in the red cell. As the red cell ages, it becomes less flexible or deformable and becomes rigid as it passes through the low oxygen tension atmosphere of the small capillaries in the body. In the absence of oxygen, hemoglobin S polymerizes into rods, causing the sickle cell shape. Sickle cells can be somewhat pointed at the ends,

Most sickled cells can revert back to the discoid shape when oxygenated. About 10% of sickled cells are unable to revert back to their original shape after repeated sickling episodes.

Echinocyte (Urchin) Echinocytes are reversible, The projections are rounded and evenly spaced around the cell. Acanthocytes have irregularly spaced thorn-like projections.

Echinocyte Uremia, Following heparin injection, Pyruvate kinase deficiency. Artificial

Elliptocytes Elliptocytes can vary in appearance from slightly oval to thin pencil-shaped forms. Less than 1% of red cells in normal blood are oval.

Elliptocytes Hereditary Elliptocytosis Thalassemia, megaloblastic anemia, iron deficiency.

Elliptocytes

Elliptocytosis

Knizocyte A streak of hemoglobin through the centre of the cell. In some hemolytic anemia cases

Keratocytes Keratocytes are cells which have been damaged due to contact with fibrin strands.

intravascular coagulation microangiopathic hemolytic anemia glomerulonephritis rejection of renal transplants.

Shistocytes Schistocytes are red cell fragments which are formed when fibrin strands come in contact with circulating red cells. The strands cut a small piece from the original cell.

Spherocytes cells which have a decreased surface-to-volume ratio. cell is thicker in diameter than normal red cells they appear to be round, darkly-stained cells without central pallor.

Spherocytes

Hereditary spherocytosis Immune hemolytic anemia Severe burns In-vitro prolonged storage of blood

Stomatocytes

Stomatocytes cup-shaped erythrocytes which have an elongated or slit-like central pallor. hereditary stomatocytosis, neoplastic disorders, liver disease and Rh null disease, in-vitro change in pH

Rouleaux & autoaglutination Forms of poikilocytosis describing a group of cells. True and false

True Rouleaux

True Rouleaux most of the red cells, in the proper viewing area, are stacked together like coins. Four or more cells make up each formation, leaving much of the field empty of cells (increased white space). Rouleaux is clinically significant when increased globulins are present, as in multiple myeloma.

False rouleaux

True rouleaux

Artifactual

Autoagglutination Cells clumping together rather than stacked like coins. Autoagglutination is caused by the presence of antibody in the plasma.

Match the following terms with their synonyms. Target cell Helmet cell Ovalocyte cell

Match the following terms with synonyms: Crenated cell Tear drop cell Sickle cell

Match the forms of poikilocytosis with the physiological/environmental condition associated with their formation : Damaged by fibrin strands Absence of abetalipoprotein Hemoglobin S

A general term describing variation in size: anisocytosis macrocytosis microcytosis poikilocytosis

helmet cell echinocyte crenated cell

hypochromic hyperchromic normochromic polychromatophilic

macrocytic/normochromic microcytic/hypochromic normocytic/normochromic normocytic/hypochromic