Microcytic Anemia Overview

Evaluation of Red Cell Morphology & Introduction to Platelets & White Cell Morphology

• Objectives: Discuss hematology stains, identify normal red blood cell morphology, define anisocytosis and poikilocytosis, define normochromic, hypochromic, microcytic, and macrocytic as they relate to red cell indices, correlate red cell indices with red cell morphology, define RBC morphological abnormalities and the most common RBC inclusions on a peripheral blood smear, list conditions showing leukocyte morphological abnormalities on a peripheral blood smear, and describe normal and abnormal platelet morphology on a peripheral blood smear.

Peripheral Blood Smear

• Reasons: Perceived clinical features, abnormalities in CBC, detection or verification of abnormalities, and information for differential diagnosis.
• Advantages: Detect or verify abnormalities, and provide information for differential diagnosis.
• Components: Red blood cells (size, shape, distribution, hemoglobin concentration, color, inclusions), platelets (count, shape, size, clumping), and white blood cells (differential, nuclear abnormalities, cytoplasmic abnormalities, abnormal inclusions).

Hematology Stain

• Nonvital (dead cell) polychrome stain (Romanowsky): Most commonly used routine peripheral blood smear stain. Wright's stain contains methylene blue (stains acidic components like DNA and RNA blue), eosin (stains basic components like hemoglobin red-orange), and methanol fixative.
• Staining Process: Staining does not begin until a phosphate buffer (pH between 6.4 and 6.8) is added.
• Causes of Staining Issues: Causes of RBCs appearing too red/WBC nuclei poorly stained include buffer or stain below pH 6.4, excess buffer, decreased staining time, increased washing time, thin smear, expired stains. Causes of RBCs and WBC nuclei appearing too blue include buffer or stain above pH 6.8, too little buffer, increased staining time, poor washing, thick smear, increased protein, or a heparinized blood sample.

Nonvital Monochrome Stain

• Prussian blue (Perl's Test): Stains specific cellular components, used to visualize iron granules (siderotic iron granules) in red blood cells (RBCs), histiocytes, and urine epithelial cells.

Supravital (living cell) Monochrome Stain

• Used to stain specific cellular components without fixatives.
• Examples: New methylene blue to precipitate RNA in reticulocytes (measuring bone marrow erythropoiesis), and neutral red with brilliant cresyl green for visualizing Heinz bodies (clinical disorders like G6PD deficiency).

Examination of Blood Smear

• Low Power Scan (10x): Determine staining quality, blood cell distribution, clumps, and abnormal cells, and find the optimal area for examination and enumeration.
• High Power Scan (40x): Determine WBC estimate, count WBCs in 10 fields, and correlate WBC estimate with the WBC count per mm³. Evaluate morphology and record abnormalities.
• Oil Immersion Examination (100x): Perform 100 WBC differential count, evaluate RBCs for anisocytosis, poikilocytosis, hypochromia, polychromasia, and inclusions, and perform platelet estimate.

Assessment Questions

• Example question on determining the corrected WBC count given laboratory results (WBC, RBC, Hgb, PLT, differential, and NRBC count).

The Normal Red Blood Cell

• Dimensions: 6-8 μm × 1.5-2 μm.
• Volume: 80-100 fL.
• Central pallor: 2-3 µm.
• Size variation: ~5% in normal patients
• Appearance: Reddish-orange; biconcave disc shape on Wright-stained film.

Assessment of Red Blood Cell Abnormality

• Identify abnormal morphology in all fields and assess if it's pathologic or artificially induced.
• Assess size abnormalities (anisocytosis) and shape abnormalities (poikilocytosis).
• Consider the red blood cell indices (RBC indices) and red blood cell distribution width (RDW).
• Determine the percentage of abnormal cells relative to the total number of cells.
• Use a qualitative or numerical grading system (1+ to 4+).

Normal and Abnormal Red Blood Cell Morphology

• Include illustrations of various normal and abnormal red blood cell shapes and inclusions (e.g., target cells, spherocytes, sickle cells).

Variations in Red Cell Distribution

• Includes normal distribution, dispersed; and also includes agglutination, in which cells appear clumped together

Variations in Red Cell Distribution: Agglutination

• Patient's plasma contains red blood cells' antibodies, typically IgM and complement).
• Cold agglutinins' formation, usually seen in cases of Paroxysmal Cold Hemoglobinuria.

Variation in Red Cell Distribution : Rouleaux

• Occurs due to elevated globulin or fibrinogen in the plasma.
• Red blood cells (RBCs) clump as stacks of coins in peripheral blood films.
• Saline dilution of the serum helps to disperse the rouleaux.
• Conditions associated include multiple myeloma, lymphomas, hyperproteinemia, Waldenstrom macroglobulinemia, and conditions with increased fibrinogen.

Blood Film Preparation

• Includes normal and abnormal red blood cells.

Variations in Size-Anisocytosis: Macrocytes

• Size ≥ 9 µm.
• MCV > 100 fL.
• Impaired DNA synthesis, leading to larger cells (megaloblastic erythropoiesis), accelerated erythropoiesis, membrane changes (increase in cholesterol and lecithin)
• Shape, pallor, and inclusions, must be evaluated for macrocytes.

Variations in Size-Anisocytosis: Microcytes

• Size < 7µm
• MCV < 80fL
• Impaired hemoglobin synthesis,
• Include ineffective iron utilization, absorption, or release, and decreased or defective globin synthesis.

Examination of Platelet Morphology

• Platelet size (2-4 μm) may vary.
• Mean platelet volume (MPV) is analogous to MCV.
• Examination for large platelets (up to 5 μm).
• Platelet granules.
• Variations associated with increased platelet turnover (e.g., bleeding disorders, hyposplenism) occur after splenectomy.
• Evaluation for very high platelet counts (in myeloproliferative disorders), and low platelet counts (thrombocytopenia).

Examination of Platelet Morphology II, III, and IV

• Very high platelet counts, and associated conditions.
• Characteristic morphological descriptions (e.g., Bernard-Soulier syndrome and Grey platelet syndrome.).
• Platelet-satellitosis, and EDTA-induced pseudothrombocytopenia; noting associated conditions.

Leucocytes: Normal and Abnormal Morphology

• Includes morphology descriptions, along with cell counts and associated conditions.

Toxic Granulation

• Dark-blue-black cytoplasmic granules seen in neutrophils.
• Associated conditions such as acute infection, drug poisoning, burns, vasculitis, and toxemia of pregnancy.

Dohle Bodies

• Small light-blue staining areas within the cytoplasm (ribosomes or rough endoplasmic reticulum).
• Conditions associated with their presence include infections, poisoning, burns, chemotherapy, and toxemia of pregnancy.

Hypersegmented Neutrophils

• Neutrophils with 5 or more lobes.
• Associated conditions, such as acquired megaloblastic anemia, inherited Undritz anomaly, and chronic infections

Pelger Huet Anomaly

• Failure of neutrophil nuclei to segment properly into 2 or fewer lobes.
• Coarse clumped chromatin.
• Either inherited or acquired, potentially associated with leukemia or secondary to drugs.

Chediak-Higashi Syndrome

• Inherited disorder causing abnormal large azurophilic granules in several cell types, including neutrophils, lymphocytes, and monocytes.
• Increased susceptibility to infection.

Alder-Reilly Anomaly

• Heavy azurophilic granules in neutrophils, eosinophils, basophils, and monocytes.
• Normal function although the granules contain mucopolysaccharides.

May-Hegglin Anomaly

• Hereditary condition with Dohle-like inclusions and abnormal platelets (giant platelets and thrombocytopenia).

Auer Rods

• Rod-like or cigar-shaped reddish-purple inclusions seen in the cytoplasm of myeloblasts and monoblasts in acute myeloid leukemia (AML).
• Represent abnormal fusion of primary granules.

Vacuolated Neutrophils

• Round clear cytoplasmic areas as a consequence of active phagocytosis.
• Associated conditions such as septicemia and severe infections.

Smudge or Basket Cells

• Disintegrating neutrophil nuclei.
• Condensed nuclear chromatin
• Do not count in WBC differential.

Hypogranular or Agranular Neutrophils

• Associated with myelodysplastic syndromes (MDS) and some myeloid leukemias.

LE Cells

• Neutrophils engulfing the nuclei of other cells, often seen in systemic lupus erythematosus (SLE).

Barr Bodies (Drum Stick)

• The second X chromosome seen in female neutrophils.

Erythtrophagocytosis

• Neutrophils or monocytes phagocytosing red blood cells.
• Associated with direct antiglobulin test positivity (DAT) and Paroxysmal cold hemoglobinuria (PCH).

Leukoagglutination

• Agglutination of leukocytes, often an in vitro artifact.

Effect of Storage on Blood Cell Morphology

• Changes in neutrophil morphology (e.g., cytoplasmic vacuoles, homogeneous nuclei, ragged margins) when blood samples are stored.
• Changes in morphology of other cells (e.g., monocytes, lymphocytes) with storage.

Heat Artifacts

• Changes in blood cell morphology due to heat exposure.

Additional Images and Illustrations

• Provide details from supplementary figures/images and illustrations (e.g., specific cell types, inclusion morphology, etc.)

Test your knowledge of microcytic anemia and its characteristics, including the features of red blood cells in blood films. This quiz covers causes of impaired hemoglobin synthesis and the implications of microcytes and hypochromia. Dive into the details of hematology to understand the visual markers and consequences of this condition.