Peripheral Blood Smears: MLT Lab Technique Guide

This lecture covers the morphological evaluation of white blood cells, platelets, and parasites in peripheral blood smears, including identification of abnormal WBC morphologies such as toxic granules (indicating severe bacterial infection), shift to left (indicating infection/inflammation), hypersegmented neutrophils (indicating megaloblastic anemia), atypical lymphocytes (indicating viral infections), and blasts (requiring immunophenotyping for definitive classification). The lecture also explains differential leukocyte count methodology, numerical abnormalities of WBCs, correction for nucleated RBCs, and commonly encountered platelet and parasite problems.

When blasts spill into the bloodstream, they can be identified on blood smears by two key characteristics: abnormal size compared to normal blood cells, and immaturity indicated by low cytoplasm-to-nucleus ratio. These morphological features help distinguish blast cells from mature blood cells and are essential for initial diagnosis.

This segment covers blast cell identification and granulocyte differentiation. Blast cells are identified by absence of cytoplasmic granulation, loose homogeneous chromatin, and visible nucleoli. The instructor demonstrates identification of basophils by their characteristic coarse, dark granulation that often obscures the nucleus. Eosinophils are differentiated from neutrophils by their coarse, orange-red granules versus the finer granulation of neutrophils. The systematic approach of first determining granulation presence is emphasized.

White blood cell morphology provides critical diagnostic information: Neutrophilia indicates bacterial infections or inflammation; Basophilia suggests allergic reactions or chronic myeloid leukemia; Eosinophilia points to parasitic infections or allergic conditions. Left shift (presence of immature band neutrophils) indicates active infection or stress. Blast cells in peripheral blood indicate acute leukemia, with >20% blasts suggesting acute leukemia requiring urgent treatment. Dysplasia (abnormal cell development) may indicate myelodysplastic syndromes. Toxic granulation appears in severe bacterial infections. Howell-Jolly bodies indicate splenic dysfunction.

The ICSH recommends a two-tiered grading system (moderate/2+ and marked/3+) for RBC morphology, requiring assessment of at least 1,000 RBCs, except for schistocytes which use a three-tiered system. Key abnormalities include: anisocytosis (RDW reflects variability), dimorphism (two distinct populations), microcytes (<7μm, MCV<80fL), macrocytes (>8.5μm, MCV>100fL), hypochromia (reduced staining), polychromasia (immature cells with residual RNA), acanthocytes (irregular spicules), echinocytes/bite cells (spleen removal of hemoglobin bodies), elliptocytes (>2x short axis), ovalocytes (<2x short axis), schistocytes (MAHA diagnostic fragments), stomatocytes (cup-shaped with slit pallor), target cells (central pallor with central staining), teardrop cells, and inclusions (basophilic stippling, Howell-Jolly bodies, Pappenheimer bodies).

RBC morphology assessment includes size (microcytic if smaller than normal lymphocyte nucleus, macrocytic if larger), color (hypochromia indicates low hemoglobin), and shape. Normal RBCs are biconcave discs 6-8.5 micrometers in diameter. Poikilocytosis includes teardrop cells, elliptocytes, schistocytes, and target cells. Inclusions include Howell-Jolly bodies (nuclear remnants), Pappenheimer bodies (iron granules), and Heinz bodies (denatured hemoglobin). Stomatocytes have a central mouth-like opening. These abnormalities indicate various conditions including iron deficiency, thalassemia, liver disease, and splenic dysfunction.

Abnormal red blood cell morphology is classified into four main categories: anisocytosis (variation in RBC size, including microcytosis, normocytosis, and macrocytosis), poikilocytosis (abnormal RBC shapes such as spherocytes, ovalocytes, burr cells, teardrop cells, schistocytes, target cells, pencil cells, elliptocytes, blister cells, keratocytes, stomatocytes, and rouleaux formation), variation in color (hypochromia and hyperchromia), and inclusions within RBCs. These morphological abnormalities provide critical diagnostic clues for identifying underlying hematological conditions, including nutritional deficiencies, genetic disorders, liver disease, and various anemias.

Abnormal red blood cell morphology encompasses variations in color and intracellular inclusions that indicate underlying pathological conditions; color variations include hypochromia (pale cells with reduced hemoglobin, seen in iron deficiency anemia and thalassemia), hyperchromia (deeply stained cells with excess hemoglobin, seen in hemolytic anemia), and polychromatia (grayish-blue cells with multiple colors from premature marrow release, seen in hemorrhage); intracellular inclusions include Howell-Jolly bodies (nuclear fragments, post-splenectomy, megaloblastic anemia), Heinz bodies (denatured hemoglobin, hemoglobinopathies, G6PD deficiency), Cabot rings (nuclear membrane remnants, megaloblastic anemia, lead poisoning), basophilic stippling (RNA granules, lead poisoning, thalassemia, liver disease), Pappenheimer bodies (iron granules, splenectomy, sideroblastic anemia), and malaria parasites (Plasmodium species causing hemolytic anemia).

Blood smear platelet counting is the most common routine method, utilizing a single smear for multiple diagnostics including leukocyte differential and RBC morphology. Count 10 fields at 100x magnification, calculate the average, then multiply by 20,000 (for monochromatic microscopes) to get platelets per microliter. Hematology analyzers automate cell counting but cannot replace manual smear analysis for differential counts, morphology evaluation, and detecting hemoparasites. The complete workflow includes: receiving samples with request forms, verifying anticoagulant presence, homogenizing and checking for clots, running analyzer after species selection, preparing and staining smears, performing manual hematocrit, and conducting platelet counts. Results are typically available within two minutes.

Manual platelet count using the slide method involves preparing a blood smear on a glass slide, staining it, and counting platelets in the 'zone of morphology' (where RBCs are in light contact but not overlapping) across 10 fields under the microscope; the total platelet count is calculated using the formula: (number of platelets counted × 15,000) ÷ number of fields counted, which provides an accurate platelet count for clinical reporting.

This video demonstrates how to manually count and identify white blood cell types from a blood smear by examining 100 cells under a microscope, focusing on nuclear segmentation patterns (such as segmented neutrophils with multiple lobes, band neutrophils with a single elongated nucleus, and lymphocytes with round nuclei) and cytoplasmic characteristics to determine the leukocyte formula.

The complete manual platelet counting workflow includes preparing the Malacé cell slide by placing the lamella correctly without air bubbles, allowing 3 minutes of incubation for cell settling, and examining under microscope starting at 10x then 40x objectives. Technicians count platelets in exactly 10 squares while distinguishing them from white blood cells. The final calculation uses the formula: platelet count per liter = number counted in 10 squares × 10,000.

The Differential Leukocyte Count (DLC) identifies specific infection types. Neutrophilia (increased neutrophils) indicates bacterial infections, trauma, or steroid use, while neutropenia occurs in aplastic anemia, sepsis, or drug reactions. Lymphocytosis increases in viral infections, tuberculosis, and blood cancers like CLL. Eosinophilia thrives in allergic conditions and remains elevated in chronic diseases. Monocytes and basophils increase in many chronic conditions. Platelet analysis reveals bleeding risk: thrombocytopenia (<1.5 lakhs) indicates dengue, ITP, or aplastic anemia, while thrombocytosis occurs in some cancers. Critical red flags include hemoglobin <6 g/dL, TLC >1 lakh suggesting leukemia, platelet count <20,000 indicating severe bleeding risk, and pancytopenia (simultaneous reduction of all blood components) indicating severe bone marrow suppression requiring immediate specialist consultation.

A peripheral blood smear is indicated when automated analyzers flag abnormalities such as monocytosis, morphology issues, or abnormal scattergrams. The smear allows examination of WBC differential, RBC morphology, and platelet appearance. Key findings include: abnormal cell shapes (poikilocytosis), inclusions, nucleated cells, and platelet abnormalities. The indices from the CBC should correlate with morphological findings observed on the smear.

Blood smear examination is a microscopic test for morphological evaluation of blood cells that detects abnormalities missed by automated counters. While automated CBC provides rapid numerical results, blood smear offers critical advantages: detecting errors, identifying morphological changes, recognizing left shift and toxic changes, estimating platelet counts, detecting tumor cells, and identifying infectious pathogens. Key findings only visible through smear include neutrophil toxic changes (toxic granulation, cytoplasmic vacuolization, Döhle bodies) and left shift, which indicate bone marrow activation and acute inflammation. These findings are essential for differentiating acute inflammation from chronic inflammation, stress patterns, and other conditions. Combined with CBC, blood smear enables accurate diagnosis of severe inflammatory conditions like pyometra, uterine rupture, and brain abscesses.

Manual examination of blood smears allows detection of parasites (like malaria), abnormal white blood cells, and other morphological changes that automated machines cannot identify. This manual review is essential for diagnosing conditions like malaria, leukemia, and other hematological disorders that require microscopic examination.

This video demonstrates practical knowledge about CBC (Computer Based Examination) systems, explaining common defects such as system lag, voltage issues, and component failures, while showing step-by-step troubleshooting procedures including system settings configuration, component replacement, and maintenance techniques to ensure proper system functionality.

Quality assurance ensures reliable automated hematology results through systematic calibration and quality control. Calibration verifies instrument accuracy and consistency, performed upon installation and every six months, with daily quality checks before testing. Troubleshooting common interferences is essential: agglutination causes dual RBC populations or right shifts, corrected by warming at 37°C; lipemia/icterus falsely elevates hemoglobin through increased turbidity, corrected by plasma replacement; hemolysis decreases RBC and hematocrit, requiring new specimens; lysis-resistant RBCs (HbS, HbC, HbF) are counted as WBCs, corrected by manual dilution or incubation; microcytes/fragments are counted as platelets, requiring microscopic review; nucleated RBCs and megakaryocyte fragments increase WBC counts, corrected by manual counting; platelet clumps are counted as WBCs, corrected by sodium citrate redraw and multiplying by 1.1; high WBC counts (>100,000/μL) cause turbidity affecting hemoglobin readings, requiring manual hematocrit verification; leukemia/chemotherapy fragments are counted as platelets, requiring blood film review; old specimens cause RBC swelling and altered automated differentials, requiring rejection based on stability criteria.