The peripheral blood smear (PBS) is a fundamental laboratory technique that provides visual confirmation of automated CBC results and enables the detection of morphologic abnormalities that analyzers cannot reliably identify. Every medical laboratory scientist must master the preparation, staining, and systematic examination of the blood smear.
Preparation of a Quality Smear
A wedge smear is prepared by placing a small drop of EDTA-anticoagulated blood (or capillary blood) near one end of a clean glass slide. A second spreader slide is held at a 30–45° angle, backed into the drop until the blood spreads along its edge, and then smoothly pushed forward to create a feathered edge. The ideal smear has a gradual transition from thick to thin, with the feathered edge occupying 1–2 cm of the slide length. The smear should be air-dried rapidly by waving the slide. Common artifacts include holes (fatty blood, slow drying), irregular thickness (uneven spreader pressure), and long tail (spreader angle too low). The smear is then fixed in absolute methanol for 30 seconds before staining.
Wright-Giemsa Staining
The Romanowsky-type stains (Wright, Giemsa, or Wright-Giemsa combination) are the standard for blood smear staining. These stains contain azure B (basic, stains nucleic acids blue-purple) and eosin Y (acidic, stains hemoglobin and eosinophilic granules orange-red). The staining protocol involves flooding the fixed smear with Wright stain for 1–2 minutes, then adding buffer solution for 2–4 minutes to promote differential staining, followed by rinsing with water. Properly stained smears show pink-red RBCs, blue-purple nuclei and RNA, and distinct granule colors. Overstaining produces dark, muddy colors; understaining yields pale, poorly differentiated cells.
Systematic Smear Examination
The examination begins at low power (10× objective) to assess overall quality, select the optimal counting area, and identify abnormalities in WBC distribution (edge effect in lymphocytosis or CLL), platelet clumps at the feathered edge, and RBC rouleaux or agglutination. The WBC differential and morphologic assessment are performed at 50× or 100× oil immersion in the counting zone — the area where RBCs overlap slightly without stacking. At least 100 WBCs are counted and classified for the manual differential. The examination covers three aspects in sequence: platelet estimation and morphology, RBC morphology (size, shape, color, inclusions), and WBC morphology and differential.
RBC Morphology Assessment
RBC shape abnormalities (poikilocytes) provide diagnostic clues. Spherocytes (small, dense, no central pallor) suggest hereditary spherocytosis or AIHA. Schistocytes (fragmented cells) indicate microangiopathic hemolytic anemia (TTP, HUS, DIC). Sickle cells confirm sickle cell disease. Target cells are seen in thalassemia, liver disease, and HbC disease. Elliptocytes suggest hereditary elliptocytosis. Teardrop cells (dacrocytes) with nucleated RBCs suggest myelofibrosis or marrow infiltration. Acanthocytes (spur cells) occur in liver disease and abetalipoproteinemia. Echinocytes (burr cells) are often artifacts but can indicate uremia. RBC inclusions include basophilic stippling (lead poisoning, thalassemia), Howell-Jolly bodies (post-splenectomy, megaloblastic anemia), Pappenheimer bodies (sideroblastic anemia, thalassemia), and Cabot rings (megaloblastic anemia, myelodysplasia).
WBC Morphology Assessment
Beyond the differential count, WBC morphologic features are noted. Toxic granulation (dark, coarse neutrophil granules), Döhle bodies (pale blue cytoplasmic inclusions), and vacuolation indicate infection or inflammation. Hypersegmented neutrophils (> 5 lobes) suggest megaloblastic anemia. Pelger-Huët anomaly (bilobed, pince-nez nuclei) is a benign inherited condition but may be acquired in myelodysplasia. Blasts are identified by high nuclear-to-cytoplasmic ratio, open chromatin, prominent nucleoli, and basophilic cytoplasm, and their presence triggers further investigation by flow cytometry. Atypical lymphocytes (large, abundant basophilic cytoplasm, indented nucleus) are characteristic of EBV infection.
Platelet Estimation and Morphology
Platelet count is estimated from the smear by counting platelets in 10 oil immersion fields and multiplying by 15–20 × 10⁹/L. This provides a rapid check of the automated count. Giant platelets, gray platelets (lacking granules), or platelet clumps are noted. The presence of megathrombocytes suggests increased platelet turnover as seen in ITP.
Reporting and Correlation
The PBS report should describe the quality of the smear, the estimated cell counts, and all morphologic abnormalities using standardized terminology. Findings are correlated with the automated CBC parameters and clinical information. The PBS often drives additional testing such as hemoglobin electrophoresis, RBC enzyme assays, flow cytometry, or cytogenetic analysis.
