Examination of a stained peripheral blood smear is a cornerstone technique in clinical pathology and biological research. It allows for the qualitative and quantitative assessment of the different cellular components of blood: red blood cells (erythrocytes), white blood cells (leukocytes), and platelets (thrombocytes). Accurate identification of these cells based on their distinctive morphological features is critical for diagnosing various hematological conditions and monitoring patient health.
Materials Required
- Light Microscope (with 10x, 40x, and 100x objectives)
- Prepared and stained peripheral blood smear slide (Wright-Giemsa stain is common and provides the necessary color differentiation)
- Immersion Oil
- Lens paper and cleaner
General Microscopy Setup and Smear Examination
- Prepare the Microscope: Ensure the light source is on and the condenser is properly adjusted for optimal illumination.
- Place the Slide: Secure the stained blood smear slide on the microscope stage.
- Initial Scan (Low Power, 10x or 40x): Begin by scanning the slide at lower magnification (10x or 40x). Your goal here is to get an overview of the smear’s quality, locate the “monolayer” area (where cells are spread thinly, typically just behind the feathered edge), and observe the overall cellularity. You can identify larger cell types (like white blood cells) at this power, but detailed identification requires higher magnification.
- Move to High Power (100x Oil Immersion): Once you’ve located a suitable area in the monolayer, rotate to the 100x objective. Add a drop of immersion oil directly onto the slide in the area you wish to examine, then carefully lower the 100x objective into the oil. Adjust the fine focus to bring the cells into sharp view. This magnification is essential for examining intracellular details like nuclear shape, chromatin pattern, and cytoplasmic granules.
- Systematic Examination: Scan the smear systematically in a meander or battlement pattern to ensure you cover different areas and encounter representative cells.
Identification Criteria and Application
Below are the key criteria for identifying common blood cell types. For each cell type, we will outline the criteria and then describe the process of applying these criteria to specifically find and identify that cell on the smear.
1. Identifying Neutrophils
Neutrophils are the most abundant type of white blood cell and are part of the innate immune system, primarily involved in fighting bacterial infections.
- Criteria for Identification:
- Size: Typically 12-15 micrometers (µm) in diameter, roughly twice the size of a red blood cell.
- Nucleus: Characteristically multi-lobed, usually with 3 to 5 distinct lobes connected by thin chromatin filaments. The chromatin is condensed and stains dark purple to blue. Sex chromatin (Barr body), appearing as a small drumstick appendage in female neutrophils, may sometimes be visible.
- Cytoplasm: Abundant and typically stains pale pink or tan. Contains numerous fine, dust-like granules that stain a neutral lilac, pink, or pale violet with Wright-Giemsa stain (hence “neutro-phil”). Specific granules are small and numerous, while azurophilic (primary) granules are larger, fewer, and stain more pink-purple.
- Key Distinguishing Features: Multi-lobed nucleus and fine, neutral-staining cytoplasmic granules.
- Process to Identify a Neutrophil on the Smear:
- Scan at Low Power (10x or 40x): Look for cells that are significantly larger than the surrounding red blood cells. They will appear as larger nucleated structures amongst the smaller, anucleated RBCs.
- Transition to High Power (100x Oil Immersion): Once you spot a potential large cell, move to 100x immersion oil objective for detailed examination.
- Examine the Nucleus: Carefully observe the nucleus. How many distinct segments or lobes does it have? Are they connected by thin strands? Does it look like 3, 4, or 5 blobs linked together? Is the chromatin dark and condensed?
- Examine the Cytoplasm: Look at the area surrounding the nucleus. What is the color of the cytoplasm? Are there small, fine granules visible within the cytoplasm? What color are these granules?
- Compare to Criteria: Does the cell have a multi-lobed nucleus (3+ lobes)? Is the cytoplasm abundant and pale pink/tan with fine, lilac/pink granules?
- Confirm Identification: If the features match the criteria (multi-lobed nucleus, pale cytoplasm, fine lilac granules, approx. twice RBC size), you have identified a neutrophil. Move on to find another example to reinforce the recognition.
Repeat the Same Process for Other Blood Cells:
Now, apply the same step-by-step approach (Scan -> Go to 100x -> Examine Nucleus -> Examine Cytoplasm -> Compare to Criteria -> Confirm) for the following cell types, using their specific criteria:
2. Identifying Basophils
Basophils are the least common type of white blood cell and are involved in allergic reactions and inflammation, releasing histamine and heparin.
- Criteria for Identification:
- Size: Similar to neutrophils, typically 10-14 µm.
- Nucleus: Usually bilobed or irregular in shape, but often obscured by the prominent cytoplasmic granules. Stains blue/purple.
- Cytoplasm: Packed with large, coarse granules that stain dark blue or purple (basophilic). These granules often overlie and obscure the nucleus.
- Key Distinguishing Features: Very large, dark blue/purple granules that often completely cover the nucleus. They are the rarest granulocyte.
- Process to Identify a Basophil: Look for large, nucleated cells. At 100x, specifically search for cells dominated by very large, dark blue/purple granules that might make it hard to see the nucleus clearly. Confirm size is similar to other granulocytes.
3. Identifying Eosinophils (Acidophils)
Eosinophils are involved in combating parasitic infections and modulating allergic inflammatory responses. They are sometimes referred to as “acidophils” due to their affinity for acidic dyes like eosin, which stains their granules red.
- Criteria for Identification:
- Size: Similar to neutrophils, typically 12-15 µm.
- Nucleus: Usually bilobed, appearing like a pair of spectacles or headphones. Stains blue/purple.
- Cytoplasm: Filled with large, uniform, spherical granules that stain bright red, orange, or pink (eosinophilic/acidophilic). These granules are distinctly larger and more prominently colored than neutrophil granules.
- Key Distinguishing Features: Bilobed nucleus and large, bright red/orange granules.
- Process to Identify an Eosinophil: Scan for large, nucleated cells. At 100x, look for cells with granules that are noticeably bright red or orange and uniform in size. Check for the characteristic bilobed nucleus, often visible despite the granules.
4. Identifying Lymphocytes
Lymphocytes are the second most common type of white blood cell and are central to the adaptive immune system (T cells, B cells, NK cells).
- Criteria for Identification:
- Size: Highly variable. Small lymphocytes are common (7-10 µm, about the size of an RBC). Medium (10-15 µm) and large (15-18 µm) lymphocytes also exist.
- Nucleus: Typically round or slightly indented, occupying most of the cell’s volume (high nucleus-to-cytoplasm ratio, especially in small lymphocytes). Chromatin is densely clumped and stains dark blue/purple.
- Cytoplasm: Scant (thin rim) in small lymphocytes, staining pale blue. May be more abundant in larger lymphocytes. Usually agranular, but sometimes contains a few scattered, non-specific azurophilic (pink-purple) granules.
- Key Distinguishing Features: High nucleus-to-cytoplasm ratio (nucleus takes up most space), dense, dark nucleus, often a thin rim of pale blue cytoplasm.
- Process to Identify a Lymphocyte: Scan for nucleated cells. At 100x, look for cells where the nucleus looks very large relative to the total cell size, almost filling it up. Observe the nucleus shape (round/indented) and chromatin pattern (dense). Note the amount and color of the cytoplasm (usually just a thin blue rim). Pay attention to size variation – compare small lymphocytes directly to nearby RBCs.
5. Identifying Platelets (Thrombocytes)
Platelets are not true cells but are small, irregular fragments derived from megakaryocytes in the bone marrow. They play a critical role in hemostasis (blood clotting).
- Criteria for Identification:
- Size: Small, typically 2-4 µm in diameter. Much smaller than red blood cells.
- Nucleus: Absent (they are anucleated fragments).
- Cytoplasm: Irregular shape, often appearing round, oval, or spiky. The cytoplasm stains bluish and contains scattered clumps or fine granules that stain reddish-purple.
- Key Distinguishing Features: Very small size, irregular shape, lack of a nucleus, granular appearance. Often found in clumps.
- Process to Identify Platelets: Scan the background area between the larger blood cells at 10x or 40x. You will see numerous tiny particles. Move to 100x. Look for the smallest structures on the slide. Are they anucleated, irregularly shaped, and do they show faint blue cytoplasm with reddish-purple granularity? Are they often aggregated in clusters? If yes, you have identified platelets.
6. Identifying Red Blood Cells (RBCs / Erythrocytes)
Red blood cells are the most numerous cells in the blood and are responsible for oxygen transport.
- Criteria for Identification:
- Size: Uniform, typically 7-8 µm in diameter. They serve as a standard size reference for other cells.
- Nucleus: Absent in mature mammalian red blood cells.
- Cytoplasm: Stains uniformly pink or red due to the presence of hemoglobin (eosinophilic).
- Shape: Typically a biconcave disc, which appears as a round cell with a lighter central area (central pallor) under the microscope.
- Key Distinguishing Features: Uniform size, lack of a nucleus, pink/red color, central pallor, most numerous cell type.
- Process to Identify Red Blood Cells: As you scan the smear, these will be the overwhelmingly dominant cells. They are easily recognizable at any magnification due to their abundance, pink/red color, and lack of a nucleus. Identify them early as they provide the crucial size reference for classifying other cell types. Observe their uniform size and the characteristic central pallor.
Tips for Accurate Identification
- Practice: Morphological identification requires practice. The more cells you examine, the better you will become at recognizing subtle differences.
- Compare: Always compare the size of white blood cells and platelets to the surrounding red blood cells.
- Focus on Key Features: Pay close attention to the nucleus (shape, lobularity, chromatin) and the cytoplasm (color, presence/absence and type/size/color of granules).
- Scan Quality Areas: Ensure you examine the smear in the monolayer where cells are spread out and not distorted by being too thick or too thin.
- Use Oil Immersion: Accurate identification of WBCs and platelets relies heavily on viewing details at 100x with oil immersion.
- Recognize Variations: Blood cells can exhibit slight variations in appearance depending on the individual, the staining technique, and the clinical condition. This guide covers the typical appearance; learning pathological variations comes with further study and experience.
Conclusion
Mastering the identification of blood cells under the microscope is a fundamental skill in various scientific and clinical disciplines. By systematically applying the criteria for size, nuclear characteristics, and cytoplasmic features, and practicing regularly, you can confidently distinguish between neutrophils, basophils, eosinophils, lymphocytes, platelets, and red blood cells. This forms the basis for more advanced hematological analysis and diagnosis.
