Overview
A peripheral blood smear (PBF — peripheral blood film) is a glass microscope slide coated with a thin layer of blood, stained and examined under a microscope. It provides a direct morphological view of red cells, leucocytes, and platelets, supplements automated analyser data, detects parasites, and identifies abnormal cells in haematological malignancies.
This lesson covers smear preparation, the principle and technique of Romanowsky staining (Wright, Leishman, Giemsa), and the interpretation of both normal and abnormal red cell morphology — an essential skill for every laboratory technologist.
Learning Objectives
After this lesson you will be able to…- Describe the principle of Romanowsky staining
- Prepare and stain a peripheral blood film correctly
- Explain red cell abnormalities seen on the smear and their clinical interpretation
- Identify normal and abnormal red cell morphology
Clinical Story
Why This MattersA child is brought in with jaundice, pallor, and splenomegaly. The automated analyser flags abnormal red cell indices, but it is the peripheral blood smear that reveals the answer — numerous target cells and sickle-shaped red cells, pointing unmistakably to sickle cell disease. Without the peripheral smear, this diagnosis might have been delayed. This is why smear examination remains the gold standard for red cell morphology despite automation.
Core Concepts
Romanowsky stains (Leishman, Wright, Giemsa, Jenner's) contain two key dyes: Azure B (basic dye) binds anionic molecules — phosphate groups of DNA and nuclear proteins — staining nuclei purplish-black; Eosin Y (acidic dye) binds cationic sites — haemoglobin and cytoplasmic proteins — staining red cells pink. This differential binding gives each blood cell component its characteristic colour. The International Council of Standardization in Haematology advocates a combination of pure azure B and eosin Y for standardised, reproducible results. Leishman and Wright stains are most widely used in routine practice.
Normal red cells appear well spread with a smooth, round outline. The inner one-third of each cell is devoid of haemoglobin and appears as a central pale area (area of central pallor). There is very little variation in size (anisocytosis) or shape (poikilocytosis) of normal red cells. All cells stain uniformly pink with eosin.
| Morphological Finding | Description | Associated Conditions |
|---|---|---|
| Anisocytosis | Variation in size (macrocytes or microcytes) | Any anaemia |
| Poikilocytosis | Variation in shape | Nutritional anaemias, thalassaemia, haemolytic anaemias |
| Macrocytes | Larger red cells | Megaloblastic anaemia, aplastic anaemia, myelodysplasia, chronic liver disease |
| Microcytes | Smaller red cells | Iron deficiency, thalassaemia, chronic disease, sideroblastic anaemia, haemoglobinopathies |
| Hypochromia | Increased area of central pallor | Iron deficiency, thalassaemia, chronic disease, sideroblastic anaemia |
| Polychromasia | Pale blue red cells (reticulocytes on fixed smear) | Haemolytic anaemias, nutritional anaemia post-treatment |
| Target cells | Central round stained area + peripheral rim of Hb, separated by non-staining zone | Liver disease, iron deficiency, thalassaemia, haemoglobinopathies |
| Spherocytes | Spheroidal cells, regular outline, no central pallor | Hereditary spherocytosis, immune haemolytic anaemia, haemolytic disease of newborn |
| Basophilic stippling | Numerous blue granules within red cells | Thalassaemia, lead poisoning, unstable haemoglobins, megaloblastic anaemia |
| Howell-Jolly bodies | Single nuclear remnants, basophilic | Post-splenectomy, pernicious anaemia |
| Agglutination | Clumping of red cells | Autoimmune haemolytic anaemia |
Wright stain is prepared by dissolving 2.5g of powder in 2.5L of methanol, shaking well, and allowing to mature for 4-5 days before use. The stain is filtered before use. The buffer water used in staining is pH 6.8, prepared from potassium dihydrogen phosphate (Solution A) and disodium hydrogen phosphate (Solution B). Giemsa stain is prepared similarly by dissolving 1g of powder in methanol warmed to 50°C, kept for 15 minutes, filtered, and allowed to stand before use.
Laboratory Principle
Romanowsky staining exploits the differential ionic charges of cellular components. Azure B (cationic/basic) is attracted to anionic molecules — the phosphate backbone of nucleic acids and acidic proteins — staining nuclei, RNA, and granules blue-purple. Eosin Y (anionic/acidic) is attracted to cationic molecules — haemoglobin, cytoplasmic basic proteins — staining red cells pink. The methanol component of Wright stain simultaneously acts as a fixative, preserving cellular morphology before the dye is applied.
Equipment Required
Reagents & Materials
| Reagent / Material | Concentration / Grade | Purpose | Storage |
|---|---|---|---|
| Wright stain | 2.5g in 2.5L methanol; mature 4-5 days before use | Romanowsky staining — routine smear | Room temperature, filtered before use |
| Giemsa stain | 1g in 100ml methanol; warm 50°C 15min | Romanowsky staining — parasite detection, supplementary | Room temperature after preparation |
| Buffered water (pH 6.8) | Mix KH₂PO₄ (9.1g/L) and Na₂HPO₄ (9.5g/L) solutions: 50.8ml A + 49.2ml B | Dilutes and washes Wright stain for optimal colour | Room temperature |
Step-by-Step Procedure
Making the Smear
Place a clean 1"×3" glass slide on the bench. Write the laboratory number on the frosted end (facing upward). Place a 2–3mm drop of blood approximately ¼" from one edge using a glass capillary tube.
Hold the base slide with one hand. With the other hand, place the edge of a spreader slide in front of the drop of blood (on the far side from the frosted end). Pull the spreader backward until it touches the drop and the blood spreads by capillary motion to almost reach the edges of the spreader.
Push the spreader slide forward at a 30° angle with a rapid, even motion. Allow the smear to air dry completely before staining.
Wright Staining
Place the dried smear on the staining rack and flood with Wright stain. Leave for 2 minutes — the methanol in the stain simultaneously fixes the smear.
Add twice the volume of buffered water (pH 7) from a plastic wash bottle. Leave for 10 minutes without allowing the stain to dry on the slide.
Wash the stain with buffered water until the smear has a pinkish tinge. Wipe the back of the slide and stand upright to dry. Examine under microscope.
Flow Diagram
Quality Control
Always stain smears as soon as possible after drying. Do not leave smears unfixed for more than a few hours — prolonged delay causes distortion of cellular morphology and dried plasma stains the background blue. The staining rack must be level, and stain must never be allowed to dry over the smear during the procedure.
If fixation delay is expected (e.g., samples sent from remote sites), fix the smear immediately in methanol at the collection point to prevent morphological distortion during transport.
Reference Values
Normal Morphology⚠️ Reference ranges may vary between laboratories. Always apply your laboratory's established reference intervals.
Clinical Interpretation
| Morphological Finding | Possible Significance | Action / Follow-up |
|---|---|---|
| Microcytes + hypochromia | Iron deficiency anaemia, thalassaemia | Order iron studies, Hb electrophoresis |
| Macrocytes (oval) | Megaloblastic anaemia (B12/folate deficiency) | Order B12, folate, LDH, bone marrow if needed |
| Target cells | Liver disease, thalassaemia, iron deficiency, haemoglobinopathies | Correlate with LFTs, Hb electrophoresis |
| Spherocytes | Hereditary spherocytosis, immune haemolytic anaemia | Osmotic fragility test, DAT (Coombs test) |
| Howell-Jolly bodies | Post-splenectomy, megaloblastic anaemia | Correlate with clinical history and B12/folate |
Common Errors & How to Avoid Them
Cause: Too large a blood drop, too slow a spreading motion, or a chip on the spreader slide edge causes thick, uneven areas where cells overlap and cannot be properly assessed.
Prevention: Use a 2-3mm drop only; spread with a rapid, even 30° angle motion; check the spreader slide for chips before use.
Cause: Staining for too long, buffer pH too alkaline, or inadequate washing causes cells to stain deep blue, obscuring detail.
Prevention: Time staining precisely; use buffer at the correct pH; wash thoroughly until the smear shows a pinkish tinge.
Cause: Delayed fixation and staining allows red cells to form rouleaux stacks on the slide that can be misread as pathological agglutination.
Prevention: Stain smears promptly; if delay is expected, fix the smear in methanol immediately after drying.
Laboratory Tips from the Bench
Examine the smear first under low power (10×) to find the zone of morphology — a monolayer of well-spread cells just ahead of the tail — then switch to 40× for detailed morphology and 100× (oil immersion) for fine cellular detail and parasite identification.
A good peripheral smear should have a gradual gradient from thick (head) to thin (tail), with the zone of morphology showing well-separated cells where each red cell has a visible central pallor but cells do not overlap.
Remember "AMTSH-PA" for key red cell abnormalities: Anisocytosis, Microcytes, Target cells, Spherocytes, Howell-Jolly bodies, Poikilocytosis, Agglutination.
Important Notes
If smears are left unfixed for a long time, there is distortion of cellular morphology and dried plasma stains the background of the smear a pale blue, interfering with interpretation. Always fix promptly if staining will be delayed.
Leishman and Wright stains are the most widely used in routine practice but give results inferior to Giemsa and Jenner's for certain applications (e.g., parasite detection). Giemsa, fixed first in methanol, is preferred for malaria smears and lymphoma diagnosis requiring fine chromatin detail.
Interactive Quiz
Test Your KnowledgeFlashcards
Tap to flipClick or tap any card to reveal the answer.
Clinical Case Study
Apply Your KnowledgeArjun presents with pallor, fatigue, and mild jaundice since early childhood. His spleen is palpable 4cm below the left costal margin. Family history reveals a sibling with similar complaints.
Microcytic hypochromic anaemia with target cells, basophilic stippling, and nucleated red cells in a child with splenomegaly and a positive family history points strongly to thalassaemia major. Haemoglobin electrophoresis is essential to confirm the diagnosis.
- →The peripheral smear provides a diagnostic cluster (target cells + basophilic stippling + nucleated RBCs) that points directly to thalassaemia
- →Red cell indices alone (MCV, MCH) cannot differentiate iron deficiency from thalassaemia — the smear is essential
- →Haemoglobin electrophoresis is the definitive test to confirm and type the thalassaemia
Frequently Asked Questions
Giemsa stain gives superior staining of intracellular parasite components and stippling (Schüffner's dots in Plasmodium vivax), enabling identification of the malaria species with greater confidence. Slides must be fixed separately in methanol first when using Giemsa, unlike Wright stain where fixation and staining occur simultaneously.
Automated analysers provide quantitative data (counts, indices) but cannot reliably identify specific morphological abnormalities such as target cells, Howell-Jolly bodies, parasites, or blasts. The smear remains the only way to directly visualise and characterise abnormal cells and inclusions, making it indispensable in haematology.
A well-stained smear shows red cells staining uniformly pink, leucocyte cytoplasm staining light pink, nuclei staining purplish-black, and granules of different leucocyte types staining their specific characteristic colours. The background should be clear with no residual stain precipitate.
Quick Revision
10-Minute ReviewKey Takeaways
- Peripheral blood smears are stained with Romanowsky stains containing Azure B and Eosin Y
- Wright and Leishman stains are most commonly used; Giemsa is preferred for parasites
- The smear should be prepared at 30°, spread rapidly and evenly, then stained promptly
- Normal red cells are biconcave, uniformly sized, with a central pallor occupying the inner 1/3
- Morphological abnormalities — anisocytosis, poikilocytosis, target cells, spherocytes, stippling, Howell-Jolly bodies — each point to specific disease categories
- The peripheral smear remains essential even in the era of automation for detecting abnormal morphology and parasites
Competency Checklist
Track Your MasteryReferences
- Bain BJ. Blood Cells: A Practical Guide. 5th ed.
- Dacie JV, Lewis SM. Practical Haematology. 11th ed.
- NIOS Vocational Course — Hematology and Blood Bank Technique, Lesson 9.