8.5 Peripheral Blood Smear Preparation and Staining, Bone Marrow Examination, and Cytochemistry
Key Takeaways
A wedge smear is made at a spreader angle of about 30° to 45°; a steeper angle, larger drop, or faster push gives a thicker smear.
Romanowsky stains are buffered at about pH 6.4 to 6.8; a smear that is too red reflects acidic conditions and one that is too blue reflects alkaline conditions or a thick smear.
The posterior superior iliac crest is the preferred site for bone marrow aspiration and core biopsy, and the sternum is used for aspiration only in adults.
Normal adult bone marrow has an M:E ratio of about 2:1 to 4:1 and a cellularity of about 100 minus the patient's age, ±10%.
Absent stainable marrow iron indicates iron deficiency, while ringed sideroblasts have iron granules encircling at least one-third of the nucleus.
Peripheral Blood Smear Preparation and Staining, Bone Marrow Examination, and Cytochemistry
Morphology of peripheral blood and bone marrow carries 20% of the Hematology blueprint. Good morphology depends on a well-made, well-stained smear and a systematic review. This section covers smear preparation, Romanowsky staining and its troubleshooting, the differential count and smear review, bone marrow aspiration and biopsy, marrow evaluation (cellularity, M:E ratio, iron stores), and the practical side of cytochemical staining.
1. Preparing the Peripheral Blood Smear
Wedge (Push) Smear
- Place a drop about 2 to 3 mm in diameter near one end of a clean slide. Bring a spreader slide back into the drop at an angle of about 30° to 45°, let the blood spread along its edge, and push forward in one smooth, steady motion.
- A good smear covers about two-thirds to three-quarters of the slide, has a smooth feathered edge, and has a monolayer area just behind the feathered edge where red cells barely touch. Morphology and the differential are evaluated there.
- Make smears from EDTA blood within a few hours of collection (commonly within 2 to 3 hours; sooner is better) to avoid storage artifacts such as crenated red cells and swollen, vacuolated white cells.
Controlling Smear Thickness
| Change | Effect on Smear |
|---|---|
| Larger drop, steeper angle, faster push | Thicker, shorter smear |
| Smaller drop, flatter angle, slower push | Thinner, longer smear |
| High hematocrit (polycythemia) | Use a flatter angle to avoid a thick smear |
| Low hematocrit (anemia) | Use a steeper angle to avoid a thin smear |
Common Smear Defects
- No feathered edge: The spreader was lifted before the end, or the drop was too large.
- Ridges or uneven thickness: Jerky pushing or uneven pressure.
- Holes or streaks: Dirty or greasy slides, or lipemic blood.
- Smudge cells: Fragile lymphocytes broken during spreading, typical of CLL. Adding a drop of 22% albumin before making the smear reduces them.
Other Preparations
- Coverslip smears (two coverslips pulled apart) give an even distribution but are rarely used now.
- Spinner (centrifugal) slides and automated slide makers produce even monolayers.
- Thick and thin smears for malaria: The thick smear concentrates blood to screen for parasites (red cells are lysed during staining), and the thin smear identifies the species. Thick smears must dry completely before staining.
2. Romanowsky Staining
- Romanowsky stains (Wright, Giemsa, Leishman, and Wright-Giemsa) combine methylene blue and its oxidation products (azures), which are basic dyes that stain acidic structures (DNA, RNA, basophil granules) blue to purple, with eosin, an acidic dye that stains basic structures (hemoglobin, eosinophil granules) orange-red.
- Wright stain contains methanol, which fixes the smear. A phosphate buffer at pH 6.4 to 6.8 is then added, and the stain develops during the buffering step.
- Troubleshooting:
| Problem | Appearance | Causes |
|---|---|---|
| Too acidic (too red) | Red cells bright red, white cell nuclei pale, eosinophil granules bright, little blue anywhere | Buffer pH too low, overwashing, too short a staining time |
| Too alkaline (too blue) | Red cells blue-gray, everything dark, neutrophil granules too dark | Buffer pH too high, thick smear, too long a staining time, inadequate washing |
| Stain precipitate | Dark granular deposit over cells, mimicking inclusions or parasites | Unfiltered or evaporated stain, slide allowed to dry during staining |
| Water artifact | Refractile spots in red cells | Moisture in the methanol or slow drying in humid air (a common problem in tropical laboratories) |
3. The Differential Count and Smear Review
- Low power (10×): Check stain quality and distribution, scan the feathered edge for platelet clumps, large abnormal cells, and microfilariae, and find the monolayer.
- Oil immersion (100×): Classify at least 100 white cells in the monolayer using a systematic pattern (the battlement or meander track) so no field is counted twice. Report relative (%) and absolute counts (relative % × WBC count).
- Red cell morphology: Grade size, color, shape, and inclusions in the monolayer.
- Platelet estimate: Confirm the analyzer's count and look for giant platelets.
- Correlate the smear with the CBC. A smear that does not fit the analyzer result is investigated before release.
4. Bone Marrow Aspiration and Biopsy
Indications
Unexplained cytopenias, suspected leukemia, myelodysplasia or myeloma, staging of lymphoma and other tumors, fever of unknown origin (marrow culture for typhoid or mycobacteria, or for Leishmania and fungi), and assessment of iron stores.
Sites
- Posterior superior iliac crest: The preferred site in adults and children for both aspiration and core biopsy.
- Anterior iliac crest: An alternative.
- Sternum: Aspiration only, in adults, because the bone is thin and the heart and great vessels lie beneath it.
- Anterior medial tibia: In infants (commonly under about 1 to 2 years of age).
Specimens and Processing
| Specimen | How It Is Obtained | What It Shows |
|---|---|---|
| Aspirate smears | Marrow drawn into a syringe; marrow particles (spicules) spread on slides; Wright-Giemsa stained | Cell morphology, differential count, M:E ratio, iron stain |
| Particle (crush) preparations | Spicules squashed between slides | Cellularity and megakaryocytes |
| Clot section | Remaining aspirate allowed to clot, fixed, and processed in paraffin | Architecture of particles |
| Core (trephine) biopsy | A Jamshidi-type needle removes a solid core, ideally at least 1.5 to 2 cm long | Cellularity, architecture, fibrosis, granulomas, metastatic tumor; decalcified (often with EDTA or formic acid) before sectioning |
| Touch imprints | Core rolled gently on a slide before fixation | Morphology when the aspirate is a dry tap |
A dry tap (no marrow aspirated) suggests marrow fibrosis (primary myelofibrosis, hairy cell leukemia, metastatic tumor) or a marrow packed with cells, and makes the core biopsy and imprints essential.
5. Evaluating the Marrow
- Cellularity: The proportion of marrow space occupied by hematopoietic cells rather than fat, judged on the core biopsy. A common rule of thumb is about 100 minus the patient's age, ±10%. It is very high in infants and falls with age.
- Myeloid-to-erythroid (M:E) ratio: Counted on the aspirate. Normal adults have about 2:1 to 4:1 (commonly about 3:1), because granulocytes have a shorter life span and are produced in larger numbers.
- Increased M:E: Infection, chronic myeloid leukemia, or erythroid hypoplasia (pure red cell aplasia).
- Decreased M:E: Erythroid hyperplasia (hemolysis, blood loss, megaloblastic anemia, thalassemia, response to treatment of iron deficiency) or reduced granulopoiesis.
- Megakaryocytes: Assessed for number and morphology, for example small hypolobated forms in myelodysplasia and increased megakaryocytes in immune thrombocytopenia.
- Differential count: Usually 300 to 500 nucleated cells on the aspirate. Blasts of 20% or more of marrow cells define acute leukemia under WHO criteria (lower thresholds apply to some genetically defined subtypes).
- Iron stores (Prussian blue / Perls stain): Storage iron in marrow macrophages is graded from absent to markedly increased. Absent stainable iron is the classic finding in iron deficiency. Ringed sideroblasts, erythroid precursors with iron granules encircling at least one-third of the nucleus, define sideroblastic anemias.
6. Practical Cytochemistry
The diagnostic reactions of each stain in acute leukemia are tabulated in the hematologic malignancy section. Practical points:
- Use fresh smears (ideally within a day or two) because enzyme activity declines over time. Fixatives differ by stain (for example, formalin-based fixatives for peroxidase and buffered formalin-acetone for esterases).
- Myeloperoxidase (MPO) and Sudan black B mark the granulocytic lineage. Sudan black B stains lipid and remains positive in older smears.
- Specific esterase (naphthol AS-D chloroacetate) stains granulocytes. Nonspecific esterase (alpha-naphthyl acetate or butyrate) stains monocytes, and sodium fluoride inhibits the monocytic reaction.
- Periodic acid-Schiff (PAS) shows glycogen, with coarse block positivity in many lymphoblasts and in abnormal erythroid precursors.
- Leukocyte (neutrophil) alkaline phosphatase (LAP) score separates a leukemoid reaction (high score) from chronic myeloid leukemia (low score).
- Tartrate-resistant acid phosphatase (TRAP) is positive in hairy cell leukemia; flow cytometry has largely replaced it.
- Every run includes a positive control smear (normal blood for MPO and esterases), because a negative reaction is only meaningful when the stain is shown to work.
A Wright-stained smear shows red cells that are bright red, white cell nuclei that are pale and poorly defined, and almost no blue staining anywhere. Which correction is most likely to fix the problem?
Shorten the staining time and wash the slide longer
Check the buffer and raise its pH toward 6.8
Make the smear thicker by using a steeper spreader angle
Fix the smear in water instead of methanol
A bone marrow aspirate from a patient with long-standing hemolytic anemia shows a myeloid-to-erythroid (M:E) ratio of 1:2. How should this be interpreted?
Normal marrow, because the normal adult M:E ratio is about 1:3
Myeloid hyperplasia caused by a superimposed bacterial infection
Erythroid hyperplasia, the expected response to hemolysis
Chronic myeloid leukemia in the accelerated or blast phase
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