10.2 Bone Marrow & Hematopoietic Preparations: Romanowsky, Giemsa & Wright Stains
Key Takeaways
- Romanowsky stains combine azure B, a basic thiazine dye, with eosin Y, an acid dye, in methanol; the azure B-eosin complex produces the purple Romanowsky effect in nuclei and azurophilic granules.
- Romanowsky buffer pH is critical: below about 6.4 erythrocytes stain too red and nuclei pale, and above about 6.8 the whole smear shifts blue-grey.
- Wright and Wright-Giemsa stains are dissolved in absolute methanol, which fixes the air-dried smear; water contamination of the methanol produces refractile erythrocyte water artifact.
- Bone marrow trephine cores must be decalcified in EDTA rather than strong acid, because acid decalcification destroys iron, nucleic acid, and many antigens.
- Storage iron and ring sideroblasts are assessed by Perls Prussian blue on an aspirate smear or clot section, never on an acid-decalcified core.
10.2 Bone Marrow & Hematopoietic Preparations: Romanowsky, Giemsa & Wright Stains
ASCP HT Exam Focus: Bone marrow is listed as its own procedure under Staining in the ASCP BOC content outline, and Giemsa and Romanowsky are named in the Summary of Stains. The highest-yield items connect a preparation type to the stain it supports and to the decalcification decision that precedes it.
A bone marrow examination is not one specimen but four simultaneous preparations, each answering a different question. Histology owns two of them outright and shares a third.
| Preparation | How it is made | Fixation/handling | Primary stain |
|---|---|---|---|
| Aspirate smear | Marrow particles spread on glass | Air-dried, then methanol-fixed by the stain | Wright or Wright-Giemsa |
| Touch imprint (roll prep) | Core rolled gently across a slide before fixation | Air-dried | Wright or Wright-Giemsa |
| Clot section | Residual aspirate allowed to clot, processed as tissue | 10 percent NBF or B-5, no decalcification | H&E plus special stains |
| Trephine core biopsy | 1.5 to 2 cm needle core of cancellous bone | Fixed, then decalcified | H&E plus special stains |
Because the core requires decalcification and the smears do not, the two preparations answer different questions. Cellularity, architecture, fibrosis, and focal lesions come from the core; individual cell morphology, blast percentage, dysplasia, and stainable iron come from the smears.
Romanowsky Dye Chemistry
A Romanowsky stain is any polychrome mixture of a thiazine dye (methylene blue and its oxidation products, the azures) with an acid xanthene dye (eosin Y), dissolved in methanol.
- Azure B is the essential thiazine. It is a cationic basic dye that binds anionic DNA, RNA, and acidic granule proteoglycan.
- Eosin Y is an anionic acid dye that binds cationic hemoglobin and eosinophil granule protein.
- The Romanowsky effect is the distinctive purple-magenta of nuclear chromatin and azurophilic granules. It is produced by an azure B-eosin Y complex formed on the tissue, not by either dye alone. A stain lacking azure B gives blue nuclei and no purple.
Named members of the family
- Wright stain: azure B plus eosin Y in methanol; single working solution plus buffer.
- Wright-Giemsa: adds Giemsa's fuller azure mixture for richer granule detail; the standard for marrow aspirates.
- May-Grunwald-Giemsa (Jenner-Giemsa, Pappenheim): sequential eosinate then Giemsa; favored in European practice.
- Leishman: a Wright variant used widely for peripheral blood.
- Diff-Quik and other rapid Romanowsky kits: three-step fixative/eosinophilic/basophilic dips used for rapid on-site evaluation of fine-needle aspirates.
Buffer pH: the single most tested variable
Romanowsky staining is buffered at approximately pH 6.4 to 6.8.
| Deviation | Appearance | Common cause |
|---|---|---|
| Too acidic (below 6.4) | Erythrocytes bright red-orange, nuclei pale blue, no purple | Acidic buffer or tap water, prolonged eosin exposure |
| Correct (6.4 to 6.8) | Erythrocytes pink-tan, nuclei purple, granules crisp | Correctly buffered water |
| Too alkaline (above 6.8) | Everything blue-grey, erythrocytes blue-green | Alkaline buffer, alkaline glassware detergent residue |
Methanol and water artifact
Wright and Giemsa stock solutions are made in absolute, water-free methanol, and the methanol in the stain is what fixes the air-dried smear. Methanol that has absorbed atmospheric water, or a slide that was not fully air-dried, produces the classic water artifact: erythrocytes with refractile, punched-out pale centers and a crenated rim. Store methanol tightly capped and replace it on a schedule.
Decalcification Decisions That Control the Stains
The trephine core is bone, so it must be decalcified - but the choice of agent determines which downstream stains still work.
- EDTA (10 to 14 percent, pH 7.0 to 7.4) is the marrow standard. It chelates calcium without acid hydrolysis, preserving iron, nucleic acids for FISH and sequencing, and immunohistochemical epitopes.
- Strong mineral acids (nitric, hydrochloric) are fast but hydrolyze DNA, strip iron, and destroy many antigens. A core decalcified in strong acid cannot be used to assess stainable iron, and the Perls stain will read falsely negative.
- Formic acid and formic-acid-based commercial reagents are an intermediate compromise; several proprietary agents are validated for IHC but should be confirmed against the laboratory's own antibody panel.
Consequence for practice: stainable storage iron and ring sideroblasts are evaluated on the aspirate smear or the clot section, which are never decalcified.
The Bone Marrow Special-Stain Panel
| Stain | Target | Diagnostic use |
|---|---|---|
| Perls Prussian blue | Ferric iron in macrophages and erythroblasts | Storage iron, ring sideroblasts of myelodysplastic syndrome |
| Reticulin (silver) | Reticular fiber network | Myelofibrosis grading (MF-0 through MF-3) |
| Masson trichrome | Mature collagen | Distinguishes collagen fibrosis from reticulin increase |
| PAS | Glycogen and neutral mucosubstance | Megakaryocytes, abnormal erythroid precursors |
| Naphthol AS-D chloroacetate esterase (Leder) | Granulocyte and mast cell esterase | Granulocytic sarcoma, systemic mastocytosis |
| Congo red | Amyloid | Marrow involvement by amyloidosis |
| Giemsa on paraffin sections | Nuclear and granule detail, organisms | Mast cells, plasma cells, Leishmania, Histoplasma |
Core sections for marrow are cut thin, at approximately 3 micrometers, because overlapping hematopoietic nuclei at 4 to 5 micrometers make differential counting unreliable.
A bone marrow aspirate smear stained with Wright-Giemsa shows bright red-orange erythrocytes, pale blue-grey nuclei, and no purple coloration of azurophilic granules. Which correction is indicated?
A hematopathologist reports that a bone marrow trephine core shows no stainable iron on Perls Prussian blue, while the concurrent aspirate smear from the same procedure shows abundant storage iron. Review of the histology worksheet shows the core was decalcified in 10 percent hydrochloric acid for 4 hours. What explains the discrepancy?
Which component is responsible for the characteristic purple coloration of nuclear chromatin and azurophilic granules in a Romanowsky-stained marrow smear?