What is a Romanowsky stain and how is it used in the microscopic examination of peripheral blood smears and bone‑marrow aspirates?

Romanowsky Stain: Definition and Clinical Applications

Romanowsky stain is a polychromatic staining method using a mixture of eosin (acidic dye) and methylene blue derivatives—particularly azure B—that produces distinctive multicolored staining of blood and bone marrow cells, enabling identification of cellular components and hematologic abnormalities. 1, 2

Composition and Chemistry

The Romanowsky family of stains includes several variants that share a common chemical principle:

• Core components: Eosin Y (acidic dye) combined with azure B and other methylene blue derivatives (basic dyes) 2, 3
• Common variants: May-Grünwald-Giemsa (MGG), Wright-Giemsa, Leishman, and Diff-Quik (a rapid modified Wright-Giemsa) 4, 3
• The "Romanowsky effect": The characteristic purple coloration of chromatin and certain granules results from azure B-eosin complexes forming in situ, not merely from simultaneous presence of both dyes 2, 5, 6

The selectivity of this purple complex formation depends on the rate of eosin entry into azure B-stained structures—only faster-staining components like chromatin, neutrophil granules, and platelets permit purple complex formation under standard conditions. 5

Primary Clinical Applications in Hematology

Peripheral Blood Smear Examination

Romanowsky stains are the reference standard for blood smear evaluation:

• Red blood cells: Appear pink-orange (acidophilic) or buff-colored under correct pH conditions 3
• White blood cell cytoplasm: Nearly transparent with clearly delineated granules 3
• Chromatin: Stains purple due to azure B-eosin complex formation 2, 5
• Neutrophil specific granules: Purple-stained 5
• Eosinophil granules: Red from eosin uptake 5
• Basophil granules: Violet 5
• Platelets: Purple 5

For detection of intracellular organisms: Wright-Giemsa stains (eosin-azure type dyes) can reveal morulae in the cytoplasm of infected leukocytes during ehrlichiosis or anaplasmosis, though this requires experienced microscopists to distinguish morulae from platelets, Döhle bodies, and other inclusions. 4

Bone Marrow Aspirate Evaluation

Wright-Giemsa staining of bone marrow aspirate smears is mandatory for morphologic assessment in acute leukemia diagnosis:

• Acute myeloid leukemia (AML): European LeukemiaNet guidelines mandate Romanowsky staining (May-Grünwald-Giemsa or Wright-Giemsa) on bone marrow and peripheral blood smears as part of standard diagnostic evaluation 1
• Acute lymphoblastic leukemia (ALL): Wright-Giemsa-stained bone marrow aspirate smears are required for morphologic assessment, with a differential count of 500 cells recommended 4
• Acute promyelocytic leukemia (APL): Romanowsky-derived stains on bone marrow aspirate smears are mandatory to identify characteristic abnormal promyelocytes with heavy granulation or bilobed nuclei 7
• Hairy cell leukemia: Wright's stain is used for peripheral blood smear review to identify characteristic hairy cells 4
• Chronic myelomonocytic leukemia (CMML): Peripheral smear examination with Romanowsky stains can reveal promonocytes and immature neutrophil precursors 8

Technical Specifications for Optimal Results

Preparation Requirements

• Rapid air-drying of cell preparations and imprints is essential 3
• Fixation: Use either methanol alone or May-Grünwald solution for 3-10 minutes 3
• Two-step staining protocol:
  • 50% May-Grünwald in buffer pH 6.8 (v/v) for 3-5 minutes
  • 10% buffered Giemsa solution for 10-30 minutes
  • Running water rinse for 1-3 minutes 3

Staining Variants and Their Advantages

Diff-Quik (modified Wright-Giemsa) offers specific advantages in certain contexts:

• Extremely rapid: Staining completed in <30 seconds 4
• Excellent cytoplasmic detail: Superior for visualizing mucin, lipid, and extracellular substances 4
• Familiar patterns: Similar to routinely processed blood and bone marrow samples 4
• Limitations: Nuclear detail less crisp than H&E, potential air-drying artifacts 4

Standard Wright-Giemsa or May-Grünwald-Giemsa provides:

• Superior nuclear detail: Better chromatin visualization 4
• Standardization: More reproducible, especially with automated slide stainers 3
• Reference standard: Remains the gold standard for hematologic diagnosis 3

Quality Control and Interpretation

Quality evaluation must be performed on red blood cells and leukocytes, not on tumor cells. 3

Critical quality indicators:

• pH control is essential: Incorrect pH causes RBCs to appear green or blue instead of pink-orange 3
• Leukocyte cytoplasm transparency: Should be nearly transparent with sharp granule delineation 3
• Automated methods: Slide stainers are the standard for reproducibility, though testing is recommended for each automated system 3

Common Pitfalls and Solutions

• Blue nuclei instead of purple: Results from inadequate eosin penetration or incorrect pH; increase staining time or adjust buffer pH to 6.8 5
• Solution instability: Acidic dye-basic dye mixtures are inherently unstable; prepare fresh solutions regularly 2
• Heterogeneity of polychrome methylene blue: Commercial dye lots vary in azure B content, affecting standardization 2
• Loss of intracytoplasmic features: H&E fixation causes loss of some vacuoles; use Diff-Quik when cytoplasmic detail is critical 4