hemestains1

HEMATOLOGY STAINS

Heme I

• Document Number: 1304

• Date: March 2025

WRIGHT GIEMSA STAIN

• Preparation:

  • The wedge smear or push prep is the most common smear preparation in hematology.

  • Fresh blood should be well mixed, collected in EDTA or by capillary method.

  • A well-dried smear is prepared for staining.

  • Smears are fixed using a methanol fixative to stabilize cellular elements.

• Dye Components:

  • Eosin Y: Anionic dye, stains the cytoplasm pink.

  • Methylene Blue: Cationic or basic dye, responsible for staining nucleus and DNA blue.

DYES IN WRIGHT GIEMSA STAIN

• Compound Dyes:

  • Utilizes a combination of dyes; at least one must be polychromatic.

  • Acidic tissue components have an affinity for basic dyes, and vice versa.

  • Important for identifying leukocytes, erythrocytes, and platelets in various specimens.

POLYCHROMATIC DYE

• Definition: A dye exhibiting multiple colors, commonly used in nuclear and cytoplasmic stains for bone marrow biopsies.

• Polychroming:

  • Involves dyes forming other dyes spontaneously or through oxidation.

  • Most often uses methylene blue (basic dye) in combination with eosin (acid dye).

ROMANOWSKY DYES

• Mixtures of methylene blue, azure, and eosin compounds.

• Common types include Giemsa and Wright's stains, often used for peripheral blood smears and tissue identification.

METACHROMASIA IN DYES

• Definition: A property where tissue components stain a different color than the dye itself; often stains blue dye as purple.

• Common examples include:

  • Mast cell granules

  • Cartilage

  • Mucin

  • Amyloid

COMMON ROMANOWSKY STAINS

• 1. Jenner

• 2. Leishmann

• 3. Giemsa

• 4. Lillie-Wright

  • Differentiation: Variations in oxidizer and staining timing.

  • Color differences noted among stains.

METHYLENE BLUE OXIDIZATION

• Requires an oxidizing agent, silver oxide, to produce colored compounds.

• Coloured Compounds:

  • Azure A (methylene violet)

  • Azure B (methylene blue)

• Often used in conjunction with Giemsa for intensifying nuclear structures.

CHEMICAL FIXATION & STAINING PROCESS

• Cells are chemically fixed with methanol.

• Staining:

  • Primarily occurs during the buffering stage.

  • Affects the hues based on the pH level (acidic or basic) - defines cellular reactions to the specific dyes.

UNDERSTANDING STAIN RESULTS

• Typical Cell Staining:

  • RBCs: Red to pink

  • Neutrophils: Dark purple nuclei, pale pink cytoplasm with small granules

  • Eosinophils: Blue nuclei, pale pink cytoplasm, with orange-red granules

  • Basophils: Dark blue nucleus, dark granules

  • Lymphocytes: Dark purple nuclei, sky blue cytoplasm

  • Platelets: Violet to purple granules

SOURCES OF ERROR IN HEMATOLOGICAL STAINING

• Thick blood smears lead to inaccurate results.

• Delayed slide preparation causes distortion.

• Smears must dry completely before staining to prevent wash-off.

• Timely staining post-preparation is critical for accurate leukocyte identification.

• Dilution and buffering processes require careful monitoring.

• Air drying is preferred over blotting the smear.

TROUBLESHOOTING STAIN ISSUES

• Stain is Too Alkaline:

  • Causes RBCs to appear bluish; may mask abnormalities.

  • Corrective Measures: Check pH, shorten stain time, and prolong buffering.

• Stain is Too Acidic:

  • Results in bright red RBCs and poorly stained WBCs.

  • Corrective Measures: Lengthen staining time, check pH, and adjust washing time.

STAINING CONTROL PREPARATION

• Control samples are preferred from women in the last trimester of pregnancy or using oral contraceptives.

• Controls should be prepared, air-dried, and stored correctly to maintain integrity.

LEUKOCYTE ALKALINE PHOSPHATASE (LAP) METHOD

• Distinguishes between noncancerous reactions and chronic myelogenous leukemia through enzyme activity.

• LAP is a cytochemical stain quantifying the enzyme across WBCs, especially in segmented neutrophils.

• Specimen Requirements: Fresh unstained peripheral blood smears, preferably heparin-anticoagulated.

LAP SCORING

• Based on enzyme activity from stained neutrophils; scoring ranges from 0 (no staining) to 4 (heavy). 100 neutrophils must be counted per slide.

• Normal LAP score ranges from 30 to 100, with increased scores indicating specific conditions such as leukemoid responses and pregnancy.

PEDAGOGICAL STRATEGY

• Adhering to meticulous practices can minimize errors in staining, and consistent scoring protocols help ensure reliability in laboratory results.