Leishman Stain - Complete Procedure

Background

• Henry's Clinical Diagnosis and Management by Laboratory Methods notes that Romanowsky-type stains include "Giemsa, Leishman's, Jenner's, May-Grunwald, MacNeal's, and various combinations."

Principle

1. Acts as a fixative - prevents cell distortion and makes cells adhere to the slide
2. Acts as a solvent for the dye components

Preparation of Leishman Stain Solution

• Leishman stain powder: 0.15 g
• Methanol (absolute, acetone-free): 100 mL
• Dissolve powder in methanol; filter before use
• Mature the stain by leaving it at room temperature for 1-2 weeks (or place in a water bath at 37°C for 24 hrs to accelerate)

• Phosphate buffer at pH 6.8-7.0 (or pH 6.4 for malaria smears)
• Alternatively, use clean distilled water

Standard Procedure (Flood Slide Method)

Step 1 - Prepare the Blood Film

• Collect blood (finger prick or EDTA anticoagulated)
• Make a thin peripheral blood smear on a clean, grease-free glass slide
• Air dry completely - do not heat fix

Step 2 - Fix and Stain (Initial Flooding)

• Place the slide horizontally on a staining rack
• Flood the slide with Leishman stain (approx. 15-20 drops)
• Allow to act for 1-3 minutes (the methanol fixes the smear simultaneously)
• Do NOT rinse

Step 3 - Add Buffer Water (Dilution Step)

• Add double the volume of buffered distilled water (or phosphate buffer pH 6.8-7.0) directly on top of the stain
• Gently rock the slide or blow on it to mix
• Allow the diluted stain to act for 10-15 minutes
• A greenish metallic sheen should appear on the surface - this confirms adequate staining

Step 4 - Wash

• Wash with a gentle stream of buffered water (distilled water) until the slide acquires a pinkish tint
• Hold the slide horizontally during initial washing to prevent uneven washing
• Do NOT wash too vigorously

Step 5 - Dry and Examine

• Drain excess water and allow to air dry in an upright position
• Do NOT blot with paper (may cause scratches)
• Examine under oil immersion (100x objective)

Simplified Procedure Summary

Staining Results (Expected Colors)

Uses / Applications

1. Routine peripheral blood smear - differential WBC count, morphology
2. Malarial parasite identification - ring forms, trophozoites, gametocytes
3. Trypanosomes in blood
4. Bone marrow aspirate examination
5. Leishmania (Leishman-Donovan bodies) - intracellular amastigotes in macrophages
6. Other haemoparasites - Babesia, microfilariae

Common Staining Problems and Corrections

Comparison with Other Romanowsky Stains

Anaplastic Large Cell Lymphoma (ALCL) - FNAC Microscopy Description

Overview

FNAC Smear - General Features

• Cellularity: Highly cellular smear
• Background: Necrotic background may be present; lymphoglandular bodies usually sparse or absent (unlike reactive lymphadenopathy)
• Discohesion: Cells tend to appear loosely cohesive or in small clusters - cohesive grouping can mimic metastatic carcinoma
• Tissue fragments: Occasional loosely cohesive aggregates of large cells packing sinusoidal spaces

The Hallmark Cells (Pathognomonic Feature)

Nuclear Features

• Large cells - significantly larger than normal lymphocytes
• Nuclei are bizarrely shaped: horseshoe-shaped, kidney-shaped, embryo-shaped, or lobulated
• Eccentric nucleus with a characteristic paracentral indentation or folded nuclear contour
• Open/vesicular chromatin (pale, finely granular)
• Multiple prominent nucleoli - often 1-3 large eosinophilic nucleoli
• Some cells show wreath-like or ring nuclei (nuclei arranged around the periphery of the cell)
• Reed-Sternberg-like cells (binucleate or multinucleate) may be seen
• "Donut cells" - cells with nuclear pseudoinclusions (nucleus appearing to have a hole)

Cytoplasmic Features

• Abundant cytoplasm - much more than typical lymphomas
• Cytoplasm is pale, amphophilic to basophilic (gray-blue on H&E/Giemsa)
• Prominent paranuclear Golgi zone - appears as a clear or slightly eosinophilic region adjacent to the nucleus (perinuclear eosinophilic hof)
• Cytoplasmic borders are distinct

Cell Populations in FNAC Smear

Morphological Variants and Their FNAC Appearance

1. Common (Typical) Pattern (~70%)

• Sheets of large hallmark cells
• Diffuse effacement of lymph node architecture
• Cells pack the sinuses - sinus infiltration pattern (in ~75% of cases)
• Most recognizable pattern on FNAC

2. Lymphohistiocytic Variant

• Neoplastic hallmark cells are overshadowed by numerous histiocytes with pale/eosinophilic cytoplasm
• FNAC can be misleading - appear as histiocyte-rich smear
• Small lymphocytes and plasma cells in background
• Hallmark cells tend to cluster around blood vessels
• Most commonly misdiagnosed variant

3. Small Cell Variant

• Predominant population of small to medium cells with irregular nuclei and pale cytoplasm
• Hallmark cells present but sparse - tend to be perivascular
• Occasional "fried egg cells" and rare signet ring cells
• Can mimic peripheral T-cell lymphoma NOS

4. Hodgkin-like (1-3%)

• Resembles nodular sclerosis classic Hodgkin lymphoma
• Mononuclear cells predominate
• Binucleate Reed-Sternberg-like cells and mummified cells
• Eosinophils and neutrophils present but less than in classic HL

5. Sarcomatoid Variant

• Spindle-shaped or bizarre sarcomatoid cells
• May mimic soft tissue sarcoma

Key Diagnostic Clues on FNAC

1. Hallmark cell morphology - horseshoe/kidney-shaped nucleus + abundant cytoplasm + Golgi zone = strongly suggestive
2. Sinusoidal infiltration - clusters of large cells mimicking metastatic carcinoma
3. Perivascular clustering of tumor cells
4. Brisk mitotic activity - numerous mitoses and apoptotic cells
5. Absence of typical lymphoid cell pattern - no germinal center cells, no tingible body macrophages

Differential Diagnosis on FNAC

Immunocytochemistry on FNAC Cell Block

Summary: FNAC Diagnostic Statement

"Smear shows a highly cellular aspirate comprising large pleomorphic lymphoid cells with hallmark cell morphology - cells having eccentric, horseshoe/kidney-shaped or lobulated nuclei, open chromatin, prominent nucleoli, and abundant pale cytoplasm with a conspicuous perinuclear Golgi zone. Reed-Sternberg-like cells and wreath cells are also noted. Brisk mitotic activity is present. Background shows scattered small lymphocytes. Findings are consistent with Anaplastic Large Cell Lymphoma. Immunocytochemistry/immunohistochemistry recommended for confirmation and ALK status."

Perinuclear Golgi Zone

What It Is

Why It Appears Pale on Light Microscopy

• It lacks the ribosomes and rough endoplasmic reticulum that give surrounding cytoplasm its basophilic (blue) color
• It is relatively devoid of RNA (which is what methylene blue/haematoxylin stains)
• The membranous cisternae of the Golgi itself do not take up standard stains strongly

Classic Example: Plasma Cell

Golgi Zone in Different Cell Types

Golgi Zone in ALCL - Why It Differs

Summary

The perinuclear Golgi zone is the pale/clear cytoplasmic halo immediately adjacent to the nucleus, representing the physical location of the Golgi apparatus. It appears pale because the Golgi region lacks ribosomes (which provide basophilia). It is most prominently seen in plasma cells (classic "clock-face nucleus + perinuclear hof"), and is also a notable feature in ALCL hallmark cells and several other secretory cell types.