Cytochemistry and Histochemistry

Cytochemistry

• Cytology examines cells from tissues, lesions, and tumors.
• Cytochemistry is the science of localizing chemical components of cells and organelles on histological sections using various techniques.
• Cytochemistry is a valuable diagnostic tool that is quick, inexpensive, and carries minimal risk to patients.
• Cytological techniques include Fine Needle Aspiration and fluid analysis, which allow diverse sample collection methods.
• Cytochemistry identifies and characterizes leukocyte populations, especially in acute leukemias.
• Myeloperoxidase (MPO) and neutrophil alkaline phosphatase (NAP) are used to differentiate between myeloid and lymphoid cells and to detect abnormalities in leukemic cells.

Histochemistry

• Histochemistry studies the chemical composition of cells and tissues using staining techniques.
• Histochemistry localizes chemical components of cells and tissues on histological sections using various techniques.
• Histochemical techniques demonstrate components like enzymes, proteins, nucleic acids, and lipids.
• Histochemistry bridges morphology and function using both anatomy and biochemistry.
• Histochemistry is used in medical diagnosis, scientific study, autopsy, and forensic investigation.

May-Grunwald Giemsa Stain

• A mix of two neutral stains.
• May-Grunwald stain = acidic stain (eosin) and a basic stain (methylene blue).
• Giemsa stain = acidic stain (eosin) and basic stain (azure of methylene).

Diff-Quik Stain

• The Diff-Quik stain is quick and easy to perform.
• The azures in the stain are basic dyes that bind acid nuclei and give blue to purple color.
• Eosin is an acidic dye that binds with alkaline cytoplasm giving pink to red color.

Papanicolaou Stain

• The Papanicolaou stain is a multichromatic cytological staining technique.
• The Papanicolaou stain was developed by George Papanicolaou.
• The principle of the Papanicolaou stain involves hydration, dehydration, and clearing.
• It allows for good nuclear detail.
• It can identify keratin in normal squamous cells versus dyplastic/carcinoma cells.

Haematoxylin and Eosin Stain

• Hematoxylin stains the acidic part of the cell, namely the nucleus (nuclear stain).
• Eosin acts as an acidic stain and binds with the basic part of the cell, namely the cytoplasm.

Special Stains in Cytology

• Some stains demonstrate cellular products
• Periodic Acid Schiff (PAS) stain: for glycogen in mesothelioma
• Mucicarmine stains: for mucinous adenocarcinoma
• Oil red O stain: for lipid in renal cell carcinoma
• Some stains demonstrate extracellular substances, such as Amyloid with the Congo red stain
• Some stains demonstrate infective organisms, such as acid-fast bacteria by Ziehl Neelsen stain.
• Some stains demonstrate DNA content and ploidy using the Feulgen stain.

Broad Classification of Special Stains

• Special stains can be used for collagen.
• Special stains can be used for muscle.
• Special stains can be used for elastic tissue.
• Special stains can be used for reticulin fibers.
• Special stains can be used for carbohydrates.
• Special stains can be used for amyloid.
• Special stains can be used for lipid.
• Special stains can be used for pigments & minerals.
• Special stains can be used for nerve tissue.
• Special stains can be used for microorganisms.
• Special stains can be used for decalcified bone.

Sudan Black B (SBB)

• Sudan black B is a lipophilic dye that stains intracellular phospholipids and other lipids.
• It to differentiate between ALL and AML.
• It can be performed on old smears in which MPO cannot be performed.
• The SBB dye binds irreversibly to an unidentified/ undefined granule in granulocytes and eosinophils.
• The reaction product of the stain is black and granular.
• Nuclei are stained blue.

Periodic Acid Schiff (PAS)

• The stain differentiates between AML (diffuse positivity) and ALL (block positivity).
• The stain is useful in identifying abnormal erythroblasts and dysplastic megakaryocytes in AML and MDS.
• The stain can be used to confirm the diagnosis of acute promyelocytic leukaemia.
• Periodic acid oxidizes 1-2 glycol groups of carbohydrates to produce stable di aldehydes, then these di aldehydes give a red reaction product when exposed to Schiff's reagent.
• In hemopoietic cells, the main source of positive reaction is glycogen.
• Granular precursors show diffuse weak positivity with neutrophils and act as internal positive controls.
• Eosinophil granules are negative with diffuse cytoplasmic positivity.
• Basophils are usually negative.
• The reaction product is red.
• Intensity ranging from pink to bright red.
• Cytoplasmic positivity may be diffuse or granular.

Types of Cytochemical Stains

• Enzymatic stains include Myeloperoxidase and Esterase
• Non-enzymatic stains include Sudan black B and Periodic acid Schiff.

Leukocyte Alkaline Phosphatase LAP

• Myeloperoxidase (MPO)
• Leukocyte Alkaline Phosphates (LAP or NAP)
• Acid Phosphates, TRAP
• Estrase
• Sudan Black B stain (SBB)
• Iron stain
• Reticulin stain
• Periodic Acid Schiff (PAS)

Stains and Their Uses

• Myeloperoxidase stains myeloperoxidase in neutrophils, monocytes and eosinophils to differentiate a myelogenous or monocytic leukemia from acute lymphocytic leukemia, and to visualize Auer rods.
• Sudan Black B stains intracellular lipid, similar to MPO.
• Toluidine Blue stains basophils and mast cells in order to identify dysplastic basophils in myeloproliferative diseases.
• Specific esterases stain neutrophil series and mast cells as a marker of cytoplasmic maturation in myeloid leukemias
• Non-specific esterases stain monocytes, acting as a monocytic component in AML
• Tartarate resistant acid phosphatase stains T-cells and granulocytes in order to diagnose T-cell ALL and hairy cell leukemia.

Basic Principle of Cytochemical Stains

• In cytochemical stains, a substrate reacts with an enzyme to produce a product.
• The visible product interacts with a chromogen.

Fixatives

• Common types of fixatives are Methanol, ethanol, acetone, formation vapor, formaldehyde, formal acetone, and formal ethanol.
• Most fixatives take 5-7 minutes
• MPO: 15 seconds
• LAP: 30 seconds
• At room temperature (LAP, SBB) – at 4°C (MPO, estate, LAP)

Types of Specimens

• Peripheral blood
• Bone marrow aspirates and imprints
• Paraffin section from bone marrow biopsy
• Aspirated and imprints of LN, spleen
• Fresh samples ensure optimal enzyme activity
• Smears from non-enzymatic stains as PAS and SBB stains may remain stable for months

LAP Stain

• Assesses cause of high peripheral leukocytosis.
• Helps differentiate between a reactive process and leukemia (chronic myelogenous leukemia, or CML).
• Differentiated cells stain more readily while leukemic cells will not when using an LAP.
• Used to generate an "LAP score."
• A high score generally indicates a "leukemoid reaction" or reactive condition (with an infection or other inflammatory process) while a low score suggests CML.
• Count 100 neutrophils and score them (0/+4), then calculate the final score by adding the total scores.
• (0) No stain
• (+1) Faint stain
• (+2) Moderate stain
• (+3) Strong stain
• (+4) Strong stain without cytoplasmic background
• Normal values are in the range of 30-185.

Ziehl-Neelsen Staining

• Mycobacterium is stained by Ziehl-Neelsen staining
• Mycobacterium cell wall contains mycolic acid.
• Phenolic acid and high temperatures increase the porosity of the membrane and help dye penetration.
• Mycolic acid resists the removal of the stain by acid and alcohol.