Importance and Uses of Periodic Acid-Schiff (PAS) Stain

• PAS helps to demonstrate glycogen, cellulose and starch. This is useful to detect glycogen deposits in liver when glycogen storage disease is suspected.
• Basement membrane of various tissues may also be visualized through the PAS stain. The PAS is most commonly used to demonstrate the thickness of glomerular basement membrane when renal disease is being assessed.
• Certain fungi in tissue samples such as Cryptococcus neoformans, Histoplasma capsulatum, Aspergillus fumiagtus, Blastomyces etc can be demonstrated by PAS stain because of high carbohydrate content in their cell wall/capsule.
• Mucin, particularly acid mucin is demonstrated by PAS. It is important un endocervical glands, intestinal glands and bronchial glands.
• PAS helps to demonstrate cerebrosides and gangliosides. It helps in diagnosis of Gaucher’s disease, Krabbe’s disease and lysosomal storage diseases etc.
• Certain pigments such as lipofuchsin and pigments of Dubin-Johnson syndrome are demonstrated by PAS stain.
• Russell bodies of plasma cells are stained by PAS.

Principle of PAS Stain

Glycol group of carbohydrates are oxidized by periodic acid to release dialdehydes. These dialdehydes on subsequent combination with Schiff’s reagent result in the formation of magenta colored complex, localized at the site of aldehyde formation.

Preparation of Reagents for PAS Stain

Periodic Acid Solution :

Periodic acid solution, used for oxidation is used in any strength between 0.5% and 2.5%. 1% solution is preferred.
Periodic acid= 1gm
Distilled water= 100ml

Schiff’s Reagent

Fuchsin Basic : 1 gm
Distilled water : 100 ml
Sodium metabisulphite : 2 gm
Conc. HCl : 2 ml
Charcoal activated : 0.3 gm

Dissolve basic fuchsin in boiling water, cool at 50°C and filter. Add sodium metabisulphite and HCl. Store at dark room at room temperature overnight. Add charcoal, shake for one minute and filter.

Procedure of PAS Stain

1. Deparaffinization: flame the slide on burner and place in the xylene. Repeat the treatment to remove the wax.
2. Hydration: Drain xylene and hydrate the tissue section by passing through decreasing concentration of alcohol baths (100%, 90%, 80%, 70%) and water.
3. Oxidation: Place the sections in periodic acid solution (1%) for 5-10 minutes.
4. Rinse: Wash in at least two changes of distilled water.
5. Treatment with Schiff’s reagent: Cover with Schiff’s reagent for 20-30 minutes.
6. Rinse: Rinse in running tap water for 5-10 minutes.
7. Counterstain: Cover with hematoxylin for 3-5 minutes. Differentiate and blue for the sections.
8. Dehydration: Dehydrate in increasing concentration of alcohols.
9. Clearing: Put slides in two xylene baths for clearing.
10. Mounting: Mount in DPX or other mounting media.
11. Observe under microscope.

Results and Intrepretation

The substances positive for PAS are:

1. Polysaccharides: Glycogen, cellulose and starch. Many leucocytes contain glycogen, capsule of fungi (Candida albicans, Histoplasma capsualtum, Cryptococcus and Blastomycosis), actinomycosis and bacteria.
2. Glycoproteins: Mucins, mucoid secrection of intestinal tracts, uterine glands, ducts, tracheobronchial tress, hormones (TSH), megakaryocytes etc.
3. Glycolipids: Gangliosides, mainly gray matter composed of fatty acids.
4. Non-carbohydrate containing substances: Unsaturated lipids, phospholipids and phosphoinositides.
5. Certain pigments and substances: Ceroid, lipofucsin, pigment in melanosis coli and Dubin-Johnson pigment.
6. Plasmogens: They are acetyl phospholipids, eg: Russell bodies.
7. Miscellaneous: Amyloid, cartilage matrix, colloid and ocular lens material.

References

1. Shariff, S., & Kaler, A. K. (2016). Principles & Interpretation of Laboratory Practices in Surgical Pathology. JP Medical Ltd.
2. Dey, P. (2018). Basic and advanced laboratory techniques in histopathology and cytology. Springer Singapore.
3. Bancroft, J. D., & Gamble, M. (Eds.). (2008). Theory and practice of histological techniques. Elsevier health sciences.
4. Löffler, H., & Rastetter, J. (2012). Atlas of clinical hematology. Springer Science & Business Media.
5. https://www.pathologyoutlines.com/topic/stainspas.html
6. https://www.labce.com/spg949466_periodic_acid_schiff_pas_diagnostic_applications.aspx

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Principle

The reticulocyte count is based on the property of ribosomal RNA to react with isotonic solution of a supravital stain such as New methylene blue or brilliant cresyl blue. Supravital stains are those which stain living material. Hence, for the detection of ribosomal RNA is reticulocytes, they should be fixed.
Blood is mixed with the stain and incubated. The RNA in the cell gets precipitated as dark blue network or reticulum. Blood smear is made and examined under microscope. As a direct count is not possible, a relative count is taken against the number of RBcs and expressed as the percentage of RBC.

Requirements

1. Specimen: EDTA whole blood/capillary blood
2. Reagent:
   New Methylene Blue
   New methylene blue = 1.0 gm
   Sodium citrate = 0.6 gm
   Sodium chloride = 0.7 gm
   Distilled water = 100 ml

   OR

   Brilliant Cresyl Blue
   Brilliant cresyl blue = 1.0 gm
   Sodium citrate = 0.6 gm
   Sodium chloride = 0.7 gm
   Distilled water = 100 ml

Procedure

1. Take 2-3 drops of dye solution in a test tube.
2. Add 2-4 drops of well-mixed blood sample and mix.
3. Stopper the tube and incubate at 37⁰C for 10-15 minutes.
4. After incubation, mix well and make a thin smear of stained blood.
5. When dry, examine the films without fixing or counterstain.
6. Count 1000 RBCs and note the number of reticulocytes among them. A dark-blue reticulum or network will present in reticulocytes.

Calculation

1. Retculocyte Percentage

This is the percentage of reticulocytes per 1000 RBCs.

2. Absolute Reticulocyte Count

The absolute reticulocyte count (ARC) is the actual number of reticulocytes in 1 L of whole blood.

3. Corrected Reticulocyte Count

In specimens with a low hematocrit, the percentage of reticulocytes may be falsely elevated because whole blood contains fewer RBCs. A correction factor is used, with the average normal hematocrit considered to be 45%.

Reference Ranges

Adults: 0.2-2%
Infants: 2-6%
Childrens upto 5 years= 0.2-5.0%

Clinical Significances

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Principle

The principle behind H & E stain is the chemical attraction between tissue and dye. Hematoxylin, a basic dye imparts blue-purple contrast on basophilic structures, primarily those containing nucleic acid moeties such as chromtatin, ribosomes and cytoplasmic regions rich in RNA. An acidic eosin counterstains the basic elements such as RBCs, cytoplasm, muscle and collagen in varying intensities of pink, orange and red.

Requirements

1. Harri’s Hematoxylin stain
   A = 1 gm hematoxylin in 10 ml ethanol
   B = 20 gm ammonium alum in hot distilled water
   Mix A & B, boil and add 0.5 gm of mercuric oxide and filter.
2. Eosin solution
   Yellow eosin = 1 gm
   Distilled water = 80 ml
   Ethanol = 320 ml
   Glacial Acetic Acid = 2 drops
3. 0.5% HCl
4. Dilute ammonia water

Procedure

1. Deparaffinization: flame the slide on burner and place in the xylene. Repeat the treatment to remove the wax.
2. Hydration: Drain xylene and hydrate the tissue section by passing through decreasing concentration of alcohol baths (100%, 90%, 80%, 70%) and water.
3. Nuclear Staining: Stain in hematoxylin for 3-5 minutes.
4. Wash in running tap water until sections “blue” for 5 minutes or less.
5. Differentiation: selective removal of excess dye from the section). Dip in 1% acid alcohol (1% HCl in 70% alcohol) for a few seconds.
6. Blueing: Rinse in running tap water. Dip in ammonia water until the sections become blue, followed by tap water wash.
7. Counterstain: Stain in 1% Eosin Y for 10 minutes.
8. Wash in tap water for 1-5 minutes.
9. Dehydration: Dehydrate in increasing concentration of alcohols.
10. Clearing: Put slides in two xylene baths for clearing.
11. Mounting: Mount in DPX or other mounting media.
12. Observe under compund microscope.

Results and Interpretation

• Nuclei : blue, black
• Cytoplasm : Pink/purplish pink
• Muscle fibres : deep red
• RBCs : orange red
• Calcium : Dark blue
• Mucin : Grey blue

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