10.0 HAEMATOLOGICAL CELLS Also refer to Giemsa (See 5.6)

10.1 METHYL GREEN - PYRONIN TECHNIQUE FOR PLASMA CELLS (Pappenheim 1890, Unna 1902)

Plasma cells are found in serous membranes, in the lining of the respiratory and intestinal tracts and in lymphoid tissue. They are plentiful in regions of chronic inflammation.

PRINCIPLE
The methyl green-Pyronin technique can be used to demonstrate malignant plasma cell tumours such as plasmocytomas.
DNA has a differential affinity for methyl green and RNA has an affinity for pyronin, thus staining the nuclei green and the RNA (plasma cell cytoplasm) red.

REFERENCES
p 42 Cook H C, Manual of Histological Demonstration Techniques.

SPECIMEN
Standard formalin fixed Paraffin section

CONTROL
Tissue with chronic inflammatory reaction.

REAGENTS
(A) pH 4.8 ACETATE BUFFER
Stock A 0.2M acetic acid (1.2 mL in 100 mL distilled water) Stock B 0.2M sodium acetate (1.6g in 100 mL distilled water) Mix A:B in ratio 2:3 (10 mL A with 15 mL B)
(B) METHYL GREEN - PYRONIN SOLUTION
Make a mixture of the following four reagents
2 % aqueous methyl green (C.I.42585) 9mL (Purchase Methyl violet free reagent or extract with chloroform until the methyl violet contaminant is removed - difficult to remove)
2 % aqueous pyronin Y or G (C.I. 45005) 4mL
Glycerol 14mL pH 4.8 acetate buffer 23mL
Stock A 0.2M acetic acid (1.2mL in 100mL distilled water)
Stock B 0.2M sodium acetate (16.4g/L or 1.6g/100mL)
Mix in ratio A:B = 2:3 Mix before use.

PROCEDURE
1. Deparaffinize and hydrate to distilled water. Rinse with pH 4.8 buffer.
2. Stain with the methyl green-pyronin solution for 25 minutes.
3. Wash with buffer and blot dry.
4. Rinse in equal parts acetone-histoclear, then in histoclear and mount in Safety Mount.

RESULTS
DNA (nuclei) Green, green-blue
RNA (plasma cell cytoplasm, Nissl substance, bacteria) Red
Some mucins Red

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PRINCIPLE
This technique is similar in principle to the methyl green-pyronin but uses instead malachite green and acridine red with similar results.
DNA has a differential affinity for malachite green and RNA has an affinity for acridine red.

REFERENCES
p 43 Cook H C, Manual of Histological Demonstration Techniques.

SPECIMEN
Zenker-acetic fixation preferred or 10 % neutral buffered formalin. 4æ paraffin sections. Treat sections with 1% acetic acid if fixative other than Zenker-acetic fixative is used.

CONTROL
Chronic inflammatory reaction in tissue.

REAGENTS
(A) MALACHITE GREEN SOLUTION
Dissolve 3 g of malachite green in 150 mL of distilled water. Filter and label.
(B) ACRIDINE RED SOLUTION Dissolve 9 g of acridine red in 450 mL of distilled water. Filter and label.

WORKING SOLUTION
Mix one (1) volume of (A) and three (3) volumes of (B).
15 mL of (A) and 45 mL of (B) is sufficient for a coplin jar.

PROCEDURE
1. Deparaffinize and hydrate to distilled water.
2. Stain in working solution for 3 minutes.
3. Rinse quickly in water
4. Dehydrate rapidly in alcohols.
5. Clear and mount.

RESULTS
DNA (nuclei) Blue - green
RNA (plasma cell cytoplasm) Red
 

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Mast cells contain heparin and histamine, usually in the form of granules which are difficult to see in an H&E preparation. They are found in connective tissue around blood vessels in many varied inflammatory conditions. Rapid fixation is essential as the granules deteriorate with time, hence they are better seen on surgical material than on postmortem material.

PRINCIPLE
Mast cell granules stain metachromatically with methylene blue.

REFERENCES
p 175 Mallory, F.B., Pathological Technique, NY, Hafner Publishing Co., 1961.
Printed in Luna , Manual of Histologic Staining Methods of the AFIP, 3rd ed, p 115

SPECIMEN
10 % neutral buffered formalin or formol alcohol. Cut standard paraffin sections.

CONTROL
Trachea.

REAGENTS
(A) POLYCHROME METHYLENE BLUE SOLUTION
Methylene blue 1 g Distilled water 100 mL Alcohol 95 % 20 mL Potassium carbonate 1 g

(B) GLYCERIN - ETHER SOLUTION
Glycerin 50 mL Calcium Chloride anhydrous 10 g Heat the above mixture until the calcium chloride dissolves completely. Cool to room temperature.

PROCEDURE
1. Deparaffinize and hydrate to distilled water
2. Polychrome Methylene Blue solution for 10 minutes.
3. Rinse in distilled water.
4. Differentiate in Glycerin-ether solution, diluted 5 to 10 times with distilled water, for 30 seconds to 1 minute, or until section is a medium blue. Be careful not to differentiate too long.
5. Wash thoroughly in water for 2 - 5 minutes, then blot with filter paper.
6. Dehydrate rapidly in absolute alcohol.
7. Clear in Histoclear, two or three changes.
8. Mount with Safe-t-mount.

RESULTS
Mast cells Red
Other cells Greenish-blue

NOTES
This technique, for the differentiation of mast cells and plasma cells, is especially useful for highly cellular specimens, such as lymph node and spleen.
 

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Toluidine blue is a polychromatic stain which stains many tissue components.

REFERENCES
p 164 Bancroft JD & Stevens A, Theory and Practice of Histological Techniques, 4th Ed. The exact origin of this method is unknown.

SPECIMEN
Standard paraffin sections or 6æ frozen sections.

CONTROL
Trachea.

REAGENTS
Toluidine blue (CI 52040) 0.25 g Sodium acetate 2 g Sodium barbiturate 3 g Distilled water to 100 mL

PROCEDURE
1. Paraffin or frozen sections may be used.
2. Bring sections to water.
3. Stain in toluidine blue solution, for 20 seconds .
4. Blot with filter paper; allow to dry and clear in histolene.
5. Mount in Safety Mount

RESULTS
Metochromatic substances Red, pink or purple
Nuclei and other components
Blue Mast cell granules Purple
 

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10.5 LUNA'S METHOD FOR ERYTHROCYTES AND EOSINOPHILS.

PRINCIPLE
The tissue is stained with a combined Haematoxylin-Biebrich Scarlet solution to demonstrate eosinophil granules against a blue background..

REFERENCES
Luna,LG, Manual of histologic staining methods of the Armed Forces Institute of Pathology, 1968:111.

SPECIMEN
10 % neutral buffered formalin. Standard paraffin sections.

CONTROL
Known positive control containing eosinophils.

REAGENTS

(A) WEIGERTS IRON HAEMATOXYLIN
Same as reagent A in Van Gieson method 2.4
1. Dissolve 1g of haematoxylin in 100mLs of 95% ethanol (filter)
2. To 4mLs of 30% aq. ferric chloride add 95mLs of distilled water. Add 1mL of concentrated hydrochloric acid.
Working Solution:
Mix equal volumes of (1) and (2) prior to use.

(B) 1% BIEBRICH SCARLET SOLUTION
Biebrich scarlet (CI 26905) 1 g Distilled water 100 mL (

C) HAEMATOXYLIN-BIEBRICH SCARLET SOLUTION (WORKING)
Weigert's iron haematoxylin solution (working) 45 mL Biebrich scarlet solution 5 mL

(D) 1% ACID ALCOHOL SOLUTION 70 % alcohol 100 mL Concentrated HCl 1 mL

(E) 0.5 % LITHIUM CARBONATE SOLUTION
Lithium carbonate 0.5 g Distilled water 100 mL

PROCEDURE
1. Deparaffinize and hydrate to distilled water.
2. Stain in working haematoxylin-biebrich scarlet solution for 5 minutes.
3. Differentiate in 1% acid alcohol until desired nuclear detail is achieved (usually eight dips).
4. Rinse in tap water to remove acid alcohol. 5. Dip Lithium carbonate solution until section turns blue and erythrocytes are bright red (usually five dips).
6. Wash in running water for 2 minutes.
7. Dehydrate in 95% alcohol, absolute alcohol, and clear in Histolene, two changes each.
8. Mount with Safety-mount.

RESULTS
Eosinophil granules Red
Erythrocytes Red
Charcott Leyden crystals
Red Background Blue

NOTES
Especially useful for phagocytosis and sometimes useful for demonstrating Negri bodies.
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PRINCIPLE
Haemoglobin stains orange-red by acid dyes such as eosin. The Dunn Thompson method is a modification of the Van Gieson stain and stains haemoglobin, red cell and haemoglobin urinary casts and phagocytosed haemoglobin an emerald green or greenish-black colour. Fixation in neutral buffered formalin is essential.

REFERENCES
p 244 Bancroft JD & Stevens A, Theory and Practice of Histological Techniques, 2nd Edition.

SPECIMEN
Fixation -10% neutral buffered formalin - essential.
Standard paraffin sections.

CONTROL
Haemoglobin is found normally within red blood cells.

REAGENTS
(A) ALUM HAEMATOXYLIN
Commercial Harris haematoxylin as used in linear stainer.
(B) VAN GIESON SOLUTION
As used in Van Gieson's stain method 2.4 100mL saturated aqueous Picric Acid 10 mL 1% aqueous fuchsin (C.I. 42685)
Boil for 3 minutes, cool and filter then add 0.25mL concentrated hydrochloric acid.
(C) 4 % IRON ALUM
4 g ammonium ferric sulphate in 100 mL distilled water (as used in reticulin fibres stain method 2.5)

PROCEDURE
1. Deparaffinize and hydrate to distilled water.
2. Alum haematoxylin - 7 minutes (up to 15 minutes).
3. Wash in tap water.
4. Mordant in 4 % iron alum - 5 minutes (up to 10 minutes).
5. Rinse in tap water.
6. Stain in van Gieson solution - 15 minutes.
7. Differentiate in 95 % alcohol - 3 minutes.
8. Rinse in absolute alcohol 9. Clear in histolene.
10. Mount in Safety Mount.

NOTE
The double haematoxylin staining produces stronger haemoglobin colouration.

RESULTS
RBCs Greenish / black
Haemoglobin casts Lighter green

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10.7 OKAJIMA'S METHOD FOR HAEMOGLOBIN

Name: Okajima's Stain
Purpose: To stain hemoglobin
Techniques:
Cut paraffin sections at 4 to 5 microns. Use control slide.

Solutions:

10% Phosphomolybdic Acid - Stock (make fresh)
Phosphomolybdic acid 10.0 gm
Distilled water 100.0 ml

7.7% Alizarin Red S - Stock (shelf life: 1 year)
Alizarin Red S (C.I. #58005) 7.7 gm
Distilled water 100.0 ml

Working Stain Solution: shelf life: 10 hours
10% Phosphomolybdic acid (stock) 10.0 ml
7.7% Alizarin red S (stock) 30.0 ml
Mix just before use. Discard after use.

Hematoxylin of choice

1. Deparaffinize and hydrate to water.
2. Place in 10% phosphomolybdic acid solution for 1 minute.
3. Wash in water.
4. Preheat 20 ml of working stain for 10 - 15 seconds in microwave.
Add slides and stain for 10 minutes.
(or 1-2 hours in non-heated solution)
5. Wash in water.
6. Counterstain in hematoxylin for 30 seconds - 1 minute (we use Gill's)
(Follow whatever you normally do after your hematoxylin, but don't
counterstain with Eosin!)
7. Dehydrate, clear, and coverslip with synthetic mounting medium.

Hemoglobin..............orange to orange red
Nuclei..................................blue
Background.....................reddish brown

Sheehan, 2nd ed., pp. 216-217

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