CYTOPATHOLOGY Archives - LaboratoryTests.org

May Grunwald-Giemsa Stain: Principle, Preparation and Procedure

Dhurba Giri — Sat, 19 Feb 2022 15:40:22 +0000

May Grunwald-Giemsa stain(MGG) is a type of Romanowsky stain, which is used routinely for staining of air-dried cytological smears, blood, and bone marrow smears. Cytological preparations made from FNAC and serous fluids are normally processed by May Grunwald-Giemsa stain. It is useful for studying cellular morphology and is superior to PAP stain to study cytoplasm, granules, vacuoles, and basement membrane.

Principle

May Grunwald-Giemsa stain is a combination of two stains: May Grunwald stain and Giemsa stain.

May Grunwald stain is alcohol-based stain composed of methylene blue and eosin.
Giemsa stain is alcohol-based stain composed of methylene blue, eosin and azure B.

The working principle of MGG stain is the same as that of other Romanowksy stains. Polychromatic Romanowksy dyes contain different ratios of methylene blue (and the reagent-related thiazine dyes, such as azure B), as the cation (+vely charged, basic) component, and eosin Y as the anion (-vely charged, acidic) component. Cation and anion components in combination produce the well-known Romanowsky effect or metachromasia.

The solvent methanol initially fixes the cells. The basic dyes carry net positive charges; consequently, they stain nuclei (because of the negative charges of phosphate groups of DNA and RNA molecules), granules of basophil granulocytes, and RNA molecules of the cytoplasm. The eosin carries a net negative charge and stains red blood cells and granules of eosinophil granulocytes. Buffer solution of pH 6.5-6.8 is used to enable the dye to precipitate and bind well with the cellular material.

Reagents

May Grunwald Stain

Stock solution:
May Grunwald dye= 0.3 gm
Methanol = 100 ml
Working Solution:
Stock solution= 20 part
Phosphate Buffer (pH 6.8)= 30 part

Giemsa Stain

Stock Solution:
Giemsa Powder= 1 gm
Glycerine= 66ml
Absolute ethanol= 66ml
Mix giemsa and glycerine, place in 60C oven for 30 minutes 2 hr. Add 66ml methanol
Working Solution:
Stock Giemsa= 50 drops
Distilled Water=50ml

Procedure

Prepare a thin smear and air dry.
Fix smears for 5-10 minutes with methanol.
Stain the smear in May Grunwald working solution for 10 minutes.
Rinse in pH 6.8 buffer.
Stain the slides with diluted Giemsa stain for 30 minutes.
Wash the smears with distilled water and let them dry.
Mount the slide with DPX and examine under microscope.

Results

Erythrocytes: Light pink to light purple
Platelets: Granules – Reddish purple
Lymphocytes/monocytes: Nuclei – Dark purple, Cytoplasm – Sky blue
Neutrophils: Nuclei – Dark blue, Granules – Reddish purple, Cytoplasm – Pale pink
Eosinophils: Nuclei – Blue, Granules – Red/orange red, Cytoplasm – Blue
Basophils: Nuclei – Dark blue, Granules – Purple

References

Dey, P. (2018). Basic and advanced laboratory techniques in histopathology and cytology. Springer Singapore.
Matutes, E., Pickl, W. F., Van’t Veer, M., Morilla, R., Swansbury, J., Strobl, H., … & Ludwig, W. D. (2011). Mixed-phenotype acute leukemia: clinical and laboratory features and outcome in 100 patients defined according to the WHO 2008 classification. Blood, The Journal of the American Society of Hematology, 117(11), 3163-3171.
Product Information May-Grünwald Giemsa, Avantor Performance Materials.

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Wright’s Stain : Preparation, Principle, Procedure and Results

Dhurba Giri — Thu, 25 Jul 2019 14:58:04 +0000

Wright’s stain is a type of Romanowsky stain, which is commonly used in hematology laboratory for the routine staining of peripheral blood smears. It is also used for staining bone marrow aspirates, urine samples and to demonstrate malarial parasites in blood smears.
Wright’s stain is named for James Homer Wright, who devised the stain in 1902 based on a modification of Romanowsky stain. The stain distinguishes easily between the blood cells and hence became widely used for performing differential WBC counts and evaluate the morphology of blood cells.

Principle

Wright’s stain is a polychromatic stain consisting of a mixture of Eosin and Methylene blue. As the Wright stain is methanol based, it doesn’t require a fixation step prior to staining. However, fixation helps to reduce water artefact that can occur on humid days or with aged stain.

Methanol fixes the cells to the slide. Eosin Y is an acidic anionic dye and methylene blue is basic cationic dye. When diluted in buffered water, ionization occurs. Eosin stains the basic components such as hemoglobin and eosinophilic granules an orange to pink color. Methylene blue stains acidic cellular components such as nucleic acid and basophilic granules in varying shades of blue. The neutral components of the cells are stained by both components of the dye, producing variable colors.

Reagents

The dye may be purchased as a powder which is then mixed to methanol or a ready-made solution may be obtained.

Staining Solution
Wright’s stain powder = 1.0 gm
Water free methanol = 400 ml
Phosphate buffer (0.15M, ph 6.5/6.8)
Potassium dihydrogen phosphate, anhydrous = 0.663 gm
Disodium hydrogen phosphate, anhydrous = 0.256 gm
Distilled water = 100 ml

Procedure

Prepare a film of blood or bone marrow on a microscopic slide and allow to air dry.
Place the air-dried smear on the slide staining rack, smear side facing upwards.
Cover the blood film with undiluted staining solution. The undiluted stain fixes and partially stains the smear.
Let stand for 2-3 minutes.
Add approximately equal amount of buffered water (pH 6.5). The diluted stain shouldn’t overflow. Mix by gentle blowing. A metallic sheen (or green ‘scum’) should appear on the slide if mixing is appropriate.
Leave for 5 minutes.
Without disturbing the slide, flood the distilled water and wash until the thinner parts of the film are pinkish red.
Allow the slide to dry at room temperature and examine under microscope.

Results

Cells	Result
Erythrocytes	Yellowish-red
Neutrophils	Nucleus: Dark purple Granules: Reddish ileac granules Cytoplasm: Pale pink
Eosinophils	Nucleus: Blue Granules: Red to orange red Cytoplasm: Blue
Basophils	Nucleus: Purple to dark blue Granules: Dark purple
Lymphocytes	Nucleus: Dark purple Cytoplasm: Sky blue
Monocytes	Nucleus: Dark purple Cytoplasm: Mosaic pink and blue
Platelets	violet to purple granules

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Sudan Black B Stain : Purpose, Principle, Procedure and Interpretation

Dhurba Giri — Sun, 18 Nov 2018 06:31:18 +0000

Sudan Black B (SBB) is a fat soluble dye which has very high affinity for neutral fats and lipids. SBB staining is useful for for the differentiation of Acute myeloid leukemia (AML) from Acute lymphoid leukemia (ALL). It is similar to that of Myeloperoxidase (MPO) staining pattern of leukocytes and monocytes. It has few advantages over MPO:

SBB can be used for staining smears more than 2 weeks old.
SBB stains both azurophilic and specific granules in neutrophils, whereas MPO stains azurophilic granules only.
There is only a little fading of the SBB stain over time.

Principle of Sudan Black B Stain

As SBB is a fat soluble dye, it stains lipids such as sterols, neutral fats and phospholipids. These are present in azurophilic and secondary granules of myelocytic and lysosomal granules of monocytic cells. During staining, the dye leaves the solvent because of its high solubility in lipids than solvent. On microscopic examination, varying degree of black colored pigments are seen in the positive reaction.

Requirements

Sample: Fresh anticoagulated whole blood or bone marrow smear may be used. The slides must be fixed as soon as possible.
Fixative: 40% formaldehyde solution vapor
Stain: SBB 0.3 g in 100 ml absolute ethanol
Phenol buffer: Dissolve 16 g crystalline phenol in 30 ml absolute ethanol. Add to 100 ml distilled water in which 0.3 g Na2HPO4.12H2O has been dissolved
Working SBB stain solution: Add 40 ml buffer to 60 ml SBB solution (The composition of working stain may slightly vary upon different products.)
Counterstain: May–Grunwald–Giemsa or Leishman stain.

Procedure of Sudan Black B Stain

Fix air dried smears in formalin vapour, formaldehyde or formalin-ethanol fixative for 10 minutes.
Wash gently in water for 5-10 minutes.
Place the slides in the working stain solution for 1 hour in a Coplin jar with a lid on.
Remove and flood the slides with 70% alcohol for 30 seconds. Tip the 70% alcohol off and flood again. Repeat this three times.
Rinse in running tap water and air dry.
Counterstain without further fixation with Leishman stain or May–Grunwald–Giemsa stain.
Air dry and examine microscopically.

Results and Interpretation

Production of black and granular pigment indicates positive reaction.

Lipids are present in azurophilic and secondary granules of myelocytic cells. Hence, these are SBB positive. The staining becomes more intense as cells mature from myeloblast to mature forms. Basophils are generally not positive but may show bright red/purple metachromatic staining of the granules.
Lipids are present in lysosomal granules of monocytic cells. Monocytic cells show variable reactions, from negative to weakly positive.
Lymphoid cells are SBB negative. However, in ALL, less than 3% of blast cells show positive reaction.

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Myeloperoxidase (MPO) Stain : Purpose, Principle, Procedure and Interpretation

Dhurba Giri — Tue, 06 Nov 2018 14:33:07 +0000

Myeloperoxidase is a lysosomal enzyme present in the primary azurophilic granules of neutrophils, eosinophils and to a certain extent, monocytes. Lymphocytes do not contain myeloperoxidase.

Purpose of Myeloperoxidase stain

Myeloperoxidase (MPO) stain is useful for differentiating the blasts of acute myeloid leukemia (AML) from those of acute lymphoblastic leukemia (ALL). It is also used to diagnose congenital deficiency of neutrophil myeloperoxidase.

Principle of MPO stain

In presence of hydrogen peroxide, myeloperoxidase present in the leukocyte granules oxidize substrates from colorless form to an insoluble blue/brown derivative at the site of the activity. Benzidine or 3,3′-diaminobenzidine or p-phenylenediamine dihydrochloride are used as substrates.

Requirements

Fixative: 10% formal ethanol or buffered formal acetone.
Substrate: Benzidine or 3,3′-diaminobenzidine or p-phenylenediamine dihydrochloride
Buffer: Sorensen’s phosphate buffer, pH 7.3
3% Hydrogen Peroxide
Working substrate: Add 30 mg DAB in 60 ml buffer, add 120 ul hydrogen peroxide and mix.
Counterstain: Hematoxylin

Procedure of MPO stain

Fix air dried smears in formal ethanol or buffered formal acetone for 60 seconds.
Rinse thoroughly in running tap water for 30 seconds.
Cover the smear with working substrate and incubate for 10 minutes.
Wash gently with running tap water for 30 seconds.
Counterstain with hematoxylin for 3-5 minutes.
Rinse in running tap water and air dry.
Examine under microscope.

Interpretation

Myeloperoxidase is present in the primary granules of myeloid cells. Early myeloblasts are negative, with granular positivity appearing progressively as they mature.
In many cases of AML (without maturation-M1, with maturation-M2 and promyelocytic leukemia-M3), Myeloperoxidase activity has been found in more than 80% blasts. Auer rods are strongly MPO positive.
Cells of monocytic series display a less intense positive reaxtion that is characterized by fine granular deposits scattered throughout the cell.
Lymphoblasts and lymphoid cells are MPO negative.

Positive MPO Reaction	Negative MPO Reaction
Neutrophilic granulocytes except blast forms	Basophils (weakly positive)
Eosinophils	Lymphocytic cell series
Monocytes except blast forms	Erythrocyte cell series

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