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

1. Dey, P. (2018). Basic and advanced laboratory techniques in histopathology and cytology. Springer Singapore.
2. 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.
3. Product Information May-Grünwald Giemsa, Avantor Performance Materials.

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]]> https://laboratorytests.org/may-grunwald-giemsa-stain-principle-preparation-and-procedure/feed/ 0 https://laboratorytests.org/reticulocyte-count/ https://laboratorytests.org/reticulocyte-count/#comments Tue, 06 Nov 2018 06:38:48 +0000 http://laboratorytests.org/?p=273 Reticulocytes are immature red blood cells (RBCs) with considerable quantities of ribosomal and mitochondrial RNA. Normally, there are a small number of reticulocytes in the peripheral blood which remain for 24-48 hours during maturation. Reticulocyte [...]

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]]> Reticulocytes are immature red blood cells (RBCs) with considerable quantities of ribosomal and mitochondrial RNA. Normally, there are a small number of reticulocytes in the peripheral blood which remain for 24-48 hours during maturation.
Reticulocyte count is the test for determinigg bone marrow function and evaluating erythropoietic activity. It is used to classify and moniter therapy for anemias. Reticulocyte number increases when there is an increase in erythropoietic activity.

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

Reticulocytosis	Reticulocytopenia
Condition where there is an increase in reticulocytes. Such as: Hemolytic anemias: Immune hemolytic anemia Primary RBC membrane defects Sickle cell diseases Enzyme defects Exposure to toxins Following hemorrhage Following treatment of anemias Physiologic increase in pregnancy and infants.	Condition where there is an decrease in reticulocytes. Such as: Iron deficiency anemia Aplastic anemia Radiation therapy Untreated pernicious anemia Tumor in bone marrow

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