Cellular iron staining assay
Cellular iron staining assay
Ferritin and ferritin (extracellular iron) in bone marrow and iron particles (intracellular iron) in naive erythrocytes interact with potassium ferrocyanide to form ferric ferrocyanide in a hydrochloric acid environment, i.e., the Prussian blue reaction. This experiment is based on the experiment guide of Mudanjiang Medical College undergraduate 5-year testing program.
Operation method
Cellular iron staining assay
Principle
Ferritin and ferritin-containing hemoflavin (extracellular iron) in the bone marrow and iron particles (intracellular iron) in naive erythrocytes interact with potassium ferrocyanide in a hydrochloric acid environment to form ferric ferrocyanide, known as the Prussian blue reaction.
Materials and Instruments
Hydrochloric acid solution Potassium ferricyanide solution Alkaline compound red storage solution Compound dye application solution Formaldehyde Move I. Experimental reagents: Caveat l. The equipment used must be clean and previously de-ironed, especially the slides. 2. the application solution for the potassium ferricyanide-hydrochloric acid reaction must be prepared ad hoc. 3. for extracellular iron examination, a smear containing bone marrow pellets should be used. 4. Intracellular iron counts should be performed on medium and late-stage erythrocytes. Intracellular iron should be counted in medium- and late-stage erythrocytes. For more product details, please visit Aladdin Scientific website.
1. 4% hydrochloric acid solution, carefully take 40 ml of concentrated hydrochloric acid (38%) and slowly add it to 340 ml of cold distilled water with stirring while adding, and keep this solution at room temperature for several months. 2. 40 g/L potassium ferrocyanide solution (can be kept at room temperature for several months). 3. Basic Fucbsin storage solution: Dissolve 1 g of Basic Fucbsin in 10 ml of anhydrous acetic acid and 5% carbonate solution and mix with 90 ml. 4. Mix with 90ml of anhydrous acetic acid and 5% carbonic acid solution. 4. Application solution for re-staining: Add 3ml of the above Basic Fucbsin storage solution to 100ml of distilled water and keep at room temperature for several days. 5. 37% formaldehyde.
Laboratory operation:
l. Dry the blood and bone marrow slices in the air, and fix the smear in formaldehyde vapor for 2-3min.
2. Mix equal amounts of 40g/L potassium ferricyanide and 4% hydrochloric acid solution, freshly prepared and heated to about 56°C. Place the fixed blood or bone marrow smears to be examined into it for 30 minutes and then remove and rinse with running water.
3. Place the re-staining application solution in the re-staining for 5min, rinse with distilled water, anhydrous ethanol rinse, distilled water rinse, and finally dry in the air and then examined with a microscope. IV. Experimental results to determine: iron can be stained into blue particles, small beads and small pieces.
Extracellular iron: observe the incompletely unfolded bone marrow granules with low magnification first, then with oil microscope.
"-" No blue iron particles visible
"+" A few iron particles or only few iron microparticles are present
"2+" Many iron particles and beads and a few small pieces.
"3+" There are many iron particles or beads and a few small pieces.
"4+" There are a lot of iron particles and beads. and many small pieces.
Intracellular iron: count 100 nucleated erythrocytes, record the percentage of positive cells (blue particles in the cytoplasm) and note the number of intracellular iron particles, size, staining, etc., with or without ring-shaped iron particles of erythrocytes. Reference value: normal human bone marrow smear in the extracellular iron is a small amount to more iron particles, iron beads, most people are "+", a small number of people for "2 +".
Young erythrocytes are positive for iron particles at a rate of 12% to 14%. Most of the middle and late juvenile cells, most of the positive juvenile erythrocytes contain 1-2 small and irregular iron particles, only a few contain 3-5 iron particles, generally do not see more than 5 iron particles in the juvenile erythrocytes and ring-shaped iron particles in juvenile erythrocytes.