Method for Argyrophilic Inclusion Positive Erythrocyte Count:
(1) Fix a thoroughly air-dried blood smear or a Wright stained smear for 3 minutes in a solution made by diluting 150 mL of formalin (37% formaldehyde) to 500 mL with 95% ethanol.
(2) Wash the fixed smear in distilled water.
(3) Cover the smear with 3 drops of silver staining solution, made from 2 volumes of 50% silver nitrate solution mixed with 1 volume of 1% formic acid in 2% gelatin solution.
(4) Drop a 40 x 24 mm coverslip on the slide to ensure an even spread of the staining solution.
(5) Allow the stain to complete in complete darkness for 30 minutes.
(6) Remove the coverslip and rinse in distilled water.
(7) Counterstain with 1% eosin in 95% ethanol for 30 seconds.
(8) Wash with tap water.
(9) Dehydrate through increasing concentrations of ethanol, clear and mount.
(10) Examine 500 erythrocytes in well-spread areas with a light microscope at 1000x magnification, counting separately the number of erythrocytes with one or more black inclusions. Only erythrocytes with distinct black granules are counted as positive. Cells with punctate or diffuse pattern of brown granules are considered negative.
argyrophilic inclusion positive erythrocyte count in percent =
= (argyrophilic inclusion positive RBCs in 500 cell count) / 500 * 100
Method for Pocked Erythrocyte Count:
(1) Add a drop of blood to 0.5 mL of 1% glutaldehyde in 0.1 M phosphate buffer with pH 7.4, then mix.
(2) Place a drop of the diluted blood on a glass slide and then apply a coverslip.
(3) Using interference contrast microscopy with Nomarski optics at 1000x magnification, count 500 erythrocytes, noting separately those cells with one or more pocks.
pocked erythrocyte count in percent =
= (number of RBCs showing pocks in 500 cell count) / 500 * 100
estimated argyrophilic inclusion positive erythrocyte count in percent =
= (1.061 * (pocked erythrocyte count in percent)) + 0.102
Interpretation:
• normal range: 0 - 3%
• abnormal if > 3%
• for argyrophilic inclusion positive erythrocyte count, test performance compared to the pocked erythrocyte count showed a sensitivity of 88.9% and specificity of 97.1%
• both tests are more sensitive than Howell Jolly body counts
