Question.

My methanol-fixed blood smears are not staining reliably with Giemsa. Some advice is needed, please.

Answer

Fixation of well dried (at RT) PB smears can vary from 1-10 minutes; automated systems tend to use about 1-2 minutes and use the methanol only once. For manual staining, most labs would fix for about ten minutes. Precautions  must be taken against absorption of water from humid air. The methanol is usually replaced twice daily, but more frequently at those times of the year when humidity is high.

The first sign of unacceptable water content in the fixing methanol will be the appearance of clear refractive spaces on the biconvave surfaces of erythrocytes: perhaps only a few cells per high-power field, but this will increase further as the water content increases, and eventually the films will lose all diagnostic value. Replacement of the methanol when you see more than say 1-2/HPF might not be a bad idea. This artifact may also be seen in some automated systems where the stain pack is not turned over very quickly. Rather than replacing the stain pack, economy of reagent can be maintained by manually fixing the slides before thay go on the machine. This is particularly so for the older Hematek grey models.

Caution. Longer fixation times are required for bone marrow smears: 15-20 minutes, and always use fresh methanol for these.

Most persons using Giemsa prefer to stain the smear first with May Grunwald or Jenner stain, either using it neat or diluting 1:2 with buffer. This pre-step improves the granule definition and clarity, and also changes the traditional reddish purple of nuclei with plain Giemsa to a blue purple as seen with Wright's stain.

The selection of Sorensen's buffer will vary form 6.4-7.2, with the lower pH being most popular with Wright's rather than Giemsa. The aim is to select a pH that produces a colour balance that readily allows the user to differentiate between normochromic and polychromic red cells and to distinguish toxic granulation when present, this is usually pH 6.8. If looking for malarial parasites, then a pH of 7.2 is preferable because it allows better contrast to detect chromatin dots, trophozioites etc.

Dilution of the Giemsa solution is best done immediately before use and will vary from 1:8 to 1:12 depending upon your protocol. As a general rule of thumb the higher dilutions require longer staining times of about 20 minutes, and the less dilute stains need between 6 and 12 minutes, depending upon tthe quality of the Giemsa. It was frequently claimed that the longer times gave better definition, but I must admit that I've seen short timed smears that are every bit as good.

For many years good quality Giemsa would be stable after dilution for 6 to 8 hours. For the last 2 or 3 yrs, however, the best you can hope for is 3 to 4 hours. After dilution the solution starts to deteriorate, with the appearance of floccules and a subsequent loss of staining ability or strength. As the time progresses you may need to compensate by increasing the staining time, but after 3 hours you will need to replace it.

Recipes for Giemsa vary, whether it be that of Hayhoe or of Dacie & Lewis, and measurements may be by weight or volume. Stock solutions that have a 50% by volume content of glycerol (Analar or USP) are the most stable. Under no circumstances ever heat your glycerol to more than 45C, even though most texts say 56C. Above these temperatures there is a risk of oxidation, even in the stock solution, I use 45C as a cut-off point to give me a safety margin. Dye content will also vary from 0.45 to 0.8%. Lillie's comments should considered here. After standing for up to 5 days, filtration to remove undissolved material is essential.

Differentiation, by giving the slides two rinses in buffer of two minutes each, is fairly standard, but you can overdo it. A single rinse of three quick dips may in fact suffice. It will depend upon your Giemsa solution and tastes. If overstaining is a problem then consider adding methanol to your buffer rinse, starting at 5% and adjusting according to results, followed by a water rinse to remove solvent.

Mike Rentsch, "Histomail," Downunder