Q. What stain is used for demonstrating Auer rods in myeloblasts? Myeloperoxidase or PAS?
A. The best stain for demonstrating Auer rods is the myeloperoxidase (MPO) stain. This stain highlights one of two main populations of granules in the neutrophil: the primary (or azurophilic) granules. Secondary (or specific) granules do not light up with MPO. I could never get that straight until I realized that the Primary granules were Purple (okay, “azurophilic,” but close enough). Using this differential staining, it is possible to classify neutrophil maturation into four distinct stages:
1. Myeloblast. This is the earliest committed stage of development. These cells will turn into neutrophils if you just leave them alone. They look like typical blasts (high n/c ratio, big nucleus with fine chromatin), and they may or may not have a very small number of tiny primary granules in the cytoplasm. (Even myeloblasts that do not have these granules have been shown using immunohistochemical markers to be of the myeloid lineage.)
2. Promyelocyte. This is the next stage in development. Primary granules start appearing in abundance at this stage. The cell is larger than it is at any other stage of development. I love this stage; it is my favorite stage (doesn’t everyone have a favorite stage of neutrophil development?) because it’s just so dang pretty. Huge cell, beautiful blue cytoplasm, and these gorgeous luminous purple granules. Yum. If I could eat any cell, I’d eat a promyelocyte. I think it would taste like grape candy.
3. Myelocyte. At this stage, the cell is a bit smaller, and there are lots of secondary (specific) granules around. These don’t stain with MPO, so they end up as sort of pale orangeish pink. They’re said to be fawn-colored, but I haven’t seen any fawn with that color fur. Then again, I haven’t seen many fawns. The number of primary granules is significantly less (because promyelocytes divide into two cells, which mature out to become neutrophils. This means that the number of primary granules in the daughter cells is significantly smaller than in the mama promyelocyte (due to dilutional effect). The nucleus gets a bit smaller, and the chromatin condenses a bit.
4. More mature cells: metamyelocytes (these are basically just myelocytes with an indented nucleus and a bit more nuclear condensation) and neutrophils (cells in which the nucleus has multiple lobes – at least three). These more mature cells (metamyelocytes and neutrophils) can’t be differentiated on the basis of MPO staining alone, like the three preceding cells can. You need to use other parameters, like size and shape of nucleus.
Cool article on this: Neutrophil Secondary-Granule Deficiency as a Hallmark of All-Trans-Retinoic Acid-Induced Differentation of Acute Promyelocytic Leukemia Cells. Miyauchi J, Ohyashiki K, Inatomi Y, Toyama K. Blood 1997; 90(2):803-813.
So what does all of this have to do with MPO staining of Auer rods? Well, since Auer rods are basically clumps of azurophilic granules which contain peroxidase and other stuff, the MPO stain works well on these structures. The PAS stain highlights glycogen (not myeloperoxidase), so it stains a bunch of different cell types, like red cells and megakaryoblasts, but it is not of much use when looking for Auer rods.
If you were looking for Auer rods, and you wanted to do a special stain other than MPO, you could do a Sudan Black B (SBB); it provides results identical to those of MPO. Or you could just look at the Wright-Giemsa-stained smear and forget about the special stains for the moment. The MPO and SBB are good in that they will highlight more of the Auer rods than you can see using just the Wright-Giemsa stain – but often, you see so many on just the Wright-Giemsa alone that you don’t need to bother with an MPO. Check out the Wright-Giemsa-stained Auer rods (tons of them!) in the above cell from a case of acute promyelocytic leukemia.
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