How do you identify all those neutrophil precursors?

Q. I’m having a hard time identifying some hematopoietic cells on smears. I have no trouble with the mature PMNs, bands, and metas, but once I get past that, it gets rather difficult. On some slides, if you don’t know the diagnosis, it can be so difficult to determine if that cell is a promyelocyte, smaller monocyte, or even a lymphocyte (if there isn’t a lot of granules). Do you have any suggestions?

A. Your question is a very good one, and I can give you a few tips that might help. A couple caveats though: some of this stuff comes with experience and with development of your “eye”; also, sometimes even seasoned hematopathologists will disagree on the nature of a cell (especially if it is malignant).

Starting with the neutrophil series, here is the way I would describe each cell type, starting at the beginning of development:

Myeloblast
This cell is a typical “blast,” meaning that it has a high nuclear-cytoplasmic ratio (a large nucleus relative to the overall size of the cell) with fine (not clumpy) chromatin and nucleoli. There is a thin rim of somewhat basophilic cytoplasm, and there may be very few fine, azurophilic, cytoplasmic granules (or none at all).

By the way, there is nothing that distinguishes a myeloblast from other hematopoietic blasts – unless it is a malignant myeloblast and you see an Auer rod in it! Unlike monoblasts, which tend to be larger blasts with abundant cytoplasm, or megakaryoblasts, which sometimes show some cytoplasmic blebbing, or erythroblasts, which are often perfectly round and have deep dark blue cytoplasm, myeloblasts are pretty “bland” and don’t have any distinguishing features. That being said, if you are looking at a normal marrow, and you see cells fitting the above description, you count them as myeloblasts. For some reason, you don’t see lymphoblasts in a normal marrow, and the other blast types are very rare. The difficulty comes when you’re looking at a case that might be a leukemia; then it can be hard to tell apart normal myeloblasts from malignant blasts (myeloid or otherwise).

Promyelocyte
This is the biggest cell in the neutrophilic series. It’s my favorite cell because it’s just so beautiful. The identifying feature of this cell is its granules, which are coarse (not fine), purple (also called azurophilic), and located in the cytoplasm as well as overlying the nucleus. These granules are called “primary” because they are the first of the two types of neutrophilic granules to appear as the cell matures. You might remember this by associating “primary” with “purple” and “secondary” with “specific” (see the myelocyte, next).

The cytoplasm in the promyelocyte is generally pretty basophilic. It’s medium to deep blue, and very pretty. The nucleus is still immature – you can still see nucleoli in it – but the chromatin is starting to become a little more coarse.

Myelocyte
The hallmark of this cell is the appearance of secondary, or specific, granulation. These secondary granules are smaller and finer than primary granules, and they are said to be “fawn-colored,” a description which I find a bit problematic, since I think of fawns as brown. To my eye, the granules are more peachy-pink. They are usually not very distinct from one another, and sometimes they just appear as sort of a “blush” in one region of the cytoplasm. As soon as you see any of this secondary granulation at all, even if it’s very faint, you must call the cell a myelocyte (it’s no longer a promyelocyte).

The myelocyte is smaller than the promyelocyte, and it has coarser chromatin (so coarse that you can’t see any nucleoli). There are less primary granules than there are in the promyelocyte (because at the promyelocyte stage, the cell can still divide – a process which dilutes out the primary granules among daughter cells), and as the myelocyte matures, the granules become finer.

In the photo above, the fourth cell from the left is an early myelocyte. If you look closely at the cytoplasm, you’ll see that there is some specific granulation (compare it to the band to its left, and the neutrophil below). As the myelocyte matures, it will become smaller, and the primary granules will become finer.

Metamyelocyte
This cell looks very much like a mature neutrophil except for its nucleus, which is larger than that of a segmented neutrophil, and which usually has an  indentation (it’s said to be kidney-bean-shaped). The cytoplasm looks like that of a segmented neutrophil (lots of specific granulation, very little primary granulation).

Band
A step beyond the metamyelocyte, the band is a bit smaller than the metamyelocyte, and the nucleus is thinner and, well, band-like. Often the nucleus appears to be U-shaped. There are no segments to the nucleus. In the photo above, the cell farthest to the left and the cell third from the left are bands. The cell farthest to the right might also be a band, with its nucleus folded over on itself (or it might be a late metamyelocyte; sometimes you catch a cell right at the junction between two stages and it’s hard to classify it as one or the other!).

Segmented neutrophil
This cell has a weird nucleus, as you know: it has hot-dog-link segments with pinched, narrowed strands of nucleus between the segments. It should have about 3-4 segments; much more than that and it becomes “hypersegmented,” a finding typically seen in megaloblastic anemia. In the photo above, the second cell from the left and the second cell from the right are both segmented neutrophils. The one at left only appears to have two lobes, which is weird. Either the remaining lobe/lobes are tucked underneath, or the patient has a disorder in which the neutrophils are not segmenting properly. You’d have to look at the rest of the smear to figure that one out.

As for monocytes and lymphocytes, there are a few things that can help you identify those cells:

Monocyte
This cell is a large cell with abundant cytoplasm and an indented or irregularly-shaped (not round) nucleus. The cytoplasm does not have specific granulation in it (which should help you distinguish it from myelocytes and metamyelocytes); it is sometimes called “dishwater” cytoplasm because it looks like a dirty gray with a hint of blue in it. Sometimes there are very fine granules and/or a bit of vacuolization in the cytoplasm.

The distinguishing feature, however, is the chromatin, which has a “raked” appearance. It looks like you took a rake and dragged it lightly across the nucleus. This is different from neutrophil chromatin, which has a “blocky” look as the chromatin matures, or lymphocyte chromatin, which is smudgy and clumpy (see below). These distinctions can be subtle, and you only get good at seeing them with experience (looking at hundreds of blood smears helps!).

Lymphocyte
Mature lymphocytes, like those you see in normal peripheral blood, have distinctive chromatin. It is both smudgy and clumpy. It looks like you took your thumb and smudged the nucleus before it dried. Sounds weird, but if you look at the chromatin of a typical lymphocyte next to that of a typical segmented neutrophil, you’ll see the difference. The chromatin of the segmented neutrophil has pretty distinct, well-defined clumps in it; the spaces in between the clumps are light in color. The chromatin of the lymphocyte also has clumps, but they are indistinct and they blend together, giving a more smudgy rather than blocky appearance.

One final bit of advice
One thing that sometimes helps if you are having trouble with a smear is to keep looking around at the cells until you get a feel for how the cells look. Find a cell that you know for sure is of the neutrophilic series, and compare it to the other cells in the smear. Do the same thing with the monocytic series (or any other series you’re having a hard time with): find a cell you can identify for sure, and then keep that “look” in mind as you’re going through the smear. It sounds funny, but every blood or bone marrow smear has its own subtle distinctions, which may have to do with the patient, or the way the smear was made, or the quality of the stain on that particular day. You have to get used to looking at each blood smear on its own and get familiar with the cells in it like they are little friends’ faces. After you look long enough, you can usually start seeing subtle differences between cells that were inapparent at first.