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.

How to identify lymphocytes in a blood smear

Q. Currently I am in a residency course to finish up my training as a medical laboratory technician; for the next two weeks I’ll be doing nothing but cell differentials in the hematology lab. Today as I was skimming the abnormal slides I found that I was having some difficulty distinguishing lymphocytes (particularly plasmacytic lymphs) from plasma cells found in the peripheral blood. Any pointers? In addition, I’m having a similar issue making the distinction from activated lymphocytes and monocytes. Pesky lymphs…

A. Those are very legitimate questions and ones that trouble even people with lots of experience from time to time. The key to both of these problems (and most problems where you’re trying to distinguish one cell from another) is to look at the chromatin.

1. Lymphocytes vs. plasma cells vs. plasmacytoid lymphocytes

Lymphocyte chromatin has a unique look in that it is clumpy and smudgy at the same time. Check out the top photo of normal lymphs – there are light and dark areas (clumping) within the chromatin, but the distinction between the two is not sharp (it’s smudgy). It’s like you licked your thumb and smudged the chromatin. Okay, that’s a weird analogy, but whatever. Plasma cell chromatin is blocky and discrete; it is sometimes arranged in a “clock-face” pattern around the edge of the nucleus. Not smudgy. Plasmacytoid lymphs have the chromatin of a lymphocyte (clumpy and smudgy) but the cytoplasm of a plasma cell (eccentric nucleus with a clearing where the golgi apparatus is).

2. Reactive (activated) lymphocytes vs. monocytes

Reactive lymphocytes – particularly big ones – can look a lot like monocytes. Again, the key is to look at the chromatin. Large reactive lymphocytes are usually immunoblasts, and as such, they have a big nucleolus (or two). In the bottom photo, there is a big reactive lymphocyte (called a Downey 3 cell) on the right. These cells also have fine chromatin (it has to be fine, or you wouldn’t be seeing the nucleolus). Monocyte chromatin is more dense (no nucleoli) and has a “raked” appearance. It is like you dragged a tiny garden rake across the nucleus. Also, the nucleus is often kidney-bean or horse-shoe shaped, or at least has a nice indentation or two. In addition to the chromatin differences, there are cytoplasmic differences (though these are less consistent): monocyte cytoplasm is typically dishwater grey with tiny dust-like granules, whereas reactive lymphocyte cytoplasm is usually light blue (either pale light blue or a relatively bright light blue) and if granules are present, they tend to be larger.

It just takes time and practice. Show everything you’re wondering about to someone who’s been in the lab a while – that’s the best way to learn. Most techs – as you no doubt know – are really nice and very knowledgeable!

Microangiopathic hemolytic anemia

MAHA

We’ve talked about a whole bunch of different hemolytic anemias over the past few weeks.

We’ve gone through the main hereditary hemolytic anemias: hereditary spherocytosis (and its less-common counterpart, hereditary elliptocytosis), glucose-6-phosphate dehydrogenase deficiency, the hemoglobinopathies (like sickle cell anemia) and the thalassemias.

We’ve also talked about immune-related hemolytic anemia (warm and cold), which is an acquired hemolytic anemia.

The last main type of hemolytic anemia on our list is microangiopathic hemolytic anemia, or MAHA for short, which falls under the acquired group of hemolytic anemias. In this type of hemolytic anemia, the red cells are ripped apart by physical trauma. Often the trauma results from red cells getting snagged as they try to pass through vessels laden with fibrin strands (there are a ton of situations in which this occurs, as we’ll see). Sometimes the trauma is due to other types of trauma (like an artificial heart valve that busts a few red cells each time it closes).

Let’s take a look at the other-types-of-trauma group first because it’s a little easier to conceptualize. There are two main causes of MAHA in this group: artificial heart valves and coarctation of the aorta. They really should call this group “macroangiopathic hemolytic anemia” because the problem is in big (macro) not tiny (micro) vessels, but they didn’t ask me. In both of these causes, red cells are getting ripped up in large spaces – either by the smashing of cells within an artificial heart valve (the old ball-and-socket valves were the worst for this; the newer models are much kinder to red cells), or by the ripping apart of red cells in turbulent blood flow (as you would get in coarctation of the aorta).

The remaining cases of MAHA are due to red cells getting snagged as they try to traverse thrombus-laden vessels. There are tons of situations in which the patient starts forming fibrin at an increased rate. If you look at Robbins, or any hematology textbook, you’ll be quickly overwhelmed by the sheer number of disorders and conditions that are associated with a microangiopathic hemolytic anemia, such as:

  1. Disseminated intravascular coagulation (DIC) – a nasty condition in which there is bleeding and clotting at the same time in the patient. Lots of things can cause DIC (like malignancy, obstetric complications, trauma, and sepsis) – and it’s complicated enough that we’ll get into it in a future post.
  2. Thrombotic thrombocytopenic purpura (TTP) – a syndrome in which the patient gets little thrombi within the microvasculature anywhere in the body, but especially the CNS and kidneys. We’ve talked a little about TTP before.
  3. Hemolytic-uremic syndrome (HUS) – a disorder often related to ingestion of food (especially raw hamburger, but also spinach, other vegetables, you name it) containing E. coli 0157:H7. The bug makes a toxin that damages endothelial cells, and for some reason, the kidneys are hit the hardest.

The blood smear is where the action is in MAHA. If you look carefully at a blood smear from a patient with MAHA, you’ll see fragmented red cells, or schistocytes. Schistocytes are smaller than normal red cells, and they have points on them. There are all kinds of permutations on this theme – some schistocytes have just one point, some look like they have little horns, some just look like little ragged red cell shards. If you look at the image above, you’ll see a whole bunch of schistocytes of varying shapes.

The most specific type of schistocyte is the “triangulocyte” (that’s really the name; would I make that up?), which is, as the name suggests, a triangular fragment of a red cell. These aren’t as common as the other types of schistocytes (there isn’t a triangulocyte in the above image). If you see one of those puppies, you better figure out what’s going on with the patient.

And that’s the main point I want to make about this type of hemolytic anemia. Given all the causes of this anemia – many of which carry a high mortality – you can’t just say the patient has MAHA, and move on to the next blood smear. You have to figure out what’s causing the hemolysis (or, rather, the clinician needs to figure it out); don’t miss this one. It could be a matter of life and death.

Photo credit: Ed Uthman at http://commons.wikimedia.org/ (DIC_With_Microangiopathic_Hemolytic_Anemia_(301920983).jpg)

Hereditary elliptocytosis

hereditary elliptocytosis

Here’s a disorder that’s very similar to hereditary spherocytosis: hereditary elliptocytosis. Patients with this disorder have numerous elliptocytes (as the name says) rather than spherocytes – but many of the features of the two diseases are similar. (more…)