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.
Q. I’m will be starting my pathology residency in about a year. Any suggestions for getting prepared for residency? I have been reviewing www.enjoypath.com and others, but wanted to get your opinion.
A. Good for you! When many people think of pathology (do many people think of pathology?), they think of surgical pathology – stuff that comes out of the operating room, biopsies, etc. But there are many other parts to a pathology residency, such as hematopathology, microbiology, forensic pathology, and blood banking. I’ll run through some of the books used in these areas, then I’ll tell you what I would have done if I knew then what I know now.
Surgical pathology: Rosai’s Surgical Pathology is probably the most commonly-used book; another good source is the set of AFIP Fascicles (there’s a fascicle on pretty much every organ system). These sources are too in-depth for you now (with one exception that I’ll mention in a minute), and probably too expensive. They’re more for reference than for reading through on a Sunday night. You’ll use them until you’re nauseated when you’re a resident though.
Hematopathology: The best source for this is the AFIP Fascicle on the subject: Tumors of the Bone Marrow. This is the exception to what I said above about reading the fascicles before residency – this one would be great to go through ahead of time. There’s a lot to learn, and if you go through it once, it will make a lot more sense when you get to it in your residency. It’s small enough that you can certainly get through it in a few months.
Microbiology: We used Koneman in our residency program, and I think it is a good textbook. It’s more than you’d want to go through ahead of time though; I’d use something like Clinical Microbiology Made Ridiculously Simple. It has nice drawings and mnemonics, which is something you need in microbiology.
Forensic pathology: A couple good ones for this are put out by DiMaio: a textbook (long) and a handbook (short).
Blood banking: We used McCullough’s Transfusion Medicine text in residency. Nice and short and readable. Here’s a fun game that you might want to try too.
I think if I had it to do over again, I would do three things:
1. Read Robbins. All of it. Maybe twice. I know, I know, it is a “med-school” textbook, but we used it all the time in residency. So did the attendings at times, by the way. It’s no small feat, but should be possible in a year, and it would prepare you well. You might even take notes on the histologic appearance of different tumors and diseases; you would have those to refer to during residency. You can look at websites too (like Webpath and Ed’s Pathology Notes) – and you should – but Robbins will give you a systematic and thorough review.
2. Read the AFIP bone marrow fascicle. I actually did this before my med school rotation in hematopathology, and I was so glad I did. It will make you shine when you get to your rotation.
3. Not worry about the other stuff. The other rotations will be easy enough to go through without advance preparation.
Good luck!
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!
Q. Can you write a post about antiphospholipid syndrome? I could not find good source which explains its pathophysiology and laboratory results
A. First, before we get into the antiphospholipid syndrome, we need to talk about antiphospholipid antibodies. As you might expect from their name, antiphospholipid antibodies are autoantibodies in the patient’s plasma that are directed against various phospholipids (there are lots of phospholipid surfaces in the body – including the phospholipid surface upon which the coagulation factors interact). There are a bunch of different types of antiphospholipid antibodies, including anticardiolipin antibodies, anti-glycoprotein antibodies, and the so-called lupus anticoagulants (which were discovered in patients with lupus).
In addition to binding to various phospholipid surfaces in the body, these autoantibodies also just happen to bind to the phospholipid part of the PTT reagent (and sometimes, the PT reagent). Then there’s not enough usable reagent in the test tube, and the patient’s specimen doesn’t clot! The coagulation tests are therefore falsely prolonged.
Antiphospholipid antibodies are sometimes called “inhibitors” because they appear to inhibit coagulation in the test tube. But here’s a weird thing: in the body, they can be associated with thrombosis!
You may be asking yourself: how do you get these antiphospholipid antibodies? And are they dangerous?
It turns out there are different answers for different patients. Children, for example, sometimes develop antiphospholipid antibodies after an infection. In this setting, the risk of thrombosis is only slightly increased; it’s usually not a big deal clinically. Adults sometimes develop antiphospholipid antibodies as part of an autoimmune disorder like lupus (in fact, antiphospholipid antibodies – in whatever clinical setting – are often called “lupus inhibitors” because of this association). In these patients, the risk of thrombosis is moderately increased. Finally, elderly adults may develop antiphospholipid antibodies in association with drugs. This is virtually always a harmless event with no increased risk of thrombosis.
Okay, so here’s where we get to the antiphospholipid antibody syndrome part. This term is used when a patient with an antiphospholipid antibody has thromboses or pregnancy-related complications (like recurrent miscarriage, pre-term labor, or pre-eclampsia). This syndrome is a serious thing. In a small number of patients, the thromboses can be widespread, leading to multi-organ system damage and death. The term is reserved for patients who are symptomatic; you wouldn’t use the term in patients who have an antiphospholipid antibody but no symptoms.
So what would you do if you think your patient might have an antiphospholipid antibody? Well, you’d need to confirm this suspicion with laboratory tests. First, order a PTT (in fact, that’s how a lot of these patients get picked up – they present with an abnormally prolonged PTT in the face of clinical evidence of thrombosis).
Then, order up a mixing study. Remember what a mixing study is? You do this test when you have a patient with a prolonged PTT and you want to know why. It’s performed by taking the patient’s (probably abnormal) plasma and mixing it with some pooled (normal) plasma – then running the PTT on this new mixed sample.
If the new PTT value is within the normal range (if it “corrects”), then you know the pooled human plasma must have supplied something to the patient’s plasma to make it clot normally. The “something” is usually a coagulation factor that the patient is missing.
If the new PTT value is still abnormal (if it’s still prolonged, and doesn’t “correct”), then you know that even though you added a bunch of normal plasma to the mix, the patient’s plasma still couldn’t clot normally. There must be some other problem with the patient’s plasma. The “other problem” is usually an inhibitor.
One caveat: some antiphospholipid antibodies do not prolong the PTT. It all depends on the particular PTT reagent your lab is using (some reagents are just more easily swayed by the antiphospholipid antibodies). So if you really feel your patient may have an antiphospholipid antibody, you shouldn’t stop investigating that possibility just because the PTT comes back normal! There are plenty of fancy lab tests that can be done to detect antiphospholipid antibodies. Just call your friendly neighborhood pathologist and see what he/she has to offer.
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