How to remember which genes are tumor suppressors vs. proto-oncogenes

molecular neoplasiaHere’s a question that I got by email yesterday and it’s such a good one that I want to share it with everyone.

Q. I love love LOVE your blog and your daily emails, and your book Clot or Bleed saved my butt for studying for my hematology exam. I was just wondering – do you have a good mnemonic or know of an easy way to remember which cancer gene mutations are proto-oncogenes and which are tumor suppressors? 

A. Thanks for letting me know you found the book and other stuff helpful! I’m so glad to hear that.

I don’t have a mnemonic for these genes (if anyone does, please comment below!). However, I think the best way to remember these is to learn what each gene product does (because then you’ll know whether it’s a proto-oncogene or a tumor suppressor gene).

For example: RAS encodes a signal-transducing protein associated with cell growth. It takes the signal from a growth receptor, and helps get that signal down to the nucleus so something can be done about getting the cell to grow. So it’s a growth-promoting gene (a proto-oncogene). If RAS is going to be a cancer-causing gene (an oncogene), it is going to have to be mutated in such a way that it is always turned on.

Here’s another example: the retinoblastoma (RB) gene. The RB gene product inhibits the cell cycle, turning off normal cell growth when necessary. So it is a tumor suppressor gene (this is a dumb name, but we can save that tirade for another time). If you’re going to cause cancer by mutating the RB gene, you’d have to mutate it in such a way that it doesn’t work well (and cells can just whiz through the G1/S checkpoint no problem). And actually, kind of like the brakes on your car, you typically need to mutate BOTH alleles of a tumor suppressor gene in order to cause tumors (if you just mutate one allele, you’ll still have some “brakes” left in the other allele).

Here is my favorite diagram (above) relating to this topic. It’s from Robbins, and it’s got all the important cancer-related genes (or at least the most important ones for you to learn) listed according to what their products do in the cell. Yay! You can see RAS up there just beneath the cell membrane, doing its job as a signal transducer. RB is down in the nucleus, acting as a cell cycle inhibitor.

A nice touch in this diagram is the color coding: all the red things are growth-promoting (so their genes would belong to the proto-oncogene category). Blue things are growth-inhibiting (so they would be encoded by tumor suppressor genes). The green things function as DNA repair mechanisms (the nice little scissors and hammer). If you look at this diagram long enough, you’ll start remembering which color things are – and if you freak out on a test, remembering the color just might get you grounded again.

I am sure that someone does have a snazzy mnemonic. But I figure that since you’re going to have to learn what these gene products do, you might as well just reason out which ones are proto-oncogenes vs. tumor suppressor genes, rather than try to memorize that list separately using a mnemonic.

It’s always best when you can get material to make sense! Cuts waaayyy down on the brute memorization – and also helps get the info into long-term memory. Maybe save the mnemonic strategy for stuff that you can’t reason out, like cranial nerve numbers, or clinical syndromes that don’t make sense, or well, pretty much all of micro.

How to read a blood smear

When you look at a blood smear, it’s best to have a plan, and it’s best to try to follow it each time.

That might sound boring – but you’ll make a much more accurate and complete assessment that way. Otherwise, the temptation is to just put the slide under the microscope, scan around to see if you see anything weird, and then focus on that (while missing some important features).

There are 10 main things you need to be sure to evaluate on a blood smear. I like to start with the red cells, move to the platelets, and save the white cells for last…but you can come up with whatever method suits you.

1. Red blood cell number

First, make sure you’re in the right part of the smear. You should be a couple medium-power fields in from the “feather edge,” which is the thin edge of the smear where the cells are all spread out and there are huge empty spaces. Just give it a quick glance and make sure the red cells aren’t either piling up all over each other, or spread out too far with lots of holes in between – like the red cells in the image above. Take a look at the RBC on the CBC and make sure it fits with what you’re seeing.

2. Red cell size

Normally, if all the red cells are roughly the same size, your eye won’t be able to tell if they’re microcytic (small) or macrocytic (large). So you have to just look at the MCV for that. What your eye can see, however, is a range of sizes. So take note and see if there are some cells that are smaller, and some that are bigger. If that’s the case, it’s called “anisocytosis” and it should be reflected in the RDW (red cell distribution width) on the CBC. The more anisocytosis (variation in size) there is, the bigger the RDW should be.

3. Red cell shape

Normally, red cells are all nice and round, like the ones in the image above. In some anemias, there are funny-shaped cells, like schistocytes (fragmented red cells), sickle-shaped cells, teardrop-shaped cells, or target cells. Your eye will naturally be drawn to these (which is why you should force yourself to follow a consistent method when looking at a smear – otherwise you just look at what your eye is drawn to!). Take note of whether there are any non-round cells, and if so, describe what kinds of shapes you see.

4. Red cell chromasia

“Chromasia” refers to the amount of hemoglobin in the average red cell. Normally, there is a zone of central pallor (the white dot in the center of the cell) that comprises about 1/3 of the diameter of the cell. Check out the cute zones of central pallor in the red cells above. These cells are called “normochromic.” If there is a huge white dot, and just a thin rim of hemoglobin, then the cells are called “hypochromic.” There really isn’t a “hyperchromic” type of red cell. Some sources call spherocytes hyperchromic – but I don’t really like that, because when we talk about chromasia, it’s almost always for the purpose of sorting out anemias (some are normochromic, some are hypochromic, and there are none that we refer to as hyperchromic). So there.

5. Reticulocytes

Take a look around and see if you see any polychromatophilic cells (these are slightly bigger than normal red cells, and they have a lilac tinge to them). These are just young red cells whose RNA has not yet been completely extruded (so they stain a bit blue). In normal blood, about 1% of the red cells are reticulocytes (because we’re always making new red cells). That equates to about 1-2 red cells per field. If you see more than that, it means the marrow is kicking out red cells at an increased rate.

6. Stuff inside red cells

Take a look and see if you see any red cells with stuff inside – like nuclei, Howell-Jolly bodies (little nuclear remnants that didn’t get extruded), Pappenheimer bodies (little iron granules), organisms (like malaria or babesia).

7. Platelet number

There should be between 7 and 21 platelets per high power field, which corresponds to a platelet count between 150 and 450 x 109/L.

8. Platelet morphology

This doesn’t usually yield much – but take a look at the platelets anyway and make sure they’re roughly of normal size, and have some nice granules inside. There are rare platelet disorders in which the platelets are abnormally large, or lack granules, or both.

9. White blood cell count

Do a quick scan of a bunch of high power fields and see how many white cells there are. There should be a few white cells per high power field. Check the WBC and see if it seems to correspond to what you’re seeing. Then, do a differential count: count a few hundred white cells (500 is best) and put them in categories (neutrophils, lymphocytes, monocytes, eosinophils, basophils). Compare this to the automated differential on the CBC, and multiply the percentages by the total WBC to get the absolute counts of each cell type. When you’re trying to determine if a patient has a normal number of a certain cell type, absolute counts are much more reliable than percentages.

10. White blood cell morphology

Finally, check the morphology of the white cells. You’ll probably do this as you’re doing your differential – your eye will be drawn to any abnormalities as you’re classifying the cells. Make sure the neutrophils and lymphocytes look normal, and keep your eye open for any weird-looking cells like blasts or circulating carcinoma cells.

Bottom line

Whatever order you decide to use, if you do it the same way each time, it will start to become automatic – and you’ll be much more likely to do a thorough, accurate job.

How to identify normal leukocytes in a blood smear


Q. I’m not sure if I can identify leukocytes correctly. Could you give me some tips? Thanks very much.

A. Sure! When you are just starting out in hematopathology, it can be a bit overwhelming. It’s really not as difficult as it seems. There are just 5 kinds of cells that you see in normal peripheral blood, and with a few guidelines, you can tell them apart pretty easily.

Neutrophils
These are the most numerous white cells in normal blood. There are two in the above image (from WebPath), one at about 3 o’clock and one at about 10 o’clock. They are part of a category of white cells called “granulocytes,” which refers to the cytoplasmic granules you see in these cells. In neutrophils, the cytoplasmic granules are mostly small, pale peachy-pink granules. These granules (called “specific granules”) are what give the neutrophil cytoplasm its pinkish color. There are also scattered larger, dark purple (or “azurophilic”) granules. These are called “primary granules” because they are the granules that appear first as the neutrophil matures. If you forget the thing about neutrophil maturation, you can remember which is which by remembering that the p words go together (primary=purple).

The nucleus of a normal neutrophil is also unique-looking. It’s segmented – pinched off into different sections, like sausage links – rather than round, like most other cells. Neutrophils are sometimes called polymorphonuclear leukocytes because there are several (poly) bodies (morpho) in the nucleus (nuclear). Rarely, you might see a “band” cell (which is the neutrophil at 10 o’clock), which is the stage of neutrophil right before the nucleus becomes segmented. Neutrophil chromatin in general is clumpy, and you can’t see any nucleoli.

Lymphocytes
These are the second most numerous type of white cell in normal blood. There’s one lymphocyte at 8 o’clock in the above image. Lymphocytes are generally a bit smaller than neutrophils, and the thing that sets them apart is their chromatin, which is both clumpy and smudgy at the same time. It looks like someone took a finger and rubbed the nucleus before the ink fully dried. Although there are clumps in lymphocyte chromatin, there aren’t discrete white spaces between the clumps, like you see in neutrophil chromatin. Check out the above image and you’ll see what I mean. Sometimes you’ll see lymphocytes that are a bit larger, with more cytoplasm and maybe a few coarse granules. T cells often have this appearance (though you really can’t tell for sure without doing some special studies).

Monocytes
Monocytes are big cells (there’s one at about 8 o’clock above) with lots of cytoplasm. The cytoplasm often has a “dishwater” appearance, meaning it is sort of cloudy and grayish. Sometimes, as in the cell above, it’s more of a pale purple color. You can see some fine purple granules scattered about as well. The nucleus is big and it’s usually indented, or horseshoe shaped. The chromatin is pretty fine (finer than neutrophil or lymphocyte chromatin), and it has a weird “raked” appearance on high power (it looks like someone messed up the chromatin by dragging a rake across it).

Eosinophils
These cells, along with basophils, are probably the easiest to spot (there’s an eosinophil at 2 o’clock above). Both eosinophils and basophils are granulocytes. The granules in eosinophils are beautiful – they are large, luminous, and reddish-orange. The word eosin comes from the Greek word eos, which means “flush of the dawn sky.” Very cool name for these gorgeous, sunrise-colored granules. The nucleus is nothing to write home about, really – it’s segmented into a few different parts, and it looks kind of like a neutrophil nucleus.

Basophils
You can tell a basophil from a mile away: it’s the cell with the big, super-dark-purple-blue granules (there’s one at 4 o’clock above). The granules are so numerous and dark that they often obscure the nucleus (which is a rather boring nucleus, usually divided into two segments). Basophils are the least numerous of all white blood cells – you may have to look several fields to find one.

And that’s it! When you start looking at different diseases (like infection, or leukemia), it gets a bit more complicated, because you often see immature cells out in the blood. But for now, you can just focus on the normal, mature white cells. Once you get familiar with these, they start looking like little friends that you happily recognize from across the street.

Why does the GFR go down in nephritic syndrome?

Q. I have a question. Why do you see a decreased glomerular filtration rate in nephritic syndrome? I read on your blog and other places that it’s due to “hemodynamic changes.” From Robbins I’m assuming this is compensatory stuff – but wouldn’t that increase GFR?

A. It’s because of what’s going on in the glomerulus! In a normal glomerulus, the capillaries are all nice and open and patent. Blood flows through the capillaries like a little river, fluid gets filtered out into the urinary space, and the GFR is normal. But in nephritic syndrome, the glomeruli are stuffed full of cells, and blood flow slows way down.

Take a look at post-streptococcal glomerulonephritis, a common cause of nephritic syndrome. In that disorder, the glomeruli are huge and hypercellular, with tons of neutrophils in there (and probably some other proliferating glomerular cells as well). The poor capillaries are compressed by all that extra stuff, and you can imagine how hard it is for the poor blood to flow through there! If the blood can’t flow through at the same rate, then the filtration of fluid from blood into urine is decreased (and the GFR slows down to a sad little dribble).

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!