Pathology Student

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!

One of the reasons our cells die is because they are inherently programmed to have only 60 to 70 doublings. That’s it. After that, they die.

Why is that?

Turns out, we have things called telomeres that protect our DNA. Telomeres are highly repetitive DNA sequences that protect the end of the chromosome during replication. During cell division, the enzymes that duplicate the chromosome and its DNA can’t continue their duplication all the way to the end of the chromosome. If there wasn’t a fix for this problem, then every time the cell divided, it would lose a bit of DNA at the end of the chromosome! Bad idea.

Telomeres protect the DNA by allowing the enzymes to travel all the way to the end of the important DNA at the end of the chromosome. The telomere doesn’t have any important genetic data in it; it’s just a disposable lengthener that is tacked onto the end of the chromosome. Each time the cell divides, the telomere gets a bit shorter. Eventually, it’s so short that it may as well not exist, and the original DNA starts getting chopped off during replication. The cell realizes that there are errors in DNA replication, and the cell cycle is arrested (via p53 and RB), leading to apoptosis.

So how come stem cells and cancer cells are able undergo an unlimited number of doublings? It turns out that they are able to replenish their telomeres after DNA replication. An enzyme called telomerase restores the telomeres to their original length, avoiding the progressive shortening and eventual cell cycle arrest that normal cells undergo.

As you might expect, the length of one’s telomeres is intimately related to the aging process. Several age-related diseases, including heart disease, are related in part to shortened telomeres. In fact, you can estimate a person’s age from the length of their telomeres! Not much use in every day life, but very useful in forensic pathology.

Perhaps Ponce de León’s fountain of youth was really just a bath spiked with telomerase.

Photo credit: Storm Crypt (http://www.flickr.com/photos/storm-crypt/3321856585/), under cc license.

Yesterday we talked about how cancer is caused by non-lethal genetic mutation. We mentioned the six particularly nasty features that cancer cells acquire when they undergo genetic mutation: autonomous growth, insensitivity to growth inhibitory signals, evasion of apoptosis, limitless replication, sustained angiogenesis, and invasion/metastasis. And we talked about the four types of genes that commonly get mutated in cancer: genes that promote growth (proto-oncogenes), genes that inhibit growth (tumor-suppressor genes), genes that regulate apoptosis, and genes involved in DNA repair. Now let’s talk about three specific genes that are commonly mutated in cancer.

1. RAS gene
This gene encodes a signal transduction protein (the RAS protein) that takes the signal from a growth receptor and transduces it to the cell nucleus so that the cell can proliferate. It’s a normal protein, just like all the others in this list; we all have it in all of our cells. It’s a proto-oncogene – meaning it’s necessary for normal cell growth and proliferation. But when it gets mutated in such a way that it’s always on (at this point it’s called an oncogene), then it’s always transducing signals, and the cell is always proliferating…which is great if you’re a cancer cell because then you’re growing like crazy. The RAS gene is mutated in up to 30% of all human tumors. It’s particularly commonly mutated in colon and pancreatic cancer.

2. Retinoblastoma gene
This gene encodes a protein that stops cells at the G1 checkpoint in the cell cycle. This is important – the cell needs to stop and see if all the DNA is okay before it progresses to mitosis. It is like a little set of brakes for the cell cycle; it falls into the tumor-suppressor gene category. If you mess up this gene (actually, you have to mess up both copies in order to get cancer), you remove the brakes from the cell cycle, and the cell proliferates like crazy. The RB gene is mutated in most cancers!

3. p53 gene
This gene is nicknamed “guardian of the genome” (kinda like the guy in the image above). It does a couple very important things. If a cell’s DNA gets damaged, the p53 protein causes a pause in the cell cycle (via RB!) so the DNA can get repaired. If the DNA gets repaired, fine. If the damage is irreparable, however, p53 tells the cell to kill itself (by apoptosis). Most human tumors have p53 mutations! It’s so critical to the health of the cell’s DNA – you can see why you’d almost have to have a mutation in it in order to get cancer.

Image credit: Lachlan Hardy (http://www.flickr.com/photos/lachlanhardy/2907399418/); under cc license.

There are different ways to answer this important and difficult question. If the underlying question is “What are the agents that can cause cancer?” then the short answer is: 1) chemical substances, 2) infectious agents, and 3) radiation. If the underlying question is “What are the molecular mechanisms that change a cell from a benign one into a malignant one?” then that’s an entirely different topic. We’ll address the first topic – cancer-causing agents – in a later post. For now, let’s take a quick look at the molecular mechanisms involved in cancer formation.

Bottom line
If you want to be pithy, you can summarize the underlying molecular cause of cancer in four words (and one hyphen): non-lethal genetic damage. In other words, a cell acquires (or is born with) a genetic mutation – and that mutation does not lead to cell death. Normally, if a cell’s DNA gets damaged, it will either get fixed or the cell will kill itself (by apoptosing). In cancer, the DNA damage does not get fixed, and the cell does not die – so it is able to pass that mutation down to all subsequent cells as it divides.

Four kinds of normal genes are targets
What kinds of genes tend to get damaged in most cancers? It turns out there are four classes of normal regulatory genes that seem to be good targets for genetic damage.

1. Proto-oncogenes. These are normal genes that every cell has; their function is to tell the cell to grow. If you mutate a proto-oncogene in such a way that it is always turned on, then the cell with that mutant gene will always be growing and dividing, and a tumor will develop! Such mutant proto-oncogenes are called oncogenes. They are dominant, meaning that mutation of a single allele can lead to cellular transformation.

2. Tumor suppressor genes. These are normal genes that every cell has; their function is to tell the cell to stop growing. You can think of them like brakes on a car. If you mutate a tumor suppressor gene in such a way that it does not work, then you are effectively removing the brakes on cellular proliferation, and the cell will grow like crazy. These guys are recessive, meaning that you generally need to mutate both copies of the normal allele for transformation to occur.

3. Genes involved in apoptosis. These are important, because if a cell can’t repair its DNA, it needs to be able to kill itself (otherwise, the damaged DNA will just get passed down to daughter cells). Mutate these, and a cell will become immortal – even if its DNA is damaged!

4. Genes involved in DNA repair. Obviously, these genes are critical too. We acquire as many as 100,000 spontaneous mutations every day! (If that’s not enough to keep you up at night, I don’t know what is.) You have to have ways of fixing these mutations, or you’d be getting cancers all the time.

What happens if you damage these genes?
There are 6 particularly nasty things that a cancer cell can learn how to do as a result of genetic damage.

1. Autonomous growth. Cancer cells are able to grow all by themselves, without any of the external signals normal cells need (like growth factors or hormones).

2. Insensitivity to growth-inhibitory signals. Cancer cells are able to ignore the normal signals telling cells to stop proliferating. They just keep on growing no matter what anyone tells them.

3. Evasion of apoptosis. Cancer cells can become immortal by learning to avoid the signals that normally induce apoptosis.

4. Limitless replication. Whereas normal cells possess a limited number of doublings (usually 60-70), cancer cells can learn how to renew themselves endlessly – kind of like stem cells or germ cells.

5. Sustained angiogenesis. For a tumor to survive, it needs to be able to grow a new blood supply. Blood can only diffuse so far, so this has to be an ongoing process if a tumor wants to grow.

6. Invasion and metastasis. These are particularly nasty characteristics of cancer cells. Benign tumors tend to be non-invasive, and they never metastasize (that’s one of the few times you can say “never” with confidence in pathology class). Malignant tumors, however, are generally invasive, and they most certainly can metastasize if they so choose.

We’ll talk later about some specific genes that are commonly mutated in many different kinds of cancer.

Photo credit: Runran (http://www.flickr.com/photos/runran/3358075794/), under cc license.

We’re back after a nice long summer break! School is back in session and hopefully Pathology Student will give you a little help along the way in your study of pathology.

Here’s a general pathology concept that is important not only for boards but for real life: wound healing. Let’s take a look at wound healing in the skin. Whether the wound is a small kitty scratch or a huge burn, we have ways of repairing the damage and restoring function to the skin. There are two types of wound healing in the skin: healing by first intention and healing by second intention (weird names, I know, but whatever).

Healing by first intention occurs in small wounds that close easily – for example, paper cuts, or small surgical incisions in which the edges are easily approximated. In this type of healing, epithelial regeneration predominates over fibrosis. That’s a fancy way of saying that there is usually minimal scarring in this type of healing. Healing is generally fast. Here’s a summary of the timeline in most wounds healing by first intention:

By 24 hours: a clot forms, neutrophils arrive, and the epithelium begins to regenerate
By 3-7  days: macrophages arrive, granulation tissue is formed, collagen begins to bridge the incision, and the new epithelium increases in thickness.

Let’s stop right here for a moment. Granulation tissue is that stuff that forms when your body is filling in the gap between your remaining tissues. The contents of granulation tissue are 1) new, fragile blood vessels, 2) fibroblasts, and 3) a loose extracellular matrix holding it all together. The whole point of granulation tissue is to provide a place for the new structures to grow that will hold the tissue together (blood vessels and collagen). That’s it. Note: granulation tissue is not the same as a granuloma (which is a collection of macrophages) or chronic granulomatous disease (in which patients have neutrophils that don’t work right, so their macrophages are left with the job of killing bacteria, and they form little granulomas all over the place). So don’t get those terms mixed up.

Weeks later: the granulation tissue is gone, collagen has been remodeled (using little metalloproteinase enzymes like collagenase), and the epidermis is full and mature (though it lacks dermal appendages in the area of the healed wound). Eventually, a full-blown scar forms.

Healing by second intention occurs in larger wounds that have gaps between the wound margins. Examples of this type of wound are: areas of skin infarction, large burns and ulcers, and extraction sockets (where the dentist has pulled a tooth. Yes, this first- and second-intention healing applies to mucosal epithelium too!). In this type of healing, fibrosis predominates over epithelial regeneration. In other words, there’s gonna be a big scar that’s more prominent than any skin regrowth that occurs. Healing by second intention is slower. There is a lot more granulation tissue (because you have a huge gap to bridge) and more inflammation (neutrophils and macrophages coming in to clean up the dead cells and debris). Therefore, there’s a greater risk of infection and inflammation-related tissue injury. Also, the wound contracts as it heals (so you don’t have to make such a big scar). As far as a timeline goes, you can’t really make a universal timeline for second-intention healing, because it varies a lot depending on how big the wound is.

It all makes sense if you can just remember: first intention = small wounds, second intention = big wounds.

The strangely beautiful photo above, aptly titled “cat launchpad,” was taken by quinn.anya and can be found at: http://www.flickr.com/photos/quinnanya/2420314228/.

Q. Immediately after an acute episode of blood loss – following a motor vehicle accident, for example – the hemoglobin level is normal. Why is that? Are the other red cell indices normal too?

A. Immediately after acute blood loss, all the laboratory red cell indices are normal! The hemoglobin, RDW, RBC, MCH, and the MCHC are all normal. Because really, although the patient has lost blood (and therefore is in trouble because he/she has fewer red cells to transport oxygen through the body), the blood that’s examined in the laboratory appears totally normal! It has the same number of red cells per unit volume, and the red cells themselves are perfectly normal (assuming the patient’s blood was normal to begin with). This is because during acute blood loss, you’re losing red cells but also the plasma that goes along with them. So the blood remaining in the patient is totally normal – it’s just that there isn’t enough of it.

There are several things you should look for when evaluating a bone marrow biopsy specimen – see if you can see them in the image above.

First, take a look at the cellularity. The white spaces are fat cells that have washed out during processing; the cells in between the fat cells are hematopoietic precursors. The ratio of cells to fat is called the “cellularity.” The marrow above is approximately 30-40% cellular. You need to know the age of the patient to estimate whether the cellularity is normal. Here is a rough guide to cellularity by age:

0-3 months: 100%
3 months – 10 years: 80%
20 years: 65%
30 years: 50%
40  years: 45%
50 years: 40%
60 years: 35%
70 years and over: about 30%

Next, take a look at the composition of the marrow. Myeloid cells make up the largest percentage of the normal marrow cellularity; erythroid cells are second most common. The ratio of myeloid to erythroid cells should be about 2:1 to 4:1. It’s easier to see these cells on an aspirate smear, but you can get a pretty good idea on the marrow section too. Neutrophils and precursors often have eosinophilic, granular cytoplasm; if you look closely, you can see the indented nuclei of metamyelocytes and segmented nuclei of mature neutrophils. Erythroblasts generally have very round, dark nuclei; earlier forms are large, and later forms are small. A few megakaryocytes (large cells with abundant eosinophilic cytoplasm and multiple nuclei) should be sprinkled throughout the marrow too. Lymphocytes normally represent about 10-15% of the marrow cellularity. The above marrow appears to have a myeloid:erythroid ratio of 2:1, and megakaryocytes are normal in number.

Finally, take a look through all the sections to see if you see anything weird, like fibrosis, metastatic carcinoma, lymphoid aggregates, or amyloid deposition. As you scan the sections, you should see evenly-distributed cellularity, with evenly-spread hematopoietic precursors. Anything that deviates from this pattern should be investigated on higher power.

Before you can really appreciate pathologic changes in red cells, you need to know what normal red cells look like.  Here is a normal blood smear image, taken at high power. There are several things to notice here.

First, the cells are nicely spread across the field. They are sometimes touching or even slightly overlapping, but they’re not all piled up on top of each other. There are occasional small empty spaces, but there are not vast barren areas the size of several red cells. By the way, you need to be in the right area of the blood smear to make these assessments. The right area – the “zone of morphology” – is a few fields in from the feather edge of the blood smear (the edge opposite the thick end of the smear). In this zone, in a normal blood smear, the cells are just barely touching, as seen in this photo.

Second, the cells appear to have a normal amount of hemoglobin. See the white dot in the center of each red cell? That’s the “zone of central pallor.” It should be approximately 1/3 the diameter of the entire red cell (cells like this are called normochromic). If it’s much larger, that means that the cell does not have enough hemoglobin (cells like this are called hypochromic) and the patient is anemic. 

Next, check out the shape of these cells: they’re all round! That’s what you’d expect. Rarely, you may see a cell that looks a bit beat up, or that deviates slightly from a perfect round appearance. But overall, there are not a significant number of funny-shaped red cells (like pointy ones, teardrop-shaped ones, or sickle-shaped ones). This shape quality is termed poikilocytosis. Normal blood has minimal poikilocytosis (most all of the cells are nice and round). Some anemias, like severe thalassemia, can have marked poikilocytosis (the cells are all kinds of different shapes).

One property that is very important, but hard to gauge visually, is red cell size. It’s pretty hard to tell, just looking, whether the cells are normal in size (normocytic), small (microcytic), or large (macrocytic). You need to look at the MCV (mean cell volume), a value reported in the complete blood count (CBC), to determine whether the cells are normal in size or not. The normal MCV is between 80 and 100 femtoliters; red cells with an MCV less than 80 are considered microcytic, and red cells with an MCV over 100 are considered macrocytic. This is an important property to assess when you’re dealing with a case of anemia, because it helps you narrow down your possible diagnoses (some anemias are microcytic, some are macrocytic, and some are normocytic).

It is possible to tell, however, how much the cells vary in size. You can see, with your naked eye, whether the cells are all pretty much the same size, or whether there is a big size range from tiny ones to big huge honkers. Normal blood has minimal anisocytosis (all the cells are pretty much the same size), whereas some anemias, like severe iron deficiency anemia, can have marked anisocytosis (there are some tiny ones and some big ones). The CBC value that reflects this variation in cell size is the RDW, or red cell distribution width. If the RDW is low, that means there is minimal anisocytosis, and all the cells are pretty much the same size. If the RDW is very high, that means there is marked anisocytosis, and the cells vary in size from small to large. This is another value that can help you out when you’re trying to diagnose an anemia. 

Finally, there’s nothing weird going on. There are no nucleated red blood cells, infectious organisms (like malarial organisms) within the red cells, Pappenheimer bodies (iron aggregates), Howell-Jolly bodies (little fragments of DNA that didn’t quite make it when the nucleus was extruded), or anything else funny-looking.

It’s a good idea to go through a checklist like this when you’re looking at each part of a blood smear – red cells, white cells, and platelets. Your eye has a tendency to jump to the nearest weird-looking thing, and in doing so, you may miss subtle but important changes unless you have a nice plan for how to approach a blood smear.