Blood cookies!

Okay, that was disturbing. But it was a lot of fun making cookies in the shape of different blood cells for our lectures on anemia and leukemia this week! (more…)

How to tell apart promyelocytes and myelocytes

Here’s a quandry you may find yourself in soon, if you have a habit of sitting at the multiheaded scope down in hematopathology.

You’re looking at a bone marrow smear, and you can differentiate between some of the myeloid cells (blasts have a high nuclear-cytoplasmic ratio; segmented neutrophils are all mature with their multilobed nuceli; metamyelocytes look kinda like mature neutrophils only with a more horseshoe-shaped nucelus.)

But two cells will give you gout or a migraine if you don’t learn a couple simple facts: promyelocytes and myelocytes. How are you supposed to tell them apart, when they can look quite similar? They’re both kinda big, they both kinda have granules…so what gives?

Let’s do a little pre-test here to see what you think about these cells, before we discuss the “official’ way of distinguishing between the two. We can leave the lymphoycte and the red cell precursors out of the discussion (top of the slide). But what’s your diagnosis on cells 1, 2, and 3? Are they promyelocytes, myelocytes, or a mixture of the two?

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Here’s the morphologic criteria from my path residency (and my histology course as a medical student) that we used to differentiate between promyelocytes and myelocytes:

  • The promyelocyte is the biggest cell in the neutrophil series.
  • It also has huge, dark purple, primary (azurophilic) granules both in the cytoplasm and overlying the nucleus.
  • However, it does NOT have the beginnings of secondary (specific, pink, salmon-colored) granulation! If you see any of that (even just a little blush of it in the cytoplasm), you HAVE to call a myelocyte.

So for our cells above:

  • Cell #2 is a pretty spectacular promyelocyte. It’s huge, it’s got tons of dark granules, and no specific granulation. It does have the beginnings of a little “hof” (a clear zone next to the nucleus) but that should not be confused with specific granulation.
  • Cell #3 is pretty clearly a myelocyte. It’s a smaller cell, and there are very few azurophilic granules left; the cytoplasmic granules are mostly just pale, specific granules.
  • Cell #1 could be a bit of a challenge because it’s a rather large cell, with abundant dark purple granulation…but it also has the clear beginnings of specific granulation in the cytoplasm. So this cell should rightly be classified as a myelocyte. It’s a pretty early one, for sure – but the presence of the specific granulation pushes it into the myelocyte category.

What’s up with factor XII?

XIIbHere’s a great coag question from a Path Student reader:

Q. I am wondering why Factor VII deficiency causes significant bleeding problems but Factor XII deficiency does not. One source I found stated that this is because the extrinsic pathway is the primary pathway in vivo and that the intrinsic pathway is of lesser importance.

However, the more I thought about it, I then wondered why Hemophilia A and Hemophilia B are so severe since they are involved in the intrinsic pathway. Why doesn’t the extrinsic pathway just pick up the slack and allow the patient to remain asymptomatic as it seems to with Factor XII deficiency?

A. It turns out that factor XII is pretty important in vitro, but not in vivo. In the test tube, XII is activated by contact factors (like HMWK), and then XIIa catalyzes the conversion of XI to XIa. In the body, though, the main thing that converts XI to XIa is thrombin (not XIIa). I don’t even include XII in my drawing of the cascade for my students, since it’s of no clinical consequence. The intrinsic side is complicated enough!

With regard to the extrinsic and intrinsic pathways: both are critical for fibrin formation in vivo. I wouldn’t say that either one is of lesser importance – they just do different things.

In vivo, the cascade starts on the extrinsic side with TF showing up and binding to VIIa. The TF-VIIa complex converts X to Xa, and things proceed from there. The weird thing is that as soon as a little Xa is made, the extrinsic pathway is turned off (by tissue factor pathway inhibitor, or TFPI)!

So then what? By this point, you already have a little thrombin around – and that thrombin goes and kicks off the intrinsic pathway. Thrombin converts XI to XIa, which converts IX to IXa, which – together with VIIIa – converts X to Xa.

Bottom line: the cascade starts with the extrinsic pathway, but that pathway gets shut off very quickly. Thrombin activates the intrinsic pathway, which proceeds to convert fibrinogen to fibrin until you need to turn it off.

Microcytosis and hypochromasia

IDA1

Q. What is the pathophysiology of microcytes in iron-deficiency anemia (IDA)? I mean I understand that hypochromasia is due to low hemoglobin content, but what makes the cells smaller? Is it something like first there is hypochromasia and then the cells shrink? Aren’t hypochromatic cells normocytic? Why don’t red cells keep shrinking as they become hypochromatic? Please help. The question is bothering me a lot. 🙂

A. First of all, you’re right: in IDA, the red cells do get smaller. Since the bulk of the red cell is composed of hemoglobin, the less hemoglobin there is in the cell, the smaller the cell volume, and the smaller the cell overall. So in iron-deficiency anemia, there is less iron around, and therefore less hemoglobin – so the cells are smaller than normal. Same thing happens in thalassemia: less hemoglobin around (though not because of iron, but because of a genetic defect in a hemoglobin chain), so the red cells are smaller.

Just to clarify: chromasia just refers to the amount of hemoglobin in the cell. Cells can be normochromic (as they are in normal blood), or hypochromic (as they are in IDA). The size of the red cell is measured separately from the chromasia. Normally-sized red cells are called normocytic, small ones are called microcytic, and large ones are called macrocytic.

You asked if hypochromic cells are normocytic – and for the reason stated above, the answer is no, they usually aren’t. They are usually microcytic, because there’s less hemoglobin in the cell, so the cell gets smaller.

Finally, to answer your last question, in iron-deficiency anemia, the red cells do keep shrinking as they become more and more hypochromic! Assuming the iron deficiency is a continuing problem, as each new wave of red cells is produced, there will be less and less iron around – and the cells will get smaller and smaller.

So when you look at a blood smear from a patient with IDA (like the one above), you’ll see some cells that are a little bigger (these are older red cells that were made when there was still a fair amount of iron around), and some that are a little smaller (these are newer red cells, made when the iron level had dropped). Check out the two cells in the center of the image: both are hypochromic, but the one in the center is about twice as big as the one to its left.

This is why you can use the RDW to help differentiate between IDA and mild-moderate thalassemia!

What does phospholipid do in the PT and PTT?

phospholipid

Q. I have a quick question about coag lab tests. In the tests that you are adding phospholipid (like the PTT), what exactly is the phospholipid doing?

A. It’s just providing a surface for the coagulation factors to sit on! Many of the coagulation factors need a phospholipid surface to sit on in order to work.

Normally, the platelets provide that surface (they have phospholipids in their membranes) – but you’ve taken the platelets out of the test tube before you do the coagulation lab tests – so you need to add them back in if you want the whole cascade to run.

You also add phospholipid in the PT (INR)! It’s part of the thromboplastin molecule. Thromboplastin is just a tissue-factor-like substance plus phospholipid, all wrapped up in one reagent.

A few of the coag tests don’t require a phospholipid surface. The TT, for example, doesn’t need phospholipid; you’re just adding thrombin and seeing how fast it can convert fibrinogen to fibrin – and that single reaction doesn’t need phospholipid to work. Also, the fibrinogen assay doesn’t require phospholipid because it just measures the amount of fibrinogen.