Pathology Student

Q. What stain is used for demonstrating Auer rods in myeloblasts? Myeloperoxidase or PAS?

A. The best stain for demonstrating Auer rods is the myeloperoxidase (MPO) stain. This stain highlights one of two main populations of granules in the neutrophil: the primary (or azurophilic) granules. Secondary (or specific) granules do not light up with MPO. I could never get that straight until I realized that the Primary granules were Purple (okay, “azurophilic,” but close enough). Using this differential staining, it is possible to classify neutrophil maturation into four distinct stages:

1. Myeloblast. This is the earliest committed stage of development. These cells will turn into neutrophils if you just leave them alone. They look like typical blasts (high n/c ratio, big nucleus with fine chromatin), and they may or may not have a very small number of tiny primary granules in the cytoplasm. (Even myeloblasts that do not have these granules have been shown using immunohistochemical markers to be of the myeloid lineage.)

2. Promyelocyte. This is the next stage in development. Primary granules start appearing in abundance at this stage. The cell is larger than it is at any other stage of development. I love this stage; it is my favorite stage (doesn’t everyone have a favorite stage of neutrophil development?) because it’s just so dang pretty. Huge cell, beautiful blue cytoplasm, and these gorgeous luminous purple granules. Yum. If I could eat any cell, I’d eat a promyelocyte. I think it would taste like grape candy.

3. Myelocyte. At this stage, the cell is a bit smaller, and there are lots of secondary (specific) granules around. These don’t stain with MPO, so they end up as sort of pale orangeish pink. They’re said to be fawn-colored, but I haven’t seen any fawn with that color fur. Then again, I haven’t seen many fawns. The number of primary granules is significantly less (because promyelocytes divide into two cells, which mature out to become neutrophils. This means that the number of primary granules in the daughter cells is significantly smaller than in the mama promyelocyte (due to dilutional effect). The nucleus gets a bit smaller, and the chromatin condenses a bit.

4. More mature cells: metamyelocytes (these are basically just myelocytes with an indented nucleus and a bit more nuclear condensation) and neutrophils (cells in which the nucleus has multiple lobes – at least three). These more mature cells (metamyelocytes and neutrophils) can’t be differentiated on the basis of MPO staining alone, like the three preceding cells can. You need to use other parameters, like size and shape of nucleus.

Cool article on this: Neutrophil Secondary-Granule Deficiency as a Hallmark of All-Trans-Retinoic Acid-Induced Differentation of Acute Promyelocytic Leukemia Cells. Miyauchi J, Ohyashiki K, Inatomi Y, Toyama K. Blood 1997; 90(2):803-813.

So what does all of this have to do with MPO staining of Auer rods?  Well, since Auer rods are basically clumps of azurophilic granules which contain peroxidase and other stuff, the MPO stain works well on these structures. The PAS stain highlights glycogen (not myeloperoxidase), so it stains a bunch of different cell types, like red cells and megakaryoblasts, but it is not of much use when looking for Auer rods.

If you were looking for Auer rods, and you wanted to do a special stain other than MPO, you could do a Sudan Black B (SBB); it provides results identical to those of MPO. Or you could just look at the Wright-Giemsa-stained smear and forget about the special stains for the moment. The MPO and SBB are good in that they will highlight more of the Auer rods than you can see using just the Wright-Giemsa stain – but often, you see so many on just the Wright-Giemsa alone that you don’t need to bother with an MPO. Check out the Wright-Giemsa-stained Auer rods (tons of them!) in the above cell from a case of acute promyelocytic leukemia.

Q. I’m confused how in megablastic anemia, cells become macrocytic due to immature nuclei when RBCs don’t have nuclei—is it referring to the erythroblast precursors before the nuclei are lost?

A. Great question. In megaloblastic anemia, cells have a hard time making DNA (because there’s a lack of B12 and/or folate) – but RNA production proceeds normally. So you end up with cells that have normally maturing cytoplasm, but slowly-maturing nuclei. This means that the cell grows pretty large before the nucleus gets mature enough to signal division (so the cells end up being larger than normal). Also, when you look at the nucleus, it looks more immature than the cytoplasm (hence the term “nuclear-cytoplasmic asynchrony). You’re right: these changes are easiest to see when you look at erythroblast precursors (which have nuclei). You can also see the same changes in neutrophil precursors (you’d have to look at the marrow to see both of these types of precursor cells).

When you look at the blood, you can’t see these precursors. But you do see macrocytes (larger-than-normal mature red blood cells) and hypersegmented neutrophils. The reason the mature red cells are bigger than usual has to do with the fact that the red cell precursors get bigger than normal before each cell division, as described above…and that translates into bigger-than-normal mature (non-nucleated) red blood cells. The reason for the hypersegmented neutrophils is less clear; it has something to do with the abnormal, asynchronous maturation going on in the neutrophil series – but how the mature neutrophil winds up with a nucleus with more lobes (or segments) than normal is kind of a mystery.

Here’s a summary of those four pesky hypersensitivity reactions you will definitely be asked questions on at some point.

Sometimes, the best way to remember things is to boil them down to as few words as possible. I think you can summarize each hypersensitivity reaction in a one or two words:

Type I – allergy
Type II – antibodies
Type III – immune complex
Type IV – T cells

Here’s a little more information:

Type I hypersensitivity is the mechanism underlying the classic allergic response. It’s also called “immediate” hypersensitivity, which makes sense to any allergy sufferer (as soon as you start petting the cat, you start sneezing). It’s caused by an antigen (from an allergen, like cat dander) binding to IgE antibodies that are bound to the surface of mast cells. The antigen bridges the IgE antibodies, triggering release of nasty mediators (like histamine) from the mast cell. The end result: vessels dilate, smooth muscle contracts, and inflammation comes in and makes itself at home.

Type II hypersenstivity is also called “antibody-mediated” hypersensitivity. Which is kind of misleading, becase it’s not the only type of hypersensitivity reaction that involves antibodies. Oh well. In this type of hypersensitivity antibodies bind to antigens on a cell surface (any cell surface). Macrophages come in and eat up the cells (they think the Fc fragments of antibodies are yummy). Complement gets activated, inflammation comes in (harming tissue) and cells end up dying. Examples of this type of hypersensitivity include: autoimmune hemolytic anemia, pemphigus vulgaris, Goodpasture syndrome, myasthenia gravis, and Graves disease.

Type III hypersensitivity is also called “immune-complex-mediated” hypersensitivity. In this one, antibodies bind to antigens, forming complexes. These antigen-antibody complexes circulate (either throughout the whole body, or within one area of the body), get stuck in vessels, and stimulate inflammation, the end result being inflammation-mediated tissue damage and necrotizing vasculitis. Examples of this type of hypersensitivity include: systemic lupus erythematosus, post-streptococcal glomerulonephritis, polyarteritis nodosa, serum sickness, and the Arthus reaction.

Type IV hypersensitivity is also called “T-cell-mediated” hypersensitivity. This type of hypersensitivity has two subtypes. In one subtype, called delayed-type hypersensitivity, helper T cells secrete cytokines that activate macrophages (which eat the antigen) and induce inflammation (which damages tissue). A good example of delayed-type hypersensitivity is poison ivy. The other subtype, called T-cell-mediated cytotoxicity, involves cytotoxic T cells coming and killing target cells (like the cells of a transplanted organ, or the pancreatic islet cells in a patient with type I diabetes).

The nice kitty image is from anomalous 4 at: http://www.flickr.com/photos/31333486@N00/2066349843/

People seem to love the quizzes! Here’s another one, in a more traditional format, this time on anemia. Answers are in the first comment associated with this post.

Q1. Which of the following red cell indices tells you how big your patient’s red cells are?

1. RBC
2. Hgb
3. MCV
4. RDW
5. MCHC

 
Q2. The RDW measures:

1. The average concentration of hemoglobin in each red cell
2. The total number of red cells
3. The percentage of blood volume that is composed of red cells
4. The variation in red cell size (all the same size vs. some big ones and some little ones)
5. The height of the average Christian Louboutin heel

 
Q3. “Chromasia” refers to:

1. How big the red cells are
2. How widely spaced the red cells are
3. How much hemoglobin is in the red cells
4. What color the red cells are
5. The age of the red cell

 
Q4. A 40-year-old female says she feels tired all the time. On exam, you note that she is tachycardic and pale. You order a CBC, which shows the following: Hgb 10 g/dL (12-16), MCV 75 (80-100). Her reticulocyte count is not increased. Which of the following is most likely?

1. She has iron-deficiency anemia
2. She has megaloblastic anemia, probably due to folate deficiency
3. She has megaloblastic anemia, probably due to B12 deficiency
4. She has a hemolytic anemia

 
Q5. A 60-year-old male has a hemoglobin of 9 g/dL. He also has the following lab results: MCV normal; LDH increased; haptoglobin decreased. Which of the following is most likely?

1. Iron-deficiency anemia
2. Megaloblastic anemia
3. Hemolytic anemia

 
Q6. You order a DAT, which comes back positive for complement, but not IgG. What is the most likely diagnosis?

1. Hereditary spherocytosis
2. Warm autoimmune hemolytic anemia
3. Cold autoimmune hemolytic anemia
4. Any of a number of non-immune causes of hemolysis

 
Q7. What causes the anemia in sickle cell disease?

1. An inability of the red cell to reduce organic peroxides.
2. An abnormal hemoglobin which polymerizes and irreversibly injures the red cell.
3. Insufficient Hgb A and excess unpaired β, γ, and δ chains.
4. Insufficient Hgb A and excess unpaired α chains.
5. Consumption of red cells by splenic macrophages.

 
Q8. A 32-year-old female presents for a routine physical. Her CBC shows the following: Hgb 9 g/dL(12-16), MCV 72 (80-100), RBC 6.4 (4.5-6.0), RDW 12.8% (12-13.5). What is the most likely diagnosis?

1. Iron-deficiency anemia
2. Thalassemia
3. Megaloblastic anemia
4. Autoimmune hemolytic anemia
5. Microangiopathic hemolytic anemia

 
Q9. On a routine physical examination of an elderly male patient with no other medical problems, you note that his earlobes and fingertips are pale and slightly bluish. A CBC shows a hemoglobin of 10.6 g/dL (12 – 16) and an MCV of 88 (80 -100). Numerous red blood cell agglutinates are seen on the blood smear, made by smart technologists in your laboratory. Which of the following statements is true?

1. The antibody bound to the patient’s red blood cells in this disorder is probably IgG
2. Complement is probably bound to the patient’s red cells
3. The spleen is the main site of red cell destruction in this patient
4. 1 and 3
5. 1, 2, and 3

Much of the time, when a patient has a neutrophilia, it is due to infection. But are there any clues on the blood smear that would make that diagnosis more definitive?

Well, yes, as a matter of fact, there are. These clues are called toxic changes and they encompass three main findings: toxic granulation (as seen above), Dohle bodies (also present above – look closely), and cytoplasmic vacuolization. If you see any of these changes, you can be quite certain that the patient has an infection.

Toxic granulation is the accumulation of big, dark granules in segmented neutrophils (or, sometimes, in earlier neutrophil precursors). It is likely due to the demand placed on the marrow to get those neutrophils out in the circulation as soon as possible in order to fight the infection. Under those conditions, the myeloblasts and promyelocytes (the dividing cells of the neutrophil lineage) say, “Okay, fine. You want segmented neutrophils out in the circulation immediately? I’ll quit spending my time dividing so much, and I’ll just mature!” This is a good strategy for getting mature neutrophils out of the marrow as quickly as possible. In the process of this kind of rushing around, the big fat dark primary granules present in the promyelocytes do not get diluted out like they normally would (when the promyelocytes are taking their time, and dividing over and over again before maturing, the dark granules get spread out among many generations of cells). Instead, they are retained in the cell, and you can see them even in the mature, segmented neutrophil descendents of these harried promyelocytes.

Cool, huh?

The two other changes are Dohle bodies (pretty sky-blue cytoplasmic inclusions in neutrophils; look closely at the above image – you can just barely make one out) and cytoplasmic vacuolization (an ominous change, by the way – if you see a lot of cytoplasmic vacuolization, and particularly if it is increasing over time, watch out.).  These changes, too, are quite specific for infection.

The term “left shift” means that a particular population of cells is “shifted” towards more immature precursors (meaning that there are more immature precursors present than you would normally see). In the neutrophil series, for example, you usually see only segmented neutrophils (and maybe a rare band neutrophil) in the blood. The earlier precursors are present in the marrow – but they grow up into mature cells before they exit the marrow and spill into the blood. The term “left shift” arose during the days when cells were counted by hand using a manual counting machine. The most mature cells (segmented neutrophils) were assigned to the right-most button, the least mature cells (myeloblasts) were assigned to the left-most button, and the other stages of cells were spread out in order in the buttons in between. In a normal blood smear, virtually all the neutrophils fell under the right-most counting button, but sometimes, it was noted that there were earlier precursors present (e.g., myelocytes, metamyelocytes, or promyelocytes). In these instances, the cells were “shifted” towards the left.

Most of the time, when you see a left shift, it means that the patient has an infection – often a bacterial one. Sometimes a left shift can occur when there is inflammation or necrosis. Beware, though, if you see nucleated red cells in addition to left-shifted neutrophils. This is called a leukoerythroblastotic reaction, and it may indicate a more serious problem.  Sometimes, a leukoerythroblastotic reaction is physiologic. If the hemoglobin is very low (for whatever reason – severe iron deficiency, massive blood loss), the bone marrow tries very hard to make new red cells and send them out into the blood as fast as possible. Sometimes, it is a little overzealous, and it lets a few red cell precursors (nucleated red cells) slip out of the marrow too. And sometimes, it is so freaked out that it starts letting neutrophil precursors (metamyelocytes, myelocytes, promyelocytes) out too! This is a normal response to a severe anemia. Sometimes, however, a  leukoerythroblastotic reaction is pathologic. If the marrow is full of something besides hematopoietic tissue – say, for example, a carcinoma, or a leukemia – then the hematopoietic cells will not have enough room and space to mature properly. They will end up leaving home before they are ready, and you’ll see both nucleated red cells and neutrophil precursors in the blood. This is an ominous sign.

One way to determine whether a  leukoerythroblastotic reaction is worrisome is to look at the hemoglobin. As mentioned above, if the hemoglobin is very low (say, below 6), then the  leukoerythroblastotic reaction is probably physiologic. However, if the hemoglobin is normal, or only slightly decreased, then there is no good reason for the patient to have a  leukoerythroblastotic reaction, and you’d better figure out what’s causing it.

Here’s an example of a common question students have in the beginning of a medical school or dental school pathology course. Unfortunately, students often feel like they “should” know the answers to certain questions – so they don’t ask. Don’t fall into this trap! You never need to feel embarassed about asking a question; everyone has things they don’t know – even professors. That’s why you’re taking the class – to learn!

On to the question.

Q. What is the main difference between a neutrophil and a monocyte? This is what I understand:  
    
     PMNs:
          fight bacteria and fungi (but they are different than NK cells–right?)
          act as antigen presenting cells
          phagocytic
          are generally the first to arrive; part of the acute inflammatory response

     Monocytes:
          act as antigen presenting cells
          can secrete cytokines and attract inflammatory cells like fibroblasts, etc.
          phagocytic
          bigger role in chronic inflammation

A. Broadly, the similarities are: neutrophils and monocytes are both phagocytes, and they both work to fight infections. But moncytes can turn into macrophages (when they get into tissues), which are very good at eating things, as well as presenting antigens. Neutrophils eat, but don’t present, antigens. One of the big differences, too, you already mentioned: neutrophils are the first to come in during an inflammatory process. Lymphocytes come next, then monocytes/macrophages come in to mop up the mess.

One note: neutrophils are phagocytes, but not antigen presenting cells. Another note: You are right, neutrophils are different than NK cells. NK (natural killer) cells are specialized lymphocytes which have functions different than those of neutrophils and monocytes.

Also: neutrophils look different than monocytes/macrophages. Neutrophils have a “busy” nucleus (that’s why they are called “polymorphonuclear” leukocytes), with several lobes. You can see one at 2 o’clock in the above photo. They also have granules, both primary (azurophilic) and secondary (fawn-colored). Monocytes have a horseshoe-shaped nucleus, with dishwater-gray cytoplasm and a few tiny granules. See the upper left corner in the above photo.