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. If the chronic leukemias have lots of mature cells, and the acute leukemias have immature cells, then how come chronic myeloid leukemia has lots of immature cells? Seems like it belongs in the acute leukemia category!

A. I think the best way to look at it is to oversimplify it a little, to get at the basics, and then put in a little detail.

The oversimplified version is this: Acute leukemias are composed of immature cells (usually blasts), whereas chronic leukemias are composed of mature cells (mostly the ones you normally see in peripheral blood).

The problem with that definition is that it doesn’t quite cover every chronic and acute leukemia. For example, AML-M2 is an acute leukemia that has at least 20% myeloblasts – but there are also a fair number of maturing neutrophils too (promyelocytes, myelocytes, metamyelocytes, and segmented neutrophils). So that doesn’t quite fit. The important thing in this AML, though, is that it does have at least 20% blasts. So you have to call it AML, even though it doesn’t quite “fit” our nice little definition.

Another example that doesn’t quite fit our neat little definition, as you noted, is CML. In CML, most of the cells are pretty mature (segmented neutrophils, metamyelocytes)…but there are some less mature ones too (myelocytes, promyelocytes). The important thing in CML is that there really aren’t very many blasts around at all; certainly not 20% or more like you’d see in AML. So even though it doesn’t quite fit, we put it into the chronic category (and it certainly acts a lot more like a chronic leukemia than an acute one!).

The underlying reason you see all these mature (and maturing) cells in CML (and in the other myeloproliferative disorders) – rather than a bunch of blasts – is that the problem has to do with a mutated, constitutively activated  growth receptor. In CML, the mutated growth receptor is produced when bcr and abl are joined together. In PV (and to some extent in ET and MF), the Jak part of the Jak-Stat pathway (a signal transduction system) is mutated. In either case, the tyrosine kinase is permanently in the “on” position, which means that growth and proliferation signals are constantly being sent to the nucleus. So the cells are dividing and proliferating even when they shouldn’t be.

These mutated tyrosine kinases don’t impair differentiation (or maturation), though, so you get uncontrolled growth of stem cells, and these bad stem cells are able to mature and progress through the normal stages of development! This is in contrast to many other leukemias, where there is increased growth but the cells are “stuck” at a certain stage of maturation (like the malignant cells in promyelocytic leukemia, which remain stuck at the promyelocyte stage).

Q. I was wondering what the difference was between labeling something as a “leukemia” vs labeling it as a “chronic myeloproliferative disorder.” I understand that leukemias are neoplastic proliferations of hematopoietic stem cells in the bone marrow, but aren’t myeloproliferative disorders the same thing? In particular, what category would chronic myelogenous leukemia be placed into? I have been grouping it with the MPDs, but then I get confused when I start to compare it to acute myelogenous leukemia, which is just labeled as a leukemia, and not a myeloproliferative disorder…?

A. You are right: leukemias are neoplastic proliferations of hematopoietic stem cells in the bone marrow. There are two big categories of leukemias: acute leukemias and chronic leukemias. The acute leukemias are divided into acute myeloid leukemia and acute lymphoblastic leukemia; the chronic leukemias are divided into chronic myeloproliferative disorders and chronic lymphoproliferative disorders.

Under these big acute and chronic categories, there are many different types of leukemia. Acute myeloid leukemia is divided into five main types: AML with genetic abnormalities (like t[8;21]), AML with FLT3 mutation, AML with multilineage dysplasia, therapy-related AML, and AML not otherwise categorized. ALL is divided into three main types: T-cell ALL, B-cell precursor ALL, and B-cell ALL (same as Burkitt lymphoma). The main chronic myeloproliferative disorders are: chronic myeloid (or myelogenous) leukemia (shown above), chronic (or idiopathic) myelofibrosis, polycythemia vera, and essential thrombocythemia. The main chronic lymphoproliferative disorders are: chronic lymphocytic leukemia, hairy cell leukemia, prolymphocytic leukemia, and large granulated lymphocyte leukemia.

I don’t know why they don’t just call the chronic myeloproliferative disorders and chronic lymphoproliferative disorders “chronic myeloid leukemias” and “chronic lymphoid leukemias,” but they don’t. Maybe it’s because one of the chronic myeloproliferative disorders is chronic myeloid leukemia, and to call the whole group of them “chronic myeloid leukemias” would be confusing. In fact, the term “chronic leukemia” isn’t really an official term either. But I like to use it because it shows that the chronic myeloproliferative and lymphoproliferative disorders really are leukemias, not some sort of benign proliferative disorders.

Q. I’m currently doing a research report on acute lymphoblastic leukaemia and I was wondering, are cytomorphology and cytochemistry important in the diagnosis of ALL? It seems like the two techniques are only important because they are able to diagnose AML and therefore, if AML is not diagnosed, by elimination, the condition is ALL. Also, for FISH and cytogenetics, why do metaphases have to be generated?

A. Cytomorphology (looking at the cells under the microscope) and cytochemistry (using stains like myeloperoxidase) are indeed important in differentiating acute myeloid leukemia from acute lymphoblastic leukemia. But that’s not all! It’s important to look under the microscope at a blood smear or bone marrow biopsy if you suspect any hematologic disorder; that’s an unspoken rule. First you look at the slides under the microscope, then you order special studies as needed to verify your presumptive diagnosis.

Diagnosing AML often involves the use of cytochemical stains. These stains are directed against certain parts of the cell. For example, the myeloperoxidase stain is directed against – you guessed it – myeloperoxidase in neutrophil granules; the non-specific esterase (NSE) stain (shown in the photo above) is directed against the NSE enzyme, which is present only in cells of the monocytic series. Cytochemical stains are useful for differentiating AML from ALL, and for subcategorizing the type of AML (some types of AML involve the monocytic series, some involve only promyelocytes, etc.). Diagnosing ALL involves more than simply ruling out AML; other studies are needed to a) confirm that the leukemia is lymphoid, and b) subcategorize the type of ALL (there are many different types, including T-cell ALL, B-cell ALL, and B-cell precursor ALL, each with their own prognosis). For this confirmation and subcategorization, immunophenotyping (looking for markers on the surface of the cell, usually using flow cytometry) is necessary.

Analysis of genetic changes is often useful in the diagnosis and prognosis of hematologic malignancies. You can look for genetic changes a number of different ways, including the two you mentioned: traditional cytogenetics and FISH (fluorescent in-situ hybridization). In traditional cytogenetic techniques, you need to get the cells into metaphase in order to see the chromosomes in their fully formed and separated state (in interphase, the chromosomes are all long and loose, forming kind of an amorphous mass referred to as “chromatin.”) After you get the chromosomes into metaphase, you take a picture of the chromosomes, cut them apart (or do it on a computer) and then sort them into their little corresponding pairs (two chromosome 1s, two chromosome 2s, etc.). The final picture, with all the chromosomes neatly lined up in order, is called a karyotype. This technique is nice because it gives you a good rough look at all the chromosomes; if there are big deletions, or translocations, or inversions, you’ll see those in the karyotype.

FISH is a little different in that you don’t have to get the cells into metaphase (although you can do so if you want). In this technique, you simply use fluorescent markers (hence the name) directed against certain genes. For example, you might use a green marker to “paint” the bcl gene on chromosome 9, and a red marker to “paint” the abl gene on chromosome 22. In a normal cell, the red and green dots would appear separated (since they are on different chromosomes). In a case of chronic myeloid leukemia, in which the malignant cells always have the 9;22 translocation, you’d see red dots right next to green dots (because the bcl gene is sitting right next to the abl gene). There are lots more uses for FISH, but this is the way it’s commonly used in hematologic malignancies.

The bottom line is that you always look at blood smears and bone marrow biopsies under the microscope. If you see what looks like a hematologic malignancy, you usually do additional studies (cytochemistry, immunophenotyping, and/or cytogenetic studies) to confirm the diagnosis and add prognostic information.

Another quiz – this time on acute leukemia. Answers and explanations are in the first comment following this post.

1. Patients with which of the following leukemias may go into DIC if given routine chemotherapeutic agents?

A. Acute promonocytic leukemia
B. Acute promyelocytic leukemia
C. Acute lymphoblastic leukemia
D. Chronic myeloid leukemia
E. Chronic lymphocytic leukemia

2. All of the following terms are technically incorrect, EXCEPT:

A. Acute lymphocytic leukemia
B. Chronic myeloblastic leukemia
C. Chronic lymphoid leukemia
D. Leukemoid reaction
E. Chronic myeloid leukemia

3. Which of the following leukemias is likely to show a panmyelosis:

A. Acute lymphoblastic leukemia
B. Acute monoblastic leukemia
C. Acute erythroblastic leukemia
D. Chronic lymphocytic leukemia
E. Chronic myeloid leukemia

4. A bone marrow biopsy shows 5% myeloblasts and some funny-looking neutrophils and precursors. The most likely diagnosis is:

A. Acute myeloid leukemia
B. Acute lymphoblastic leukemia
C. Myelodysplastic syndrome
D. Bacterial infection
E. Chronic myeloid leukemia

5. While looking around a blood smear, you notice a blast with an Auer rod in it. This patient has:

A. A bacterial infection
B. No disease, unless 20% of the nucleated cells have Auer rods
C. A myelodysplastic syndrome
D. Acute myeloid leukemia
E. Acute lymphoblastic leukemia

6. Acute lymphoblastic leukemia:

A. Often has a good prognosis
B. Never occurs in children
C. Is classified according to morphologic appearance
D. Is only diagnosed when 20% or more of the nucleated cells are lymphoblasts
E. Is an indolent disease

7. Which of the following is a GOOD prognostic indicator in acute lymphoblastic leukemia?

A. Age less than 1
B. A WBC >10,000
C. B-lineage immunophenotype
D. Normal cytogenetics
E. Age >10

Just as there are many different types of myeloid cells (neutrophils, red cells, monocytes, eosinophils, basophils), there are many different types of acute myeloid leukemia (AML). Two types of AML are composed almost entirely of cells of the monocytic series: acute monoblastic leukemia and acute monocytic leukemia. In both of these types of AML, at least 80% of the leukemic cells are from the monocytic series (monoblasts, promonocytes, and monocytes). In acute monoblastic leukemia, most of these cells are monoblasts, and in acute monocytic leukemia, most of these cells are promonocytes. Promonocytes have a very characteristic appearance, as shown above. They have nuclei that show a delicate folding pattern, almost like a piece of tissue paper that has been crumpled a bit. If you had a case of acute leukemia and most of the cells looked like this, you would think about acute monocytic leukemia – and you’d get an NSE to prove it.

Some cases of acute leukemia are composed entirely of undifferentiated-appearing blasts. These cases are difficult (or impossible) to diagnose morphologically (under the microscope) – you really need special tests like immunophenotyping in order to make a definitive diagnosis. Other cases of acute leukemia have obvious morphologic clues. Acute promyelocytic leukemia (APL) falls into the latter category. It is composed of malignant promyelocytes, which often have a distinctive appearance. But the really characteristic finding in APL is the faggot cell, so named because it contains a ton of Auer rods all piled up on each other, resembling a bundle of sticks (or faggot). When you see these, you can make the diagnosis of APL based on morphology alone, without waiting for molecular or cytogenetic studies (which will show the characteristic t(15;17) of APL – but which take some time to perform).

Making an immediate, morphologic diagnosis is critical in cases of APL, because these patients cannot be given routine acute myeloid leukemia chemotherapeutic agents. The malignant promyelocytes of APL contain lots of granules which have strong procoagulant activity. If you give the patient typical acute leukemia treatment, the promyelocytes will burst, the nasty granules will be released, and the patient will be at high risk for disseminated intravascular coagulation (DIC), a very dangerous syndrome in which patients bleed and clot all over the body. However, there is a drug called all-trans retinoic acid (or ATRA) that works great for patients with APL because it causes the malignant promyelocytes to mature (into myelocytes, then metamyelocytes, then neutrophils). Then the patient can be treated with regular chemotherapy without risk of DIC.

When you’re faced with an acute leukemia composed entirely of blasts, one way to figure out the identity of those blasts is to use cytochemical stains. These stains are applied to slides – either smears (blood or bone marrow) or sections (of bone marrow) – and are read under the microscope. There are several different cytochemical stains that are commonly used in hematopathology, one of which is the non-specific esterase (or NSE) stain. Monocytes and their precursors (promonoblasts and monoblasts) stain a pretty red color with this stain. Neutrophils and their precursors (including myeloblasts), and all the other types of cells in the bone marrow, for that matter, are negative. 

So if you have a leukemia composed of a sea of undifferentiated blasts, you could stain the blood or marrow smears (or the bone marrow section, though it’s harder to see individual cell morphology on a section) with NSE. If the cells stain red, then you’re dealing with a leukemia composed of monocytic cells – probably a monoblastic leukemia, if all you’re seeing is blasts. It’s a quick and easy way to help you diagnose the leukemia.

Some types of acute leukemia are composed of only blasts (no differentiating neutrophils, no monocytic precursors, just a sea of blasts). In those cases, look for Auer rods. A blast with an Auer rod can only be a myeloblast! It cannot be a lymphoblast, or a monoblast, or any other kind of blast. So if you see blasts with Auer rods, you know it is some type of acute myeloid leukemia. Remember, though, that the converse is not true: just because you don’t  see Auer rods, that does not mean that the blast is not a myeloblast. Some myeloblasts have Auer rods, and some don’t. So if you see Auer rods, it is an AML. If you don’t, it still could be an AML.