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 abl gene on chromosome 9, and a red marker to “paint” the bcr 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 abl gene is sitting right next to the bcr 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.