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!

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.

When people talk about immunodeficiency states, they’re usually talking about secondary immunodeficiencies, like AIDS. The primary immune deficiencies really don’t get much press. Which is unfortunate, because although they are much less common than secondary immune deficiencies, they still occur, and it’s important to understand them for that reason alone. Plus, they are very testable – either on board exams, or on class exams.

Time is short, though, and you need to know the basic points for each one without having to wade through a lot of chapters in a textbook. So, without further ado, here is a short, bullet-pointed list of the main disorders, with particular emphasis on the part of the immune system that is affected, and the clinical manifestations of the disease.

X-linked agammaglobulinemia

• Pre-B cells can’t differentiate into B cells
• Patients have no immunoglobulin (Ig)
• X-linked (so seen only in males)
• Presents at 6 months of age (when maternal Ig runs out)
• Patients get recurrent bacterial infections
• Treatment: intravenous pooled human Ig

Common variable immunodeficiency

• Group of disorders characterized by defective antibody production
• Affects males and females equally
• Presents in teens or twenties
• Basis of Ig deficiency is variable (hence the name) and often unknown
• Patients more susceptible to infections, but also to autoimmune disorders and lymphoma!

Isolated IgA deficiency

• Most common of all primary immune deficiencies
• Cause is unknown
• Most patients asymptomatic
• Some patients get recurrent sinus/lung infections or diarrhea (IgA is the major Ig in mucosal secretions)
• Possible anaphylaxis following blood transfusion (patients have anti-IgA antibodies, and there is IgA in transfused blood!)
• Increased incidence of autoimmune disease   (who knows why)

Hyper-IgM syndrome

• Patients make normal (or even increased) amounts of IgM
• But can’t make IgG, IgA, or IgE!
• X-linked in most cases
• Patients also have a defect in cell-mediated immunity
• Patients have recurrent bacterial infections and infections with intracellular pathogens (e.g., Pneumocystis jiroveci)

DiGeorge syndrome

• Developmental malformation affecting 3rd and 4th pharyngeal pouches
• Thymus doesn’t develop well
• Patients don’t have enough T cells
• Infections: viral, fungal, intracellular pathogens
• Patients may also have parathyroid hypoplasia
• Treatment: thymus transplant!

Severe combined immunodeficiency

• Group of syndromes with both humoral and cell-mediated immune defects
• Patients get all kinds of infections
• Lots of very different genetic defects
• Half of cases are X-linked
• Treatment: bone marrow transplantation

The best way to remember these might be to make a little chart, with the diseases in one column, and subsequent columns for transmission (X-linked or not),  immunologic defect (e.g., no immunoglobulin production), and clinical features (e.g., infant with recurrent bacterial infections).

Photo credit: Wikimedia (http://commons.wikimedia.org/wiki/File:Antibody_je2.png) via cc license.

Systemic lupus erythematosus is one of a few diseases that have earned the name “the great imitator.” It is a chronic, systemic illness with many, many possible symptoms in many different organ systems, and widely varying disease courses in different patients. We’ll go through a short summary of lupus here and save the more detailed information for other posts.

The underlying cause of lupus is not known. To manifest the disease, you most likely need have two things: 1) a genetic predisposition, and 2) some environmental trigger (sun exposure, drugs, etc.). Whatever the underlying cause, patients with lupus all have autoantibodies against self antigens. All patients with lupus have anti-nuclear antibodies (antibodies against some part of the cell nucleus – like DNA, histones, RNA-bound proteins, or nucleoli). Some patients also have anti-RBC, anti-lymphocyte, anti-platelet and/or anti-phospholipid antibodies as well.

The autoantibodies cause damage by forming immune complexes with their corresponding antigens (in the case of anti-nuclear antibodies, there must be some type of cell destruction to expose the corresponding nuclear antigens!), and circulating around the body lodging in places they should not be (like glomeruli, skin, joints, the pericardium, etc.). Remember what type of hypersensitivity reaction this is? (See the bottom of the post for the answer. But try to remember before you look!)

Organ systems commonly affected (with common manifestations) are:
1. Renal system (wide variation in manifestations, from painless hematuria, to lupus nephritis, to end-stage renal failure. One histologic hallmark is membranous glomerulonephritis with a “wire-loop” appearance due to immune complex deposition.)
2. Skin (the classic manifestation is a malar, or “butterfly,” rash, but scaly patches, ulcers, and other skin lesions can occur)
3. Nervous system (headache, mood disorders, seizures, psychosis, focal neurologic deficits)
4. Musculoskeletal system (arthritis of small joints of hands, muscle pain)
5. Cardiovascular system (pericarditis, endocarditis (called Libman-Sacks endocarditis), atherosclerosis)
6. Hematopoietic system (anemia, thrombocytopenia, leukopenia, antiphospholipid antibody syndrome)

There are two main forms of lupus: discoid lupus and systemic lupus erythematosus. Patients with discoid lupus have only skin (not systemic) involvement, and they do not have anti-DS DNA antibodies (an antinuclear antibody frequently present in patients with systemic lupus). Systemic lupus erythematosus is just what the name says: a systemic form of the disease. It is the more common form, unfortunately.

The prognosis is difficult to predict for individual patients. Some patients have very few symptoms; rarely, the disease course is acute and rapid. Most patients suffer a relapsing and remitting course over a period of many years (the overall 10 year survival is 80%). Acute flare-ups can be treated with steroids – but as of now, there is no cure.

————–
The type of hypersensitivity reaction that is characterized by immune complex deposition is type III.

Photo credit: NIH (http://www.niehs.nih.gov/health/topics/conditions/lupus/index.cfm), under cc license.

The MHC (major histocompatibility) complex is a collection of genes on chromosome 6. It’s organized into three regions: the class I region, the class II region, and the class III region. Class I genes encode glycoproteins expressed on the surface of nearly every nucleated cell in the body. These glycoproteins, called MHC I receptors, present antigens (proteins made within that particular cell) to CD8+ T cells. Class II genes encode glycoproteins expressed only on the surface of antigen-presenting cells (macrophages and dendritic cells). These glycoproteins, called MHC II receptors, present antigens (which that antigen-presenting cell has eaten and processed) to CD4+ T cells. The MHC III region encodes a bunch of things, including complement proteins and cytokines.

So what?

Well, your T cells can’t recognize antigen unless it’s shown to them by an MHC receptor (the books say “presented in the context of an MHC receptor” but I like simpler words better). Helper (CD4 +) T cells recognize antigens only when they are presented by MHC II receptors; cytotoxic (CD8 +) T cells recongize antigens only when they are presented by MHC I receptors.

How are you supposed to remember which cells have MHC I receptors and which have MHC II receptors, and which T cell subset recognizes each one? I think of it like this. The MHC I receptor is kind of a low-class, ordinary, run-of-the-mill receptor (hence the common-sounding “I” designation). It’s present on virtually all nucleated cells in the body. The MHC II receptor is a high-class, specialized, advanced kind of receptor (hence the higher “II” designation). Only certain kinds of cells (antigen presenting cells) get to have this receptor. I’m sure that’s not why they named them that – but it helps me remember the numbers if I think of them in this way.

As to the cells that recognize each type of receptor – if you can remember the above, then you can figure out which type of T cell will recognize each receptor. Cytotoxic T cells recognize that type of MHC that is present on all cells in the body: the type I MHC receptor. It has to be this way, because any cell in the body can become infected, and cytotoxic T cells need to have a way to recognize and kill these cells. Helper T cells, on the other hand, recognize only that type of MHC that is present on antigen presenting cells: the MHC II receptor. Which makes sense, because helper T cells need the cytokines released by antigen presenting cells to help them do their job. So they lock onto the MHC II receptor on antigen presenting cells, and while they’re there, they get nice cytokine signals telling them to grow and differentiate.

Image credit: NIH.

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/