Indirect antiglobulin test

IAT

Yesterday, we looked at the direct antiglobulin test, or DAT. Today, we’ll take it one step further and look at the indirect antiglobulin test, or IAT (which is really just the DAT with an extra step thrown in).

This test is the basis for two important assays in transfusion medicine: (1) the antibody screen (in which serum samples from recipients and donors are screened for the presence of antibodies to red cell antigens), and (2) the cross-match (in which the recipient serum is combined with the potential donor red cells in the laboratory as a final check to make sure there will be no antibody-antigen mismatch in the transfusion).  These tests are done to avoid hemolytic transfusion reactions (which can happen if your patient has a red cell antibody, and you give red cells with the corresponding antigen).

The point of this test is to find out if your patient has antibodies against red cells – either antibodies against any red cell antigen at all (which is what the antibody screen looks for), or antibodies against the particular unit of red cells you have chosen to give the patient (which is what the cross-match is for). The DAT is looking to see if your patient’s red cells are coated with antibody; the IAT is looking to see if your patient has antibodies in his or her serum.

Here’s how it’s done:

1)    Mix a little of your patient’s serum (shown as grey antibodies on the left hand side of the diagram) with a little of the donor red cells (shown as red cells with little blue antigens below the second test tube).
2)    Centrifuge tubes and check for agglutination (depicted in the diagram in the center panel, showing red cells with antibodies attached). Sometimes, the reaction is so strong that you can see agglutination with the naked eye at this point! Usually not, though.
3)     Incubate at 37° C for half an hour, then check again for agglutination again. Sometimes heating the test tube will aid in the agglutination process.
4)    Wash the cells (to remove any plasma proteins that could interfere).
5)    Add anti-human globulin (Coombs’ reagent, discussed under the DAT) (depicted as blue antibodies under the fourth test tube).
6)     Incubate and check again for agglutination (shown as the final panel, way on the right hand side of the diagram).

If there is agglutination in the tube (in any of the steps), you know that your patient must have an antibody to one (or more) of the antigens on the donor red cells in that tube. You can’t give that particular unit of red cells, obviously!

Note: The very nice depiction of the IAT above was created by A. Rad, and can be found on Wikimedia commons at: http://commons.wikimedia.org/wiki/File:Coombs_test_schematic.png.

Direct antiglobulin test

Coombs_test_schematic

The direct antiglobulin test (also called the Coombs’ test, or the DAT) is an important one for you to know. It’s used mostly in one particular setting: when you have a patient with a hemolytic anemia (one in which the red cells are getting busted open) and you want to know if the hemolysis is immune-related or not. As explained below, the DAT is positive in immune hemolytic anemias and negative in non-immune hemolytic anemias.

The whole point of the DAT is to find out whether there are antibodies and/or complement bound to the surface of the patient’s red cells. In an immune hemolytic anemia, the patient may have antibody, or complement, or both bound to his or her red cells. Since you can’t see the antibody or complement under the microscope, you need a way to determine whether these molecules are present – and that’s what the DAT is for.

Here is how it is done. A small amount of a reagent called Coombs’ reagent, or anti-human globulin (AHG) is added to the patient’s blood in a test tube. This reagent (depicted as blue antibodies in the diagram above) consists of antibodies directed against human antibodies. These antibodies are raised by injecting human antibodies into another animal (a rabbit, or a mouse, or some other non-human), and then collecting the anti-human-antibody antibodies the animal makes (the animal sees the human antibodies as foreign substances, and it makes its own antibodies against them). You also add some antibodies directed against complement to the patient’s blood sample (these are not depicted above).

The cool thing about the Coombs’ reagent is that if the patient’s red cells are coated with IgG, the Coombs’ reagent will bind to this IgG on the red cells, bridging the gap between adjacent red cells, and causing the red cells to clump together (see the right hand side of the diagram above)! You can see this clumping with the naked eye. The same principle works for the anti-complement antibodies; if there is complement bound to the red cells, the anti-complement antibody will bind to it, and the red cells will clump together.

So: if you see clumping in the test tube, the DAT is positive, and that means your patient has an autoimmune hemolytic anemia. The next thing to figure out is which kind of autoimmune hemolytic anemia it is – but that’s the subject of another post.

Note: The very nice depiction of the DAT above was created by A. Rad, and can be found on Wikimedia commons at: http://commons.wikimedia.org/wiki/File:Coombs_test_schematic.png.

What does normal parathyroid tissue look like?

parathyroid

Might as well admit it: the histology of the parathyroid glands is highly forgettable. I’m not sure why this is – perhaps because we don’t talk about it very often, perhaps because it’s so banal – but I do know that it’s one of those things that needs a lot of repetition to become permanent.

The parathyroid glands are four dinky little things (3-4 mm, about 35 mg each) usually located on the posterior surface of the thyroid gland. They exist in two pairs; the upper pair is derived from the fourth branchial cleft and descends with the thyroid gland, and the lower pair is derived from the third branchial cleft and descends with the thymus.  Their main function is the regulation of serum calcium levels. They do this by secreting a hormone called parathormone (or PTH) when serum calcium levels go down. PTH does all kinds of things (it activates osteoclasts to chew up bone, increases renal reaborption of calcium, increases renal conversion of vitamin D to its active form, and increases calcium absorption from the gut) but the bottom line is that it raises the serum calcium.

It’s the histology that’s like a blank spot in most medical students’ (and physicians’) minds. The parathyroid is composed of two types of cells: chief cells (small, round, bland cells) and oxyphil cells (large cells with abundant eosinophilic cytoplasm). The chief cells are the secretors of PTH, and they make up the bulk of the cellularity of the parathyroid. Scattered throughout are small islands of oxyphil cells (you can see one at 12 o’clock in the image above). There is a varying proportion of fat, too, that increases with age (like every other part of the body, it seems).

That’s it! Now go memorize it! Maybe this is the final repetition of parathyroid histology that will stick in your brain forever.

What’s ringworm?

athletes-foot

Ringworm is a superficial fungal infection of the skin, nails, or hair. The causative agent is not a worm, but a type of mold called a dermatophyte (“skin plant”). (more…)

What causes nephrotic and nephritic syndrome?

coke

Okay, we talked about how to remember the components of nephrotic syndrome and nephritic syndrome. But what causes these syndromes?

Let’s boil it down to the top causes.

Nephrotic syndrome can be caused by renal diseases or systemic diseases (like diabetes). We’ll just discuss the renal diseases here. All of these diseases are characterized by a loss of foot processes (look it up in a physiology textbook if you’ve forgotten what these are). The three main renal diseases are:

1. Minimal change disease. This is the number one cause of nephrotic syndrome in children. The pathogenesis is unknown. It’s called minimal change (or sometimes “nil”) disease because under light microscopy, the glomeruli look pretty normal! Prognosis is good.

2. Focal segmental glomerulosclerosis. This disorder can be primary, or it may be associated with other conditions (like HIV, or heroin use). It’s called focal segmental glomerulosclerosis because if you look at a kidney biopsy, some (“focal”) glomeruli show partial (“segmental”) hyalinization. The pathogenesis is unknown, and unlike minimal change disease, the prognosis is generally poor.

3. Membranous glomerulonephritis. This type of glomerular disease is really an autoimmune reaction against some unknown renal antigen. Immune complexes are formed and are deposited along the glomerular basement membrane, which appears thickened on light microscopy. If you look at an electron micrograph, you’ll see subepithelial deposits, or “spikes.”

There are two main causes of nephritic syndrome. Both are immunologically mediated, and are characterized by proliferative changes and inflammation in the glomeruli. These causes are:

1. Postinfectious glomerulonephritis. This used to be called post-Streptococcal glomerulonephritis, because it most commonly occurs in children following a case of Strep throat. There’s a rather crude, but useful, mnemonic for this disease: sore throat (Strep infection), face bloat (edema), pee coke (patients often have brown-colored urine). Immune complexes are formed (the antigen is unknown, but probably is some type of Streptococcal protein) and deposited in the glomerular basement membrane (you can see the deposits on electron microscopy; they look like subepithelial humps). On light microscopy, the glomeruli look big and hypercellular, with lots of inflammatory cells. Recovery occurs in most children.

2. IgA nephropathy. This is also called Berger disease. It’s the most common glomerular disease worldwide, and it’s one of the most common causes of recurrent hematuria. It usually occurs in children or young adults, and it presents as hematuria following an upper respiratory infection. Patients with this disease produce abnormally high IgA levels; following an infection, you can see this IgA in the mesangium of the glomerulus (using immunofluorescent stains for IgA). Once IgA gets trapped there, complement is activated, and we all know what that does (bad stuff). The prognosis is variable.

There are, of course, other causes of nephrotic and nephritic syndrome (nothing is ever that easy!). But if you remember the main causes listed above, that should serve you very well.

Note: the photo of coca-cola bottles was taken by DeusXFlorida and can be found at http://www.flickr.com/photos/8363028@N08/3029152878/.

What is cryptorchidism?

orchid Cryptorchidism (from the Greek kruptos, hidden, and orkhis, testicle) is a term describing incomplete testicular descent. Normally, the testis descends into the pelvis by the third month of gestation, and then through the inguinal canal into the scrotum during the last two months of gestation.   (more…)