Pathology Student

Hemolytic disease of the newborn (HDN) is a disease in which there is hemolysis in a newborn or fetus caused by blood-group incompatibility between mother and child.

There are a bunch of related terms:

• Immune hydrops (Hydrops means accumulation of edema fluid in the fetus during intrauterine growth. It is not specific to HDN, but can occur in many different fetal conditions including cardiovascular conditions, chromosomal disorders like Down syndrome, non-immune fetal anemia, twin-twin transfusion, infections, tumors, and metabolic disorders. Whew.)
• Hydrops fetalis (When the accumulation of fluid – from whatever cause – is severe and generalized, it is called hydrops fetalis.)
• Erythroblastosis fetalis (Erythroblastosis means that early red cell precursors are showing up in the peripheral blood. This can happen in any severe anemia, not just HDN.)

Mechanism of HDN

1. Fetus inherits blood group antigens (usually Rh D antigen or ABO antigens) from the father that are foreign to the mother.
2. Fetal blood gets into mom’s circulation (either during last trimester of pregnancy, when cytotrophoblast is no longer present, or during childbirth).
3. Mom makes antibodies to these blood group antigens.
4. Antibodies cross the placenta, attack baby’s red cells, causing hemolytic anemia and its consequences.

The consequences of hemolysis are numerous. One such consequence is extramedullary hematopoiesis. If the anemia is mild, extramedullary hematopoiesis in the liver and spleen may produce enough red cells to maintain normal numbers.

Other consequences are not so happy. If the anemia is severe, the heart and liver may suffer hypoxic injury, resulting in circulatory and hepatic failure. Liver failure causes decreased protein levels (proteins are synthesized in the liver) and a reduction in oncotic pressure in the circulation. Heart failure causes an increase in venous pressure (blood is backing up behind the failing heart). If severe enough, the combination of reduced oncotic pressure and increased venous pressure leads to generalized edema and ascites, a condition called hydrops fetalis, which can be fatal. Lesser degrees of edema can also occur.

If hemolysis is severe, jaundice can occur due to accumulation of unconjugated bilirubin. Unconjugated bilirubin is water insoluble; it binds to lipids in the brain (the blood-brain barrier in the fetus is poorly developed), causing serious damage to the CNS, termed kernicterus. The affected brain is enlarged, edematous, and yellow.

Rh-mediated HDN

Rh-mediated HDN most often involves the D antigen (sometimes it involves E or c; rarely it involves e or C). The baby inherits the D antigen from father, and mom is D negative (same as saying “Rh negative”). Fetal blood gets into mom’s circulation (through trauma, ruptures in the placenta during pregnancy, medical procedures carried out during pregnancy that breech the uterine wall, or childbirth). As a result, mom makes anti-D antibodies (the amount of antibody made depends on dose of antigen received from baby! Mom only makes anti-Rh antibodies when the she has received more than 0.5- 1 mL of Rh + cells.). Just like any other developing antibody, IgM appears first, and IgG appears later. This is important because IgG can cross the placenta, but IgM can’t. So HDN is uncommon in a first pregnancy. But if the mom gets pregnant again, and the fetus inherits D again, mom will now make IgG antibodies, and HDN can happen then.

Rh-mediated HDN is diagnosed using a direct antiglobulin test (DAT). This test will be positive in the baby (baby’s cells are coated with mom’s antibodies). An indirect antiglobulin test  will be positive in mother (though if the mother has received Rhogam at 28 weeks – keep reading – the IAT will be artificially positive!). Administration of anti-D antibody (Rhogam) at 28 weeks and again within 72 hours of delivery to Rh-negative moms prevents HDN in the current pregnancy (and, if mom has not produced anti-D yet, protects future pregnancies too) by coating any circulating D+ fetal red cells before mom is able to make any anti-D antibodies! The incidence of Rh-mediated HDN has gone way down since Rhogam was developed.

In order to determine the appropriate dose of Rhogam to give the mom, you have to quantify the amount of fetomaternal hemorrhage. This is done using either the Kleihauer-Betke test or an immunophenotyping assay. We’ll discuss these tests in our next post.

ABO-mediated HDN

ABO incompatibility occurs in 20-25% of pregnancies…but laboratory evidence of hemolytic disease occurs only in 1 of 10 such infants, and the hemolytic disease is severe enough to require treatment in only 1 in 200 cases.

There are a number of reasons why ABO incompatibility is rarely serious:

1. Most anti-A and anti-B antibodies are IgM (hence they don’t cross the placenta).
2. Neonatal RBCs express A and B poorly (the expression of A and B antigens increases as the baby grows).
3. Many cells other than red cells express A and B antigens and thus sop up some of the transferred antibody.

ABO hemolytic disease occurs almost exclusively in infants of A or B type born of group O mothers. Normal anti-A and anti-B antibodies are IgM and therefore don’t cross the placenta. For reasons not understood, however, some group O women have IgG anti-A and anti-B even without prior sensitization! In this situation, a firstborn child may be affected. Fortunately, even with transplacentally acquired antibodies, lysis of infant red cells is minimal.

ABO incompatibility is diagnosed with same tests as Rh incompatibility (DAT, IAT, Kleihauer-Betke test). There’s no effective protection against ABO incompatibility reactions! Good thing they’re not very common.

Treatment of HDN

It’s obviously way better to prevent HDN than to try treat it once it’s developed. That’s why the mother’s blood type is determined very early in pregnancy, and Rhogam is administered if mom is D negative.

If HDN does develop, there are several options for treatment. Minimally affected newborns can be treated with phototherapy (as in the photo above). Light oxidizes unconjugated bilirubin (toxic) to water-soluble, readily-excreted dipyrroles (harmless). Severely affected fetuses can be treated by total exchange transfusion of the infant (through umbilical vein). The mother can be treated with plasmapheresis (which removes antibody). High-dose intravenous immunoglobulin can be used too – but the best dosage and timing are not well-defined.

Photo credit:  treehouse1977 (http://www.flickr.com/photos/treehouse1977/3310309612/in/photostream/), under cc license.

Sometimes we (okay, I) get so caught up in describing pathologic mechanisms that real-life examples get the short end of the stick. Let’s look at some real diseases in which the underlying problem is a hypersensitivity reaction.

Type I (allergic) hypersensitivity
The big example (obviously) of this type of hypersensitivity is allergy. Pollen, cat dander, peanuts – they all have the same mechanism and this is it.

Type II  (antibody-mediated) hypersensitivity
There are a ton of diseases that have an underlying type II hypersensitivity reaction going on. Here’s a partial list:

1. Autoimmune hemolytic anemia: the patient makes antibodies to red cell antigens for some reason (not a good thing to do) which end up causing hemolysis.
2. Pemphigus vulgaris: the patient makes antibodies against the proteins connecting epithelial cells together, resulting in epithelial cell discohesion and bullae formation.
3. Goodpasture syndrome: the patient makes antibodies that react against proteins in both the glomeruli and the alveoli, leading to nephritis and lung hemorrhage.
4. Myasthenia gravis: the patient makes antibodies that bind to the acetylcholine receptor (on the muscle end plate), preventing acetylcholine from binding and doing its job; the end result is muscle weakness.
5. Graves disease: the patient makes antibodies that bind to the TSH (thyroid-stimulating hormone) receptor on thyroid epithelial cells, causing excessive stimulation of the receptor (just the opposite of what happens in myasthenia gravis!), leading to excessive production of thyroid hormone (hyperthyroidism).

Type III (immune-complex-mediated) hypersensitivity
There are a ton of diseases in this category too.

1. Lupus: the patient makes antibodies that bind to certain nuclear antigens; complexes lodge anywhere they please but especially in the kidneys, skin, and joints.
2. Post-streptococcal glomerulonephritis: in fighting a strep infection, the patient makes an antibody that reacts against the strep bug but also cross-reacts with some antigen in the glomerulus; antigen-antibody complexes lodge there and cause nephritis.
3. Serum sickness: after injection of foreign proteins into the patient (e.g., like they did in the old days when they used horse serum in vaccines!), the patient makes antibodies against the foreign proteins, and the resulting complexes lodge anywhere they want, but especially in the joints, kidneys, and vessels.
4. Arthus reaction: after injection of foreign protein into the skin, the patient (the original patient in Arthus’ 1903 experiments was a poor little bunny – but the same thing happens in humans) makes antibodies against this protein, and the resultant complexes stay in the skin, eliciting a nasty localized vasculitis and a big owie on the skin.

Type IV (T-cell-mediated) hypersensitivity
There are two kinds of this one (after all, there are two kinds of T cells!).

Delayed-type hypersensitivity
A good example of this is poison ivy exposure (patient gets exposed to poison ivy, helper T cells respond and some become memory cells; upon repeat exposure the memory T cells rush to the site, activating macrophages and causing inflammation). Another example is the Mantoux test; same principle.

T-cell-mediated cytotoxicity
Type I diabetes is a good example of this one. In this disease, cytotoxic T cells kill pancreatic islet cells. That’s not very nice. They are supposed to be killing infected cells, or tumor cells – not the patient’s own normal cells.

Photo credit: Joe Shlabotnik (http://www.flickr.com/photos/joeshlabotnik/506346418/in/photostream/).

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/

We’re back after a nice long summer break! School is back in session and hopefully Pathology Student will give you a little help along the way in your study of pathology.

Here’s a general pathology concept that is important not only for boards but for real life: wound healing. Let’s take a look at wound healing in the skin. Whether the wound is a small kitty scratch or a huge burn, we have ways of repairing the damage and restoring function to the skin. There are two types of wound healing in the skin: healing by first intention and healing by second intention (weird names, I know, but whatever).

Healing by first intention occurs in small wounds that close easily – for example, paper cuts, or small surgical incisions in which the edges are easily approximated. In this type of healing, epithelial regeneration predominates over fibrosis. That’s a fancy way of saying that there is usually minimal scarring in this type of healing. Healing is generally fast. Here’s a summary of the timeline in most wounds healing by first intention:

By 24 hours: a clot forms, neutrophils arrive, and the epithelium begins to regenerate
By 3-7  days: macrophages arrive, granulation tissue is formed, collagen begins to bridge the incision, and the new epithelium increases in thickness.

Let’s stop right here for a moment. Granulation tissue is that stuff that forms when your body is filling in the gap between your remaining tissues. The contents of granulation tissue are 1) new, fragile blood vessels, 2) fibroblasts, and 3) a loose extracellular matrix holding it all together. The whole point of granulation tissue is to provide a place for the new structures to grow that will hold the tissue together (blood vessels and collagen). That’s it. Note: granulation tissue is not the same as a granuloma (which is a collection of macrophages) or chronic granulomatous disease (in which patients have neutrophils that don’t work right, so their macrophages are left with the job of killing bacteria, and they form little granulomas all over the place). So don’t get those terms mixed up.

Weeks later: the granulation tissue is gone, collagen has been remodeled (using little metalloproteinase enzymes like collagenase), and the epidermis is full and mature (though it lacks dermal appendages in the area of the healed wound). Eventually, a full-blown scar forms.

Healing by second intention occurs in larger wounds that have gaps between the wound margins. Examples of this type of wound are: areas of skin infarction, large burns and ulcers, and extraction sockets (where the dentist has pulled a tooth. Yes, this first- and second-intention healing applies to mucosal epithelium too!). In this type of healing, fibrosis predominates over epithelial regeneration. In other words, there’s gonna be a big scar that’s more prominent than any skin regrowth that occurs. Healing by second intention is slower. There is a lot more granulation tissue (because you have a huge gap to bridge) and more inflammation (neutrophils and macrophages coming in to clean up the dead cells and debris). Therefore, there’s a greater risk of infection and inflammation-related tissue injury. Also, the wound contracts as it heals (so you don’t have to make such a big scar). As far as a timeline goes, you can’t really make a universal timeline for second-intention healing, because it varies a lot depending on how big the wound is.

It all makes sense if you can just remember: first intention = small wounds, second intention = big wounds.

The strangely beautiful photo above, aptly titled “cat launchpad,” was taken by quinn.anya and can be found at: http://www.flickr.com/photos/quinnanya/2420314228/.