Weatherly's Woes - Normocytic Anemia

NORMOCYTIC ANEMIA

Normo- (normal) -cytic (cell)

Normocytic Anemia refers to anemia where the RBCs are regular sized, meaning that the MCV is normal (between 80 and 100). Up until now, every anemia in this document has originated from some problem within the bone marrow. In microcytic anemia the production of Hgb was interrupted, and in macrocytic anemia the production of DNA was halted. Normocytic anemia is typically not due to a problem with the bone marrow. There are two major causes: hemorrhage and hemolysis.

Hemorrhage is self-explanatory. If you bleed out half of your RBCs, the remaining RBCs will remain the same size.

Hemolysis, or “breaking of blood,” is the destruction of RBCs. It’s categorized by where the destruction occurs. RBCs are either killed in the blood vessels (intravascular) or in the spleen / liver / bone marrow / lymph nodes (extravascular). These four organs are collectively called the reticuloendothelial system. They all have special fenestrated blood vessels that hide macrophages, and the point of those macrophages is to eat any RBCs that are old or broken. The spleen is the largest contributor to the reticuloendothelial system -- which is why splenomegaly is often seen with hemolytic conditions.

Intravascular Hemolysis

Intravascular Hemolysis is when an RBC explodes inside of a blood vessel. When the RBC dies, its Hgb spills out into the plasma. The free Hgb floats through the blood, and eventually passes out into the urine (hemoglobinuria), turning it brown (this can damage the kidneys). Since Hgb is toxic to nephrons, there are mechanisms in place to clean up Hgb before it can reach the kidneys. Plasma contains a small amount of haptoglobin, a sort of vascular janitor, that binds to free Hgb. The haptoglobin carries the lost Hgb to the spleen, where it can be destroyed and recycled. Low haptoglobin is a sign of intravascular hemolysis, because the haptoglobin is consumed as it binds to the spilled Hgb. Normal haptoglobin levels are seen in extravascular Hemolysis (as well as in health). Note that some of the free Hgb will deposit into tissues and cells, where it’s stored for long periods of time as a pigmented molecule called hemosiderin. This brown molecule is a common finding in tissues previously swollen with blood (like the legs in heart failure).

Extravascular Hemolysis

Extravascular Hemolysis is like taking your car to the auto shop. Every few thousand miles, you have to take it to the shop to get a routine inspection. The mechanic can give your car a clean bill of health, or can make a few repairs, or can even condemn the car if the damage is bad enough. So, in this metaphor, your car is the RBC, the spleen is the auto shop, and the splenic macrophages are the mechanics. The splenic macrophages inspect every RBC that passes through the sinusoids of the spleen, and based on the presence or absence of certain membrane proteins, will either ignore the RBC, eat a little chunk off of the RBC, or will consume the RBC entirely. When it eats the old / broken RBC, it breaks it down in a controlled and sanitized manner, just like a good seasoned mechanic. That means that there won’t be any Hemoglobin spilling out. Extravascular hemolysis is a more orderly and controlled process than intravascular hemolysis.

Findings of Hemolysis

All the symptoms are due to corpses of RBCs.

RBCs are gobbled up in the Spleen → Splenomegaly
Hgb spills out, which becomes bilirubin → Jaundice
Some Hgb spills into the urine → Hemoglobinuria & Kidney injury

HEMOLYSIS

Hemo- (blood) -lysis (break)

Hemolysis is the pathological breakdown of red blood cells. But how do you know if your patient’s anemia is a hemolytic one in the first place?

Well, you can look at the patient. In addition to the usual signs and symptoms of anemia (pallor, fatigue, breathlessness), you may see additional signs that indicate that the anemia is hemolytic, such as jaundice (from bilirubin, a yellow-colored breakdown product of the heme molecule), or splenomegaly (some types of hemolytic anemia are accompanied by a big spleen). But these signs are non-specific; they can occur in disorders other than hemolytic anemia. For good, definitive proof of a hemolytic process, you need to look at lab results.

First, you should see signs that the red cells are being destroyed. These include:

Hemoglobinuria (as evidenced by brown urine)
High unconjugated bilirubin in the serum (this occurs when red cells are being destroyed faster than the liver can process the bilirubin)
High lactate dehydrogenase (LDH is an enzyme present in all cells, including red cells; when you bust open red cells, their LDH is released)
Low haptoglobin (in intravascular hemolysis)

You should also see signs of accelerated erythropoiesis, like an increased reticulocyte count. Reticulocytes are young red cells that are a little bigger and have more RNA than mature red cells. We all have a small percentage of reticulocytes in our blood, but if there is hemolysis, the number of reticulocytes should increase (like a warring nation calling up teenaged soldiers).

MICROANGIOPATHIC HEMOLYTIC ANEMIA (MAHA)

Sharp little blood clots

In microangiopathic hemolytic anemia (MAHA), red blood cells are sliced into ribbons as they pass through a meshwork of tiny fibrin-rich clots that form inside of capillaries and small vessels. The torn RBCs have a characteristic angular, fragmented appearance termed schistocytes. There are several causes, here are the big three.

Disseminated Intravascular Coagulation (DIC) is nasty condition in which there is bleeding and clotting at the same time. See the DIC page.

Thrombotic Thrombocytopenic Purpura (TTP) is a rare but deadly hematologic disorder. In this syndrome, the patient gets little thrombi within the microvasculature everywhere in the body. “Thrombotic” (they’re clotting) + “Thrombocytopenic” (platelets are consumed) + “Purpura” (they have purpura, or tiny skin bleeds, due to the clotting). TTP is autoimmune, so it, predictably, occurs more often in young adult females. TTP is typically caused by antibodies gobbling up a little known protein called ADAMTS13. ADAMTS13 is a vWF metalloprotease. vWF is stored in endothelial cells as long bundled chains. But cells can’t use the unwieldy multimers, they have to chop them up first. Enter ADAMTS13, which cuts them apart. Without ADAMTS13, there’ll be an excess of vWF multimers, which don’t function properly, leading to platelet adhesion and aggregation. For whatever reason, TTP loves to hit the kidneys and CNS. Thus, patients suffer from acute kidney injury and neurological symptoms in addition to the purpura. Also fever (for some reason). TTP only affects primary hemostasis (because there’s extra vWF around). It doesn’t touch the coagulation cascade! That’s a really important piece of information, because it means that the INR / PTT won’t be affected! The bleeding time, a cumbersome test, would be abnormal (prolonged). Mortality of a TTP flare is surprisingly high. Treat with plasmapheresis (to clean up the existing antibodies) and steroids (to suppress the production of new antibodies).

Hemolytic Uremic Syndrome (HUS) is an autoimmune sequelae of a gastrointestinal infection from E. coli 0157:H7, typically spread by eating undercooked ground beef. This strain of E. coli. makes a toxin (Shiga-like toxin) that damages endothelial cells, and for some reason, the kidneys are hit the hardest. “Hemolytic” (hemolysis) + “Uremic” (happens in the kidney). Child → eats undercooked beef → the beef is infected with E. coli 0157:H7 → releases Shiga-like verotoxin → damages the endothelium (also gives a nasty bloody diarrhea) → exposes vWF → microthrombi form (mostly in the kidney) → hemolysis. HUS typically occurs several weeks after the episode of bloody diarrhea. Treatment is supportive.

The blood smear is where it’s at for MAHA. If you look at the blue circles, you’ll see fragmented red cells, or schistocytes. Schistocytes are smaller than normal red cells, and they have jagged pointy edges on them. There are all kinds of possible shapes - some schistocytes have just one point, some look like triangles and others resemble helmets.

MACRO-angiopathic Hemolytic Anemia is when RBCs are crushed outside of the capillaries. The most common example is a mechanical heart valve. Unlike a normal valve, the metal valve is so hard that it crushes a handful of red cells every time it closes. The old ball-and-socket model valves were the worst for this, the newer models are much kinder. It can also occur in coarctation of the aorta, apparently due to the highly turbulent flow, as well in long-distance runners (each step pops a few RBCs in the foot).

PAROXYSMAL NOCTURNAL HEMOGLOBINURIA

Paroxysmal (the intermittent severe fit of a disease) Nocturnal (night) Hemoglobinuria (Hgb in the urine)

Paroxysmal Nocturnal Hemoglobinuria is a rare, chronic intravascular complement-mediated hemolysis causing vessel thrombosis and hemoglobinuria (visible to the naked eye as dark urine) that presents in the morning.

How does the complement system normally recognize healthy human cells (to avoid them)?

Human cells are studded with flagging proteins that tell complement “Chill out! I’m a good guy!” Two of these flag proteins are Membrane Inhibitor of Reactive Lysis or MIRL (CD59) and Decay Accelerating Factor or DAF (CD55). MIRL and DAF disable C3 and C5 of the complement cascade. MIRL and DAF are anchored to the membrane by GPI (glycosylphosphatidylinositol). GPI is encoded by the PIG gene (phosphatidyl inositol glycan). Whew.

So what’s the problem in PNH?

A mutation of PIG in a myeloid stem cell. Without PIG, the anchoring protein GPI doesn’t work. Without it, DAF and MIRL can’t be on the membrane of blood cells. Without DAF and MIRL, blood cells are vulnerable to complement.

What activates complement?

(A lot of things) But for our purposes, know that complement is activated by acidosis. When we sleep our breathing becomes shallow, resulting in a mild respiratory acidosis (retention of carbon dioxide). Hemolysis occurs continuously through the night. Dark morning urine occurs when hemoglobin and other cellular debris spills into the blood, and then urine, overnight.

Are only red cells affected?

Nope. All myeloid cells are affected. Red cells, white cells and platelets all express MIRL and DAF. Patients can have pancytopenia. When platelets are killed, they release aggregating factors (ADP/TXA2), which leads to thromboses all over the body (strokes, heart attacks). In PHN, this can lead to some unusual blood clots. For example, clotting of the hepatic vein (Budd-Chiari Syndrome).

Diagnosis?

Flow cytometry is the test of choice, looking for a lack of CD55 (DAF). There is an older test called the sucrose test which would also be positive (sucrose activates complement and precipitates hemolysis).

Treatment?

Eculizumab, the C5 inhibitor.

The timeline of dark urine. It’s darkest in the morning, because the RBC destruction takes place overnight!

G6PD DEFICIENCY

Favism

Glucose-6-Phosphate Dehydrogenase Deficiency is an X-linked recessive genetic hemolytic anemia caused by a deficiency in, dur, G6PD - an early enzyme in the pentose phosphate pathway. It’s one of the most common diseases in the world! About 10% of black individuals have it, and it’s also common in Mediterranean populations. How on earth could an X-linked recessive disease become so common? Because it conferred a survival advantage by protecting red cells from P. falciparum malaria.

What’s the purpose of the pentose phosphate pathway?

G6PD uses Glucose-6 phosphate to make NADPH, which replenishes glutathione. Glutathione is our body's main antioxidant molecule. Therefore, G6PD-D patients are at risk for oxidative damage. The blood is a highly volatile environment, and there is a lot of oxidative stress. Red cells use glutathione to protect themselves. No G6PD means no NADPH which means no replenished Glutathione which means oxidative damage!

What is the pathophysiology?

The G6PD enzyme increases the longevity of RBCs. Red cells live about 120 days. So normally cells only need G6PD to last for 120 days. But in G6PD-D the enzyme denatures before day 120 (the exact day is variable, G6PDD is a highly variable condition). In severe cases (Mediterranean variant), G6PD will degrade after about 30 days, but in a mild case (African variant) after 90 days. Thus the older red cells have less glutathione than the younger cells. When something oxidizes the blood, the older red cells are killed. The young cells are easily able to use glutathione to shield themselves. In the more deadly Mediterranean variant, most of their red cells have lost G6PD, so they are susceptible to massive anemia in an oxidizing situation.

How does oxidation harm the red cell?

Oxidative stress damages Hgb → Heinz bodies → Heinz bodies damage the RBC membrane → intravascular hemolysis → Splenic macrophages remove the Heinz bodies from survivors (extravascular) → Bite cells

How do you oxidize blood?

Infection - The most common oxidant stress
Drugs - Nitrofurantoin, Primaquine, Chloroquine ,Dapsone, Sulfonamides. High Yield!
Fava beans - beans are mildly oxidizing, so this only affects the Mediterranean type

Clinical findings?

After exposure to some oxidative stress, the patient will unexpectedly develop flank pain (Hgb is nephrotoxic) and their urine will turn dark (hemoglobinuria). Jaundice may occur.

Lab findings?

Normocytic anemia with reticulocytosis (healthy marrow remember). On a blood smear, bite cells and Heinz bodies can be seen. But Heinz cells need a special stain (supravital stain) to be visualized. Enzyme studies confirm the disease, but NOT during an acute attack (because the defective RBCs are all dead).

HEREDITARY SPHEROCYTOSIS

Sphero- (spherical) -cyte (cell)

Hereditary Spherocytosis is an Autosomal Dominant genetic disorder affecting the plasma membrane of RBCs. The proteins that tether the membrane to the cytoskeleton are defective.

Which proteins are involved in the membranous architecture?

Mostly spectrin. But some other players are ankyrin, band 2 and band 3.

How is the membrane affected by the loss of spectrin?

Little blebs form on the surface of the red cell. This is really only a cosmetic problem at first. But when the blebby red cell passes under the gaze of the splenic macrophages, something happens. The macrophages are notoriously the fashion police of the hematologic world. They nibble off the unsightly blebs. As more and more blebs are excised, the RBC is forced into a spherical shape due to the loss of the plasma membrane. It reminds me of a face lift, you snip off some extra skin, and the wrinkles disappear! When the cells become too spherical, the spleen destroys them, producing a normocytic extravascular hemolytic anemia. Spherocytes are perfectly functional at carrying oxygen, but they’re clunky and can’t maneuver through the splenic sinusoids, like a Hummer trying to navigate a small alley.

Clinical signs and symptoms?

The symptoms are pretty much the same as all the other hemolytic anemias: anemia, jaundice, splenomegaly and bilirubin gallstones. Interestingly, the jaundice is often seen as early as the first week of life.

Lab findings?

Normocytic anemia
Spherocytes on the blood smear (spherocytes are also seen in warm autoimmune hemolytic anemia). Spherocytes can be identified by the loss of central pallor.
↑ MCHC (mean corpuscular hgb concentration) because the spherocytes lose a little bit of cytoplasm when the blebs are excised. This is very helpful diagnostically because it’s the only anemia with a high MCHC
↑ Osmotic Fragility - this is a test that’s reserved for investigating spherocytosis. The lab tech puts red cells into a hypotonic solution. Water rushes into the red cells. Normal red cells turn into spherocytes. The spherocytes are already full, and they explode.

Treatment?

Splenectomy. Afterwards, the patient will have weird shaped red cells that are still functional. Removing the spleen removes the site of red cell destruction, and prevents anemia.

Hereditary Elliptocytosis is a milder variant. The red cells become elliptical instead of spherical. This is a very benign condition. They won’t have anemia, although you might expect to see some splenomegaly.

Spherocytes

Sickle Cell Disease

Sickle Cell Disease is a tragic condition. I saw it often while working in the ER. “Sicklers,” as they’re called, shuffled in and out begging for powerful pain medications. That’s because SS causes repeated, extraordinary bouts of pain anywhere in the body. It’s described as one of the most painful conditions in all of medicine. The only way to soothe their 11 / 10 pain is with high dose opioids. That has led to a predictable second tragedy, rampant opioid addiction. Many people with sickle cell are jobless, with sunken eyes and hollow affects. They walk a long and difficult road. Above all else, you should take them seriously and treat their pain with respect! Sickle Cell Anemia causes a plethora of complications all over the body. They have contact with almost every single medical specialty. Despite some new advances in treatment, most don’t reach old age.

Causes of Sickle Cell

It’s a bit of a mouthful, but here goes nothing. It’s caused by a homozygous autosomal recessive mutation of genes encoding for beta globin chains. Specifically, it’s a missense point mutation, where Glutamic acid (a hydrophilic molecule) is replaced with Valine (hydrophobic). This new, mutated hemoglobin has an unusual property -- it polymerizes into rods (which then crystallize) whenever the RBC is deoxygenated. These stiff rods force the RBC into a thin, elongated “sickle” shape. Upon re-oxygenation, most sickle cells return to normal, but a few will remain permanently distorted. SCA is only seen in Black people, which is true of most genetic blood disorders. It’s theorized to protect against malaria.

Anemia

Hemoglobin is made of heme and globin. Each globin is made of four subparts. The beta globin is mutated in sickle cell anemia. We refer to the mutated hemoglobin as HbS (S for sickle) instead of the healthy HbA (A for adult). The problem with HbS is that it polymerizes when stressed (hypoxia, dehydration, acidosis) which changes the shape of the RBC into a sickle. Sickling reverses when the stressor resolves, but it still incurs some permanent damage in the RBC membranes. The splenic macrophages notice the membranous damage and eat the RBCs, which produces a mixed hemolytic anemia (some of the RBCs simply lyse within the circulation). Patients tend to have a normocytic anemia at baseline. But if something adds to the red cell damage (like a parvovirus infection), this can precipitate an acute worsening of their anemia, termed an Aplastic crisis because their marrow is too weak to respond.

Diagnosis and Treatment

Using Hgb electrophoresis, you can determine the exact % breakdown of the different Hgb types. The majority of their hemoglobins will be HbS. Acute treatment of a pain crisis is with oxygen (oxygen inhibits sickling) and pain medicine. Chronic treatment is with Hydroxyurea, which works by increasing the numbers of fetal hemoglobin (HbF) via mysterious mechanisms. Newborns do not have any HbA. Instead, they mostly use HbF for the first 6 months of their lives. HbF doesn’t have any beta globin. Therefore, sickle cell symptoms don’t begin until patients are 6 months old! Keep vaccinations (especially for encapsulated organisms, like the pneumococcal vaccine) up to date. They also require folic acid supplements because folate is depleted by the accelerated erythropoiesis. Many patients require repeated blood transfusions.

Sickle Cell TRAIT

Sickle Cell Trait is the heterozygous form of SCA. Only one of their beta globin genes is mutated. Less than half of their hemoglobins are in the HbS form, which means that they CANNOT actually sickle. Well, except for one place. It’s the most hyperosmotic location in your entire circulatory system: the renal medulla. Recall that the osmolarity there skyrockets up to like 1200. The extreme hypertonicity of the renal medulla results in sickling and microinfarcts, which causes microscopic hematuria and a decreased ability to concentrate the urine.

Vaso-Occlusive Crises

The underlying problem with sickling is recurrent, disseminated blockages in blood flow. Sickle cells have a tendency to clump up against the endothelium and cause a traffic jam. Every part of the body can undergo vaso-occlusion, but there are a few organs with high-yield associations. Keep in mind that occlusions cause infarction, and infarctions are hot sites for infection. Most patients undergo several crises a year, usually 5 or 6.

PYRUVATE KINASE DEFICIENCY

Baby anemia

PK Deficiency is an inherited enzymatic disease that causes hemolytic anemia. PKD is most severe during childhood. The diagnosis is usually made soon after birth.

Pyruvate Kinase is the last step in glycolysis, and it produces Pyruvate and 1 ATP. RBCs are dependent upon glycolysis for ATP and lactate (to make further ATP), since they lack mitochondria.

In PK Deficiency, the RBCs suffocate a few days after being created. Without ATP, the Na-K Pumps stop working and water slowly leaks out of the cell. This leads to the characteristic RBC morphology of PKD, the spiky echinocyte, aka the Burr Cell. Eventually the echinocyte will die, resulting in hemolytic anemia.

Echinocyte (Burr cell)

AUTOIMMUNE HEMOLYTIC ANEMIA

AIHA is an umbrella term for the autoimmune causes of hemolytic anemia. The two flavors of AIHI are IgG-mediated and IgM-mediated. So the patient is either pumping out tons of IgG antibodies or IgM antibodies, which in AIHA are like heat seeking missiles for healthy red blood cells.

The Coombs test looks for autoimmune hemolysis

If the blood sample clots (or "agglutinates") then it's a positive test.

If the sample doesn't clot then it's a negative result.