AssessmentsIntrinsic hemolytic normocytic anemia: Pathology review
USMLE® Step 1 style questions USMLE
A 35-year-old man comes to the primary care office complaining of mild fatigue and shortness of breath. The patient has a past medical history significant for episodes of jaundice and intermittent right upper quadrant abdominal pain, which he has never been to the doctor for before. Temperature is 37.2°C (98.9°F), pulse is 72/min, respirations are 18/min, and blood pressure is 128/88 mmHg. Physical examination shows splenomegaly. Laboratory tests are obtained, and the results are shown below.
Peripheral blood smear shows hexagonal crystals and target cells. This patient’s disease is most likely caused by a substitution of glutamic acid with which of the following amino acids?
Contributors:Kaylee Neff, Jake Ryan, Maria Emfietzoglou, MD, Robyn Hughes, MScBMC, Stephen A. Robinson, MSMI, CMI, Darren Miller
On the hematology ward, there’s a mother with her daughter, Kyra, a five -year old that has developed jaundice and complains of easy fatigability. She is an adopted child with an unknown family history. Clinical examination reveals a palpable spleen. Next to her, there’s a 35-year-old person of African descent, called Darnell, who started trimethoprim-sulfamethoxazole for treatment of acute prostatitis a few weeks ago. Recently, he developed jaundice, dark urine, back pain and fatigue. There’s also a father who brought Billy, his 13-year-old son, to the emergency department because of a painful and prolonged erection. CBC is ordered for all of them and it shows low hemoglobin with normal MCV and reticulocyte count index over 2%. They also have increased LDH. Now, Kyra also has an increased MCHC and spherocytes on peripheral blood smear, while Billy has sickled cells.
Although their symptoms are very different, they all suffer from anemia, which is defined as lower than average levels of hemoglobin, typically below 13.5 g/dL in adult men and below 12.0 g/dL in adult women.This level varies based on the age for children. Now, anemias can be broadly grouped into 3 categories based on mean corpuscular volume, or MCV, which reflects the volume of a red blood cell. So microcytic anemia is where the MCV is lower than 80 fL, normocytic, with an MCV between 80 and 100 fL, and macrocytic, with an MCV larger than 100 fL. Normocytic anemias can be further classified as hemolytic when there’s increased destruction of RBCs, or hemolysis, and non-hemolytic when there’s decreased production of RBCs from the bone marrow. When there’s hemolysis, the bone marrow revs up and starts pumping out immature RBCs called reticulocytes, but when there’s a bone marrow problem reticulocyte count is low. So for your exams, it’s important to know that in hemolytic anemias there’s an increased reticulocyte production index of over 2%, while in non-hemolytic anemias it’s lower than 2%.
Alright, now hemolytic anemias can be classified as intrinsic and extrinsic hemolytic anemias. In intrinsic hemolytic anemias, the RBCs are destroyed due to RBC membrane defects, like in hereditary spherocytosis and paroxysmal nocturnal hemoglobinuria, or PNH; enzyme deficiencies, like in glucose 6 phosphate, or G6PD, deficiency and pyruvate kinase deficiency; and hemoglobin abnormalities, like in sickle cell anemia. Now, in extrinsic hemolytic anemias, the RBCs are normal but are later destroyed via extrinsic mechanisms such as autoantibodies directed against RBCs. In this video, let’s focus on intrinsic hemolytic anemias.
Now, we can also divide intrinsic hemolysis into intravascular, meaning RBCs are destroyed within the vasculature, or extravascular, meaning that they are removed by macrophages in the spleen and liver. Hereditary spherocytosis and pyruvate kinase deficiency cause extravascular hemolysis, PNH causes intravascular, while G6PD deficiency and sickle cell anemia can cause both intravascular and extravascular hemolysis. There are findings that can help identify the type of hemolysis. In intravascular hemolysis, hemoglobin that is released inside the vessels gets bound by a protein called haptoglobin and because they’re removed together, haptoglobin decreases. Also, when haptoglobin gets overwhelmed, the rest of hemoglobin goes via the blood through the kidneys and into the urine resulting in hemoglobinuria. Now, when hemoglobin is inside the renal tubules, the cells lining the renal tubules reabsorb hemoglobin. The heme component of hemoglobin contains iron which is stored as hemosiderin in tubular cells and after a few days, when tubular cells slough into urine, there’s hemosiderinuria. Hemoglobinuria and hemosiderinuria can damage the kidneys causing back pain. Okay, now in extravascular hemolysis, RBCs are destroyed outside the vessels and so, haptoglobin is normal and there’s no hemoglobin or hemosiderin in the urine. RBCs are usually destroyed in the spleen causing splenomegaly or the liver causing hepatomegaly.
Alright, now whenever there’s RBC lysis, an intracellular enzyme called lactate dehydrogenase, or LDH, spills out directly into the plasma and builds up in the blood. Hemoglobin also spills out of the cell and breaks up into globin and heme. Heme is converted into unconjugated, or indirect, bilirubin which is then taken up by the liver cells and eventually secreted out with bile. If all of a sudden, your body starts breaking down more RBCs than the liver cells can handle, the excess bilirubin stays in the blood and cause jaundice where the bilirubin deposits in the skin and the eyes, causing them to turn yellow. Also, when there’s too much bilirubin in the bile, it can form pigmented gallstones. Some of the bilirubin is converted to urobilin which is what gives urine that yellow color, but if there’s too much of it, the urine becomes a much darker, tea-like color.
Okay, so first let’s look at intrinsic hemolytic anemias caused by RBC membrane abnormalities which include hereditary spherocytosis and PNH. Hereditary spherocytosis is an autosomal dominant disorder characterized by defects in the spectrin and ankyrin proteins found in the RBC membrane. This results in abnormally shaped RBC that are more spherical instead of the normal flexible biconcave disks. The spherocytes get trapped and destroyed in the spleen resulting in chronic, mild extravascular hemolysis.
Next is paroxysmal nocturnal hemoglobinuria, PNH, a genetic disorder caused by a mutated PIG-A gene in myeloid stem cells. This gene encodes for a protein called phosphatidyl inositol glycan A that is needed to synthesize another protein called GPI anchor. GPI anchor is found in the membrane of all types of blood cells and serves to anchor proteins like decay accelerating factor, or DAF, also known as CD55, and CD59, to the cell membrane. These proteins normally inactivate the complement and so they protect the cells from complement lysis. So, a high yield concept for your exams is that in PNH, the complement stays activated and causes intravascular hemolysis.Another important fact to remember is that since PNH affects stem cells, it can cause aplastic anemia or pancytopenia. It also affects other blood cells like platelets and can cause formation of blood clots and thrombosis. So, an important clue to help you identify this disorder is that the patient can have thrombosis, hemolytic anemia, and reduced blood cell counts. In some cases, it could even lead to leukemia!
Alright, moving onto intrinsic hemolytic anemia due to enzyme defects. The most common is G6PD deficiency, an X-linked recessive disorder characterized by decreased levels of an enzyme called G6PD. It almost exclusively manifests as a disease in males, while females are carriers and is more common in individuals of African, Mediterranean and Asian descent. G6PD deficiency leads to hemolysis by making RBCs susceptible to damage caused by free radicals. But first things first. Free radicals, which are products of metabolism, can destroy RBCs but normally, a molecule in our body called glutathione neutralizes them. Glutathione needs to be in the reduced state where it can donate protons and electrons to the H2O2 and convert them into water. This causes glutathione to become oxidized, so before it can get back to work, an enzyme called glutathione reductase uses NADPH to reduce the oxidized glutathione, and NADPH becomes NADP+. So to replenish the supply of NADPH, we have G6PD, which reduces NADP+ back to NADPH. Okay, so in G6PD deficiency, low levels of G6PD causes low levels of NADPH, leading to low levels of reduced glutathione and increased susceptibility to hemolytic episodes caused by free radicals. Hemolysis usually happens in response to certain triggers like infections, metabolic acidosis, and foods and drinks like fava beans, soy products, red wine, and others. A high yield fact is that certain medications also act as oxidant stressors like the antimalarials, primaquine and chloroquine, painkillers like aspirin and ibuprofen, quinidine that is used to treat arrhythmias, and other medications that contain sulfonamide like the antibiotic trimethoprim-sulfamethoxazole. An interesting fact is that G6PD deficiency protects against Plasmodium falciparum that causes malaria since it makes the parasite-infected RBC more susceptible to oxidants, which will also kill the malaria parasites.
Another less common enzyme defect is pyruvate kinase deficiency. This is an autosomal recessive disorder characterized by decreased levels of an enzyme called pyruvate kinase. Pyruvate kinase is an enzyme involved in glycolysis, which is when glucose gets processed in order to generate energy in the form of adenosine triphosphate, or ATP. So, deficiency of this enzyme makes RBCs deficient in ATP Without ATP, sodium potassium ATPase pumps stop working. And because the cell membrane is more permeable to potassium than sodium, potassium leaks out. This makes the intracellular fluid hypotonic, so water moves out of the cell and the cell shrinks. These dehydrated RBCs can form tiny, uniform projections, turning into echinocytes or Burr cells. And these abnormally shaped RBCs get trapped and destroyed in the spleen, resulting in extravascular hemolysis. Now, another high-yield fact is that pyruvate kinase-deficient RBCs show enhanced oxygen delivery. That’s because the block in glycolysis results in the buildup of a metabolic intermediate called 2,3-bisphosphoglycerate or 2,3-BPG for short. 2,3-BPG has a strong affinity for hemoglobin, so within tissues, it competes with oxygen, thus reducing oxygen-hemoglobin affinity, allowing more oxygen to be released from hemoglobin to the tissues.
Okay, let’s move on to hemolytic anemia due to hemoglobin defects. The most common ones are sickle cell disease, also called sickle cell anemia, and hemoglobin C or HbC disease . These are autosomal recessive disorders caused by mutated genes that encode for abnormal adult hemoglobin called hemoglobin S for sickle, or HbS, and hemoglobin C or HbC. There’s substitution of glutamic acid in the sixth position of the beta globin chain, with valine in the case of HbS and lysine in the case of HbC. A mutation in both copies of the gene is needed to get the disease. If the person has just one copy of the mutation and one normal hemoglobin A gene, or HbA for short, then they have an HbS or HbC trait and they’re said to be a carrier. Some individuals have HbSC disease, meaning that they have one of each mutant gene. An important fact to remember is that, like G6PD deficiency carriers, sickle cell and HbC carriers are also protected against malaria, probably because infected RBCs get removed by the spleen. Now in individuals with sickle cell disease, when there’s acidosis, hypoxia, or dehydration, HbS changes its shape, and aggregates with other HbS proteins to form long chains that distort the RBC into a crescent shape, that looks like a sickle. In individuals with HbC disease, HbC is less soluble, so it aggregates into crystals, which build up in red blood cells, making them more rigid. And since there’s less soluble hemoglobin, there’s a relative membrane excess, so red blood cells start resembling a shooting target with a bullseye, with a dark center of hemoglobin, a ring of pallor and an outer band of hemoglobin.
In both cases, this leads to both intravascular and extravascular hemolysis, resulting in increased RBC breakdown, causing jaundice and gallstones, as well as anemia. There’s also increased compensatory erythropoiesis in the bone marrow, leading to new bone formation, and extramedullary hematopoiesis leading to hepatomegaly. In the case of Sickling, there’s also vaso-occlusion, where RBCs get stuck in capillaries, and that leads to tissue ischemia and pain. Often times it’s an emergency, called sickle cell crisis or vaso-occlusive crisis.
Alright, now whatever the cause of hemolytic anemia, all of them can present with symptoms of anemia like fatigue, pallor and shortness of breath, and symptoms of hemolysis like jaundice, dark, tea-colored urine, and back pain due to kidney damage.
Each of these diseases also have specific symptoms that’s special to them and these will help you identify them. In hereditary spherocytosis, because hemolysis is extravascular and occurs in spleen, there’s often splenomegaly. For the exams, remember that individuals with hereditary spherocytosis have chronic hemolysis since birth and thus, they can develop bilirubin stones and cholelithiasis at an early age. Another important fact to remember is that anemia is usually mild but can be severe if there’s folate deficiency or aplastic crisis due to parvovirus B19 infection. In this case, reticulocyte count will be low.
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