Intrinsic hemolytic normocytic anemia: Pathology review

Chapters:

Watch video only

Case Study0:00–1:32

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.

Pathology1:32–3:32

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.

Intrinsic Hemolytic3:32–6:28

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.

Membrane Defects6:28–8:31

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.

Enzyme Deiciencies8:31–12:49

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.

Hemoglobin Defects12:49–15:33

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.

Symptoms15:33–20:48

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. Now, in paroxysmal nocturnal hemoglobinuria, these individuals present with dark urine most commonly in the first morning urine since urine collects and concentrates during the night.
They can also present with episodes of venous thrombosis that could lead to pulmonary embolism, and hepatic vein thrombosis, also known as Budd-Chiari syndrome.
Next are enzyme defects, starting with G6PD deficiency. For the test, remember that most of the patients with G6PD deficiency are completely asymptomatic until exposed to an oxidative stressor and develop symptoms of acute hemolysis and anemia.
For pyruvate kinase deficiency, what you need to remember is that it typically presents with jaundice in a newborn. And since hemolysis is extravascular and occurs in spleen, there’s often splenomegaly.Let’s move on to hemoglobin defects, and specifically sickle cell disease.
During sickle cell crises, the pain starts suddenly, can last from hours to days, and is excruciating. It can occur within bones of the hands and the feet, called dactylitis, in the hip, causing avascular necrosis, in the penis, causing priapism, in the cerebral vasculature, causing a stroke, mental status changes, and Moyamoya disease, named for the “puff-of-smoke” that you see on radiographs from the collateral blood vessels that develop over time to bypass blocked arteries.
If it happens in the kidneys, it can lead to necrosis and cause hematuria or proteinuria, while in the lungs, it can lead to acute chest syndrome presenting with chest pain, shortness of breath or cough.
Sickling in bones can lead to bone necrosis predisposing to osteomyelitis. For the exams remember that the most common cause of osteomyelitis in children with sickle cell disease is Salmonella while Staphylococcus aureus and Escherichia coli are also common.
Okay, now bone marrow compensation causes the medullary cavities of the skull to expand, which causes enlarged cheeks and a ‘hair-on-end’ appearance on X-ray, while extramedullary hematopoiesis presents with hepatomegaly.
Additionally, a large portion of circulating blood volume can get trapped in the spleen causing it to massively enlarge, which is known as splenic sequestration and it’s a life-threatening complication.
There’s usually a rapid drop in hemoglobin, leading to hypovolemic shock over a few hours. Another high yield fact to remember is that RBCs can clog up the spleen, which can lead to splenic infarcts and auto-splenectomy.
Biopsy of the spleen will reveal significant fibrosis and atrophy, and since the spleen plays an important role in immunity against encapsulated bacteria, the person will be susceptible to Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis infections.
Now, chronic complications of sickle cell disease, include growth and developmental delay, learning and behavior issues, pulmonary hypertension, and chronic kidney disease.
Alright, now sickle cell carriers are basically asymptomatic unless they’re exposed to high altitude or dehydration. However, in sickle cell carriers sickling can occur in the renal medulla, since the renal cortex gets all the blood and the medulla is more hypoxic and acidotic, causing papillary necrosis and isosthenuria, or inability to concentrate their urine.
Then, for HbC disease, symptoms are nonspecific and might be even absent, except for mild symptoms of hemolytic anemia, especially under stress.
And with HbSC disease symptoms are similar to sickle cell disease, but less frequent and less severe.Moving on to diagnosis.
In hereditary spherocytosis, the peripheral blood smear shows spherocytes that are slightly smaller than normal, round, dysmorphic RBCs with more intensely stained cytoplasm and no central pallor.
The mean corpuscular volume, MCV can be normal or slightly decreased, the mean corpuscular hemoglobin, or MCH, is normal meaning that every RBC has the correct amount of hemoglobin, but the mean corpuscular hemoglobin concentration, or MCHC, is increased due to membrane loss and RBC dehydration.
So, for the test remember that increased MCHC can be helpful in making the diagnosis. Spherocytes have increased sensitivity to lysis in hypotonic solutions known as osmotic fragility test, but they’re not specific for hereditary spherocytosis and are also present in autoimmune hemolytic anemia.
However, the Coombs test is positive in autoimmune hemolysis and negative in hereditary spherocytosis which can help distinguish these two disorders.
The treatment is a splenectomy to prevent hemolysis in the spleen. In Paroxysmal nocturnal hemoglobinuria, PNH, there are findings of intravascular hemolysis such as low haptoglobin, hemoglobinuria, and hemosiderinuria.

Diagnosis20:48–27:16

The gold standard test is flow cytometry which shows deficiency of CD55 and CD59. Treatment includes eculizumab which is an antibody against the complement protein C5.Now, to diagnose G6PD deficiency the peripheral blood smear can be very helpful.
Free radicals damage hemoglobin molecules which then precipitate inside the cells and are seen as dark inclusions, called Heinz bodies, when stained with crystal violet stain.
The spleen macrophages notice these Heinz bodies and try to remove them by taking a bite out of the cells, leaving these RBCs partially devoured, so we call them bite cells.
The diagnosis is suspected if there is a history of recent exposure to an oxidant like starting a new medication or eating fava beans followed by a hemolytic episode.
But the definitive test for G6PD diagnosis is an enzyme assay to detect the levels of G6PD. The assay shouldn't be done during the hemolytic episode since the cells with low enzyme level are dead and the levels of G6PD will be falsely normal.
Given the high risk of hemolysis, it’s important to diagnose G6PD deficiency as early as possible and so newborns in many developed countries are tested a few days after birth.
Likewise, diagnosis of pyruvate kinase deficiency starts with a peripheral blood smear, which may show the characteristic echinocytes or Burr cells.
But definite diagnosis comes with an enzyme assay showing low levels of pyruvate kinase. Once again, the assay shouldn't be done during the hemolytic episode since the the levels of pyruvate kinase will be falsely normal.
In severe cases, the treatment is a splenectomy to prevent hemolysis in the spleen. Hemoglobin defects can be diagnosed with prenatal testing for hemoglobin gene mutations.
A sample of the fetal DNA can be obtained through either chorionic villus sampling or amniocentesis. In some settings, hemoglobin defects are tested for in newborn screening by using hemoglobin electrophoresis.
In a newborn with sickle cell disease, it will show an intense band of hemoglobin F, or HbF, and another lighter band of HbS instead of the adult hemoglobin A, or HbA.
A newborn with sickle cell trait will have an HbF, as well as two separate HbS and HbA bands. A newborn with HbC disease will have an intense band of HbF and another lighter HbC band, while a newborn with HbC trait will have an HbF and two separate HbC and HbA bands.
And a newborn with HbSC disease will have an HbF, as well as an HbS and HbC band. If newborn screening is not done, then the diagnosis is made later on.
A peripheral blood smear can show target cells with hemoglobin C crystals in HbC disease or cells that are sickled in sickle cell disease.
Individuals with sickle cell disease often have a spleen that is nonfunctional or absent, the smear will also show Howell-Jolly bodies, which are basophilic nuclear remnants in RBCs.
These are normally not seen, because a healthy spleen would instantly filter and remove these RBCs. Finally, the diagnosis will be confirmed with hemoglobin electrophoresis which is the most specific test.
In this case, a healthy individual would have an intense band of HbA, an individual with sickle cell disease would have an intense band of HbS, and an individual with sickle cell trait would have two, almost equal, bands of HbS and HbA.
Similarly, someone with HbC disease would have an intense band of HbC, someone with HbC trait would have two, almost equal bands, of HbC and HbA, while someone with HbSC disease would have two, also equal bands, of HbS and HbC.
Additionally, there’s a screening test for sickle cell trait, known as the sickle prep, or Sickledex, but it cannot distinguish trait from homozygous disease.
Treatment of sickle cell disease includes hydroxyurea, which reactivates the synthesis of fetal hemoglobin to replace the abnormal hemoglobin S - so there’s a lower chance of sickling.
Hydration can also help both during sickle cell crises as well as for prevention. In contrast, HbC disease typically needs no treatment.All right, as a quick recap, Intrinsic hemolytic anemias occur when RBCs are abnormal.
There are RBC membrane defects, like in hereditary spherocytosis and PNH, enzyme deficiencies, like in G6PD deficiency and pyruvate kinase deficiency, and hemoglobin abnormalities, like in sickle cell anemia.
Hemolysis can be extravascular like in hereditary spherocytosis and pyruvate kinase deficiency, intravascular like in PNH, or both like in G6PD deficiency and sickle cell anemia.
A diagnosis can be made based on clinical presentation and lab findings. The diagnosis can often be confirmed with more specific tests like osmotic fragility test, enzyme assay and hemoglobin electrophoresis.
Now, back to the patients! All of them have normocytic hemolytic anemia.
Kyra most likely has hereditary spherocytosis. Her young age, unknown family history, splenomegaly, increased MCHC and the presence of spherocytes on peripheral blood smear are important clues for the diagnosis which can be confirmed with the osmotic fragility test.
A Coombs test should be done to rule out autoimmune hemolytic anemia. Now, Darnell most likely has G6PD deficiency due to his history of a hemolytic episode after taking a medication that contains sulfonamide.

Review27:16–28:10

Also, G6PD deficiency presents almost exclusively in males and is common in individuals of African descent like Darnell.
To confirm the diagnosis an enzyme assay for G6PD must be ordered. Meanwhile, Billy has priapism and due to the presence of sickled cells on peripheral blood smear, he most likely has sickle cell anemia.
A hemoglobin electrophoresis should be ordered to confirm the diagnosis. lab findings.
The diagnosis can often be confirmed with more specific tests, like the osmotic fertility. Test enzyme, S A and hemoglobin electrophoresis.

Summary28:10–29:18

Now, back to the patient's, all of them have normocytic, hemolytic. Anemia Kyra, most likely has hereditary spherocytosis.
Her young age, unknown family history, splenomegaly increased, MCHC in the presence of spirits sites on peripheral blood.
Smear are important clues for the diagnosis, which can be confirmed with the osmotic for Agility Test. A Coombs test.
Should also be done to roll out autoimmune, hemolytic. Anemia.
Now Darnell, most likely has a G6PD deficiency. Do is history of a hemolytic episode after taking a medication that contains sulfonamide also G6PD deficiency presents, almost exclusively in males and is common, and individuals of African descent, like, her know to confirm.
The diagnosis is enzyme a safe for G6PD must be ordered. Meanwhile, Billy has priapism and due to the presence of Sickle cells on peripheral blood smear.
He, most probably has sickle cell. Anemia, hemoglobin electrophoresis should be ordered to confirm the diagnosis.