Transcribed by Jacqueline HeathFebruary 21, 2014

Erythrocytes Lecture Part II by Dr. Sitara

[11] RBC AssessmentOk so red blood cells can be assessed by the number, size, color, shape, distribution in the blood smear or red cell incisions.

[12] RBC Assessment- numberSo the number is measured by the CBC (complete blood count) but it can also be seen in the blood smear. Increased number of red blood cells is called erythrocytosis or polycythemia and decreased number of red blood cells is called erythropenia or erythrocytopenia and its usually seen in anemia. So here what you have in the bottom of this slide, in the middle you have a normal blood smear. Please look at just the red blood cell and ignore this, which is a basophil. We will talk about it in the next lecture. So the number of red blood cells, here is normal, here we have polycythemia, a lot more than in this slide, and here we have anemia where there are a lot less red blood cells than in a normal slide/smear.

[13] RBC Assessment size and color The red blood cells are also assessed by their size, remember the MCV, the mean cell volume is the one that shows whether the cells are small or big. You can also see that in a blood smear, if in a normal smear the red blood cells should be the same size, but if the size is variable, this is called anisocytosis. Normal sized RBC are called normocytic. When they are small they are called microcytic and when they are large they are called macrocytic or in some cases people use megalocytic.The color shows its of course obvious in the blood smear but not in the automated blood count and that shows the amount of hemoglobin. A normal RBC is called normochromic. But when its pale its hypochromic and when its darker its hyperchromic and here is a schematic where you see the difference in the color and in the size, which makes it a little easier for you to understand the differences in terminology.

[14] Size and color variationsSo here are some real slides. These two- top and bottom- show anisocytosis, it means that they show different sizes of RBCs. So here is number one. Its normal. Also here is a normal cell. Number two here is small much smaller than a normal one. And also here, and number three is a lot bigger than a normal erythrocyte, a normal red blood cell. Look how much bigger this one is in comparison to this. And then this middle three show differences in sizes. This person here has normocytic a normal size of RBCs. Here there are some smaller RBCs like for example this one here is much smaller, this is also smaller, its a different type of cell we will see later. And also these are microcytic blood smear where the RBC are bigger. And these panels on the right, differences in color. This is normochromic, normal color of RBCs. Look how much paler these RBCs are.Some of them like this one looks like its empty, looks like it doesnt have any hemoglobin. Look how much more intense the color is in this bottom slide.And look how much more intense the color the color is in this bottom side, there is no central pallor, so these cells have much more hemoglobin than the normal middle slide.

[15] RBC Assessment - shapeWhen the shape is different/variable, this is called poikilocytosis. I just said that this shape is biconcave and here are some pretty different shaped cells. You have a spherocyte which lacks the middle flattened part and you have a elliptoyte and ovalocyte and dacrocyte, this is called a tear-dropped cell. Target cell, stomatocyte that has a slit in the middle keratocyte also called a bite cell because it looks like someone took a bite out of it. A echinocyte is a crenated cell, it has a lot of little projections all around its surface. An acanthocyte can be confused with this one but it has less projections that are a little sharper and more pointed and sickle cell which we already know from sickle cell anemia. And a boat-shaped cell and so on. So here are some pictures of these different shaped RBCs. These are spherocytes, there is no central pallor, they are like sphere, really. The dacrocytes here, here are the tear-drop cells. There are sickle cells in this slide and also some boat cells. This slide has lymphocytes and ovalocytes, stomatocytes with a slit in the middle. Im not sure if maybe it will be easier for you to remember stoma in greek means mouth, and it looks like they have a mouth-like appearance with a slit in the middle. Target cells are pretty obvious, with a target in the middle. These are the keratocytes with a bite in the middle. And here there are echinocytes and acanthocytes.

[17] RBC Assessment Distribution There is also the differences in distribution in the blood smear. In some cases what is called rouleaux is formed. It means that, as you can see in this slide, the cells form like a stack of coins. This is due to an increase in high molecular weight plasma proteins. This changes the electric charge in the surface of the RBCs and they segment more rapidly, which is why they form stacks like coins. It happens in some pathological conditions associated with infection or inflammation. Another RBC distribution condition is RBC agglutination, where red cells get stuck together. Its not quite like rouleaux, its not like many RBCs form like a stack of coins, but they are 2 or 3 stacked together. This is usually due to the presence of an antibody on the surface of RBCs that makes them sticky, so they stick to each other. These are only seen in pathological conditions, not in normal individuals.

[18] RBC Assessment - inclusionsAlso the RBCs are assessed by the presence of inclusions. These are three types of inclusions. Pappenheimer bodies are small basophilic inclusions in red cell periphery. These are usually made of iron. There are also some inclusions called basophilic stippling. These are a basophilic inclusion throughout the cells. These are not made of iron. These are completely different. And they are the Howell-Jolly bodies which are large round, densely stained inclusions on the edge of the cell, like you see here. And this is a nuclear remnant, so its a little part of the nucleus that just stays there. Again, these are only seen in pathological conditions.

[19] no titleThis table is just a filler, you dont need to know. There are different types of cells, different red cell morphology and in which diseases you can find them. The ones you need to know are sickle cells for sickle cell anemia, thats a given. And target cells, are the main characteristic of a blood smear of a thalassitic person. You will see in a minute when I go through that.

[20] Disorders of RBCsOk, so RBC disorders. Increased number of RBCs is called erythrocytosis or polycythemia. This is caused by increased production of RBCs by the bone marrow and decreased number of RBCs in the blood is a cause for anemia. Now the decreased number of RBCs can be caused by many causes. For example blood loss can be caused after trauma or surgery. Or chronic, like gastrointestinal bleeding in cases of ulcers. The decreased number of RBCs can also be cued to decreased production of RBCs and these are some conditions that this happens, like iron deficiency, B12 folica acid, sideroblastic anemia, or it can also be caused by the RBCs are destructed. There is an increased destruction of RBCs like in hemolytic anemia or in hemolytic anemia or G6PD deficiency.

[21] Polycythemia/ErythrocytosisSo in polycythemia, the hematocrit, the packed cell volume is increased. Polycythemia can be absolute or relative. Absolute is divided into primary and secondary. Primary is over production of RBCs-real over production of RBCs. This is seen in the condition of polycythemia (rubra) vera. Secondary polycythemia means that its caused by an increase in erythropoietin (Epo) which is a protein that is produced by the kidney and its actually what determines the production of RBCs in the bone marrow. The kidneys sense that the oxygen levels in the blood are low, and they produce this Epo which then is transported into the blood, reaches the bone marrow and tells the bone marrow to make more RBCs. So its Epo in the kidneys that determines the rate of RBC formation. The increase in Epo is usually due to hypoxia, this can be in cases of high altitude or cigarette smoking or several other diseases like COPD or renal and heart disease. When you treat the underlying disease, the cause, then the polycythemia is treated. Polycythemia can also be relative. It means that its not really due to over production of RBCs but its due to dehydration and fluid loss. For example in cases of diarrhea, vomiting, burns, there is less plasma because the individual has lost fluid and more cells. It looks lie there are more cells because they are more in proportion to the plasma level. Thats why its not really over production, it just looks more because the person has lost a lot of fluids.

[22] Polycythemia Rubra Vera Polycythemia Rubra Vera, which is the condition of primary polycythemia is a rare condition and its a myeloproliferative disease. It is due to increased RBC proliferation in bone marrow. The cause is not known but recently patients with polycythemia Ruba Vera have tested positive for the mutation in JAK-2 gene, so testing for the JAK-2 gene can be used as a diagnostic test for patients with this condition. From the clinical features its easier to recognize, the individuals have red complexion, gum bleeding. This is interesting to you as future dentists. And youre not going to examine their (splints?), but its a clinical feature. Now how its treated, its treated by therapeutic phlebotomy, which is blood drawing. Because the patients have such an overproduction of RBCs, b continuous and regular blood drawing, the doctor can bring the hematocrit into normal levels. Also beaus the blood has so many RBCs, its thick, so its good that the patients takes some blood thinners like the aspirin so that it doesnt give the individual clotting problems. [23] AnemiaAnemia is caused by a decreased production of RBCs. Almost 1/3 of the world population is anemic, so its a very common condition. The rate of RBC production fails to match the rate or RBC extraction. The cause of anemia is blood loss (bleeding), acute trauma or surgery or chronic, for example in GI bleeding or menstruation. Also cause of anemia is insufficient RBC production by the bone marrow. In this case the anemia can be classified as microcytic, or low MCV or macrocytic (high MCV), so small cells or big cells. It can also be caused by significant RBC concentration in the RBCs. It can also be caused by accelerated RBC production that leads to hemolysis in hemolytic anemia. What is the cause of anemia can be distinguish by the reticulocyte count, which is decreased when there is a decrease in RBC production, but the reticulocyte count is increased when there is an increase in RBC destruction.

[24] Decreased RBC Production: iron deficiency anemiasSo a little about individual disorders. Iron deficiency anemia. It is very common in menstruating and pregnant women. And it occurs when insufficient iron stores are in the body. That can be mainly due to poor diet, iron-low diet, or due to poor iron absorption or utilization. The body iron stores are depleted by prolonged bleeding. I dont think really you should memorize this, but because its a microcytic anemia, please remember that the MCV is low. And subsequently the MCH is low because smaller cells have less hemoglobin. So the cells in the blood smear are hyperchromic and microcytic. When the individual takes iron supplements, the iron deficiency is corrected. Also if the individual takes a vitamin C supplement, it helps to increase iron absorption.

[25] Decreased RBC production: microcytic anemiasSideroblastic anemia is another type of microcytic anemia. This is due to an abnormal incorporation of iron into the heme group of hemoglobin. In this case there is a toxic accumulation of iron in mitochondria and what you see is this here, like ringed sideroblasts so the iron forms a ring around the periphery of RBC. Sirderoblastic anemia can be hereditary due to deficiency of an enzyme involved in heme synthesis. It can be secondary like in drug-induced medication imposed or alcohol and lead poisoning because some drugs and alcohol inhibit enzymes that are important for heme synthesis or it can be idiopathic meaning that the cause is not known. This is mostly in older people. Of course the major characteristic is in the blood smear, the formation of this ring around the periphery of sideroblasts. The management only in very severe cases, a bone marrow transplantation is given. Management you dont need to know for the exam.

[26] Decreased RBC production: anemia of chronic diseaseOk anemia of chronic disease. Its the second most common anemia after iron deficiency anemia. It is usually caused by chronic infection or inflammation such as in rhemathoid arthritis. The mechanism for this type of anemia is that the iron transport is blocked from the storage site where the ion is kept to the developing RBC in the bone marrow. Anemia of chronic disease is usually associated with renal failure and decreased erythropoietin production. In this case, in the blood smear, you see a marked rouleuz formation due to increased plasma protein concentration. Once you treat the underlying cause of the disease, the anemia is also treated.

[27] Decreased RBC production: microcytic anemias - ThalassemiasThalassemias are a group of microcytic anemias that are inherited, they are genetic disorders caused by reduced or absent production of hemeblobin A. it has taken its name by the combination of two Greek words, thalassa which means sea nad haima which means blood. Its taken it name because its most common in the Mediterranean sea region. There are two types of thalassemia, alpha and beta, depending on what genes are affected. So alpha thalassemia there is a deletion of 1, 2, 3 or all 4 alpha globin genes. In beta thalassemia, there are mutations in the beta globin gene cluster. It is the most common genetic disorder, and approximately 2 million people in the US affected by thalassemia.

[28] Alpha ThalassemiaAlpha Thalassemia is the deficiency or no synthesis of alpha globin chain. The severity of this disease depends on the number of alpha globin genes are deleted. If only one is deleted, these individuals are silent carriers. They have no symptoms, they are asymptomatic and you can only identify them by doing a pedigree and DNA analysis. If two alpha globin genes are deleted this is a condition called alpha thalassemia trait. Its also asymptomatic or there is a very mild hypochromic microcytosis. Also, these individuals are identified by pedigree and DNA analysis. They dont have anything that gives it away that they have a severe form of anemia.

[29] Alpha Thalassemia Hemeglobin H diseaseWhen three and four alpha globin genes are deleted, this is a different story. When three alpha globin genes are deleted, it results in a condition called hemoglobin H disease. And the four alpha-globin deleted results in a condition called hemeglobin Barts hydrops fetalis. Hemoglobin H disease is caused by production of hemoglobin H. This type of hemoglobin contains four hemoglobin chains. It is moderately to severe hypochromic anemia due to reduced hemoglobin synthesis and hemolysis. And in the blood smear, of course because its microcytic, you see smaller cells, and target cells, please remember in thalassemia target cells grow together. There aer some tear-drop cells and in this slide here you see some inclusions, precipitated hemoglobin H which is a sign/cuase of hemolysis. This is one of the normal stains, provided the methylene blue stains.

[30] Alpha thalassemia major Hb Barts Hydrops Fetalis Even more severe is the alpha Thalassemia major which is also called Hemeglobin Barts Hydrops Fetalis for a name. Its the most severe form of alpha thalassemia, in which there is a complete deletion and absence of alpha globin chains. Instead, they have hemoglobin Barts, which are alpha 4. This is incompatible with life. The babies are usually still birth by the third trimester or shortly after delivery. They die because there is fluid build up in multiple organs that leads to cardiac failure. This condition also leads to toxemia in the mother which is carrying the fetus with this condition.

[31] Alpha thalassemia major (Cooleys anemia)Beta thalassemia major is also known as Cooleys Anemia. Its the most severe form of this type of thalassemia. Both beta globin genes are affective. That leads to reduced or absent hemoglobin A1, which is the major form of our Hb. Instead, they have increased levels of Hb A2 and fetal Hb, which can be up to 98%. For an adult to have 98% of its Hb as Hb F, it is not a good thing. In a blood smear, again its microcytic, small cells, hyperchromic, less Hb, target cells, there are some nucleated cells, like this one that has not come out of the red cell, and some cells are also fragments, as you can see here are a fragment of the red cell. The patient doesnt have any symptoms until after 6 months of age because until birth the person also has Hb F. He/she would. But after 6 months when the Hb F has not switched to normal Hb, then the person starts showing symptoms. Individuals with this condition are bound to have regular lifelong blood transfusions, iron chelation therapy because with regular transfusions, they get overloaded with iron, which cannot be excreted normally, so they need iron chelation to remove the iron. And it can be more efficiently treated with blood marrow transplantation but early in childhood. Unfortunately the prognosis is not good. The average life is 17 years, and some individuals stay alive until 30 but eventually they lose the battle due to cardiotoxicity.

[32] beta thalassemia minor (Trait)Beta thalassemia minor or beta thalassemia trait is a milder form. Only one beta globin gene is affected, so it is moderately reduced Hb A1. There is also increased Hb A2 and Hb F but not to the extent that it happens in beta thalassemia major. The patients are asymptomatic. They have no symptoms. But what can give it away is that they have high RBCs which is different from iron deficiency. So thats something that distinguished beta thalassemia from iron deficiency. They may have high RBC count. In the blood smear, they are hypochromic microcytic, you can see how many more RBCs are in this slide, and there are some basophilic stippling like a form of inclusion. There is no form of treatment for beta thalassemia minor, but individuals have to go through genetic counseling because if both patients have beta thalassemia minor, they have one in four chances to have a child with beta thalassemia major, two in four chance to have beta thalassemia trait and only one in four chances to have a healthy child. If both parents have beta thalassemia minor, they should really go through genetic counseling.

[33] Decreased RBC production: macrocytic anemiasNow we are going to move to macrocytic anemias. They are the anemias that result in larger size of red blood cells. In the CBC, what gives it away is increased MCV. They are caused by impaired DNA synthesis even though RNA synthesis is normal. They are usually due to a deficiency in vitamin B12 or folic acid. This can be caused by a dietary reason like inadequate dietary intake, for example in vegans, poor diet, if the individual does not eat fresh fruits and veggies, or overcooked food that destroys the nutrients. It can also be caused by intestinal malabsorption due to the lack of intrinsic factor which is important for the absorption of vitamin B12, but can be due to hysterectomy or alcoholism or some drugs can cause intestinal malabsorption. Also due to increased requirements during pregnancy. There is a small difference between B12 and folic acid in how they manifest. B12 takes years to develop the deficiency because there are usually sufficient body stores. But folic acid develops relatively fast and body stores only last for about three months.

[34] Macrocytic Anemias B12 and folic acid deficiencyIn blood smear, the cells are much bigger. Look how much bigger this blood cells is compared to this. There are oval cells, tear drop cells, and some circulating nucleated red cells in severe cases. If the individual takes supplements of B12 and folic acid, the deficiency is corrected. However for folic acid, there is prophylactic treatment because folic acid deficiency is associated with some neural crest defects like spina bifida, so usually the women at the child bearing age have to take preventative folic acid to eliminate the possibility of having folic acid deficiency during pregnancy.

[35] Increased RBC destruction hemolytic disordersIn the cases of increased RBC destruction, we have the hemolytic disorders. The lifespan of RBCs is reduced. And there is a compensatory increase in rate of erythropoiesis. The cause for this can be incompatible blood transfusion, can cause lysis of RBCs, or cancer, or some drugs can cause this. Hemolysis can be intravascular or extravascular, Extravascular is the most common form. The RBCs are prematurely extracted by the macrophages in the spleen, liver and bone marrow. Intravascular is not as common and it involves the breakdown of RBCs within the blood vessels. Thats why its called intravascular. Hemolytic disorders can be inherited or acquired. Inherited usually are caused by an intrinsic defect and acquired is usually caused by an extrinsic defect.

[36] Increased RBC destruction hemolytic disordersIntrinsic defects can be structural or functional within the red cell. It can be a defect in red cell membrane or red cell metabolism or hemoglobin synthesis. And extrinsic defect is usually an abnormality in the red cell environment. It could be an immunologic abnormality or mechanical injury or an infectious organism that invade RBCs and destruct them.

[37] hemolytic disordersIn hemolysis, there is usually anemia but sometimes there is an increased rate of erythropoiesis so the body can try to catch up with increased destruction. Individuals with hemolytic disorder have jaundice because the RBCs break down and jaundice is caused by a byproduct of RBC breakdown, which is called bilirubin. Cholelithiasis is gall stones, and also this is caused by increased bilirubin, which is a byproduct of RBC lysis.Spleonomegaly. Passing of Hb in the urine called hemoglobinuria. Hemegobinemia is when the RBC slides there is too much Hb in the blood that exceeds the binding capacity of this molecule called haptoglobin and it cannot be cleared from the body, so there is too much Hb in the blood. It exceeds the binding capacity of this molecule called haptoglobin and it cannot be cleared from the body. So there is too much Hb in the blood. And also there are some skeletal changes because the bone marrow expands. In order to accommodate the increased RBC production to compensate for the increased destruction of the RBCs. Lab findings, uh the marrow is hyperlasic, there is reticulocytes increased because of the increased bone marrow activity and nucleated red cells. Treatment, blood transfusion, folic acid replacement, because folic acid is a component of the cell membrane so since the RBCs are destructed, if you give folic acid it maintains the red cell membrane intact, and splenectomy. Please do not worry about the lab finding and treatment.

[38] increased RBC destructionOk hereditary spherocytosis is one type of increased red blood cell destruction, its the most common inherited red cell membrane abnormality. Its transmitted in an autosomal dominant manner but it also may be autosomal recessive. Inheritance, the defective gene encodes for the red cell cytoskeletal protein spectrin. And the major site of hemolysis is the spleen.

[38] increased RBC destruction G6PD deficiencyThe next two disorders I am going to discuss we have already discussed in genetics. So the G6PD deficiency and hemolytic anemia. These are the two most important forms of hemolytic anemias. Glucose-6-phosphate dehydrogenase (G6PD) is a red cell enzyme its role is to protect the red cell protein from endogenous or exogenous oxidant stress. The mechanism by which it works it that it converts glucose to ribose 5 phosphate, which is a molecule important for nucleic acids, and this chemical reaction, this conversion leads to NADPH production. NADPH is a very important molecule because it prevents the build-up of free radicals within the cells. In cases of G6PD deficiency, this is the enzyme deficiency, the advantage is that individuals with this condition are resistant to malaria, but they have increased susceptibility to oxidant stress, and that leads to hemolysis. Its the most common enzyme deficiency, about 400 million people are infected and its inherited in an X-linked recessive disorder, therefore it affects males more than females because its in the one X chromosome that the males have.

[39] contThere are several triggers for the hemolysis to occur. That can be ingestion of certain drugs that induce oxygen stress on the RBCs. Ingestion of fava beans because they contain this molecule called devicine and that also creates free radicals inside the RBCs. Or it can be various viral infections which activate the neutrophils and that causes hemolysis. In a blood smear, there are bite cells, not sure if you can see here, Heinz bodies, which are the red cell inclusions that are composed of denatured Hb, but what you see in this slide, where the arrows point are this weird looking RBCs that they have, they look like Hb is push to one side, and these are red cells like it says in this slide, with Hb retracted into half of the red cell membrane. So that is a typical feature of G6PD deficiency.

[40] Sickle cell diseaseLast but not least, sickle cell disease. Its a genetic disorder, which is a result of change in the Hb structure due to mutation in the Hb gene. You remember this slide from November. Normal Hb has glutamine or Glutamic acid in position 6 of the Hb chain. But a person with sickle cell disease, this is substituted by a different amino acid, valine, and gives rise to this different type of Hb, HbS. Now what this substitution does is that it changes the actual RBC shape from biconcave to sickle shape. Thats why its called sickle cell disease. The reason it does this is that Hb becomes more rigid. The Hb molecule. So it precipitates and doesnt allow this biconcave shape to form. Thats another picture of sickle cells in comparison to normal RBCs.

[41] sickle cell cont Now because normal RBCs, because of their shape, they are flexible to move freely, but the RBCs that have the sickle shape are not as flexible and gets stuck in the capillaries, which is actually really painful, and finally they get destroyed, and thats the hemolysis part. That can cause organ damage, organs such as the spleen, kidney and liver. Its not as common as G6PD deficiency, there are more than 70 thousand Americans affected by sickle cell disease. The sickle cell trait, the individuals are carriers, there are a lot more individuals with a sickle cell trait, and the carriers are resistant to malaria because the parasite cannot invade these oddly shaped RBCs. Their membranes are porous, the sickle cells, the nutrients leak out so the parasites dont have anything to feed on and they die. And also because of the increased destruction, the parasites goes away with the lysed cell.

[43] sickle cell contSo in the blood smear, the cells are hypochromic because it is a type of anemia. Sickle cells are the given here. There are other, boat cells, this is a nucleated RBC. Howell-Jolly bodies, Papenheimer bodies, and it is usually detected by Hb electrophoresis, which shoes that they dont have Hb A but have Hb S. Signs and symptoms, the patient has some pain crisis due to veno-occlusions, there is a syndrome called hand and foot, the hands and feet are inheriting more in this pain crisis, it can also lead to a stroke or myocardial infection. There are many triggers but usually there are infection and dehydration.

[44] sickle cell contThe patient can be given bone marrow transplantation or antibiotics to prevent the infections, some analgesics to control pain, but it also and be given a drug called hydroxyurea which increases the Hb F in the individual and decreases the incidence of cell sickling.

On Thursday were going to go through the different WBCs and WBC disorders- benign and malignant disorders. Thank you.