Donath-Landsteiner Antibodies

The history of the DL antibody goes back to the 1900’s. It was one of the first recognized forms of immune mediated hemolysis and responsible for inducing Paroxysmal Cold Hemoglobinuria (PCH). PCH is a transient condition, meaning that it comes on when immunoglobulins (Antibodies) are formed in response to a viral, bacterial, or spirochete infection. Its history will suggest that there is an association between PCH and syphilis. In over 90% of the cases of PCH in early history, the patient was co-diagnosed with syphilis. Throughout the 1900’s the condition began to evolve and is now seen most commonly in children following some sort of infection. Although it should be noted that PCH is not limited to those of adolescent age. So what really is the Donath-Landsteiner antibody and how does it contribute to PCH?

Clinical Presentation

Paroxysmal Cold Hemoglobinuria (PCH) is an autoimmune hemolytic anemia (AIHA). Autoimmune meaning that they are antibodies that have cross-reacted to attack the individuals own cells. Hemoglobinuria means that there will be hemoglobin present in the blood, which suggests intravascular hemolysis. PCH is one of the more common intravascular hemolytic anemias. Typical patients present with fever, chills, abdominal and back pain, and pronounced hemoglobinuria. PCH typically presents in children following and upper respiratory infection or immunization. These patients often have a rapidly progressing anemia with hemoglobins that can fall as low as 2.5 g/dL. Peripheral blood smears show significant red blood cell agglutination and anisocytosis and poikilocytosis. Anisocytosis indicating variance in size of the red blood cells and poikilocytosis indicating variance in structure to the red blood cells. Schistocytes, spherocytes, and polychromasia are common findings. The spherocytes and polychromasia are indicative of the bone marrow trying to replenish the red cell population as best it can so it forces out immature erythrocytes into the peripheral blood. Its an effort to sustain the hemoglobin as best it can. One distinguishing peripheral blood smear finding in patients with PCH is erythrophagocytosis. Lets break this word down. Erythro- short for erythrocyte meaning red blood cells. Phagocytosis is mediated by neutrophils and monocytes as a way to kill foreign pathogens. In the case of erythrophagocytosis in PCH, neutrophils are characteristically seen engulfing red blood cells, which is diagnostic for AIHA.

The Donath-Landsteiner Antibody

The DL antibody, although being recognized as an cold autoantibody, is an IgG antibody that has developed P antigen specificity and it is a biphasic hemolysin. What that means is that when someone has the DL antibody and is exposed to cold temperatures, it will bind to the individuals red blood cells through the P antigen, but does not cause hemolysis until the coated red blood cells are heated to 37 degrees Celsius as they (RBC:antibody complex) travel from the peripheral fingertips and toes to the core of the human body.   At cold temperatures, the IgG molecule is able to recruit complement (C3), and at the higher temperatures, activates the membrane attack complex (C5-C9) and lyses the red blood cells. One very interesting piece of information regarding the difference between Cold Agglutinin Syndrome (CAS), another autoimmune hemolytic anemia caused by Anti-I, is that the hemolysis from PCH is stronger and more severe because of the DL antibodies ability to detach from lysed red blood cells and reattaching to other cells. 

Laboratory Diagnosis

There are a few different ways to pinpoint PCH in the blood bank. One is by use of a Direct Coombs test (DAT). This test provides information regarding the type of hemolysis, whether it be acquired or inherited. It also tests for antibodies that have are bound in vivo. The most common DAT result in PCH is red blood cells coated with C3d causing a positive reaction. This is sensitive in 94-99% of cases. The other way to diagnosis DLAIHA (Donath-Landsteiner Autoimmune Hemolytic Anemia) is by the indirect DL test. This process involves collection of a fresh serum specimen that is strictly maintained at 37 degrees Celsius from collection all the way through to testing. If the sample is allowed to cool or is refrigerated, there could potentially be autoadsorption of the DL anti-P antibodies onto the patients autologous red blood cells. This could cause a false negative result. Upon testing, the patients serum is mixed with P antigen positive, group O red blood cells, and fresh donor serum. The fresh donor serum is added because the complement level within the patients may be low due to consumption. The patient and donor serum mixture is incubated in a melting ice bath (O degrees Celsius) for 30 minutes, then warmed to 37 degrees Celsius for one hour. The specimen is then centrifuged and examined for hemolysis. If hemolysis is present then this constitutes a positive result for DL antibody.

Indirect-Donath-Landsteiner-test-Tube-1-OP-red-cells-suspension-patients-serum

Indirect DL test: As you can see in tubes 1 and 4, the presence of hemolysis indicates a positive test result for the DL antibody.

Treatment

There is unfortunately no cure for PCH, and very little reliable treatment options for those with the DL antibody. It is recommended to avoid cold climates as much as possible and when inside to have the temperature at 30 degrees Celsius to keep the hemoglobinuria low. This doesn’t treat the PCH, but it will minimize the recurrence and induced anemia. Steroids have been through extensive trials for treatment of PCH and there are mixed results. Theory is that steroids are better at clearing red blood cells coated with IgG, and less effective at clearing red blood cells that are coated with complement. More aggressive treatment such as splenectomy and Rituximab, which is an monoclonal antibody that targets the transmembrane protein CD20 present on B cells has been found effective for those patients with refractory PCH.

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Iron Deficiency and Microcytic Anemias

Iron is an essential element for oxygen transport within hemoglobin. Oddly enough it is the element that is missed the most in regards to adequate intake and proper nutrition. Over 1.62 billion people in the world are effected by anemia, which is most commonly caused by iron deficiency. Iron deficiency can be caused by chronic blood loss, and is most common in women and teenagers from loss of blood due to menses. Iron loss leads to increased fatigue and depression, pallor, and dry and splitting hair. It can also lead to confusion cognitive effects. Hemoglobin is made of four polypeptide chains, two of which are alpha, and two are beta that come together to form a tetramer heme group with iron located in the middle. Ferrous iron within each heme molecule reversibly binds to one oxygen molecule. With iron deficiency, there becomes a hemoglobin deficiency. A decreased hemoglobin lowers oxygen-carrying capacity leading to anemia. Anemia by definition is a reduced oxygen-carrying ability. Tissue hypoxia can wreak havoc on almost every cell of the body, and can shift the oxygen dissociation curve in an unfavorable direction. The structure of hemoglobin and its function and key elements can be reviewed here.

To understand iron deficiency its important to recognize important aspects of iron metabolism and transportation in cells. Review the Iron Absorption and Metabolism article here for that information. There are also laboratory values that give a good picture of the iron status within the body that one should pay attention to. Transferrin; which is measured as the total iron binding capacity (TIBC) indicates how much or how little iron is being transported throughout the body. Serum iron is an important indicator of the tissue iron supply, and finally serum ferritin gives a picture of iron storage status within the bone marrow and cells.

Iron Deficiency Anemia

There are three stages within iron deficiency. Each comes with their own classic picture of laboratory results and worsen from stage to stage. In the first stage, there is storage iron depletion. This is mild and the patient may not even feel a difference physically. The patients hemoglobin is normal, normal serum iron, and TIBC. There is however decreased ferritin which indicates that there is decreased storage of iron. The second stage of iron deficiency is characterized by transport iron depletion. The hemoglobin may or may not be abnormal, but there is increased TIBC, and decreased serum iron. An increased TIBC, means that there are more substrate (iron) binding spots within the transferrin molecule. This implies that less iron is binding, which when coupled with a decreased serum iron makes sense. The patient may experience mild anemia which comes with increased fatigue and pallor. A peripheral blood smear will most often start to exhibit anisocytosis and poikilocytosis. These reference indicators represent abnormal sized red cells and abnormal shaped red blood cells respectively. A good indicator is an increased RDW, an increased RDW indicates some degree of anisocytosis. This is accurate because the red blood cell is realizing the loss of this oxygen-carrying capacity so its trying to release red blood cells as fast it can from the bone marrow to compensate for the loss, and as a result these red blood cells will appear smaller in diameter and hypochromic. Hypochromasia indicates that there is less hemoglobin within the cell and there is more of a central pallor. The thought is that even though there is less hemoglobin within each cell, if the bone marrow can produce more of these red blood cells than normal then that equals out. This leads to a microcytic anemia, micro meaning small. Stage three of iron deficiency is often referred to as functional iron deficiency. Within this stage there is an unmistakable decrease in hemoglobin, serum iron, and ferritin. There is also a large increase in TIBC.

The overall effect of iron deficiency anemia on the body and on the bone marrow is ineffective erythropoiesis. The red cell production within the bone marrow is compromised. As a result, the bone marrow becomes hypercellular with red cell precursors reducing the M:E (Myeloid:Erythroid) ratio.

Iron-deficiency_Anemia,_Peripheral_Blood_Smear_(4422704616)

This picture depicts how a peripheral blood smear would illustrate iron deficiency anemia. The red cells are smaller and there is more of a central pallor to them, indicating a loss of hemoglobin. This is also called hypochromia.

normalbloodsmear

This picture depicts a normal peripheral blood smear. The red blood cells are larger in size and they have more color to them.

Anemia of Chronic Disease

Anemia of chronic disease is another form of microcytic anemia similar to iron deficiency anemia. It usually arises from a chronic infection or from chronic inflammation, but its also associated with some malignancies. A buildup in inflammatory cytokines alters iron metabolism. IL-6, which is an inflammatory cytokine inhibits erythrocyte production. It also increases hepcidin production. Hepcidin blocks iron release from the macrophages and the hepatocytes by down-regulating ferroportin. Without ferroportin there is no transportation of iron throughout the body and no production of hemoglobin or red blood cells. Laboratory findings will usually demonstrate low serum iron, low TIBC, low transferrin, and an increased to normal ferritin. The reticulocyte count is also normal, and sometimes increased. Reticulocytes are released from the bone marrow in times of red cell shortages to compensate.

This is just a brief overview of iron deficiency anemia and other microcytic anemias. This is just the beginning, follow and look forward to more in-depth reviews of each microcytic anemia. Key differences to look for is the TIBC value. In iron deficiency anemia the TIBC is increased and in anemia of chronic disease the TIBC is decreased. Ferritin is increased in anemia of chronic disease because the stored iron can’t be released from cells and the bone marrow due to the increased hepcidin production. Also the degree of anemia is mild compared to the more severe iron deficiency anemia.

 

 

Thalassemias

PIT11

The major hemoglobin that is present in adults is hemoglobin A (HbA). This is a heterotetramer that consists of one pair of alpha-globin chains and one pair of beta-globin chains. Alpha-globin chains are encoded by two copies of the alpha gene present on chromosome 16. Beta-globin chains are driven by one gene on chromosome 11. Normally there is tight regulation of the production of alpha and beta-globin chains and the ratio of production, but sometimes that regulation can be interrupted. This offsets the balance of globin chains being produced. These types of hematological disorders are coined thalassemias. They are a quantitative defect characterized by reduced or absent production of one and rarely two of the globin chains.

Alpha thalassemia is largely due to the inadequate production of alpha globin chains, which leads to an excessive production of either gamma-globin chains or beta-globin chains. In the fetus alpha thalassemia leads to excess gamma chains and in adults it largely leads to excess beta chains. In neonates the absence of alpha-globin chains is incompatible with life, leading to hydrops fetalis or hemoglobin Barts and absolute death after delivery. Hb Barts cannot deliver the oxygen to the tissues because its affinity to oxygen is too high. The hydronic state is reflected in the fetus by heart failure and massive total body edema. Excess beta-globin chains are capable of forming homotetramers and precipitate that leads to a variety of clinical manifestations.

Beta thalassemia is an inherited hemoglobinopathy in which production of beta-globin chains is impaired. There are different classifications corresponding to the degree of reduction in the beta chains. Beta thalassemia major is due to mutations that completely stop all production of beta-globin chains. These are individuals who are homozygous for the disease. They lose the ability to make HbA and because of this will experience severe manifestations and are transfusion-dependent for the rest of life. Symptoms typically begin during late infancy (6-12 months), but some newborns are asymptomatic because the major hemoglobin in newborns is HbF (4A:4G) which is constructed by gamma-globin chains and not beta-globin chains. Beta-thalassemia major presents with pallor, jaundice, and bilirubin in the urine which indicates hemolysis. Hepatosplenomegaly is present as well as heart failure. Failure to thrive and recurrent infections are also other signs. There is so much hemolysis because of the faulty hemoglobin present in the red cells that the bone marrow can’t keep up with production so extra medullary hematopoiesis occurs that results in skeletal abnormalities in the face and long bones. Iron overload is often a symptom of late untreated disease which can affect almost every organ in the body. Mortality is upwards of 85% by age five if untreated. If treated the survival rate is only 60 years of age if lucky.

Beta thalassemia major is also called transfusion-dependent beta thalassemia. There is also a subtype called non-transfusion-dependent beta thalassemia otherwise known as beta thalassemia intermedia. These individuals present with a less severe phenotype of the disease. There is significant variability with the clinical findings in individuals with beta thalassemia intermedia; from osteoporosis to thrombosis to diabetes mellitus. Some individuals will develop hepatosplenomegaly and extramedullary hematopoiesis and some won’t. Also some individuals will have to become transfusion-dependent, but that is typically in the late decades of life.

Anemia is a severe clinical manifestation of both alpha and beta thalassemia. The pathophysiology of beta thalassemia causes excess alpha-globin chains to precipitate in the developing erythrocytes in the bone marrow. This causes inclusion bodies. The inclusion bodies create oxidative stress and damages the cellular membranes. Apoptosis gets activated downstream and the red cell precursors are subsequently phagocytized and destroyed in the bone marrow by activated macrophages. This is also called ineffective erythropoiesis. The bone marrow in an effort to compensate releases these red cell precursors into the peripheral blood riddled with these inclusion bodies. These cells are subsequently sequestered by extravascular hemolysis by the RES which  further contributes to the anemia. The red cells that survive are microcytic and hypochromic and have a significantly shortened life span. Severe tissue hypoxia is seen due to the increased HbF as a compensatory mechanism. HbF has an increased affinity for oxygen and causes a shift to the left on the oxygen dissociation curve.

Typical laboratory findings for an individual with beta thalassemia is a slightly decreased red cell count and a marked decrease in hemoglobin of usually about 2-3 g/dL (12.5-16.5 g/dL). There will be marked anisocytosis (microcytosis) and poikilocytosis, target cells, basophilic stippling, slight increase in reticulocytes and nucleated red cells.

The pathophysiology for anemia associated with alpha thalassemia is associated with precipitation of HbH. Remember HbH is formed when there is decreased production of the alpha-globin chains so there is an excess of beta-globin chains. The precipitation of HbH creates inclusion bodies, typically called Heinz Bodies. These inclusions are recognized by the RES and remove the red cells via extravascular hemolysis.

Laboratory findings for an individual with alpha thalassemia is very similar to that of an individual with beta thalassemia. Decreased hemoglobin, marked anisocytosis (microcytosis) and poikilocytosis, target cells, basophilic stippling, and reticulocytes and NRBCs. The HbH inclusions can be see seen using a cresyl blue stain.

Thalassemias are a quantitative hemoglobinopathy meaning that there is a deficiency or an excess of production of globin chains leading to clinical manifestations. They are inherited and some subtypes can significantly elevate mortality. It is important to diagnose early and to treat early.