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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Thyroid 101

The thyroid gland is one branch of the endocrine system that is located in the neck that has two lobes connected by an isthmus. The hypothalamus secretes Thyrotropin-releasing hormone (TRH) which stimulates the anterior pituitary to secrete Thyroid-stimulating hormone (TSH). TSH acts on the thyroid gland to secrete the hormones triiodothyronine (T3) and thyroxine (T4). Within the circulation T4 is converted to its active metabolite T3. There are two different types of thyroid cells. Follicular cells produce the thyroid hormones T3 and T4. The parafollicular cells produce calcitonin. Calcitonin causes calcium reabsorption and maintains calcium homeostasis.

Synthesis of Thyroid Hormones is from iodine and tyrosine. Triiodothyronine (T3) has three atoms of iodine per molecule and thyroxine (T4) has four atoms of iodine per molecule. The hormones are created from thyroglobulin which is a protein within the follicular space that is originally created within the rough endoplasmic reticulum (RER). Thyroglobulin contains 123 units of tyrosine with reacts with iodine within the follicular space. A sodium-iodide symporter pumps iodide actively into the cell where it enters the follicular lumen from the cytoplasm by the transporter pendrin. In the colloid, iodide is oxidized to iodine by the enzyme called thyroid peroxidase. Iodine is very reactive and iodinates the thyroglobulin at tyrosyl residues in its protein chain. This forms the precursors of the thyroid hormones monoiodotyrosine (MIT), and diiodotyrosine (DIT). The adjacent tyrosyl residues are then paired together and subsequently the entire complex re-enters the follicular cell by endocytosis. Proteolysis liberates triiodothyronine and thyroxine and they enter the blood stream. Of the hormones secreted from the gland, 80-90% is T4, and only about 10-20% is T3. The production of T3, and T4 is primarily regulated by thyroid-stimulating hormone (TSH) which is released by the anterior pituitary gland. The thyroid hormones provide negative feedback to the thyrotropes TSH and TRH; when the thyroid hormones are high, TSH production is suppressed, and when levels are low, TSH secretion is increased.

After secretion, there is a very small percentage of the thyroid hormones that travel freely in the blood and that are metabolically active. Most are bound to thyroxine-binding globulin (TBG), transthyretin, and albumin. They act upon their respective tissues by crossing the cell membrane and binding to intracellular nuclear thyroid hormone receptors, which bind with hormone response elements and transcription factors to modulate DNA transcription. This modulation is what drives protein synthesis within the target tissue to actively project its physiological function within on the tissue and body.

Calcitonin is secreted by the parafollicular cells which helps maintain calcium homeostasis. Calcitonin is produced in response to high blood calcium. This causes inhibition of the release of calcium from the bone by decreasing the activity of osteoclasts. Osteoclasts are cells which break down bone. Bone is constantly reabsorbed by osteoclasts and created by osteoblasts, so calcitonin is effectively stimulates movement of calcium into bone. The effects of calcitonin are opposite of those of the parathyroid hormone (PTH) produced by the parathyroid gland.

Function

The primary function of the thyroid gland is the production of the thyroid hormones that have downstream metabolic, cardiovascular, and developmental effects. The basal metabolic rate is increased which effects all tissues. Gut adsorption and motility is increased. The generation, uptake by cells and breakdown of glucose is increased. The thyroid hormones also increase the breakdown of fats which increases free fatty acids, but contrary to believe, thyroid hormones decrease cholesterol levels.

There is an increase in cardiac output, as well as rate of breathing, intake and consumption of oxygen and increase in oxidative respiration within the mitochondria. These factors combine increase vascular pressure and elevate the bodies temperature.

The thyroid hormones are important for normal development. The cells of the developing brain are a major target for the thyroid hormones. They play a crucial role in brain maturation during fetal development. The thyroid hormones also play a role in maintaining normal sexual function, circadian sleep rhythm, and thought patterns.

The overarching effect is an augmented flight-or-fight response. It increases the release of the catecholamines which drives sympathetic innervation.

Clinical Significance

Hyperthyroidism is an excessive production of the thyroid hormones, most commonly a result of Graves Disease. Graves disease, also known as toxic diffuse goiter is an autoimmune disease that results in an enlarged thyroid. The exact cause is unknown, however it appears to be a combination of both genetic and environmental factors. An antibody, called the thyroid-stimulating immunoglobulin (TSI) which mimics the effect of TSH. This causes an increased T3 and T4, and an increased radio iodine uptake. Laboratory tests will show a decreased level of TSH, because there is negative feedback on the pituitary, but because of the antibody, production of the thyroid hormones continuous. One of the classic findings of Graves disease is exophthalmos, which is bulging of the eyes. This is often accompanied by irritability, muscle weakness, sleeping problems, increased heart rate and blood pressure and unintentional weight loss. Patients may complain of being “hot” all the time, illustrating a poor tolerance of heat.

Hypothyroidism is an underactive thyroid gland which results in decreased secretion of thyroid hormones. One of the most common causes is an autoimmune disorder called Hashimotos thyroiditis or chronic lymphocytic thyroiditis. The disease is characterized by gradual destruction of the thyroid gland. There are various antibodies that have been identified targeting against thyroid peroxidase, thyroglobulin, and TSH receptors. There is activation of cytotoxic T-cells in response to a cell mediated immune response affected by helper T-cells that drives thymocyte destruction. Cytokine release recruits macrophages within the gland to further drive destruction. Early on in the disease there may be no clinical evidence or symptoms of Hashimotos, but as the disease progresses, so does the clinical presentation. The most common symptoms are fatigue, weight gain, feeling cold, joint and muscle pain, depression, and bradycardia. This disease is about seven times more common in women than in men. Diagnosis is from TSH and T4 levels, imaging, along with other clinical symptoms. The thyroid gland may become firm, large and lobulated. There is lymphocytic infiltration and fibrosis that is seen.

These are not all the causes of hyper/hypothyroidism, but these are the most common, and in most cases, the most severe.