The Antibody

An antibody or immunoglobulin is a large Y-shaped protein produced primarily by plasma cells of the humoral immune system. They are used to recognize and neutralize any foreign antigens or pathogens. An antibody is identical to the B-cell receptor of the cell that secretes it except for a small portion of the C-terminus of the heavy-chain constant region. The difference is that a B-cell receptor C-terminus is a hydrophobic membrane-anchoring sequence and on an antibody, the C-terminus is a hydrophilic sequence that allows its secretion. The Y-portion of the consists of two arms that vary between the different antibody molecules, otherwise known as the V-region. The V-region is involved in antigen binding. The C-region is far less variable and is the part of the molecule that interacts with effector cells and other molecules. All antibodies are constructed in the same way paired from heavy and light polypeptide chains joined by disulfide bonds so that each heavy chain is linked to a light chain and the two heavy chains are linked together.

There are two types of light chains, lambda and kappa. A given immunoglobulin has one or the either, never both. In humans the ratio of kappa to lambda; the two types of light chains in immunoglobulins is 2:1. The class, and the effector function of an antibody is defined by the structure of its heavy chain. There are five main heavy-chain isotypes. The five major immunoglobulin classes are IgM, IgD, IgG, IgA, and IgE. IgG is the most abundant immunoglobulin and has several subclasses (1, 2, 3, and 4 in humans). The distinctive functional properties are conferred by the carboxyl -terminal part of the heavy chain, where it is not bonded with the heavy chain.

Each chain of the immunoglobulin consists of a protein domain. Each protein domain consists of a series of similar, but not identical sequences about 110 amino acids long . The light chain is made up of two domains, and the heavy chain consists of four. The variable or V-domain of the heavy and light chains together consist of the V-region of the antibody allowing it to bind specific antigens. The constant domains of the heavy and light chains together make up the C-region. The V-region or the Y of the molecule, where the antigen binding activity takes place is called the Fab fragments. Fab stands for fragment antigen binding. The other part of the molecule, the constant region (C-region) contains no antigen-binding activity, and is called the Fc fragment. Fc stands for Fragment crystallizable. This is the part of the molecule that interacts with effector molecules and cells.

The immunoglobulin molecule is flexible. There is a hinge region that links the Fc and Fab regions of the molecule, allowing independent movement of the two Fab arms.

Recap

To recap. An antibody molecule is made up of four polypeptide chains, comprising of two identical light chains and two identical heavy chains, which can be thought of as forming a flexible Y-shaped structure. Each of the four chains has a variable (V) region at its amino terminus, which contributes to the antigen-binding site, and a constant (C) region, which determines the isotype of the immunoglobulin. The light chains are bound to the heavy chains are non-convalent disulfide bonds. The V-regions of the light and heavy chains pair together to form the Fab region on the arms of the Y-structure. The trunk of the Y-structure, consisting of the carboxyl-terminal domains of the heavy chains make up the Fc fragment. The Fc fragment determines the different isotype of the immunoglobulin and interacts with different effector molecules. There is a hinge region joining the Fab and Fc regions allowing the antibody independent movement to maximize its antigen binding capabilities.

 

 

 

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Bilirubin Metabolism

Bilirubin is a metabolite of heme. It serves as a means to excrete unwanted heme, which is derived from various heme-containing proteins such as hemoglobin, myoglobin, and various P450 enzymes. Bilirubin is also notable for providing the color to bile, stool, and to a lesser extent the urine. Its produced by a two-stage reaction that occurs in cells of the RES (reticuloendothelial system). The RES includes the phagocytes, mainly being the macrophages, the Kupffer cells in the liver and the cells in the spleen and bone. Heme is taken up into these cells and acted on by the enzyme heme oxygenase, liberating the chelated iron from the heme structure and releasing carbon monoxide. The carbon monoxide is excreted via the lungs. The reaction yields a green pigment known as biliverdin. Biliverdin is then acted on by the enzyme biliverdin reductase which produces bilirubin. Bilirubin consists of a yellow pigment. Bilirubin is derived from two main sources. The majority, about 80% comes from heme which is released from senescent red blood cells. The other 20% originates from other heme-containing proteins found in the liver and muscles.

Synthesis

Bilirubin is toxic to tissues, therefore it is transported in the blood in its unconjugated form bound to albumin. For that reason, only a small amount of the free form is present in the blood. If the free fraction increases, bilirubin with invade and cause damage to the tissues. Excess unconjugated bilirubin can cross the blood-brain barrier and cause kernicterus in neonates. The unconjugated bilirubin is taken up by hepatocytes where the albumin bond is broken. Inside the hepatocyte, the bilirubin is bound to cytoplasmic proteins ligandins and Z proteins. The primary function of these proteins is too prevent the reflux of bilirubin back into the circulatory system. Unconjugated bilirubin is lipophilic. Its conjugation with glucuronic acid renders it hydrophilic, therefore it can be eliminated utilizing bile. Conjugated bilirubin synthesis occurs in a two step reaction. First glucuronic acid is synthesized from cytosolic glucose which then attaches to uridinediphosphate (UDP) via the enzyme UDP-glucose-dehydrogenase. This forms UDP-glucuronic acid. This compound has an affinity for bilirubin for which then the glucuronic acid is transferred to the bilirubin which is catalyzed by glucuronyl transferase. Conjugation of bilirubin takes place in the endoplasmic reticulum of the hepatocytes and the end result is an ester between the glurcuronic acid and one or both of the propionic side-chains of bilirubin.

Pathways in bilirubin metabolism

Metabolism

Once bilirubin is conjugated it is excreted with bile acid into the small intestine. The bile acid is reabsorbed in the terminal ileum for enterohepatic circulation, the conjugated bilirubin is not absorbed and instead passes into the colon. In the colon, the bacteria metabolize the bilirubin into urobilinogen, which can be oxidized to form urobilin, and stercobilin. Urobilin is excreted by the kidneys to give urine its yellow color and stercobilin is excreted in the feces giving stool its characteristic brown color. There can be traces levels of urobilinogen present in the blood.

Toxicity

Unconjugated hyperbilirubinemia in a neonate can lead to an accumulation of unconjugated bilirubin in the brain tissue. The neurological disorder is called kernicterus. The blood-brain barrier is not yet fully developed and bilirubin can freely pass into the brain interstitium. In cases of liver impairment, biliary drainage is blocked, and some of the conjugated bilirubin leaks into the urine, turning it a dark amber color. In cases of hemolytic anemia, there is increased hemolysis of red cells causing an increase in unconjugated bilirubin in the blood. In these cases, there is no problem with the livers mechanism to conjugate the bilirubin, and there will be an increase in urobilinogen in the urine. This is the difference between an increased urine bilirubin, and an increased urine urobilinogen.

Ketosis and Ketoacidosis

Ketosis is a metabolic state in which the bodies energy supply comes from ketone bodies in the blood in contrast to a state of glycolysis in which there is adequate glucose breakdown. In most cases, ketosis results from a high metabolism of fatty acids which are converted to ketone bodies. Ketone bodies are formed from ketogenesis when liver glycogen stores are depleted. Most cells in the body utilize both ketone bodies and glucose for energy, and while in ketosis the body works to maintain normal metobolism so it ramps up gluconeogenesis. Gluconeogenesis is glucose synthesis used to go through glycolysis.

Most of the time ketosis is a short interval of time, although long-term ketosis may be a result of fasting or a dietary insufficiency of carbohydrates. In glycolysis, high levels of insulin are released which promotes storage of fat and delayed release of fat from adipose tissue. In ketosis, fat reserves are readily available and are consumed. For this reason, ketosis has become one of the more recent diet fads as a way to burn fat quickly and lose weight.

Ketoacidosis

Although similar, ketosis is not ketoacidosis. Ketoacidosis is a physiological life-threatening situation due to insulin deficiency. Ketone bodies are acidic, and acid-base homeostasis in the blood is normally maintained through bicarbonate buffering, respiratory compensation, and renal compensation. Prolonged excess of ketone bodies can overwhelm the normal compensatory mechanisms and cause a state of acidosis when the blood pH falls below 7.35.

There are multiple precipitating factors that leads to ketoacidosis, which is most prevalent in patients with type 1 diabetes. Ketoacidosis in the case of a patient with type 1 diabetes is deemed diabetic ketoacidosis (DKA). In established type 1 diabetes, patients often forget to take insulin, with non-compliance being the bigger issue. This does not rule out other causes of ketoacidosis, as those are still prevalent. Acute major illnesses such as a myocardial infarction, cerebrovascular accident, sepsis, or pancreatitis. Certain drugs that affect carbohydrate metabolism such as glucocorticoids, diuretics, or anti-psychotic agents can cause ketoacidosis. General malnutrition associated with physiological problems can also lead to ketoacidosis. Such disorders lead to psychological starvation, which leads to ketone production and if prolonged ketoacidosis.

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Clinical Presentation

The clinical presentation of DKA is a two headed monster. The earliest symptoms of marked hyperglycemia is polyuria, polydipsia, and unexplained weight loss. As the duration of hyperglycemia continues neurological symptoms, including lethargy, focal signs and obtundation develop. Further progression can lead to a coma. The other head is the extent of the metabolic acidosis due to the excess ketone bodies. As the acidemia worsens accompanied with it is abdominal pain which can sometimes be severe. The electrolyte imbalance and metabolic acidosis causes delayed gastric emptying and an ileus (obstruction of the bowel). Vomiting and nausea are common.

Diagnostic Evaluation

The initial laboratory evaluation of patients with suspected DKA should include a serum glucose to establish whether or not the patient is hyperglycemic or not. Its helpful to measure the serum electrolytes and calculate the anion gap, BUN, plasma creatinine, and a plasma osmolality. This gives a broad picture of the metabolic state of the patient. Urinalysis is commonly performed along with urine ketones measured by dipstick method. Serum ketones are also measured to assess whether or not the patient is undergoing ketogenesis. An arterial or venous blood gas can be helpful to determine whether the serum bicarbonate is substantially reduced, which presumptively leads to metabolic acidosis. This also aids in determining hypoxia if it is present.

Findings

Hyperglycemia and hyperosmolality are the two primary laboratory findings in patients with DKA. Patients with DKA have a high anion gap metabolic acidosis. Serum glucose often times exceeds 350-500 mg/dL. Three ketone bodies are produced and accumulate in DKA; acetoacetic acid, beta-hydroxybutyric, and acetone. Acetoacetic acid is the only true ketoacid. A serum ketone measurement gives levels of beta-hydroxybutyric, while a urine dipstick measures the presence of acetoacetic acid using the nitroprusside method.

Other findings that may or may not be present are leukocytosis, and lipidemia. The majority of patients with hyperglycemic emergencies present with leukocytosis, which is proportional to the degree of ketonemia. Patients with DKA also present with marked hyperlipidemia. Lipolysis, primarily caused from insulin deficiency, and to a lesser extent elevated levels of lipolytic hormones including catecholamines, GH, ACTH, and glucagon. Lipolysis releases glycerol and free fatty acids into circulation which causes insulin resistance and serves as the substrate for ketoacid generation in the hepatocyte mitochondria.

To recap, ketosis is a dietary manipulation that if done right can lead to results. Ketoacidosis is a life-threatening metabolic state that requires immediate medical care.

This discussion will be continued with the next article focusing on ketoacid generation.

DARA-T Workup

Daratumumab (Darzalex) is an IgG1k monoclonal antibody directed against CD38, which is over expressed on the plasma cells in patients with multiple myeloma. Daratumumab binds to CD38 and causes apoptosis through antibody-dependent cellular cytotoxicity or complement-dependent cytotoxicity. In 2015 the FDA approved daratumumab for the treatment of refractory multiple myeloma. Refractory meaning that patients have received at least three previous treatment protocols that failed to show sustained efficacy or any efficacy at all. Recently in May of 2018, the FDA approved daratumumab for first line therapy in combination with bortezomid, melphalan, and prednisone. The names of the drugs aren’t important, what is important is that this monoclonal antibody approach has become more common and now has moved into first line therapy meaning that more patients are going to receive this treatment. Its no secret that patients with multiple myeloma when undergoing treatment and throughout the course of the disease progression need blood component transfusions.

Typing and screening patients that are receiving daratumumab is extremely difficult and time consuming. The daratumumab not only binds to the CD38 on the malignant lymphoma cells, but it also binds to the red cells who express CD38. This causes interference in transfusion testing. Part of normal pre-transfusion testing is an antibody screen. An antibody screen is important as it tells the transfusion team if there are any alloantibodies. Alloantibodies are antibodie directed towards red cell antigens on the donor cells. If a patient has an alloantibody, it makes selecting red cells for transfusion difficult. Additional testing must be done to select antigen negative donor cells for the antibody that the recipient or the patient has. Daratumumab causes the antibody screen and corresponding antibody panel panreactive, including a positive autocontrol. This may mask any additional clinically significant alloantibody that the patient may have.

The blood bank team must perform testing prior to the patient receiving this daratumumab. The clinical team must be in communication with the blood bank. Before the patient receives the medication, the team must get a baseline type and screen. Normally they are negative, but in the off chance that they have an alloantibody, the blood bank can identify the antibody before daratumumab interferes with testing. Other testing must include a complete phenotype of the patients cell. A complete phenotype will identify all the antigens that are present on the patients cells. This tells the blood bank and clinician vital information. If the patient does NOT have the antigen present on their red cells, there is a chance that they can produce an antibody towards that antigen on donor cells making it hard to find correct donors for transfusion. For example, if the patient is negative for the E antigen, they may or may not develop an antibody towards the E antigen, so in the event that the donor red cells have the E antigen present, the patients antibody will attack those cells and cause a transfusion reaction. For the characteristics of different transfusion reactions, reference transfusion reactions.

Once the daratumumab has been given there are techniques that must be followed to obtain a sample that is suitable for testing. An enzyme called dithiothreitol (DTT) is used to negate the binding of DARA-T to CD38 on the red cell surface. This will allow for an antibody screen to be run. Unfortunately, DTT destroys the Kell antigen on the red cell surface. Kell is a clinically significant antibody in transfusions so its important to know whether or not if the patient has the antigen or not. Patients treated with DTT, MUST have Kell negative donor units, because of the risk of developing an anti-K antibody and not being able to identify it.

 

The Precipitation Curve

This article will review basic immunology principles by defining key terms and explaining different techniques and phenomenons.

Key Definitions

Sensitization is the basic reaction of an antigen and an antibody binding. During an antigen:antibody reaction, the antigen or the antibody can be measured using a variety of methods. Each method has its advantages and disadvantages.

These reactions are sensitive and there are multiple external factors that affect the effectiveness of the reaction. The temperature, pH and concentration of the reactants effect the reaction itself. The length of incubation also affects the reaction. This principle applies to doing an indirect antiglobulin test for pre-transfusion testing. The reaction needs to incubate at 37 degrees celsius for a minimum of 15 minutes to properly allow the IgG antibodies to react and form a complex with their specific antigen.

The antigen:antibody reaction has three distinct phases; the primary phenomenon is the initial combination of a single antibody binding to its corresponding single antigen. The secondary phenomenon is where these single antibody:antigen reactions create a lattice formation to create large molecules which are easily detectable. The tertiary phenomenon is the effect that these immune complexes have within the tissues; this could be inflammation, phagocytosis, deposition of the immune complexes, immune adherence, and chemotaxis.

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The primary reaction of an antigen and an antibody depends on two defining characteristics; affinity and avidity. Affinity is the initial force of attraction that an antibody has for its specific antigenic epitope or determinant. Avidity is the sum of all attractive forces between an antigen and an antibody. The stronger the chemical bonds that hold the antibody:antigen complex together, the less likely that the reaction will reverse.

Precipitation involves the combination of a soluble antibody with a soluble antigen which produces insoluble complexes.

Agglutination is the process which particulate antigen aggregate to form visible complexes if the specific antibody is present.

Complement fixation is the triggering of the classical complement pathway due to the combination of the antigen with its specific antibody.

The Precipitin Curve

Precipitation reactions are dependent on the amount of antigen and antibody present in the test system. The precipitin curve is a graphic representation of these reactions that occur when the concentration of one reactant is constant for every test sample, while the concentration of the second reactant is increased serially in the test samples. The two reactants can be interchangeable, so the constant in any given reaction can either be the antigen or the antibody. For the purpose of this article, the antibody is going to be the constant. The addition of low concentrations of antibody allows the formation of soluble immune complexes, however as the concentration of the antigen is increased, precipitation is observed. The precipitin is the insoluble complexes. The antigen concentration continues to rise until the maximum amount of precipitin is reached. This point is called the equivalence point. The equivalence point is where there is optimum proportions of antigen and antibody to result in lattice formations to form insoluble immune complexes. When antigen concentration continues to rise past the equivalence point, the precipitin observed decreases. The curve is classed into three regions.

The early stage of the precipitin curve before the equivalence point is called the prozone and it is a zone of antibody excess. In the zone of antibody excess, there is insufficient antigen to form the large immune complexes comprised of extensive cross-linking. Its because of this principle that there will be false negative reactions. As more antigen is added, these complexes are able to form and it reaches the equivalence point.

The late stage of the precipitin curve is called the postzone and it is the zone of antigen excess. When there is an increasing amount of antigen added beyond the zone of equivalence, there is a gradual decrease in the amount of precipitin observed, until finally there is zero precipitation observed. There is free antigen is the solution. At this point all the antibody binding sites are saturated by multiple antigens and as a result there is less cross-linking leading to soluble immune complexes. This also leads to a false negative reaction.

To recap on what has been learned; There is a precipitation curve that represents the proportion of antigen and antibody concentrations, one being constant, and the other being added in serial additions. The postzone is the zone of antibody excess, resulting in the inability to form cross-linked immune complexes resulting in false negative reactions. The prozone is the zone of antigen excess which also leads to a failure to form cross-linked immune complex. The prozone, just like the postzone, results in a false-negative reaction.