| Effect of Enzymes and Dithiothreitol (DTT) Antibody Effect of Enzymes Effect of DTT Anti- D Ficin and Papain Enhanced DTT Resistant Anti-C Ficin and Papain Enhanced DTT Resistant Anti-c Ficin and Papain Enhanced DTT Resistant Anti-E Ficin and Papain Enhanced DTT Resistant Acid Resistant Anti-e Ficin and Papain Enhanced DTT Resistant Anti-Cw Ficin and Papain Enhanced DTT Resistant Anti-K Ficin and Papain Resistant DTT Sensitive Anti-k Ficin and Papain Resistant DTT Sensitive Anti-Kpa Ficin and Papain Resistant DTT Sensitive Anti-Jsa Ficin and Papain Resistant DTT Sensitive Anti-Fya Ficin and Papain Sensitive DTT Resistant Anti-Fyb Ficin and Papain Sensitive DTT Resistant Anti-Jka Ficin and Papain Enhanced DTT Resistant Anti-Jkb Ficin and Papain Enhanced DTT Resistant Anti-Lea Ficin and Papain Enhanced DTT Resistant Anti-Leb Ficin and Papain Enhanced DTT Resistant Anti-P1 Ficin and Papain Enhanced DTT Resistant Anti-M Ficin and Papain Sensitive DTT Resistant Anti-N Ficin and Papain Sensitive DTT Resistant Anti-S Usually Ficin and Papain Sensitive; Some Variable DTT Resistant Anti-s Usually Ficin and Papain Sensitive; Some Variable DTT Resistant | View Page |
| When to Use an Enzyme Panel - Results on a Regular Panel Rule-outs can be done using screen cell I and panel cells 4 and 8 (highlighted in green) Antibodies ruled out using these panel and screen cells: C, e, Kpb, Jsb, Jka, Leb, M, P1 and Lub Performing an enzyme panel could help further identify the suspected antibodies. Antibodies needing rule out: D, c, E,K, k, Fya, Fyb, Jkb, Lea, N, S, s If these antibodies are present, a stronger reaction will be seen on the enzyme panel: D, c, E, Jkb, Lea. If these antibodies are present, there will be no reaction on the enzyme panel, since the antigens are destroyed by enzymes: Fya, Fyb, N, S, s. | View Page |
| Reactions with an Enzyme Panel D, E, and Lea did not react with the enzyme panel cells (in green). If they had been present, the reactions would have been enhanced. Fya, Fyb, N, S, and s did not react with the enzyme panel cells (some are shown in green).Looking at the enzyme panel results, we can see the reaction pattern of c (in yellow) and the pattern of non-reaction for Fya (in pink). Suspected multiple antibodies are c and Fya. Fya will not react on the enzyme panel since the Duffy antigens are destroyed by enzymes. Enzymes will enhance the reaction of anti-c. | View Page |
| Case Study Four- Selected Cell Panel Cells 5 and 8 can be used for rule-out cells. Jkb, Lea, M, N, and s still need more rule-out cells. P1, C, E and Fya have no cells for rule-out.Running an enzyme panel would help to enhance Jkb, Lea,P1 C and E if these antibodies are present.If M,N,s and Fya are present, no reaction would be seen because these antigens are destroyed by enzymes. | View Page |
| Products Used to Facilitate Antibody Identification Monospecific anti-human globulin (IgG) enables sensitized red cells to cross-link so that agglutination is visible.Enhancement media are sometimes used to further promote agglutination and reduce incubation time. Low ionic strength saline (LISS) is the most common enhancement media. LISS reduces the ionic strength in the testing sample and causes reduction of the zeta potential. It increases antibody uptake and decreases incubation time. Polyethylene glycol (PEG): brings red blood cells (RBCs) closer together and concentrates antibodies by removing water molecules from the testing sample. It is the most sensitive of the enhancement media; strengthening almost all clinically significant antibodies. However, it will also enhance some clinically insignificant antibodies as well. Centrifugation should be avoided when PEG is used. PEG can cause aggregates to form if the sample (red cell - serum mixture) with PEG added is centrifuged. Reaction readings should only be done at the AHG phase. 22% albumin: reduces zeta potential, bringing the RBCs closer together and enhancing agglutination. Albumin does not contribute much to antibody uptake. Longer incubation time is needed with this media than with the previously discussed media. Detection of some IgG antibodies can be enhanced with enzyme test methods. Proteolytic enzymes (papain and ficin) denature some RBC antigens and remove negative charges from the RBC membranes. This reduces the zeta potential, bringing the cells closer together. Enzyme techniques are particularly useful in the identification of Rh antibodies and antibodies in the Kidd, Lewis, P and I systems. However, enzymes destroy some antigens including Fya, Fyb, M, and N. The effect of proteolytic enzymes on the S and s antigens are variable. | View Page |
| History In the past, an AMI was primarily diagnosed by evaluating symptoms at patient presentation, ECG measurement, and results of enzyme assays that were considered cardiac enzymes. The enzymes, creatine kinase (CK), lactate dehydrogenase (LD), and aspartate aminotransferase (AST) were assayed several times a day often for several days to observe peak concentration and return to normal level for each enzyme. The first assay result was the baseline level or baseline concentration. Isoenzymes of CK and LD were later added for AMI diagnosis. All three of these enzymes are found in other tissues, making the diagnosis difficult and lengthy. In the 1980s, CK isoenzyme, CK-MB, though not totally cardiac specific, became the benchmark marker for an AMI. None of these enzymes are in any of the current recommendations except for CK-MBCurrent diagnosis, monitoring, and screening relating to heart disease includes measurement of lipids, proteins, enzymes, and other biomolecules. Risk stratification for cardiac and vascular disease is an additional role for measurement of these analytes. The physiological changes in the development of heart disease are better understood and the role of the clinical laboratory is greatly expanded.Today's markers are significant because of their location in the myocyte, the kinetics of their release in myocyte damage, and their rate of clearance from peripheral blood. | View Page |
| Stains and Dyes Substance Stain or DyeCommentsProteinsPonceau SCoomassie Brilliant BlueSilverSpecific for ProteinsSilver is a biohazardLipoproteinsSudan Black BOil Red O-EnzymesEnzyme substrate and achromagen or fluorescent dyeReaction catalyzed by enzyme and color or fluorescence detectedHemoglobinNot neededIs intensely coloredNucleic Acids (DNA/RNA)Ethidium Bromide (EtBr)SyBr GreenSilver EtBr is CarcinogenicSyBr Green is new - Introduced in 1995Silver is a biohazard | View Page |
| Densitometry After electrophoresis, a stained gel is passed through the optical system of a densitometer to create an electrophoregram, a visual diagram or graph of the separated bands. A densitometer is a special spectrophotometer that measures light transmitted through a solid sample such as a cleared or transparent but stained gel. Using the optical density measurements, the densitometer represents the bands as peaks. These peaks compose the graph or electrophoregram and are printed on a recorder chart or computer display. Absorbance and/or fluorescence can be measured with densitometry.An integrator or microprocessor evaluates the area under each peak and reports each as a percent of the total sample. If the electrophoresis is for separation of serum proteins, the concentration of each band is derived from this percent and the total protein concentration. If the electrophoresis is for separation of enzymes, the enzyme activity of each band is derived from this percent and the total enzyme activity. The densitometer scan below depicts the separated bands from a serum sample electrophoresis. The SPIFE 3000, Helena Laboratories, electrophoresis splits the beta zone into two fractions for easier detection of small beta-migrating monoclonal gammopathies. The densitometer scan from this electrophoresis shows five bands with two peaks in the beta band. Recall the order of protein fractions from left to right is: Albumin, alpha 1, alpha 2, beta, and gamma. | View Page |
| Blotting Techniques Blotting techniques were developed to discriminate fragments of nucleic acids. These techniques involve several processes; electrophoresis is one of the processes and is used to separate fragments of DNA and RNA. In Southern blotting (named after Edward Southern) restriction enzymes cut fragments of DNA are separated by AGE or PAGE, transferred to a membrane or blot, and visualized by hybridization with labeled probes.Northern blotting (not named after an inventor but by analogy to Southern blotting) separates RNA. RNA molecules are shorter and have defined lengths; cutting by restriction enzymes is not required. Denaturing conditions are required because of RNA secondary structures. After membrane blotting, the separated types of RNA are visualized with staining or labeled probes.Western blotting (again not named after an inventor but by analogy to Southern blotting) does not separate nucleic acids; it separates proteins in a mixture. The proteins are usually separated with PAGE, transferred to the membrane and visualized with a labeled antibody against the proteins of interest. | View Page |
| What is the function of the majority of HIV's genes? | View Page |
| Function of HIV Genes HIV consists of nine genes. Three of the genes provide genetic information for the capsid proteins, envelope proteins, and viral enzymes. The other six genes are regulatory genes, controlling functions such as uncoating of the HIV genome and the penetration of host cells.
Gene NumberAbbreviationGene Function1gagcapsid proteins2polviral enzymes3envenvelope proteins4vifregulatory gene5tatregulatory gene6vpuregulatory gene7nefregulatory gene8vprregulatory gene9revregulatory gene
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| Spread of Infection (2) At this time an enzyme called protease, using enzymes and proteins from preliminary protein molecules, forms capsomere segments which unite to form an icosahedral capsid.The capsid then changes into a bullet-shaped capsid and surrounds the viral RNA.Next some of the host cell's membrane joins with the viral glycoproteins gp120 and gp41 to form the spikes.Last, part of the host cell's surface membrane encloses the virus and becomes the envelope. | View Page |
| Spread of Infection (1) The proviral DNA provides genetic coding that instructs cellular enzymes to construct new HIV genomes, capsid proteins, and reverse transcriptase molecules.All of these are assembled near the edge of the host cell. | View Page |
| Penetration and Infection After penetration of the cell membrane by the gp41, the HIV capsid enters the cell's cytoplasm. Next, cellular enzymes strip away the capsid so that the HIV genome is released. Also stripped away are proteins p24 and p17. Protein 24 coats the HIV genome and protein 17 lines the inside of the capsid. | View Page |
| HIV is a Retrovirus In most cellular biochemistry, DNA is used as the template for the synthesis of RNA. In HIV however, RNA is the template for the synthesis of DNA. That is why the enzyme is called reverse transcriptase. Because of the enzyme's activity, HIV is known as a retrovirus - retro implying reverse. | View Page |
| DNA Replication from RNA Once the capsid and p24 and p17 have been stripped away, an enzyme complex known as reverse transcriptase is released.One of the enzymes in this complex is DNA polymerase. It synthesizes a single-stranded DNA copy using one of the HIV-RNA molecules as a template.Another enzyme in this complex, called ribonuclease, then destroys the original RNA molecules while the DNA polymerase synthesizes another single-stranded DNA molecule, this time using the first DNA copy as the template.The result is a double-stranded DNA molecule. | View Page |
| Staphylococcus aureus Virulence Factors S. aureus is the most pathogenic member of the genus Staphylococcus; it possesses several factors that contribute to its virulence: Structural components of its cell wall function as a protective barrier, aid in adherence to mucous membranes, and allow the organism to resist phagocytosis. The production of several different toxins Enterotoxins A, D, F (TSST1) Exfoliative toxin ( causing scalded skin syndrome Cytolytic toxins (causing cell & tissue damage). Production of enzymes Catalase – distinguishes staphylococci from streptococci Coagulase – distinguishes S. aureus from other staphylococci Hyaluronidase & lipase – aid in skin colonization/infection spread Beta-lactamase – breaks down the beta-lactam antibiotics, e.g., penicillins, cephalosporins, carbapenems and monobactams. | View Page |
| Beta-lactam antibiotics and S. aureus Antibiotics inhibit bacterial growth by interfering with one or more cellular processes. Beta lactams are a large group of cell wall active antibiotics used to treat a wide variety of infections. S. aureus cell wall synthesis is dependent on the proper functioning of a number of enzymes. The beta-lactam antibiotics exert their effect by binding with one specific type of enzyme, transpeptidase, thus interfering with its ability to catalyze the final stage of peptidoglycan synthesis, resulting in defective cell wall formation. The beta-lactams comprise four main groups of antibiotics; all have the beta-lactam ring as their basic chemical structure: Penicillins (penicillin, oxacillin/methicillin, ampicillin and piperacillin) Cephalosporins Carbapenems Monobactams The spectrum of antimicrobial activity is dependent upon the particular structural modification of the beta-lactam ring. The transpeptidases are commonly referred to as penicillin-binding proteins (PBPs). Different bacterial species have distinct PBPs, resulting in very specific drug interactions. | View Page |
| The pathogenicity of Staphylococcus aureus, as well as the frequency with which this organism produces infections, can be attributed to: | View Page |
| Beta lactam antibiotics interfere with cell wall synthesis by: | View Page |
| Beta-lactams and Methicillin Resistant Staphylococcus aureus Methicillin Resistant Staphylococcus aureus (MRSA) is resistant to the beta-lactam antibiotics. The term methicillin-resistant is historically used to describe resistance to any of this class of antimicrobials even though methicillin is no longer the drug of choice. The acronym MRSA persists and is used interchangeably with ORSA – oxacillin-resistant Staphylococcus aureus. Oxacillin/methicillin resistance implies resistance to all penicillins, cephalosporins, monobactams, carbepenems and beta-lactam/beta-lactamase inhibitor combinations. S. aureus intrinsically produces beta lactamase enzymes that breakdown beta lactam antibiotics (i.e., penicillin); these are designated PBP 1 - 4. The beta-lactam resistance of MRSA is determined by the production of a novel penicillin binding protein called PBP 2' (PBP 2a), that has a reduced binding affinity for beta-lactam antibiotics. This allows MRSA strains to continue cell wall synthesis due to the uninhibited activity of PBP2' even in the presence of otherwise inhibitory concentrations of beta-lactam antibiotics.PBP2' is encoded by a mecA gene located on the MRSA chromosome and is widely distributed among Staphylococcus aureus as well as coagulase-negative staphylococci. The mecA gene is carried by a novel mobile genetic element, designated staphylococcal cassette chromosome mec – SCCmec that is integrated into the bacterial chromosome. The mecA gene is believed to have originated in some coagulase-negative staphylococcal strains and was then transferred into S. aureus, giving rise to MRSA. It is likely that SCCmec serves as the carrier of the mecA gene moving across staphylococcal spp. as these mecA genes have never been found without the presence of a SCCmec-like structure. Phylogenetic analyses of international collections of MRSA and methicillin-susceptible S. aureus isolates have revealed that methicillin resistance has arisen in five distinct lineages designated SCCmec I – V, which differ in both size and genetic composition. In recent years, the gene has continued to evolve so that many MRSA strains are currently resistant to several different antibiotics. | View Page |
| The increased resistance of MRSA strains to beta lactam antibiotics is due to: | View Page |
| Which are true statements regarding hospital-associated methicillin-resistant Staphylococcus aureaus (HA-MRSA) and community-associated MRSA (CA-MRSA)? | View Page |
| During phagocytosis the neutrophilic granules release digestive enzymes into the vacuole to kill or destroy the phagocytized particle. | View Page |
| Primary Function of Neutrophils The primary function of neutrophils is phagocytosis, the ingestion and destruction of microorganisms or other foreign particles. For this reason, neutrophils are classified as phagocytes. When a neutrophil is faced with a microorganism or foreign particle, phagocytosis begins. The following steps are carried out by the neutrophil during phagocytosis:The neutrophil extends pseudopods around the foreign material and engulfs it. Digestive enzymes present in the neutrophilic granules are released into the vacuole containing the foreign particle, and the particle is destroyed. In most cases a mild infection enhances the function of neutrophils while a severe infection impairs their function. | View Page |
| Basophil Function and Lifespan Basophils serve as mediators of inflammatory responses, especially hypersensitivity reactions. IgE binds to the membrane receptors on basophils and degranulation is initiated. The enzymes released are vasoactive, bronchorestrictive and chemotactic (especially for eosinophils), so basophils seem to play a role in inducing and maintaining allergic reactions.The granules of basophils contain histamine, heparin and peroxidase. After degranulation occurs, basophils can synthesize more granules. The release of large numbers of these granules can cause anaphylactic shock and death. Basophils circulate in the blood for a short time and make up only a small percentage (0.5%) of the cells in circulation. They do not migrate to the tissues under normal conditions but may be seen when inflammation resulting from hypersensitivity to protein, contact allergy or skin allograft rejection is present. Basophils are sometimes increased in patients with chronic myeloproliferative disorders. | View Page |
| Glossary of Terms A through M. Antibody - A modified type of serum globulin synthesized by lymphoid tissue in response to antigenic stimulus. By virtue of specific combining sites each antibody reacts with only one antigen. Anucleate - Having no nucleus. Azurophilic granules - The well-defined large reddish granules (lysosomes) which may be present in large lymphocytes. They are called "azurophilic granules" because they stain blue with the azure stains which were originally used. Basophilic granules - Specific granules present in the cytoplasm of basophils. These granules are large and stain purple-black due to their strong affinity for basic stain. B-cell - Bone marrow derived lymphocytes which produce humoral antibodies. Biconcave - Having two concave surfaces. Cellular Immunity - The capacity of a small proportion of lymphoid population to exhibit response to a specific antigen. Chromomere - The centrally located granular portion of the platelet. Clone - A population of cells descended from a single cell. Delayed Hypersensitivity - (part of cellular immunity) that develops slowly over a period of 24-72 hours after an antigenic stimulus. It consists of an accumulation of cells around small vessels and/or nerves. Example: Tuberculin skin test reaction. Digestive Enzyme - A substance that catalyzes or accelerates the process of digestion. Eosinophilic Granules - Specific granules present in the cytoplasm of eosinophils. These granules are large, refractile spheres which stain reddish-orange due to their strong affinity for acid stain. Erythrocyte (red blood cell, RBC) - One of the elements found in peripheral blood. Normally the mature form is a non-nucleated, circular, biconcave disk adapted to transport respiratory gases. Fixed Macrophage - A phagocyte that is non-motile. Free Macrophage - An ameboid phagocyte present at the site of inflammation. Graft Rejection - A transplanted tissue that is rejected by the body's antibodies. Graft vs. Host Reaction - A complication that occurs when an implanted piece of tissue, which contains antibodies, rejects the host's tissue. Granulocyte - A leukocyte which contains granules in its cytoplasm, i.e., neutrophilic, eosinophilic, or basophilic granules. Half-life - is the length of time it takes for half of the cells circulating at a given time to leave the blood for the tissues. Hemocyte - Any blood cell or formed element of the blood. Hemostasis - A mechanism of the vascular system to arrest an escape of blood. It involves an interaction between blood vessels, platelets, and coagulation. Heparin - A mucopolysaccharide acid which, when present in sufficient amounts, functions as an anticoagulant by inhibiting thrombin. Histamine - A powerful dilator of capillaries and a stimulator of gastric secretions. Humoral Immunity - Acquired immunity produced after response to an antigenic stimulus in which B cells produce circulating antibodies. Hyalomere - the clear, blue non-granular zone surrounding the chromomere of a platelet. Immune Response - The interaction of a cell and an antigen that results in a proliferation of the cell and a capacity to produce antibodies. Isotonic Fluid - A fluid whose elements have an equal osmotic pressure. Leukocyte (white blood cell, WBC) - One of the formed elements of the blood; involved primarily with the body's defense. Lysosome - A microscopic body within cell cytoplasm; contains various enzymes, mainly hydrolytic, which are released upon injury to the cell. Megakaryocyte - A giant cell of the bone marrow from which platelets are derived. Mononuclear - A cell having a single nucleus. | View Page |
| Match the characteristics with the cells. | View Page |
| Which type of leukocyte transforms into a macrophage, has ?digestive enzymes within its granules, and ?is phagocytic in tissues? | View Page |
| Glossary of Terms A through M. Antibody - A modified type of serum globulin synthesized by lymphoid tissue in response to antigenic stimulus. By virtue of specific combining sites each antibody reacts with only one antigen. Anucleate - Having no nucleus. Azurophilic granules - The well-defined large reddish granules (lysosomes) which may be present in large lymphocytes. They are called "azurophilic granules" because they stain blue with the azure stains which were originally used. Basophilic granules - Specific granules present in the cytoplasm of basophils. These granules are large and stain purple-black due to their strong affinity for basic stain. B-cell - Bone marrow derived lymphocytes which produce humoral antibodies. Biconcave - Having two concave surfaces. Cellular Immunity - The capacity of a small proportion of lymphoid population to exhibit response to a specific antigen. Chromomere - The centrally located granular portion of the platelet. Clone - A population of cells descended from a single cell. Delayed Hypersensitivity - (part of cellular immunity) that develops slowly over a period of 24-72 hours after an antigenic stimulus. It consists of an accumulation of cells around small vessels and/or nerves. Example: Tuberculin skin test reaction. Digestive Enzyme - A substance that catalyzes or accelerates the process of digestion. Eosinophilic Granules - Specific granules present in the cytoplasm of eosinophils. These granules are large, refractile spheres which stain reddish-orange due to their strong affinity for acid stain. Erythrocyte (red blood cell, RBC) - One of the elements found in peripheral blood. Normally the mature form is a non-nucleated, circular, biconcave disk adapted to transport respiratory gases. Fixed Macrophage - A phagocyte that is non-motile. Free Macrophage - An ameboid phagocyte present at the site of inflammation. Graft Rejection - A transplanted tissue that is rejected by the body's antibodies. Graft vs. Host Reaction - A complication that occurs when an implanted piece of tissue, which contains antibodies, rejects the host's tissue. Granulocyte - A leukocyte which contains granules in its cytoplasm, i.e., neutrophilic, eosinophilic, or basophilic granules. Half-life - is the length of time it takes for half of the cells circulating at a given time to leave the blood for the tissues. Hemocyte - Any blood cell or formed element of the blood. Hemostasis - A mechanism of the vascular system to arrest an escape of blood. It involves an interaction between blood vessels, platelets, and coagulation. Heparin - A mucopolysaccharide acid which, when present in sufficient amounts, functions as an anticoagulant by inhibiting thrombin. Histamine - A powerful dilator of capillaries and a stimulator of gastric secretions. Humoral Immunity - Acquired immunity produced after response to an antigenic stimulus in which B cells produce circulating antibodies. Hyalomere - the clear, blue non-granular zone surrounding the chromomere of a platelet. Immune Response - The interaction of a cell and an antigen that results in a proliferation of the cell and a capacity to produce antibodies. Isotonic Fluid - A fluid whose elements have an equal osmotic pressure. Leukocyte (white blood cell, WBC) - One of the formed elements of the blood; involved primarily with the body's defense. Lysosome - A microscopic body within cell cytoplasm; contains various enzymes, mainly hydrolytic, which are released upon injury to the cell. Megakaryocyte - A giant cell of the bone marrow from which platelets are derived. Mononuclear - A cell having a single nucleus. | View Page |
| During phagocytosis the neutrophilic granules release digestive enzymes into the vacuole to kill or destroy the phagocytized particle. | View Page |
| Phagocytosis in a Neutrophil When a neutrophil is faced with a microorganism or foreign particle, phagocytosis begins.
The neutrophil extends pseudopods around the foreign material and engulfs it.
Digestive enzymes present in the neutrophilic granules are released into the vacuole containing the foreign particle, and the particle is destroyed.
In most cases a mild infection enhances the function of neutrophils while a severe infection impairs their function. | View Page |
| Basophils as Mediators of Inflammatory Responses Basophils serve as mediators of inflammatory responses, especially hypersensitivity reactions.IgE binds to the membrane receptors on basophils and degranulation is initiated.The enzymes released are vasoactive, bronchorestrictive and chemotactic (especially for eosinophils), so basophils seem to play a role in inducing and maintaining allergic reactions.The granules of basophils contain histamine, heparin and peroxidase.After degranulation occurs, basophils can synthesize more granules.The release of large numbers of these granules can cause anaphylactic shock and death. | View Page |
| Other Factors Affecting Drug Absorption and Distribution In addition to protein availability, other factors may affect drug absorption and distribution in the body as a whole or at specific sites within the body. The following table highlights some of these other factors. Factor Discussion Regional blood flow Reduced area blood flow can be seen in diabetics and enhanced blood flow can be seen in tumors. Lipid solubility of the drug The more lipophilic a drug is, the more likely it will enter the central nervous system. The integrity of the GI tract In a diseased gut, an orally-administered drug may not be absorbed as expected. Age Drug kinetics and dispositions change throughout life. In general, metabolism of drugs is reduced in the elderly. Genetics Mutations or deletions in drug metabolizing enzymes can greatly affect a drug's disposition. | View Page |
| Unexpected Concentrations TDM provides a quantitative measure of the circulating concentration of a drug. The physician determines if the dosage of the drug needs to be adjusted based on this information.If a drug concentration is determined to be outside the therapeutic range, it may be for one of the reasons listed in the table below. Reason Discussion Noncompliance Patients may (intentionally or unintentionally) not take the drug. TDM can thus help monitor compliance. Dosing errors The dose may have been erroneous or inappropriate given the patient's condition. Malabsorption The TDM result will reveal if the drug cannot be absorbed well through the gut and an alternative route of administration will be needed. Drug interactions Many drugs interfere with the absorption or metabolism of other drugs. These interactions will be revealed by TDM. Kidney or liver disease Any pathology that affects elimination will cause an elevation in a drug level that will be unmasked by TDM. Altered protein binding Changes in serum proteins can lead to big changes in the amount of free drug in serum. Variations in the genetics of drug-metabolizing enzymes can also affect drug concentrations in the body. This is the field of pharmacogenomics that will be discussed later in the course. | View Page |
| Why TDM? However, every patient is unique. Changes in the gut (if the drug is taken orally), genetic variations in the liver's metabolizing enzymes, and the status of organs (like the kidneys and liver) all affect how a drug will be handled by an individual. TDM helps to ensure that a dosing regimen is appropriate for a given patient. | View Page |
| Chemiluminescence Chemiluminescent assays use antibodies that are conjugated to enzymes, such as peroxidase or alkaline phosphatase. These enzymes, mixed with chemiluminescent substrates, produce light in the visible spectrum. A direct relationship exists between the amount of drug that is present in the sample and the light units that are produced and measured by the luminometer in the instrument. Assays that use chemiluminescence are more sensitive than immunoassays that rely on the generation of a colored product. | View Page |
| Individualized Medicine It has been said that we live in a new era of "individualized medicine." One of the primary drivers for this idea is the emerging field of pharmacogenomics (PGx). PGx is the study of how individual variations in the human genome affect responses to medications. The term "pharmacogenetics" is also used for this discipline (people in the field use both terms); however, the term 'pharmacogenomics' is becoming more popular since we now know the entire human genome. The primary reason that individuals metabolize and respond to drugs differently is the inter-individual differences in receptor proteins and enzymes that metabolize the drugs. Mutations in these receptor proteins and enzymes can give rise to very different responses to drugs. In PGx, these mutations are referred to as variants. | View Page |
| Polymorphism and CYP450 To discuss PGx, we must first define two terms - polymorphism and cytochrome P450 (CYP450).A polymorphism is a variation in a gene (allele) that affects at least 1% of the population. CYP450 refers to a family of enzymes found predominantly in the liver. CYP450 enzymes work on a variety of substrates (drugs), altering their chemical structures to facilitate excretion in the urine and feces. There are many known polymorphisms in CYP450 enzymes. | View Page |
| CYP450s Many CYP450 enzymes have been characterized, and the substrates (drugs) that each can recognize have been worked out to a large extent. These subfamilies of CYP450 enzymes have all been associated with polymorphisms that can affect drug disposition: CYP1A2, CYP2C9, CYP2C19 and CYP2D6. | View Page |
| Enzyme Abnormalities and Drugs The following is a list of enzymes for which known mutations have been associated with clinical effects. Enzymes Substrates (Drugs) Acetylaldehyde dehydrogenase Alcohol Acetylcholinesterase Succinylcholine Alcohol dehydrogenase Alcohol Dihydropyrimidine dehydrogenase Fluorouracil CYP2C9 Warfarin, phenytoin, losartan CYP2C19 Diazepam, omeprazole (Prilosec) CYP2D6 Many antidepressants, opioids, antiarrhythmics Glucose-6-phosphate dehydrogenase Aspirin, quinidine N-acetyltransferase Procainamide, isoniazid Thioprine methyltransferase 6-mercaptopurine UDP-glucuronosyl transferase Acetaminophen, tolbutamide, irinotecan | View Page |
| CYP450 Induction and Inhibition Variables other than mutations also affect CYP450 enzymes. Many drugs are able to induce CYP450 enzymes, and CYP450s can be inhibited by a variety of substances. For example, CYP2D6 can be inhibited by the common medications cimetidine (Tagamet) and fluoxetine (Prozac). Since many patients are on multiple medications and since dietary and environmental factors can change, CYP450 expression levels cannot be solely predicted based on their genotype. Some CYP450 inducers and inhibitors are listed in the table on the following page. | View Page |
| Genotype versus Phenotype Genotyping can give us a definitive profile of a given CYP450 enzyme's mutations. But since there are dozens of mutations usually associated with each enzyme, a complete characterization of a CYP450 is not always realistic. Without complete sequencing of the entire allele, it may not be possible to entirely rule out a mutation in a patient who shows none of the more common polymorphisms. If we consider the number of possible mutations and the possible presence of inducing/inhibiting substances, phenotyping for drug metabolism may sound more reasonable than genotyping. | View Page |
| The Bottom Line By knowing a patient's disposition to specific drugs, the physician should be able to start the patient on an appropriate regimen rather than perfecting treatment based on trial and error. Drugs whose metabolism may prove to be problematic can be avoided, and second-line therapies that are metabolized by different, unaffected enzymes can be chosen. Clinical chemists, pharmacologists, and physicians need to translate knowledge of CYP450 polymorphisms into clinically-validated treatment algorithms. Dosing recommendations for PM, EM, IM and UM patients are beginning to appear in the literature for various classes of drugs, and the FDA is encouraging the incorporation of pharmacogenomic testing in the development process for new drugs. | View Page |
| CYP2D6 CYP2D6 has received the most attention: It is estimated that about 25% of common drugs are metabolized by CYP2D6. CYP2D6 accounts for only about 1% of all CYP450 enzymes, but it is important in the metabolism of about 100 drugs. There are more than 80 genetic variants that have been described in the CYP2D6 gene. The normal, wild-type allele displays normal metabolic activity whereas some of the variant forms have enhanced or diminished activity. The variants can be grouped generally according to the resulting alterations in protein function. The groupings correlate with four major enzyme metabolic capacities (phenotypes): poor, intermediate, extensive (normal), or ultra-rapid metabolizers. | View Page |
| DNA Polymerase Polymerases are enzymes that synthesize DNA from an existing template. Polymerase requires a primer, nucleotides, and magnesium in order to function. In early PCR methods, the DNA polymerase was inactivated during the denaturation step. This required new polymerase to be added during each cycle of PCR. This problem was solved by the discovery of an enzyme termed Taq polymerase. Taq polymerase is isolated from the thermophilic organism Thermus aquaticus, a natural bacterium found in thermal springs. The Taq polymerase has optimal activity at 72°C but it can survive in temperatures up to 95°C. Today there are several other kinds of thermostable enzymes that are available for PCR, such as Pfu and Tli DNA polymerase. However, Taq is the DNA polymerase that is used most often in PCR procedures. | View Page |
| DNA Isolation There are several methods for extracting genetic material from cells. Different methods use slightly different techniques or different combinations of reagents. However, the basic process is generally the same. The desired cells are put into a tube and mixed with a special cocktail. The cocktail usually includes detergents, enzymes, and salts. The sample is disrupted to cause cell lysis, or the breaking open of the cells. The detergent breaks down the membrane lipids, enzymes remove the proteins, and the salt pulls out the DNA. Centrifuging the sample will cause the DNA to form a pellet at the bottom of the tube. The remaining cocktail can be poured off and the DNA pellet can be resuspended for further use. Today, there are systems that will isolate and purify DNA automatically. Automated techniques can often work on more than one sample at a time, require little intervention, and produce more purified samples than manual techniques. | View Page |
| If a cocktail that includes detergents, enzymes, and salts is used for DNA isolation, which one of these ingredients will pull the DNA out from the sample? | View Page |
| Investigating weak antibodies In this case the patient's antibody has disappeared from the plasma by adsorbing to transfused donor red cells. It is detectable but unidentifiable in the post-transfusion red cell eluate. Several trial and error procedures exist to enhance weak antibodies. Which methods will enhance the reactivity of a given antibody depend on its characteristics. Methods to investigate weak antibodies include: Use a higher plasma to red cell ratio (add more antibody-containing plasma or eluate) Increase incubation time (if consistent with manufacturer instructions, if applicable) Use enzyme-treated panel red cells (enzymes enhance IgG antibodies in Rh and Kidd blood systems but denature some antigens, e.g., Fya, Fyb, S) Try alternative antibody detection methods, e.g., if using LISS routinely, try polyethylene glycol (PEG) or column agglutination methods such as gel, providing they have been validated for use in the TS laboratory. | View Page |
| When the patient's plasma was non-reactive with panel cells, and very weak and unidentifiable in the post-transfusion RBC eluate, no attempt was made to try to enhance the weak antibodies.We now know that the patient has anti-Jka and that it disappeared rapidly from the patient's plasma after transfusion with two group O Rh-negative RBC. Consider the question below, then click on the question to receive the answer. | View Page |