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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

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.

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.

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.

Prompt testing (within two hours of urine specimen collection) or refrigeration of the specimen is necessary because the glycolytic enzymes from the cells and bacteria, if present, will decrease the sugar in the urine.

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

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.

These methodologies use principles that amplify or multiply the target of interest, usually incorporating an enzyme to produce millions or billions of copies in a relatively short time.Some enzymes used in amplification include:DNA ligase DNA polymerase RNA polymerase Reverse transcriptase Alkaline phosphatase Cleavase Note: the steps in amplified testing will vary depending on the target amplified, requirement for thermal cycling, and detection techniques.

Diastase is often used in conjunction with the PAS staining procedure to specifically identify glycogen granules in tissue samples. Amylase enzymes in a malt extract are used to digest the glycogen, which is then washed out of the tissue sample. It is standard procedure to cut and process two serial sections at the same time. One section is treated with diastase to digest and wash out the glycogen. The other section is left untreated. Both sections are then stained with the PAS procedure for comparative microscopic analysis.

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 Number Abbreviation Gene Function 1 gag capsid proteins 2 pol viral enzymes 3 env envelope proteins 4 vif regulatory gene 5 tat regulatory gene 6 vpu regulatory gene 7 nef regulatory gene 8 vpr regulatory gene 9 rev regulatory gene

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.

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.

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.

When HPV infects host cells, several HPV DNA-coded proteins initiate cellular changes. Two such areas in the genome are the open reading frames E1 to E7 and the late open reading frames L1 and L2. The proteins encoded by E1 to E7 regions of the genome are responsible for HPV-gene regulation and cell transformation. Proteins resulting from L1 and L2 form the viral shell.E6 and E7 encoded proteins are the most important HPV proteins in malignancy transformations. These viral proteins work together to convert normal host cells to malignant cells. E6 proteins interact with intracellular protein p53 and E7 proteins interact with intracellular retinoblastoma (Rb) protein. Intracellular proteins p53 and Rb regulate cellular growth. Both p53 and Rb are tumor suppressor proteins.When chromosomal damage occurs in normal cellular growth, p53 halts cellular growth and allows DNA repair enzymes to repair damage. Rb also halts cellular growth in DNA damage by inducing apoptosis (cellular death). When HPV E6 proteins bind to p53 and HPV E7 to Rb, mutations accumulate, unchecked cellular growth occurs, and a state of chromosomal instability results. This instability and unregulated cellular growth increases the chance of forming malignant cells. Viral E1, E2, and E5 encoded proteins may also damage cellular processes when HPV infects cells and can lead to malignant transformations.

Specialists in immunohematology perform all testing prior to blood transfusions and work to prevent transfusion infections. They also investigate any post-transfusion reactions. This specialty includes all lab procedures performed in the specialty of histocompatibility. Specialists in clinical chemistry analyze body fluids such as blood, urine, and spinal fluid to determine the chemical makeup, including the amount of carbohydrates, proteins, enzymes, and trace elements. The special covers urine microscopics and chemical evaluation of the liver, kidneys, lungs, heart, and other vital organ systems. This specialty also covers all testing performed in the specialties of radioassay and blood gas analysis. Specialists in blood banking can perform all immunohematology testing as well as testing from the specialties of clinical chemistry, hematology and serology/immunology that relates to donor blood. Clinical laboratory personnel who are licensed in the specialties of immunohematology, clinical chemistry, hematology, and serology / immunology may perform all tests in the blood banking specialty.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Semen is a substance produced by the male reproductive organs. It is composed of spermatozoa in a semi-viscous fluid. Structures within the male reproductive tract that are involved in the production of semen include: Testes and epididymisProstateSeminal vesiclesBulbourethral glandSemen is produced as a combination of secretions from the different regions of the male reproductive tract. Each fraction differs in chemical composition and function. The combination of these fractions during ejaculation results in the optimal environment for transporting sperm to the endocervical mucus in the female. Spermatozoa are produced in the testes. They mature in the epididymis. The testes also produce testosterone and inhibin.Fluid from the seminal vesicles accounts for approximately 70% of semen volume. The seminal vesicles are the source of fructose in semen. Fructose is used by the spermatozoa as an energy source.The prostate gland supplies about 20% of the volume of semen. Its fluids include acid phosphatase and proteolytic enzymes that lead to coagulation and subsequent liquefaction of semen. The prostate also contains most of the IgA found in semen.The bulbourethral gland produces mucoproteins that make up about 5% of the volume of semen.

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.

Important events that occur in an immune-mediated hemolytic transfusion reaction (HTR) include: Antibody Binding to Red Blood Cells Antibodies may be either IgM or IgG class. IgM antibodies activate complement and lead to intravascular hemolysis where free hemoglobin is released into the plasma. IgG antibodies rarely activate complement but they are often involved in effecting phagocytosis. The concentration of the antibody is directly related to the severity of the HTR. Activation of Complement The end result of complement activation is red cell lysis. Activation of Mononuclear Phagocytes and Cytokines Sensitized red cells are removed from circulation by mononuclear phagocytes. Macrophages in the spleen and Kupffner cells in the liver are active in this process. Activation of Coagulation Antibody-antigen complexes may initiate coagulation and cause disseminated intravascular coagulation (DIC). Shock and Renal Failure Hemolysis can be intravascular or extravascular. In intravascular hemolysis, free hemoglobin, RBC stroma, and intracellular enzymes are released into the blood stream. This results in hemoglobulinemia and hemglobinuria which can lead to kidney damage. In extravascular hemolysis, there is no release of free hemoglobin. Sensitized red cells are removed from the circulation by the monocytes and macrophages in the reticuloendothelial system.