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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.

C is enhanced: reactions seen on cells 1,2,and 5 E is enhanced: reactions seen on cells 3 and 6 P1 is enhanced: pattern of reactivity at IS phase matches that of P1. Jkb not reacting: cells 7,8 and 10, so not present Lea not reacting: cell 7, so not present M, N, and s will not react with enzyme treated cells. The enzyme panel was used to remove the potential reactivity of these antibodies to help further identify other antibodies present.Another selected cell panel can be run to eliminate M,N and s.

A similar procedure that is also widely used is called Colorzyme®.(Ref7) This system uses horseradish peroxidase rather than FITC as the marker on the secondary antibody. This technique offers the same advantages as the IFA procedure but also has the added benefits of being more photo-stable and not requiring a fluorescent microscope. The Colorzyme® ANA Test utilizes the indirect enzyme antibody technique. Patient serum samples are incubated with antigen substrate to allow specific binding of autoantibodies to cell nuclei. If ANA's are present, a stable antigen-antibody complex is formed. After washing to remove non-specifically bound antibodies, the substrate is incubated with an anti-human antibody reagent conjugated to horseradish peroxidase. When results are positive, there is the formation of a stable three-part complex consisting of enzyme antibody bound to human antinuclear antibody that is bound to nuclear antigen. This complex can be visualized by incubating the slide in an enzyme specific substrate. The reaction between the enzyme labeled antibody and enzyme specific substrate results in a color reaction on the slide visible by standard light microscopy. In positive samples, the cell nuclei will show a bright bluish purple staining with a pattern characteristic of the particular nuclear antigen distribution within the cells. If the sample is negative for ANA, the nucleus will show no clearly discernible pattern of nuclear staining. The cytoplasm may demonstrate weak staining while the non-chromosome region of the mitotic cells may demonstrate a darker staining. The photo to the right demonstrates the 4 basic ANA patterns (clockwise from top left): Homogeneous, Speckled, Centromere, and Nucleolar. (Additional photos of these patterns will be seen in subsequent sections.)

Currently, the most effective treatment for TTP is therapeutic plasma exchange (TPE). Fresh frozen plasma (FFP), preferably cryoprecipitate-poor plasma (that lacks von Willebrand factor), is used as the replacement fluid in the treatment. The exchange takes place over several days until the patient's platelet count stabilizes above 100 x 109/L.The logic of TPE is to rid the circulation of plasma containing ultra-large vonWillebrand factor (vWF) multimers. vWF is a large multimeric protein that is made by megakaryocytes and endothelial cells. It is is a key factor in platelet adhesion and also is responsible for carrying Factor VIII into the circulation. vWF binds glycoproteins Ib, IIb, and IIIa. The largest multimer is called ultra-large vWF and in normal plasma, it is cleaved into smaller fractions (necessary for balanced coagulation activity) by an enzyme processed by the gene, ADAMTS13. In patients with TTP, the enzyme activity is < 5% of normal and therefore, these ultra-large vWF molecules get into circulation, resulting in excessive platelet aggregation and microvascular thrombus formation.Therapeutic plasma exchange has decreased TTP mortality rate from 90% to 15% since the treatment first came into use as the standard primary treatment of TTP in the 1970's. TPE does not cure TTP, but it arrests the manifestations of the disease until spontaneous remission occurs.

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.

The analysis for glucose on a chemical reagent strip is a double-sequential enzyme reaction, utilizing the glucose-oxidase/peroxidase method. In the first reaction, glucose oxidase catalyzes the oxidation of glucose to gluconic acid and hydrogen peroxide. Then, the peroxidase catalyzes the oxidation of a chromogen by the hydrogen peroxide to form a colored product. The chemical reagent strip glucose pad is then analyzed and recorded at the set interval stated by the manufacturer.This method does not react with lactose, fructose or galactose. Study the dipstick color chart in your laboratory to become familiar with the range of color changes. NOTE: The urine specimen should be analyzed while at room temperature for these enzyme reactions to occur properly.

The test for glucose is a double sequential enzyme reaction, utilizing the glucose-oxidase/peroxidase method. In the first reaction, glucose oxidase catalyzes the oxidation of glucose to gluconic acid and hydrogen peroxide. Then, the peroxidase catalyzes the oxidation of a chromogen by the hydrogen peroxide to form a colored product. This method does not react with lactose, fructose or galactose. Study the dipstick color chart to become familiar with the range of color changes. The urine specimen should be at room temperature for these enzyme reactions to occur properly.

Enzyme-based methods are most often used to detect/monitor urine glucose and copper reduction methods are used to detect all reducing substances. Most enzyme tests use the enzyme glucose oxidase. which is impregnated on a dipstick along with a chromagen. These enzyme-based dipstick tests are specific for glucose.

Improper collection of the blood specimen that is used for testing can cause false prolongation of PT or aPTT results. The following table covers several preanalytical variables that may affect PT or aPTT test results Preanalytical Variable Cause of False Elevation of PT and or aPTT Test Result Corrective Action Blood collection tube is inadequately filled. Improper ratio of blood to anticoagulant. Excess anticoagulant causes prolonged PT or aPTT result. Recollect specimen ensuring proper fill to achieve a blood to anticoagulant ratio of 9:1. Patient has a hematocrit level above 55% Improper ratio of blood to anticoagulant. Excess anticoagulant causes prolonged PT or aPTT result. Prepare a specimen collection tube that contains less anticoagulant. Refer to your laboratory's procedure for the proper amount of anticoagulant. Specimen is clotted. Coagulation factors have been activated; insufficient levels left in the plasma. PT and aPTT results will be affected. Recollect the specimen. Specimen collected from an arm with a heparin lock or from a heparinized vascular access device (VAD). Heparin contamination will prolong the aPTT. Collect the blood from a vein rather than a VAD. If blood must be drawn from the VAD, flush it first with 5 mL of saline, and discard the first 5 mL of blood before collecting the specimen. Patient is receiving heparin therapy. Heparin will prolong the aPTT If the patient is being evaluated for possible factor deficiencies or coagulation inhibitors, use a heparin digesting enzyme as a pretreatment before testing the PT or aPTT. .

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

Lipoproteins differ in size and density as well as in their content (what they tend to carry). They also can differ in their origination (where they are made). Another significant difference between lipoprotein molecules is the proteins they have on their surfaces. These proteins, known as apolipoproteins, are the major identifying characteristics of a lipoprotein. There are many different apolipoproteins and we are continually learning more about them. Apolipoproteins have multiple roles. One role is that these amphipathic (detergent-like) proteins increase the overall solubility of the lipid particle, helping it to dissolve in the aqueous environment of the blood. Apolipoproteins can also function as enzyme co-factors (receptor ligands) and facilitate the transfer of their lipid cargo to specific cells. Thus, the apoliproteins are the smart or working-end of the lipoprotein particle. The apolipoproteins dictate where the particles will dock and where they can bind, and in so doing the apolipoproteins regulate lipid metabolism in the body.

In the clinical laboratory, we routinely measure the cholesterol content of high-density lipoprotein and low-density lipoprotein particles and not the apolipoproteins on the particles or the number of particles. Proprietary detergents and reagents are used in assays for HDL-C and LDL-C to separate lipoproteins, allowing the cholesterol content of specific lipoproteins to be measured. For example, HDL-C is commonly measured using a solution of dextran sulfate and magnesium to selectively precipitate HDL from the other lipoproteins present in the sample. Once isolated, the HDL particles are 'dissolved' and the amount of cholesterol in them is determined photometrically using a color-producing enzyme reaction. LDL-C can be measured directly or can be estimated using the HDL-C, triglycerides and total cholesterol (TC) values. The Friedewald formula is often used to calculate LDL: LDL-C = TC - (HDL-C)- (Triglycerides/5). The important point to consider here is that traditional LDL-C and HDL-C measurements only tell us how much cholesterol is associated with each lipoprotein particle class. We are now learning that the number and size of the particles are important as well. The number of LDL particles appears to be more strongly predictive of cardiovascular disease than the LDL-C content, and small dense LDL are known to be more atherogenic than larger, less dense LDL particles.

Fibrin strands woven into the clot structure are cleaved into soluble fibrin fragments and then removed by macrophages. The action of fibrinolysis also serves to restore blood flow into the area that had been sealed off, helping to promote further healing. Fibrinolysis is mediated by a proteolytic enzyme called plasmin (plasminogen is the inactive precursor form of plasmin that is found in plasma). Plasmin takes on fibrinolytic properties after activation, digesting both fibrin and fibrinogen. Inhibitors act to control the process, serving as a check and balance system for fibrinolytic activities.

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.

Detection techniques can vary in both direct and amplified methodologies and can include labeling either the probe or the target molecule of interest:Chemiluminescence: Release of light energy at the end of a chemical reaction that is detected by a luminometer; uses a label such as acridinium ester. Electrophoresis: movement in a matrix such as a gel that is caused by an electrical field.Enzyme: Uses enzyme and substrate principles to label the appropriate target or probe; can be combined with fluorescence or dyes for detection.Fluorescence: Molecules that emit light at a longer wavelength when excited at a shorter wavelength. Detection techniques include fluorescent staining of nucleic acids as well as fluorescent labeled probes that are measured in a fluorometer or with fluorescent polarization.Radioactivity: Uses a labeling technique where the radioactive label is then measured in a scintillation counter. The earliest assays utilized radioactive decay.

Anti-D causes the most severe HDFN. Symptoms and laboratory findings in HDFN due to anti-D typically include:1. Anemia: Cord Hb can be less than 10 g/dL (100 g/L) and as low as 3–5 g/dL (30–50 g/L).2. Jaundice (icterus gravis): Jaundice occurs after delivery, as fetal bilirubin is cleared by the mother during pregnancy. Extravascular fetal red cell destruction by maternal antibody produces high bilirubin levels. The newborn, who is unable to produce adequate amounts of the liver enzyme glucuronyl transferase, is unable to conjugate the bilirubin into its water-soluble, excretable form.3. Kernicterus: If indirect bilirubin levels reach approximately 20 mg/dL (340 mmol/L) the fat soluble unconjugated bilirubin deposits in the fat-rich brain cells causing brain cell damage. Cerebral palsy, deafness, mental retardation, and other serious disorders can result.4. Hydrops fetalis: Gross edema occurs in severely affected infants, and often results in stillbirth or death soon after birth. Liver failure and hypoproteinemia also play a role in this syndrome.5. Enlarged organs, e.g., liver, spleen and heart6. Laboratory findings include a positive direct antiglobulin test (DAT), low hemoglobin (as above), increased reticulocyte count, and increased nucleated red cells.

Before ABO HDFN is considered as a possible cause of jaundice and anemia in the newborn, other causes should be considered, for example, erythrocyte membrane defects or red cell enzyme deficiencies. The diagnosis of ABO HDFN in the laboratory differs from diagnosing Rh and other types of HDFN in which clinically significant antibodies must be identified. Diagnosis may be difficult, because the DAT on the newborn's red cells is unreliable. Indeed, many labs do not routinely do a DAT on infants born to Rh positive females, since many will be positive in the absence of clinically significant hemolysis. Cord blood is often retained (e.g., for 7 days) should the infant develop signs of HDFN and required testing.If ABO HDFN is possible, based on incompatible ABO blood groups and a positive DAT, and the mother's antibody screen is negative, many laboratories do not investigate the positive DAT as would be done for unexpected antibodies like anti-D or anti-K (the laboratory does not perform an elution on the newborn's red cells). Instead, the infant's plasma is tested against group A1 (or B cells) and group O screen cells using the indirect antiglobulin test (IAT). A positive reaction with A1 or B cells, but not group O cells, would suffice to report a case of possible ABO HDFN.

Sample Type Required: Deparaffinized and re-hydrated tissue sections on positively charged slides. Fixative: 10% Neutral Buffered Formalin Step Reagent Time Technical Notes 1 Malt Diastase Solution 1 hour at 37C Solution should be preheated to 37C. Do not heat the solution above 40C as enzyme activity can destroyed at higher temperatures. Insufficient heat may cause incomplete digestion of glycogen. 2 Distilled Water 3 Changes Rinse slides gently to remove diastase. 3 Periodic Acid 0.5% Solution 5 Minutes 4 Distilled Water 3 Changes 5 Schiff Reagent 15 Minutes Gluteraldehyde should be avoided as a tissue fixative since it is a dialdehyde that will react with this reagent and give false-positive PAS staining. 6 Potassium Metabisulfite 0.55% Solution 1 Minute in 2 Changes While some technicians omit this reagent step in the protocol with no problems, metabisulfite rinses may be necessary to remove any excess leucofuchsin left over after exposure to the Schiff reagent. Highly chlorinated water can cause these excess molecules to nonspecifically stain other tissue elements in the section. 7 Running Tap Water Up to 10 Minutes Warm to hot running water develops the PAS stain and gives a more intense staining reaction than cold water. Expected Results Glycogen will be absent from tissue section The following elements will show positive PAS (Rose Red) staining Neutral Mucins Some Epithelial Mucins Basement Membranes Fungal WallsPost Staining Procedure: Tissue sections should be rinsed well in distilled water, dehydrated with 95% and absolute alcohols, cleared and cover-slipped.

The Human Immunodeficiency Virus type-1 (HIV) belongs to the Family Retroviridae.The core of a retrovirus (including HIV) contains RNA, which encodes the genetic information of the virus, and an enzyme known as reverse transcriptase. Reverse transcriptase enables the virus to convert RNA to double-stranded DNA (the opposite of the normal process of transcription where double-stranded DNA is converted to single-stranded RNA). Because of this reverse process, HIV is known as a retrovirus; retro implying reverse.

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.

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.

Cervista HPV (Third Wave Technologies, Hologic, Inc, Madison, WI)Two assays are available:1. Cervista HPV HR Screening Test14 HR-HPV Types: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, or 682. Cervista HPV 16/18Identifies and differentiates HR Types 16 and 18, referred to as HPV-16/18 genotyping testIn the Cervista HPV DNA assays, two hybridizations and an enzyme cleavage occur in a primary reaction. The secondary reaction is detection with signal amplification using fluorescence resonance energy transfer (FRET). Prior to hybridization, the HPV DNA (if present) is isolated, denatured, and added to microplate wells.

Three separate loci (ABO, Hh, and Se) contain the genes that control the location and occurrence of the A and B antigens. Hh and Se genes are closely linked on chromosome 19. The precursor substance is acted upon by the H gene and is converted to H substance. The product of the H gene is an enzyme fucosyltransferase, responsible for attaching fucose to the terminal galactose of the precursor substance on the RBC membrane and thus forming H substance. There are only two recognized alleles at this locus: the active form, H, and an amorph, h. The H gene is a high-incidence gene. People who inherit hh are extremely rare. Since the h gene is amorphic, it does not act on the precursor substance.

Some organisms are present in infections in very low numbers, which may be undetectable with direct staining methods. These organisms may also prove to be difficult to recover with currently available culture methods.Although non-culture antigen methods have been developed to address some of these difficulties (examples include direct fluorescent antibody (DFA) and enzyme immunoassay (EIA) methods), the sensitivity of these methods has not always been desirable.Molecular methods offer the prospect of:Detecting nonviable organisms that did not survive transport Detecting organisms difficult/impossible to cultivate Detecting organisms present in low numbers Providing better detection capability than other non-culture methods

Reverse transcriptase PCR (RT-PCR) was developed to amplify RNA targets (RNA viruses such as HIV, HCV, and influenza are key examples). Essentially, the method entails an initial step of transcribing a portion of the RNA genome into complementary DNA (cDNA) which is then amplified through PCR.PCR depends on the Taq Polymerase enzyme; RNA is not an efficient substrate for this enzyme. This is why the target of interest (if present) is first transcribed into complementary DNA (cDNA), which can then be amplified. RT-PCR ProcessAfter RNA is released from cellular material through extraction, an aliquot of the extracted sample is added to a reaction mixture which contains reverse transcriptase enzyme, primers specific for the target of interest, and nucleotides.If the target is present, primers anneal to the RNA strand.Reverse transcriptase enzyme synthesizes a complementary DNA strand, extending from the primer.The temperature is raised to 95o C, and the RNA/DNA strands are denatured.The temperatures are lowered, allowing primers to anneal to the newly formed cDNA.Polymerase enzyme synthesizes a new DNA strand, extending from the primer.Multiple cycles geometrically increase the number of copies of DNA.RT-PCR can be performed as one or two step procedures. In a one-step procedure, the reverse transcriptase is performed in the same reaction tube as the polymerase chain reaction. In a two-step procedure, transcription of the RNA to cDNA is performed first. Transcription occurs between 40o C and 50o C, depending on the properties of the reverse transcriptase enzyme utilized; products of that reaction are then amplified in a separate reaction.

Toxin assaysThe most common laboratory tests for the detection of C. difficile are enzyme immunoassays (EIA) for the detection of C. difficile toxin A and toxin B. The immunoassays are simple to perform, provide rapid results, and are easily incorporated into the workflow of most laboratories. Sensitivities of these tests do NOT compare favorably to culture, cell cytotoxicity neutralization assay (CCNA), or molecular methods. There are many test kits commercially available for detection of C. difficile toxins. Results are available in 15 minutes to 2 hours, depending on the assay. Initially, toxin A was thought to be the toxin responsible for the majority of the effects of C. difficile disease, so most early kits only detected toxin A. With the realization that there are strains that produce aberrant or no toxin A (A-) that are known to produce infection, and more recently toxin B negative (B-) strains, it is now recommended to use kits detecting BOTH toxins.Glutamate Dehydrogenase (GDH) assaysPublished studies have indicated that toxin immunoassays, by themselves, may not provide adequate sensitivity of detection. GDH assays initially attracted attention as a possible means to provide a rapid but more sensitive means for screening for C. difficile.GDH is an enzyme produced by C. difficile. EIAs negative for the GDH antigen have been associated with high negative predictive values. However, positive results are not necessarily associated with a toxin producing strain. A second assay on GDH positive samples is required to confirm the presence of a toxigenic strain. Initially, CCNA assays were recommended as the confirmatory method of choice; molecular methods (PCR for the toxin gene) were subsequently explored for this purpose.

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.

Some literature in recent years has suggested that assays for GDH (glutamate dehydrogenase - an enzyme produced by C. difficile) could provide a more sensitive means of screening for C. difficile.Published studies have indicated that toxin immunoassays, by themselves, may not provide adequate sensitivity of detection. Several investigators have examined the utilization of a two step algorithm. This first step is an enzmye immunoassay for the GDH antigen.A negative result for GDH has been associated with a high value for prediction of a true negative result; however, a positive result is not necessarily associated with a toxin producing strain. A second assay on positive samples for detection of toxin production is required in these algorithms.

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.

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.

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

When discussing PGx, we classify a person according to his/her phenotype (metabolic capacity for a given enzyme).A poor metabolizer (PM) is a person who lacks the functional enzyme and therefore exhibits decreased metabolism of drugs. This person would require lower doses of a drug that is metabolized by that enzyme. A PM who receives a standard dose is more likely to experience unwanted side effects or toxicity. A PM can also experience diminished effects with drugs that need to be metabolized to active compounds by the enzyme in question.An ultrarapid metabolizer (UM) will require a higher dose than usual since he/she will eliminate the drug more quickly. A UM may be resistant to standard treatments, and it may take some time to adjust the dosage before therapy is achieved.An intermediate metabolizer (IM) has one wild-type (normal) copy of the gene and one absent or dysfunctional copy. The IM group is very heterogeneous.A person with normal enzyme activity is referred to as an extensive metabolizer (EM). This person should respond to standard dosages of a drug. Most people are EM's. This is the population in which most dosing regimens have been worked out in clinical trials.

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.

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.

PCR can be modified for the amplification of RNA with one additional step prior to the PCR process -- the addition of a retrovirus enzyme called reverse transcriptase. Reverse transcriptase is used to create a copy of DNA using the original RNA specimen. Though there are thermostable polymerases that have reverse transcriptase capabilities, they are not commonly used. Reverse transcriptase PCR (RT-PCR) is used for the detection of viruses, such as HIV, that have an RNA genome. RT-PCR methods provide early detection of infection, even before the formation of antibodies. Therefore, it is a particularly useful method for HIV and hepatitis C virus(HCV) detection in blood bank nucleic acid testing. In addition to testing for HIV and HCV, RT-PCR is also used for detection of Mycobacterium tuberculosis, cytomegalovirus (CMV), influenza A virus, and other microorganisms where the target is RNA.RT-PCR is commonly combined with real time PCR.

The arrow points to a microcyte with normal hemoglobin content (one-third of central pallor). Since many of the other cells in this field are normal or larger than normal, the mean corpuscular volume (MCV) would be within the normal range although the diameter and volume of this individual cell would be lower than normal. This type of microcyte can be seen in some hemolytic anemias and the rare enzyme deficiency, pyruvate kinase deficiency anemia.

Round cells in semen are of two types: immature sperm (germ cells) and white blood cells (WBCs). These cells can be differentiated by examining a stained smear at 1000X magnification. A more precise identification can be achieved by detecting peroxidase activity. The presence of immature germ cells could indicate testicular damage; increased numbers of WBCs may indicate inflammation of the accessory glands.

If the specimen is more viscous than normal, it may be difficult to dilute it or to load it onto counting chambers in the undiluted condition. In this rare situation the semen may need to be manipulated to reduce the viscosity before a count is done. One method to do this is to repeatedly pipet the specimen up and down with an equal volume of culture medium. Care must be taken to avoid foaming. Other methods include enzyme digestion, for example with bromelain at a concentration of 1 gm / liter, or addition of a small amount of emulsifier, such as Alevare or chymotrypsin. Any manipulation of this type must be recorded on the report sheet. Calculation of the number of sperm per milliliter will also have to be corrected for any dilution.

The 2009 H1N1 virus, like other influenza A viruses, is an enveloped, single-stranded RNA virus. Influenza viruses are members of the family Orthomyxoviridae and are divided antigenically into 3 major types, A, B, and C. Influenza A viruses have the ability to undergo continuous antigenic changes, whereas Influenza B viruses reassort to a much lesser degree and influenza C viruses are antigenically stable. Due to their ability to undergo antigenic changes, influenza A viruses can cause more infections with greater morbidity in the human population than influenza B or C viruses. Influenza A viruses are subtyped based on two glycoprotein spikes expressed on their surface:1) Hemagglutinin (HA)2) Neuraminidase (NA)HA, a viral attachment protein, helps to facilitate the attaching of the virus onto ciliated epithelial cell receptors in the respiratory tract. NA is an enzyme that facilitates the release of virus particles from the infected cell surface.