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Drug Information and Courses from MediaLab, Inc.

These are the MediaLab courses that cover Drug and links to relevant pages within the course.

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Laboratories Individuals

Chemical Screening of Urine by Reagent Strip
False Positive Protein Results

A urine specimen that has remained at room temperature for an extended period of time may produce a false-positive protein result on a reagent strip. A false positive may also occur in the presence of bacterial contamination, alkaline medication, or quaternary ammonium compounds such as disinfectants or drugs, and with skin cleansers containing chlorhexidine.

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False Positive Ketone Results

False positive ketone results may be seen in patients after BSP or PSP dye injection due to the phthaleins. The presence of L-DOPA metabolites, some urine preservatives (e.g. 8-hydroxyquinaline), or high levels of phenylketones will also cause false positive results. Antihypertensive drugs such as methyldopa and captopril also may produce false positive results.

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Clinical Significance

No blood is found in the urine of healthy individuals although samples from menstruating females, frequently, but not always, test positive for blood. Hematuria is associated with renal or genital urinary disorders in which the bleeding is the result of irritation to the involved organs or trauma. Examples include renal calculi, pyelonephritis, glomerulonephritis, tumors, trauma or exposure to toxic chemicals or drugs and/or strenuous exercise. Hemoglobinuria may be due to the lysis of red cells within the urinary tract. If it is caused by intravascular hemolysis, the hemoglobin is then filtered through the glomeruli. In the normal individual, the hemoglobin molecule attaches to haptoglobin and in this way bypasses the kidney filtration system. When the hemoglobin/haptoglobin system is overwhelmed, as in cases of hemolytic anemia, severe burns, transfusion reaction, infection or strenuous exercise, hemoglobin passes into the urine.

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False Positive Urobilinogen Results

A false positive urobilinogen reaction may occur with the dipstick method when substances known to react with Ehrlich's reagent such as sulfonamides and p-aminosalicylic acid are present in the urine. Drugs that contain Azo dyes, such as Azo Gantrisin®, have a gold color that masks the reaction, causing a false positive reaction. Atypical color reactions may be obtained in the presence of high concentrations of p-aminobenzoic acid. The dipstick urobilinogen test cannot detect porphobilinogen in a urine specimen. Porphobilinogen is a molecule formed during the synthesis of the heme portion of hemoglobin.

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False Negative

False negative results may occur in the presence of significant levels of protein or glucose and in urines with high specific gravity which may crenate the white blood cells causing them to be come unable to release esterases. Some drugs such as Cephalexin (Kelfex®), Cephalothin Keflin®) or high concentrations of oxalic acid may also cause decreased test results. Tetracycline may cause decreased activity, and high levels of the drug may cause a false negative reaction. Large amounts of ascorbate may cause false negative results.

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CLIA Chemistry / Urinalysis Review
Identify the urine sediment elements shown by the arrow:View Page
Which of the following enzymes is the most sensitive indicator of liver damage associated with alcohol ingestion:View Page
Thin-layer chromatography is particularly useful as a tool in the identification of:View Page

CLIA Hematology / Hemostasis Review
The WBC indicated by the arrow in this illustration is exhibiting:View Page
Pelger-Huet anomaly is characterized by:View Page

Confirmatory and Secondary Urinalysis Screening Tests
The Ictotest®

False positive results can occur in screening procedures for bilirubin due to color interference from large amounts of blood in the urine, very concentrated urine or drugs that discolor the urine such as Pyridium. Because of this it is important to verify positive bilirubin results with a confirmatory test such as the Ictotest®.

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Current Topics in Clinical Microbiology
MIC susceptibility tests should also be performed against other select beta lactam antibiotics on important S. pneumoniae isolates from blood cultures and other sterile body fluids.View Page
ESBL Activity

Illustrated is the picture of the surface of a disk diffusion test including a ceftazidime disk (left) and a combintation ceftazidime/clavulanic acid disk (right).Observe in the photograph that the zone of inhibition around the the combination ceftazidime/clavulanic acid disk (right) is at least 5 mm larger than around the clavulanic acid disk (left).This observation that the presence of clavulanic acid, a beta-lactamase inhibitor, has resulted in such a large increase in the zone of inhibition indicates that an extended spectrum beta lactamase (ESBL)is being produced.When an organism is producing an ESBL, the susceptibility to individual cephalosporins cannot be predicted, thus requiring that each drug must be tested individually.It may be important to detect ESBL-producing stains of K. pneumoniae and E. coli as treatment failure may occur if the wrong cephalosporin is selected.

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Extended Spectrum Beta Lactamases

In follow-up to the observations of the ESBL screening test, the following antibiotic susceptibility profile was later reported: Ampicillin = R; Cefazolin = R; Cefoxitin 1 = S; Ciprofloxacin 0.25 = S; Gentamicin 1 = S; Ceftazidime 32 = R; Imipenem The susceptibility of the 2nd generation drug cefoxitin, with resistance of the 1st generation cefazolin and the 3rd generation ceftazidime, is another way in addition to the screening test in which ESBL activity may be detected. It is recommended that clinical microbiologists check the antibiotic susceptibility profiles for possible ESBL activity of clinically significant isolates of K. pneumoniae and E. coli.Most automated systems have built in methods for automatically detecting an ESBL isolate, or provide an "alert" that such a strain may be present.

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Vancomycin Resistance

Vancomycin and ampicillin resistance among Enterococcus species, particularly E. faecium have been on a steady increase.The disk diffusion screening test is used in many laboratories to detect vancomycin resistant strains. Note in the upper photograph that no zone of inhibition is seen around either the vancomycin or the ampicillin disk, indicating resistance to both drugs.Vancomycin-resistant Enterococci (VRE) have been divided into three phenotypes--Van A, Van B, and Van C.Vancomycin-resistant strains of E. faecalis and E. faecium are commonly of the Van A phenotype, demonstrating high level resistance (MIC's higher than 64 ug/mL), as illustrated by total resistance of the test strain in the E test and the VA disk, as illustrated in the lower photograph.The strain shown in the lower photograph, however, is ampicillin susceptible at the level of 1 ug/ml (see lower set of yellow arrows), indicating that this drug may be effective in treating the urinary tract infection.

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Most Eikenella cellulitis infections result from:View Page

Department of Transportation (DOT) Regulated Urine Specimen Collection Training
Federal drug testing custody and control form (CCF)

The federal drug testing custody and control form (CCF) must be used to document every urine collection required by the Department of Transportation drug testing program. At the present time, these include the: Federal Motor Carrier Safety Administration (FMCSA) Federal Aviation Administration (FAA) Research and Special Programs Administration (Pipeline) (RSPA) Federal Transit Administration (FTA) Federal Railroad Administration (FRA) United States Coast Guard (USCG)

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Non-federally regulated custody and control form

The Non-Federally Regulated Custody and Control Form is most often used in clinics and hospital emergency rooms when drug abuse is suspected, or by companies participating in their state's drug-free workplace program. Be aware that some states mandate the use of a special CCF for their drug-free workplace program. There are significant differences between the Federally Regulated CCF and the Non-Regulated CCF. You are strongly encouraged to review the difference between the two. Unless there are extenuating (which we will discuss next), remember that the two forms are not interchangeable. The Federally Regulated CCF can be used only for urine collections required by the Department of Transportation drug testing program.

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Exceptions

It should also be understood that a Federally Regulated CCF need not be used in every situation involving a company regulated by the Department of Transportation. For example, if an interstate truck driver slips and falls while standing on the loading dock of a trucking terminal, and the trucking company for which the driver works requires a post-accident drug screen, a Non-Federally Regulated CCF would be appropriate for this collection. The driver was not involved in a safety sensitive assignment. On the other hand, if the driver had an accident while driving, then a Federally Regulated CCF must be used since the driver was in a safety sensitive position when the accident occurred.

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Custody and Control Form

While the Federally Regulated CCF must be used exclusively for DOT drug screen collections, there are extenuating circumstances when another form of CCF may be used. For example, where a post-accident collection must be made and the collector does not have time to obtain a Federally Regulated CCF. In this situation, a Non-Federally Regulated CCF may be used. The collector should note in the "Remarks" section why the Non-Regulated CCF was used.The use of a Non-Regulated CCF for a Federally Regulated collection is not a reason for the laboratory to refuse to test the specimens nor the Medical Review Officer (MRO) to release the results. The use of a Non-Regulated CCF for a Federally Regulated drug screen is a "correctable flaw," which may include the collector detailing the reason for using a Non-Federally Regulated CCF in a "Memorandum for Record."

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Intent of this program

This program is intended to provide guidance and training to those individuals who will be conducting Department of Transportation (DOT) regulated urine specimen collections. While this program is more than just an overview, obvious restraints prohibit an in-depth discussion of every procedure or problem that might be encountered.This program only serves as a training program. It does not represent final authority. Every effort has been taken to keep this course up-to-date with current regulations. However, if anything you see in this program conflicts with the federal regulations (49 CFR 40), the federal regulations prevail and must be followed.Training to qualify as a drug screen collector must include the flawless completion of five mock collections. These mock collections must include the following scenarios and must be performed in the presence of a qualified collector: Two uneventful collections. One collection in which the quantity of specimen is not sufficient. One collection in which the temperature of the specimen is out of range. One collection in which the donor refuses to sign either the donor certification on the pink copy of the CCF or refuses to initial the security strips.

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Five areas having prerequisites for proper collection

Regardless whether you are collecting a Federally Regulated or a Non-Federally Regulated urine drug screen, there are five areas which demand specific prerequisites or conditions prior to performing a proper collection. These are: Requirements for the collection site. Supplies needed to conduct a collection. Criteria that must be met by collectors. Complete and accurate documentation. Proper identification of the donor.We will explore each of these in more detail over the next several pages.

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Collection site security requirements

All collection sites must meet the following security requirements: Must be able to prevent unauthorized access to the site during collection. Ensure that the donor does not have access to items that could be used to adulterate or dilute the specimen (e.g. soap, water, cleaning agents, etc.) Secure faucets, toilet tank tops, and other appropriate areas with tamper-evident tape if necessary. Ensure that the donor is at all times under the supervision of the collector or other collection site personnel. Provide for the secure handling and storage of specimens. (Specimens should be stored at 4-6º C. The refrigerator used should not be readily accessible to the general public and should be used only for the storage of urine drug screens and other clinical specimens. The refrigerator should be marked with a biohazard sign. No food or drink should ever be placed in the refrigerator.)

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Acceptable forms of identification

One of the most important aspects of a urine drug screen collection is the correct identification of the donor. It is the responsibility of the donor to provide the collector appropriate identification upon arrival at the collection site.Acceptable forms of identification include: A photo identification such as a driver's license, an employee badge, or any other picture ID issued by either a federal, state, or local government agency. Identification made by an employer or a representative of the employer. In this latter case, the employer or employer representative can describe the donor to the suitability of the collector via a phone call.

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If there are several donors waiting to have a drug screen and two or more restrooms are available at the collection site; it is acceptable for the collector to process more than one donor at a time.View Page
Collector applies tamper-evidence seals

After dispensing the urine specimen into the specimen vials, the collector, not the donor, removes the tamper-evidence seals from the control form and places them on the specimen vials. Seal "A" goes over the primary vial containing 30 mL; seal "B" goes over the secondary vial containing 15 mL. (When doing a Non-Regulated drug screen, since only one vial would be used, "A" would be the appropriate tamper-evidence seal to use.)The seal must be centered over the lid and down the sides of the vial to ensure that the lid cannot be removed without destroying the seal.

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Donor refuses to complete paperwork

Refusal to Complete Donor Certification on Pink Copy of CCF or Initial Security Strips.If the donor refuses to complete the donor certification on the pink copy of the CCF, or refuses to initial the security strips after they have been affixed to the specimen vials, this is not considered a refusal to test. Do not debate with the donor. It is the responsibility of the collector to note the fact in the "Remarks" section of the CCF. Failure to do so may result in a Fatal Flaw. The MRO may not release the results of the drug screen unless the collector has noted in the "Remarks" section why the donor certification was not completed.Refusal to Provide ID or Social Security NumberIf the donor refuses to provide the collector with an ID or Social Security Number, this is not considered a refusal to test. The collector must make a notation of the fact in the "Remarks" section of the CCF. Failure to do so may result in a Fatal Flaw. After making the notation, the collector continues with the collection.

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Shy bladder

The term "shy bladder" refers to a situation where the donor is unable to provide the sufficient amount of urine required for a drug screen.If the donor indicates upon arrival at the collection site that he or she cannot provide a specimen, the collector should begin the collection process anyway and have the donor make an attempt to provide a specimen. If after an attempt the donor cannot provide a specimen or can only provide a specimen of insufficient volume, the donor must be instructed not to leave the collection site and to do so will be considered a refusal to test. The donor should be monitored either by the collector or by another member of the collection site staff. The donor should be encouraged to drink up to 40 ounces of fluid reasonably distributed over a period of up to three (3) hours, or until the donor can provide a sufficient amount of urine, which ever comes first. If no specimen is provided on the first attempt, the same collection container may be used for the next attempt. The donor may keep possession of the container during the waiting period. The same CCF is used.

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Observed collection scenarios

Scenario 1:A donor was asked to wash her hands prior to picking out the drug screen collection kit. The collector noticed that the donor was washing only one hand.Collector's Response:The collector would tell the donor that washing only one hand was an indication of possible interference with the testing process and that it could be interpreted as a refusal to test.Scenario 2:The donor is asked to remove his hat before going into the restroom. As he reluctantly did so, it was noticed that he was trying to conceal that a container was hidden inside the hat.Collector's Response:The collector must tell the donor that a directly observed collection will be conducted and explain the reason why.

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Responsibilities and requirements for collectors

Under the new Department of Transportation rules for the collection of urine for drug testing, a lot more responsibility has been placed on the collector. It is imperative that you know, understand, and stay current with the rules and regulations. Do the very best you can to make every collection "error free."The examination that follows simply tests your grasp of the concepts of urine collection. It does not qualify you as a Drug Screen Collector!

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Fundamentals of Hemostasis
Laboratory Tests of Hemostatic Function – Prothrombin Time

The prothrombin time is a screening test that helps to assess the functionality of both the extrinsic and common pathways. The effectiveness and presence of factors I, II, V, VII, and X are assayed in this diagnostic test, as they are all found in the aforementioned pathways. The results of the prothrombin time are used in conjunction with other diagnostic tests, as well as the clinical picture of the patient, to determine any hemostatic abnormalities which may be present. In addition to being an integral part of the coagulation disorder assessment process, the PT is also used to determine therapeutic effectiveness of oral anticoagulants, by monitoring drugs such as Warfarin, Coumarin, and Dicoumarol. Prothrombin time test results are reported as the number of seconds needed for a clot to form in the patient specimen using the laboratory's instrument/reagent system, and as the International Normalized Ratio (INR).

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Which of the following statements is incorrect?View Page
Tests of Hemostatic Function - Platelet Function Assay

A platelet function assay (PFA) is a screening test for the evaluation of platelets/primary hemostasis. Common clinical applications include the following: Preoperative evaluation of platelet function Determining the presence of drug-induced platelet dysfunction Determining platelet functionality in high-risk pregnancy Evaluation of patients with suspected inherited or acquired platelet disorders such as von Willebrand disease Evaluation of a bleeding patientA PFA instrument is able to differentiate between drug-induced platelet defects and other platelet defects. PFA tests are superior to the bleeding time test. The bleeding time is often not reproducible and, in spite of attempts at standardization, remains prone to variations in test results between persons performing the test. It is also relatively insensitive to platelet function. The bleeding time cannot be used to identify patients who may have recently ingested aspirin or non-steroidal anti-inflammatory drugs or patients who may have a platelet defect attributable to these drugs. The bleeding time is used to assess platelet function, but may be affected by platelet quantity. NOTE: Aspirin, and some other drugs, may falsely prolong bleeding times. Patients must be asked about aspirin use, and be aspirin free for 7-10 days prior to testing, for valid results.

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Anticoagulation Therapy - Oral Anticoagulant Therapy

The therapeutic use of oral anticoagulants is typically the long-term solution for the patient in terms of managing situations of thrombosis. Warfarin, a dicumarol derivative, is one of the most popular oral anticoagulants used today. While heparin is administered intravenously and acts to inhibit thrombin, warfarin is given orally, taken in pill form, and functions as a Vitamin K antagonist. In earlier discussions, it was mentioned that certain clotting factors are considered to be vitamin K dependant. They require vitamin K molecules for their action to occur. Vitamin K dependant factors include factor II, VII, IX, and X. Vitamin K dependant metabolic processes involved with these coagulation factors are inhibited by drugs such as warfarin. The chemical structure of warfarin and similar anticoagulants enables them to bind competitively with free vitamin K. The prothrombin time (PT/INR) is used to monitor oral anticoagulant therapy.

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HIV Safety for Florida
Mutations

Genetic mutations in HIV are well known and are very likely, considering the presence of two RNA molecules per virus. Either or both RNA molecules can mutate. These mutations potentially lead to drug resistance or encourage the virus to evade the body's immune response. Mutations have created three major groups of HIV - M, N, and O. M is found in 99% of all the HIV cases in the world. N and O are primarily found in West African countries. N, though, infects only a very small number of individuals. The M group has subgroups lettered A to J. Subgroup B predominates in North America.

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HIV: Structure and Replication
Mutations

Genetic mutations in HIV are well known and are very likely, considering the presence of two RNA molecules per virus. Either or both RNA molecules can mutate. These mutations potentially lead to drug resistance or encourage the virus to evade the body's immune response. Mutations have created three major groups of HIV - M, N, and O. M is found in 99% of all the HIV cases in the world. N and O are primarily found in West African countries. N, though, infects only a very small number of individuals. The M group has subgroups lettered A to J. Subgroup B predominates in North America.

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Introduction to Bone Marrow
Ring Sideroblasts

This slide shows a marrow aspiration smear with numerous ring sideroblasts. Normal red cell precursors have only one or at most two granules of iron in their cytoplasm. These abnormal red cell precursors have numerous iron containing granules in their cytoplasm indicating abnormal iron incorporation. This iron is actually incorporated into mitochondria. Ring sideroblasts can be seen in idiopathic sideroblastic anemia, and in sideroblastic anemia induced by drugs, lead poisoning, and alcohol abuse.

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Hypocellular Bone Marrow Biopsy

This biopsy section was taken from a patient who has very few cellular elements in the marrow. Notice that over 90% of the marrow is composed of fat. If all of the cellular elements are decreased, the patient's condition is said to be pancytopenic or aplastic. There are numerous causes for aplasia, including drugs such as chloramphenicol, chemotherapy and inheritance (Fanconi's Anemia).

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Introduction to the ABO Blood Group System
ABO Antibodies and Aging

ABO antibodies are not usually produced by an infant until 3 to 6 months of age. Antibodies found in the sera of newborns are almost always IgG, passively acquired from the mother. Thus, serum testing of newborns is not performed. Anti-A and anti-B titers are highest at ages 5-10 years and then they gradually decrease. Thus, in elderly patients, ABO antibodies may be difficult to detect. In patients with hypogammaglobulinemia, some leukemias, lymphomas or patients who are taking immunosuppressive drugs, the expected antibodies may be weak or even absent, reflecting the low levels of gamma globulin in the patient’s serum. As previously mentioned, these and other ABO typing discrepancies must be resolved before true ABO type can be determined.

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Laws and Rules of the Florida Board of Clinical Laboratory Personnel
Description of Specialties (4)

Specialists in cytogenetics detect chromosome abnormalities and genetic disorders. Cytogenetics counseling may only be performed by an individual licenses in the cytogenetics specialty at the director level. Specialists in molecular genetics analyze DNA and RNA to find disease-related genotypes, mutations, and phenotypes in order to detect or predict disease and identify carriers. Specialists in histocompatibility test to determine tissue compatibility, prevent infections, and investigate and post-transplant problems. Techniques include blood typing, HLA typing, HLA antibody screening, disease markers, flow cytometry, crossmatching, HLA antibody identification, lymphocyte immunophenotyping, immunosuppressive drug assays, allogenic, isogeneic and autologous bone marrow processing and storage, mixed lymphocyte culture, stem cell culture, cell mediated assays, and assays for the presence of cytokines. Specialists in andrology and embryology examine gametes and embryos, including production, morphology, number, and motility, to address issues of fertility and infertility.

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License on probation

An individual whose license that has been put on probation for violating the laws of the Board may be subject to any or all of the following requirements, as assigned by the Board: Practice only under direct supervision of a licensed clinical laboratory personBe reviewed on a quarterly basis by his / her supervisor, with reports submitted to the BoardSubmit a personal quarterly report to the Board describing the individual's progressComplete additional continuing education requirementsConsult a psychiatristNot consume alcohol or use any controlled or illegal substancesAttend AA or NA meetings weeklyUndergo and pay for random drug testingPay an administrative fineFailure to comply with all conditions of the probation will mean that the individual's license will be suspended or revoked.

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Kickback and Inducement Violations

Offering or taking a bribe, kickback, bonus, commission, or inducement is against the rules of the Board and against the law. Many companies give away small promotional items, such as pens or note pads, to promote their products. This is legal, but be cautious about accepting more valuable items. This could be seen as a bribe. All of the following are serious violations of Board, state, and federal rules:Participating in any commissions, bonuses, kickbacks, inducements, or split-fee arrangements from physicians, health care providers, suppliers, hospitals, nursing homes, other clinical laboratories, pharmacies, and other facilities.Exploiting or influencing a patient for financial gain, including promoting, selling, or withholding services, drugs, or referrals.

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Capability Violations

The accuracy and safety of patient testing depends on the capability and honesty of clinical laboratory personnel. If an individual's ability to perform testing is influenced by illness, injury, drug use (legal and illegal), or alcohol use, he or she may no longer practice. The Board can order a doctor's exam to determine if illness, injury, drugs, or alcohol is a factor. The individual can get his / her license back after recovery and proving that the condition is no longer a problem. If an individual commits a crime in any state relating to matters of honesty (such as filing false reports or advertising false services), that individual's Florida license may be suspended. Other licensed personnel who know that an individual is practicing despite being under the influence of drugs or alcohol, is physically or mentally incapable, has been convicted of a lab-related crime, or is not competent to perform his / her duties are required to report the individual to the Board. The following are violations of Board rules:Continuing to practice after becoming unable to safely perform testing because of illness or use of alcohol or drugs, or another mental or physical condition. Continuing to practice after being judged mentally or physically incapable.Being convicted of any crime relating to activities of clinical laboratory science or involving dishonesty or lack of morals. Failing to report to the Board that one has been convicted of a crime (as listed above), been judged mentally or physically incapable, or had a licensed revoked in another state. Knowingly allow an unqualified person to perform clinical laboratory duties.

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Which of the following are violations of Board rules?View Page
If you know that a colleague has a drug problem that affects his / her work, you must report the colleague to the Board, your supervisor or director, or to another authority.View Page

Mycology: Hyaline and Dematiaceous Fungi
The differentiation between Aspergillus species and Scedosporium species may be difficult when only hyphal elements are observed in stained tissue sections. It is important to obtain a culture to make this differentiation when possible because Scedosporium species, in contrast to Aspergillus species, tend to be resistant to:View Page

Mycology: Yeasts and Dimorphic Pathogens
This photomicrograph is an acid-fast stained smear prepared from a yeast colony growing on ascospore agar. A helmet-shaped, red-staining, acid fast yeast cell is seen in the center of view at the tip of the arrow, against the background, blue-staining blastoconidia. The presumptive identification of Hansenula anomala was made. Predisposing conditions that may indicate that this isolate is more than a contaminant include:View Page

OSHA Bloodborne Pathogens
Spread of HBV in the community(1)

HBV is spread in the community through: Sexual contact Drug abusers sharing contaminated needles An infant's exposure to its mother's body fluids

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Pharmacology in the Clinical Lab: Therapeutic Drug Monitoring and Pharmacogenomics
Introduction

Therapeutic drug monitoring and pharmacogenomics are both pharmacy-related areas within the clinical laboratory. Although each is considered a sub-discipline within laboratory medicine, the two fields overlap significantly. In this course, we will provide an overview of each of these laboratory sub-disciplines and discuss the utility, rationale, and practice of each one.

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Basic Pharmacokinetics

In order to discuss TDM and PGx we need to also introduce the concept of pharmacokinetics. Pharmacokinetics is the study of drug disposition in the body: how and when drugs enter the circulation, how long they remain in the blood, and how they are eliminated. TDM is the clinical assessment of a drug's pharmacokinetic properties. Physicians and pharmacists need to establish that a drug is present at an effective concentration but not at a toxic concentration. The next few pages will describe some of the factors that determine a drug's disposition in the body. These factors ultimately decide the need for therapeutic drug monitoring.

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Drug Metabolism

The liver plays a major role in converting lipophilic nonpolar molecules (drug molecules) to more polar, water-soluble forms through a series of enzymatic reactions. Drug molecules can be modified by either phase I or phase ll reactions. Phase I reactions alter chemical structure by oxidation, reduction, or hydrolysis. Phase ll reactions conjugate drugs to create products that are water-soluble.

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Protein Binding

Most drugs are bound to proteins when they circulate in the body. Albumin is a major drug-binding protein in serum. Albumin is an alkaline protein, so acidic and neutral drugs primarily bind to it. If albumin binding sites become saturated, acidic and neutral drugs can bind to lipoproteins. Alkaline drugs tend to bind to globulins, particularly to the globulin, alpha-1 acid glycoprotein. Only free, unbound drugs are able to bind drug receptors and have therapeutic effects. An equilibrium exists in the systemic circulation between a free and protein-bound drug and between a free and receptor-bound drug. This is illustrated in the image to the right.

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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.

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Given what you have learned thus far, which of the following statements below do you think is true?View Page
Steady State

Most drugs are not given as a single dose but are part of a regimen. It is the physician's responsibility to prescribe a drug so that the concentration of that drug reaches a safe and effective level. The dosing-goal for the prescribing clinician, if multiple doses of a drug will be given, is for both the peak and the trough drug levels to be consistently within the therapeutic range. If a drug is given at intervals that are the same as its half-life, it will take about 5 half-lives to reach steady state.

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Why TDM?

Pharmacologists determine a drug's pharmacokinetic characteristics empirically during clinical drug trials. From these studies, they are able to determine the solubility and distribution, the average half-life, the levels of protein binding, and the effective concentrations needed for treatment.

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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.

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TDM for all drugs?

Can all drugs benefit from TDM? Not really. For TDM to be effective and useful, one or more of the following should apply: The effective concentration and toxic concentrations must be well-defined. The pharmacokinetics of the drug are known to be variable. The drug is given chronically. There is the potential for drug-to-drug interactions. The drug exhibits high protein binding. The toxicity will mimic the indication for the drug; toxicity may not be visible during an exam but will only be revealed with TDM. The patient is pregnant, very young, or elderly. Compliance or history with the drug is poor.

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Examples of Drugs That are Monitored by TDM

Four major classes of drugs are frequently monitored by TDM: Antibiotics Anticonvulsants Immunosuppressants Cardiac medicationsThere are other drugs that are monitored by TDM that are not included in any of the above classifications, but the majority of TDM testing is performed for drugs that are included in one of these four categories.

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TDM for Theophylline

Theophylline is used as a bronchodilator for treatment of moderate to severe asthma and chronic obstructive pulmonary disease (COPD). TDM is needed for theophylline because the kinetics of the drug are highly variable. It has a narrow therapeutic window, and overdose can result in elevated heart rate, arrhythmia, and CNS excitability. Clearance of the drug is increased in children, smokers, persons with cystic fibrosis, and persons with hyperthyroidism. Elimination is slowed in congestive heart failure and in the elderly.

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A physician needs to prescribe a drug with a narrow therapeutic window. He is concerned about possible toxic effects. To assess the upper concentration of such a drug, which time for drawing the specimen do you think makes the most sense?View Page
Sampling

Ideally, a drug level would be monitored frequently and consistently, providing the clinician with a detailed pharmacokinetic profile over time. In reality, serum samples are often measured only during relatively infrequent clinic visits, meaning that many days or weeks may pass before a drug concentration 'snap-shot' is taken.

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Albuterol is a fast-acting bronchodilator used acutely during asthma attacks. Which of the reasons below explains why TDM for albuterol is not available or common?View Page
Laboratory Methods

Immunoassay is the most common technique used by clinical laboratories for therapeutic drug monitoring. Antibodies that recognize drugs can be developed. Although most drugs are much too small to evoke an immune response, scientists can conjugate drugs to immunogenic proteins to produce antibodies that recognize drug-specific epitopes. There are several methods that utilize the principals of immunoassay for detection and quantification of therapeutic drugs in serum. Some of these methods are: Particle-enhanced turbidimetric inhibition immunoassay (PETINIA) Fluorescence Polarization Immunoassay (FPIA) Chemiluminescent assays

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Protein Availability and Drug Dosing

Drug-binding proteins in serum can fluctuate in disease states. For example, if albumin levels fall, as can occur in liver failure or nephrotic syndrome, less albumin will be available for drug binding; a subsequent dose may produce a toxic concentration of free drug.The image on the right illustrates the loss of equilibrium between a protein-bound drug and a free drug when drug-binding proteins are diminished.Doses of drugs that are highly protein-bound may need to be adjusted in patients with lower drug-binding protein levels. Examples of some common drugs that are highly protein-bound include thyroxine, warfarin, diazepam, heparin, imipramine and phenytoin. �

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Therapeutic Drug Monitoring Definition

Therapeutic Drug Monitoring (TDM) is a branch of clinical chemistry that specializes in the measurement of medication levels in serum. TDM requires quantitative measurements of drugs and/or their metabolites.

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Drug Concentration Over Time

When a drug enters the body, it reaches a peak concentration that starts to fall as the drug is eliminated. The figure on the right shows a typical kinetic with a drug given intravenously (IV).

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Half-life

The amount of time it takes for a drug's concentration in the body to decrease by 50% is called the drug's half-life (t1/2).The longer a drug's half-life, the slower it is removed from the body. Most drugs are eliminated from the body in 1 to 3 days, but some drugs with longer half-lives can still be detected in the body weeks after the initial dose. The figure below illustrates a typical kinetic pattern for an oral drug.

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Bioavailability

Bioavailability refers to the amount of drug that actually reaches the circulation. It is calculated by comparing (in the same subjects) the area under the serum concentration - time curve (AUC) of an equivalent dose of the intravenous form and oral form. This is illustrated in the diagram on the right.For IV drugs, the bioavailability is 100%For oral medications, the bioavailability will be less than 100%, due in part to any of these reasons:* Oral drugs take longer to enter the circulation.* Oral drugs have slower absorption and distribution than IV drugs.* The amount of drug that is absorbed can depend on the status of the GI tract (stomach pH, presence of food, integrity/health of the intestines, speed of the GI tract, etc.)For oral drugs to be effective, bioavailability typically should be greater than 70%.Not all of a drug taken orally is able to have a pharmacologic effect; the dose would need to be higher than an IV dose.Since the absorption of an oral drug is slower than an IV drug and the drug takes longer to enter the circulation, clearing the drug will also most likely take a longer time.

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Steady State Example

If the drug Gentamicin has an elimination half-life of 12 hours and is given every 12 hours, the drug should reach steady state after 5 half-lives (60 hours). Notice in the diagram that this kind of dosing results in a 'sawtooth' pattern. Peaks correspond to the times right after the drug is taken; troughs correspond to the times right before the next dose.

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Peak and Trough Sampling Times

To assess drug concentrations during the trough phase, blood should be drawn immediately before the next dose. To assess peak levels, the time for drawing depends on the route of administration: Oral: One hour after drug is taken (assumes a half-life of > two hours) IV: 15-30 minutes after injection/infusion Intramuscular (IM): 30 minutes - one hour after injection

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Drug Elimination

Most water-soluble drugs are eliminated from the body through hepatic metabolism. renal filtration, or a combination of the two.An alteration in renal function will have a major effect on the clearance of the drug or its active metabolite(s). Decreased renal function results in elevated serum drug concentrations.

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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.

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Why TDM?

Every drug has a sub-clinical concentration (a concentration at which effective therapy won't be achieved) and a toxic concentration (a concentration at which the drug will be harmful to the patient.)For some drugs, the range between the minimum effective concentration and the toxic concentration is large. These drugs are thus relatively safe. Other drugs have a very narrow therapeutic window and need closer monitoring. This is the role of TDM.Medications with narrow therapeutic windows, like the anticonvulsant carbamazepine (Tegretol), should be closely monitored since elevated doses can cause serious conditions such as agranulocytosis.

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When is TDM Not Useful?

TDM is not useful for these drugs or in these specific situations: Intracelluar drugs that need to be converted to active forms (like AZT) Drugs in which the effects last much longer than the serum concentrations of the drugs; examples include antineoplastics (cancer chemotherapies) and warfarin Narcotic pain medications where continued use can lead to tolerance such that the levels needed for pain relief in one person would be toxic to another person

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Alternative to TDM

Some drugs are more efficiently monitored by determining their effects rather than by measuring the serum drug level. Warfarin dosing, for example, is better monitored by measuring the Prothrombin time (PT) and International Normalized Ratio (INR).

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TDM for Anticonvulsants

Anticonvulsants typically have narrow therapeutic windows. When levels are too low, the risk for seizure remains present. Drug levels that are too high can depress the central nervous system and may even lead to coma. Examples of anticonvulsants that are monitored by TDM include: Carbamazepine (Tegretol) Valproic acid (Depakene) Phenytoin (Dilantin) Phenobarbital Primidone (Mysoline)

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TDM for Immunosuppressants

Drugs used to inhibit the immune system are part of standard treatment after transplant surgeries. Regarding the use of TDM, there are some reports of hepatotoxicity and nephrotoxicity with some agents, but the main reason for TDM is to ensure that concentrations are adequate to suppress the immune response and prevent rejection. Examples of immunosuppressants that are monitored by TDM include: Cyclosporine Methotrexate Tacrolimus FK778

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TDM for Cardiac Medications

Inotropics (drugs used to increase the pumping ability of the heart) and antiarrhythmics may need TDM. The cardiac glycoside inotropics digoxin and digitoxin have narrow therapeutic windows. Overdose can cause vomiting, diarrhea, confusion, visual disturbances, and cardiac arryhthmias. Examples of cardiac medications that are monitored by TDM include: Digoxin Digitoxin Procainamide N-Acetylprocainamide (NAPA) -the metabolite of procainamide Quinidine

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PETINIA

Particle-enhanced turbidimetric inhibition immunoassay (PETINIA) is a homogeneous competitive immunoassay.Antibody fragments and drug-latex particles will bind to form aggregates that increase the turbidity of the solution. Free drug from the sample competes for the antibody fragment, thereby decreasing the rate of particle aggregation. The rate of aggregation is inversely proportional to the concentration of drug in the sample.

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FPIA

Fluoresence polarization immunoassay (FPIA) is also a homogenous competitive immunoassay. In this system, fluorescein-labeled drug competes with unlabeled drug from the patient's serum sample for binding sites on an antibody reagent. The patient's sample, presumably containing the therapeutic drug that is being monitored, and the fluorescein-labeled drug are added to a chamber containing antibody for that drug. The labeled and unlabeled drug will compete for binding sites on the antibody. The greater the amount of drug in the sample, the fewer the number of binding sites that are available for the labeled analyte, leaving a greater number of small, free fluorescein-labeled molecules in the solution.When the chamber is excited with plane polarized light, fluorescein will absorb the light and emit it at a higher wavelength as fluorescent light. A small, free fluorescein-labeled drug rotates randomly and faster than it would if it were bound to antibody, interrupting the light and leading to less emission of light. The larger antibody-drug-fluorescein complexes rotate slower and emit more light in the measured plane. A lower level of drug in the patient's sample results in greater emission of polarized light because there are more antibody-drug-fluorescein complexes present to produce light in the measured plane. A higher level of drug in the patient's sample results in a lower emission of polarized light. This inverse relationship between the concentration of the drug and the polarization units (signal) is illustrated in the image below.

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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.

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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). Pharmacogenomics (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.

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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.

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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.

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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

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Metabolizers

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.

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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.

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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.

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TDM and PGx

Can we use therapeutic drug monitoring (TDM) to assess PGx?TDM of the drug in question can also tell us a good deal about a drug's metabolism and will also take into account all the other variables at play (co-medications, diet, impaired organ function, etc.) However, unlike genotyping and probe-drug testing, therapeutic drug monitoring must be performed during therapy, not before. So, in fact, TDM is not really used to predict therapy in PGx but serves as a confirmation of PGx findings. TDM and genotyping should be considered complementary and can be used in tandem to, first, predict and then verify appropriate serum drug levels.

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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.

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A patient is taking cimetidine for a stomach ulcer. This drug inhibits CYP2D6. The patient is now prescribed amphetamine for narcolepsy. Amphetamine is metabolized by CYP2D6. What would you predict?View Page
A person who is classified as a UM would need __________ of a drug metabolized by that enzyme.View Page
Warfarin Metabolism

The first specific PGx testing application most labs are likely to encounter is that used in patients taking warfarin. Recent studies have revealed that the variations seen in patients taking the anticoagulant warfarin are due to PGx factors. The consequences of incorrect warfarin dosing are obviously serious, with inadequate doses predisposing patients to thrombosis and higher doses placing them at risk for hemorrhage. The United States' Food and Drug Administration (FDA) recently approved updated labeling for Coumadin (warfarin sold by Bristol-Myers Squibb). The new labeling suggests that physicians incorporate PGx information into warfarin-dosing regimens for patients. Manufacturers of generic warfarin products are now adding similar labeling.

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Warfarin cont.

The genes involved in warfarin metabolism are CYP2C9 and vitamin K epoxide reductase complex subunit 1 (VKOR). Warfarin owes its anticoagulant action to its inhibition of VKOR. This enzyme recycles vitamin K, a critical element for the clotting factors II, VII, IX, and X, as well as for proteins C, S, and Z. There are six CYP2C9 alleles that are known to cause prolonged metabolism of warfarin: CYP2C9 *2, *3, *4, *5, *6, and *11. (Polymorphisms in CYP450 genes are denoted with asterisks.)One-third of the patients that receive warfarin metabolize it differently than expected and experience a higher risk of bleeding.Genetic testing for the two most common polymorphisms (CYP2C9*2 and *3) as well as for VKOR may be able to reduce the variability associated with warfarin dosing response. Labs performing PGx testing can provide general warfarin dosing recommendations based on the patient's genotype analysis. The lab report will indicate whether a patient has a normal, mild, moderate, high, or very high sensitivity to warfarin. For example, a patient who has one CYP2C9 normal wild-type allele (CYP2C9 *1), one polymorphism (CYP2C9*3), and also a VKOR polymorphism is predicted to have a moderate sensitivity to warfarin. This patient should have frequent INR monitoring and possible warfarin dose reduction. It is important to recognize that knowing a genotype does not necessarily guarantee accurate dose prediction; other drugs and/or environmental or disease factors can also alter CYP2C9 activity. Therefore, monitoring the INR is still very important.

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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.

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Genotype versus Phenotype

Phenotyping involves measuring the metabolism of a probe drug. For example, with CYP2D6, dextromethorphan or debrisoquine can be given to a patient to see how well the drug is metabolized. Both these drugs are safe and extensively metabolized by CYP2D6. By measuring the parent drug and the metabolite in urine, the metabolic capacity of a CYP450 enzyme can be estimated. Such testing is complex and tedious, however, and has not become routine in clinical laboratories. Therefore, genotyping is likely to be the main tool that is used for assessing the PGx of a patient.

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References

Clinical Chemistry: Theory, Analysis, Correlation, 4th Edition. Lawrence A. Kaplan, Amadeo Pesce, Steven Kazmierczak. New York: Mosby, 2002.FDA Clears Genetic Lab Test for Warfarin Sensitivity. FDA News. U.S. Food and Drug Administration. Available at http://www.fda.gov/bbs/topics/NEWS/2007/NEW01701.html. Accessed June 3, 2008.Goodman & Gilman's The Pharmacological Basis of Therapeutics, 11th Edition. Laurence Brunton, John Lazo, Keith Parker. McGraw-Hill, 2005.Tanaka E, Terada M, Misawa S. Cytochrome P450 2E1: it's clinical and toxicological role. J Clin Pharm Ther. 2000 Jun;25(3):165-75.The Chemistry of Mind-Altering Drugs: History, Pharmacology, and Cultural Context. Daniel Perrine, American Chemical Society Publication, 1996.Tietz Textbook of Clinical Chemistry and Molecular Diagnostics, 4th Edition. Carl A. Burtis and Edward R. Ashwood, eds. Philadelphia: WB Saunders, 2005.

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Phlebotomy
Plasma drugs and toxins

Drugs and toxins including therapeutic drugs and drugs of abuse may be present in the plasma. Other substances too numerous to mention are also present in plasma.

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Plasma components

Plasma is the liquid portion of the blood. It contains many substances including:Water Electrolytes Sugars Proteins Lipids Drugs & Toxins

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Peak and trough levels

A trough level is drawn immediately before the next dose of the drug is administered.A peak level is drawn 1 to several hours after the drug is administered (depending on the drug).

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Collection times

Communication with the patient’s nurse is critical, so that you can collect the trough specimen just before the next dose of drug is administered, and at the correct peak time.Collection times of trough and peak levels are often recorded in a log in the patients room.

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Introduction

Physicians need to know the blood concentration of certain drugs in order to select the best dose for their patients.As a phlebotomist, you might be asked to draw peak (highest), and trough (lowest) levels of various therapeutic drugs.

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Red Cell Disorders: Peripheral Blood Clues to Nonneoplastic Conditions
Cells as shown in this iron-stained bone marrow preparation are found in each of the following conditions except:View Page
G6PD deficiency

A ten-year-old boy came to a physician's attention because of recent jaundice and icteric sclerae. The immediate laboratory work revealed: Hct 24%(normal 36%-47%), MCV 79.5 fl (normal 78-95fl),RDW 13%(normal 11.5-15.0%). His blood smear findings are reflected in these photomicrographs. Note particularly the spherocytes in the upper picture. Some resemble a half-blister with the other half of the cell containing solidly-staining hemoglobin. These are called eccentrocytes. When present, they should trigger a search for red cell hereditary G-6PD deficiency and the oxidant that triggered hemolysis. These morphological findings are only clues; specific testing for G-6PD deficiency should be performed. The blue arrows in the upper photomicrograph are directed toward solid-staining spherocytes in which the cell membrane is beaded by inclusions wrapped within the cell membrane, suggesting the remains of denatured hemoglobin. Included on the smear is a target cell, several acanthocytes, a smudge cell, and a few schistocytes. The lower photomicrograph is supravital staining of affected red blood cells, verifying the presence of Heinz bodies. This disorder was first recognized during the Korean war in 10% of black American soldiers given the antimalarial drug primiquine.

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Heinz body formation

Heinz bodies are 1-3 um particles of denatured hemoglobin settling eccentrically, usually close to the red cell membrane. They are found in erythrocytes in unstable hemoglobin disorders, acute drug induced hemolysis, and following splenectomy. Their formation may be exaggerated by in-vitro incubation of a fresh blood sample with phenylhydrazine. Heinz bodies, as pictured here, are identified using a supra-vital stain, such as new methylene blue or cresyl violet. Bite cells, visible with Wright-Giemsa staining, are visual reminders that the spleen is functional and has pitted the aberrant chunk of hemoglobin from the circulating erythrocyte.

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Schistocytes vs. bite cells

Schistocyte is a general term for a fragmented red blood cell that may assume various shapes, some with horn-like projections (keratocytes), triangle-forms (triangulocytes), and helmet shapes, as illustrated in the upper photograph. Schistocytes are formed when erythrocytes are forced through a vessel blocked with interlacing fibrin strands and the red cells are sliced into fragments. True schistocytes are devoid of central pallor. These damaged cells continue to circulate while healing their torn edges. Finally, they are removed by the spleen. Bite cells (lower photograph) appear when an abnormal hemoglobin aggregate (Heinz body) is nibbled out of a red cell's cytoplasm by the spleen leaving a bitten apple appearance. Glucose 6-PD deficiency secondary to chemical poisoning or injury by oxidant drugs are settings for Heinz body formation, and the telltale bite cells remain as evidence. Hemolytic anemia associated with severe liver disease is another setting where bite cells are formed.

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The Urine Microscopic: Microscopic Analysis of Urine Sediment
Cuboidal Cells

Increased numbers of cuboidal cells are found in renal transplant rejection, acute tubular necrosis (diuretic phase), injuries that interrupt blood flow to the kidney, and acute glomerulonephritis accompanied by tubular damage. Ingestion of various drugs and chemicals may cause significant tubular shedding of these epithelial cells. Cuboidal cells are easily seen in urine in cases of salicylate intoxication.

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Renal Tubular Epithelial Cell

Another type of epithelial cell is the renal tubular epithelial cell. The proximal and distal convoluted tubules are the sites of origin for one form of these cells. They occur singly and are large (14-60 microns). Papancolaou stain is useful in distinguishing renal tubular cells from other mononuclear cells in urine. Increased numbers of proximal and distal convoluted renal epithelial cells are seen in cases of acute tubular necrosis and certain drug or heavy metal intoxication.

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Summary of Abnormal Crystals

The characteristics of the more common types of abnormal crystals are summarized in the table below. Crystal Color Significance Leucine Yellow Metabolism Tyrosine Colorless–yellow Liver disease (rare) Cystine Colorless Cystine metabolism Cholesterol Colorless Renal tubular disease Bilirubin Gold-orange Increased bilirubin High doses of ampicillin, sulfonamide drugs or other drugs may also result in urine crystal formation. It is important to check the patient’s current medications when unusual crystals are found in the urine specimen.

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Tuberculosis Awareness for Healthcare Workers
Fundamentals of TB infection control

TB infection controls include: standardized anti-tuberculosis treatment regimens in the initial phase of therapy; rapid drug susceptibility testing; directly observed therapy in which a health professional watches a patient swallow each dose of medication and records the date that the administration was observed; improved infection control practices.

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Variations in White Cell Morphology - Granulocytes
Conditions Associated with Hypersegmented Neutrophils

There are a number of conditions in which hypersegmented neutrophils may be seen, such as megaloblastic anemias that include folic acid deficiency and pernicious anemia. Individuals who are receiving chemotherapy or have long-term chronic infections may also have hypersegmented neutrophils.The cells seen in these conditions would be classified as pathological since the body is responding abnormally as a result of either a deficiency of a component needed for DNA production or because of the toxic effect that chemotherapy drugs have on DNA.

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Conditions Associated with Hyposegmented Neutrophils

The presence of hyposegmented neutrophils can be an acquired phenomenon, as a result of severe infection, burns, malignancy, chemotherapy or other drugs such as sulfonamides. When the causative agent has been removed, the cells will return to normal. Percentages of neutrophils affected will vary in this condition. Hyposegmented neutrophils as an aquired phenomenon are known as pseudo-Pelger-Huet cells. These are considered pathological.

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May-Hegglin Like Inclusion

Another example of a May-Hegglin-type body. This smear was from a case of pseudo May-Hegglin caused by drugs. Bizarre appearing platelets can also be seen in cases of pseudo May-Hegglin.

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May-Hegglin Bodies

May-Hegglin anomaly is an autosomal dominant condition characterized by the presence of pale blue inclusions in neutrophils, giant platelets, and sometimes thrombocytopenia. Inclusions can also occur in eosinophils, basophils, and monocytes. May-Hegglin inclusions resemble Dohle bodies but are larger and more prominent. Acquired forms of this anomaly may also occur as a result of the use of cytotoxic drugs. May-Hegglin bodies seen under these circumstances are considered pathological.

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White Cell and Platelet Disorders: Peripheral Blood Clues to Nonneoplastic Conditions
Select the letter representing the cell that may be seen in increased numbers in the peripheral blood smear in immediate hypersensitivity reactions:View Page
The upper photograph of this bone marrow section also reveals distinct hyperplasia with total replacement of the fat. The lower photograph is a Wright/Giemsa stain. Calculate the M:E ratio of the distribution of myeloid and erythroid cells in the lower photograph. The peripheral white blood count was 18,500/cumm. The most likely associated condition is:View Page


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