QUICK REVIEW Q & A'S TABLE OF CONTENTS INTRODUCTION. —— ee Seen GENERAL LABORATORY PRACTICE REVIEW CLINICAL CHEMISTRY REVIEW. CLINICAL MICROBIOLOGY REVIEW HEMATOLOGY REVIEW IMMUNOLOGY REVIEW «rss - = W245 IMMUNOHEMATOLOGY REVIEW... : eee 273 URINALYSIS AND BODY FLUIDS REVIEW aoeeee = e329 MANAGEMENT AND EDUCATION REVIEW 7 365 ‘TOPICAL INDEX. a : oo : 383 vilGENERAL LABORATORY PRACTICE REVIEW CREDENTIALING Compare certification and secreditation, Certification and accreditation are both Voluntary non-governmental redentialir for achievement of high standards, Insti futons are accredited. Individuals are certified The Joint Commission on Accreditation of Heath Cate Organization (JCAHO), which accredits the and the College of Am: rican Pthologists (CAP), which acres clnial laborator entire hospital, ries, How often does the College of American Pathologists inspect acredited laboratories? Every to years, CAP inpestions ae pee reviews, Namea non-governmental agency that accredits blood banks, ‘The American Association of Blood Ban ‘What isthe diterence between certification and licensure? Ty process by which a non quirements and demonstrat andar process by which some sates gran pe ‘occupation ‘governmental agency grants reeopnition ted entry-level competency by examina mission to an individual or organization t ‘© an individual who has n, Licensure ig a ‘0 engage in a given Which states require licensure of clinical laboratory personnel? California, Florida, Georgia, Hi Sal Lousiana, Montana, Nevada North Dako, Rhode Island, Te West Virginia, Other sates req mnessee, and ube licensure of clinical laboratories but not laboratory perscnnel, Name several professional organizations tha certify lineal laboratory personnel, The Amerisan Society for Clinical Pathology (ascp), the National Credentialing Agency for Labotatory Personnel (NCA), American Medical Technologists (AMT), and the American Associaton of Bioanalysis (AAB),QUICK REVIEW Q & aS A hospital ad reads “Medical Technologist, [ASCP or equivalent”, Which ofthe following credentials would be equivalent: CLS(NCA), CLT(NCA), MLT(AMT), POLT(SCLT), CPT(NPA)? CLS(NCA), NCA uses the title Clinical Laboratory Scientist instead of Medical Technologist and Clinical Laboratory Technician instead of Medical Laboratory ‘Technician. REGULATORY ISSUES 2 4 9. Which federal agency issues regulations to ensure employes safety? “The Occupational Safety and Health Administration (OSHA), what reporting is required for serious accidents that rest in fatalities oF hospitalization of three or more employees? ‘They must be reported 10 OSHA within 8 hours ‘Which federal agency regulates ‘blood banks? “The Food and Drug Administration (FDA), The FDA has authority over blood banks besause blood is considere= oth a biologic and a drug. Which agency regulates market entry of instruments and reagents? “The Food and Drug Administration (FDA). Which federal agency regulates facilities that use radioisotopes? “The Nuclear Regulatory Commission (NRC). Which federal agency regulates packaging, labeling, and transportation of biological products? ‘The Department of Transportation (DOT). Which federal agency regulates disposal of toxic chemical and biohazardous wastes? “The Environmental Protection Agency (EPA). State or local regulations may apply as well Which federal agency issues mandatory guidelines for laboratories performing forensic toxicology? “The Substance Abuse and Mental Health Services Administration (SAMHSA), formerly known as the Natios= Institute on Drug Abuse (NIDA). What isthe federal legstation that regulates almost all einieal Jaboratories in the U.S.? “The Clinical Laboratory Improvement Amendments of 1988 (CLIA 88), It establishes regulations Gualifcations, competency assessment, patent est management 8 control, quality assurance, testing.GENERAL LABORATORY PRACTICE REVIEW 10. Which laboratories are subject to regulation under CLIA "88? All laboratories that examine human specimens for diagnosis, prevention, or treatment of disease, There are exemptions for the Veterans Administration, the armed forces, and the Substance Abuse and Mental Health Services Administration, Work done for legal or employment eligibility purposes and research not used for patient care are also exempt. 11, What are the levels of testing complexity under CLIA ‘88? Waived, provider-performed microscopy, moderately complex, and highly complex. 12, Laboratories of which complexity level are required to perform quality control and participate in proficiency testing? All except waived. 13, What is the complexity level of most of the analyses performed in clinical laboratories? Approximately 75% ate classified as moderately complex. 14, What is CLSI? ‘The Clinical and Laboratory Standards Institute, an organization that issues standards of laboratory practice developed through a voluntary consensus process. It is not a regulatory agency. CLSI was formerly known as the National Committee for Clinical Laboratory Standards (NCCLS). 15, Whats the role of the Centers for Disease Control and Prevention (CDC)? Itissues standards and guidelines for hospitals and laboratories, primarily related to infection control and safe work practices. Its role is advisory; itis not a regulatory agency and does not have enforcement authority LAB SAFETY 1 Which bloodborne pathogens are of greatest concern to laboratorians? 4 Hepatitis B (HBV) and Human Immunodeficiency Virus (HIV). Hepatitis C (HCV) does not appear to be transmitted efficiently through occupational exposure to blood. ‘Which bloodborne pathogen ean be transmitted by contact with environmental surfaces? HBV. It has been demonstrated to survive in dried blood on environmental surfaces for at least 1 week. Which clinical specimens are potentially infectious? Whole blood, plasma, serum, pus and purulent fluids, semen, vaginal secretions,cerebrospinal fluid, synovial fluid, pleural fluid, peritoneal fluid, pericardial fluid, amniotic uid, and breast milk, ‘What are OPIM? (Other potentially infectious materials (besides blood), such as pus and purulent fluids, semen, vaginal secretions, cerebrospinal fluid, synovial fluid, pleural fluid peritoneal fluid, pericardial fluid, amniotic fluid, and breast milk5. 1 10. 12. 13. QUICK REVIEW Q & A'S Which specimens are usually not infectious, unless visibly bloody? Urine, feces, sputum, nasal secretions, sweat, tears, and vomitus. What are the routes of entry for occupationally-acquired bloodborne pathogens? Percutaneous exposure or contamination of non-intact skin or mucous membranes (eyes, nose, mouth). ‘What is the most common route of exposure to bloodborne pathogens for healthcare workers? Percutaneous, by contaminated needles. What is the risk of HIV transmission after # percutaneous exposure to HIV-infected blood? Approximately 0.3%, The risk increases with exposure to larger quantities of blood. The risk after a mu membrane exposure is estimated to be approximately 0.09%, An MLT student sticks himself with a dirty needle while performing phlebotomy on a combative pz ‘What should he do? Follow the laboratory's exposure control plan. A typical course of action would be to purge the puncturs =<" slight pressure, then immediately and thoroughly wash the wound with soap and water, apply 70% alcc=: * supervisor, and complete an incident report What follow-up should be provi Jed for an employee following an occupational exposure? HIV, HBV, and HCV testing of the source patient (after consent is obtained), appropriate clinical and evaluation of the employee, and post-exposure prophylaxis (PEP), if indicated, According to the CDC, which profession has the largest number of documented cases of occupatic acquired HIV? ‘Nurses. Clinical laboratory technicians are second. Fifty-seven health care workers have been docume! contracted HIV from occupational exposure and another 139 cases are considered possible cases of oct exposure, No new documented cases of occupationally-acquired HIV/AIDS have been reported since D=::~°= 2001 Explain the concept of Universal Precautions. All specimens should be handled as if they are infectious. The rationale is that a patient can be infected "~~ symptoms or laboratory evidence of infection. Altemative concepts in infection control are called Body $ Isolation (BSI) and Standard Precautions. These latter terms define all body fluids and substances as in: health care workers must routinely use appropriate barrier precautions to prevent skin and mucous me ‘exposure when contact with blood or other body fluids is anticipated When should gloves be worn? When performing phlebotomy and when handling blood or other potentially infectious materials, bioh: waste, or contaminated equipment. Gloves should never be washed and re-used,M4 15, 16. 1. 18. 19. 20, a. n, 28 u GENERAL LABORATORY PRACTICE REVIEW ‘A.new employee develops an itchy rash from wearing latex gloves. What should she do? Notify her supervisor. She probably has a latex allergy and should be provided with non-latex gloves. ‘When should face shields be worn? Whenever spraying, splashing, or spatter of blood or body Mus is possible. Face shields protect the mucous membranes ofthe eyes, nose, and mouth ‘What s the proper way to dispose of sharps? {In a sharps container, Needles should not be recapped or broken. Syringes should be discarded with needles atached. ‘What are the design requirements for sharps containers? ‘They must be puncture resistant, closable, leakproof, and either color coded or labeled with a biohazard label When should hands be washed? ‘After contact with blood or body fluids, between patients after removing gloves, and before leaving the la. Handwashing isthe single most effective method to prevent the spread of infection. [Name the disinfectant that should be used to clean up spills of blood or body fluids. A freshly prepared 10% solution of sodium hypochlorite (household bleach). ‘What is biomedical waste? ‘Any solid or liquid waste that may present a threat of infection, It should be disposed of in a biohazard container. How often does OSHA require employee safety training? Upon hiring and annually thereafter Under OSHA regulations, who must provide and launder employee lab coats? ‘The employer, at no expense tothe employee, Employees should not take ab coats home for laundering because of the risk of taking infectious agents into the home Under OSHA regulations, which immunization must the employer offer to the employee free of charge? Hepatitis B. Ifthe employee refuses, a signed declination must be placed in his her personnel file. The employee ‘maintains the right to request the vaccine inthe future, There currently is no vaceine for HIV or hepatitis C. ‘What isthe Needlestick Safety and Prevention Act? ‘A measure that mandates that employers who are subject othe provisions of the Bloodborne Pathogens Standard put safer needles into the hands oftheir employees. The act was passed in 1999 and became effective on April 18, 2001 Safer needles include retractable eles ani meddle With Bin safety shields.28. 26. 21. 28. 29. 30. 31 32, 33, 34, 38, QUICK REVIEW Q & A'S How can laboratories reduce the risk of injury due to bre age of glass? By using plastic evacuated tubes and discontinuing the use of glass capillary tubes. What is the Hazard Communication Standard? ‘An OSHA standard that requires employers to inform employees about hazardous substances in the workp: educate and train them in safe and proper handling, The standard is commonly known as the “Right to Kno — What is a chemical hygiene plan? A plan to minimize personnel exposures to hazardous chemicals. I is required under OSHA's Occupationa’ Exposures to Hazardous Chemicals in Laboratories, also known as the “Laboratory Standard”, ‘A new employee spills a chemical in the chemistry lab. He is uncertain if the chemical is hazardous or #:* 0 handle the spill. The supervisor is attending a meeting in another building. What should the employee ¢: Consult the material safety data sheet (MSDS) for the chemical, This will give information about the chemi: hazardous properties, first-aid procedures, and information on spill and disposal procedures, MSDS are ob from the manufacturer and must be readily available to employees at all times. What types of chemicals may pose physical hazards to the laboratory environment? Flammables, combustibles, oxidizers, reactives, and explosives. What is a flammable? A chemical with a flashpoint below 100° F., The flashpoint isthe lowest temperature that produces suflicie=: =» 8 form an ignitable mixture atthe surface of a liquid. A heat source, such as an electrical spark or open flame present to ignite the vapor. Examples of flammables are acetone, xylene, ethanol, methanol, and isopropan: What is a combustible? ‘Acchemical with a flashpoint between 100° F and 200° F. What is an.oxidizer? A cheinical that promotes combustion in other materials, for example, nitric acid and sulfuric acid. Oxidize=: - use not be stored near organic acids What is a reactive? ‘A chemical that is unstable when subjected to heat, shock, or pressure, Sulfuric acid is an example of a ree: ‘Name several explosives that may be found in a clinical lab. Pieric acid, sodium azide, and compressed gases, Picric acid becomes shock-sensitive when dehydrated Powerful explosive than TNT. Name several categories of chemicals that pose health hazards. Initants, corrosives, sensitizers, toxic agents, and carcinogens.—_— GENERAL LABORATORY PRACTICE REVIEW 36. Whatis an irritant? ‘chemical that causes reversible inflammation of tissue, for example, formaldehyde 37, What isa corrosive? { 4 ‘A chemical with a pH <2 or > 12. These chemicals cause ireversible damage to tissue upon contact and may also produce injury upon inkalation, Examples are sufi ci, hydrochloric acid, and sodium hydroxide 1 38. Atwhat level should corrasives be stored? Below eye level. There is less chance thatthe botles willbe dropped and broken, For the same reason, sock battles cof concentrated aids shouldbe transporte in bote carers. 38, What sa sensitizer? ‘Acerca o which a person may develop an allergy aftr repeated exposure, such as bleach or formaldehyde, 40. Whatisa toric agent? ‘Achemicl that an cause serious effets ifevena small amounts inhaled, ingested, or contacted, 41, Whatisa carcinogen? ‘Acheital tat ean cause cancer, for example, bene, potassium crore and formaldehyde 42, Whatis a mutagen? A chemical that induces chromosomal changes 43 What isa teratogen? A cheanical hat induces congenital malfommatins | 44. AnMLT is asked to organize the laboratory's chemical storage room. She begins to arrange chemicals in alphabetical order so that they wll be easy to locate and inventory, Why is this nota satisfactory sytem? | Stcould result in an unsafe situation in which chemicals that reac are stored next o each other. Chemials should be ‘organized according to reatvity, Inorganic acids should be separated ftom onganic acids and oxidizers should be separated fom reducing agents, 43, What are threshold limit values (TLVs) and where can they be found? ‘The maximum amount of axe sbstanceto which a person can be exposed without adverse effec. This information i ‘can be found in the MSDS. 46, Name chemical used in histology that requires environmental monitoring, i Formaldehyde. OSHA's Fomaldehyde Slandard requires monitoring of formaldehyde exposure, engineering Il contol, personnel protective equipment, taining, and an emergency ation plan47, 48, 49. 50. 51. 32. 53, 54; 56. QUICK REVIEW Q & A'S ‘Name several ignitable chemicals commonly used in # clinical laboratory. Xylene, ether, acetone, and alcohols, {A jar of a chemical is labeled with the following National Fire Protection Association’s (NFPA) label: red diamond 3, blue diamond 1, yellow diamond 0, Explain how to interpret this label ‘The NFPA Hazard Idemtfication System uses four diamonds to communicate information about the hazards associated with a chemical. The red diamond represenis its fie hazard, the blue diamond its health hazard, the yell> diamond its reactivity, and the white diamond communicates special hazard information, for example, ifthe chem:: isa corrosive or oxidizer. Numbers from 0-4 in each diamond indicate the degree of hazard, (0 = minimal, 1 2= moderate, 3 = serious, 4 = severe.) In the example given, the chemical isa serious fire hazard (flashpoint below 100° F) and a slight health hazard, Work should be performed in fume hood and personal protective equipment should be worn, How should concentrated acids be diluted? ‘The acid should be slowly added to the water. “Always Add Acid” ‘What labeling is required for a 24-hour urine container that contains an acid preservative? The name of the chemical and any precautions the patient should take. For example, ifthe container contains 10% HCL, the label should state thatthe urine should be collected in another container and poured into the 24-hour collection container slowly to avoid splashing of the acid preservative. When should a fume hood be used? When working with flammable, toxic, or corrosive chemicals. Flammables should not be stored in a fume hood an.= should not be cluttered with items that could block the vents How often must fume hoods and biological safety hoods be inspected? ‘Annually. ‘When working with a flammable, what should be kept at a safe distance? Sources of heat and electrical equipment that could ignite the flammable. Where should flammables be stored? Ina flammable safety cabinet or can. How should ether be stored? Inan explosion proof reigerator. These refrigerators have all wiring and switches on the outside to eliminate the risk ofa spark igniting flammables, Most laboratories have changed to procedures that do not use ether, in order eliminate the risk and expense. ‘What is the only type of chemical waste that should be flushed down the drain? ‘Water soluble chemicals. They should be followed by copious amounts of water. Strong acids and bases should be neutralized before disposal3. 60. a. 8 sng commpaile classes, and poked wp by am EPAclvenstd Same bacteria aent that forms expose ss many etal and poses a risk of explosion iit builds up in drain pipes. Sou acide. is someines used es baste agen cherixal reagents. metic ils buildup in dein pies, even tise of wrench may rest ina vie exposion. The we of zs shoul be avoided or minimized a oft exlosve potential end beast eye EBSD fon shoul be taken when a corrosive chemical comes in contact with skin or eyes? ‘The site shouldbe fished with copious mounts frunning water frat east 15 minutes. Ifeomacts extensive, @ safety shower shouldbe used. How often should eyewashes be checked? “Te American Natl Sana Insite (ANSD ous at eyevases be checked weekly They mus ler 1S int steam of eid wae and must alow the ands tobe fie to hold the eyelids open for thorough flushing of theeye. How should gas cylinders be stored and transported? “hy shold aay be hai rie sere a censored sing aba ca. Valve protein caps shoud refuting anspor ascyindrs ae presuize I ey mocked over, the ylinder valve can be broken and the force ofthe escaping gas can propel the eylnder throug the sir like a missile Which type of ire extinguisher shoud be used fo eatngish resin comptes or operating etic ‘equipment? CO; (BC) and dry chemical (ABC) fire ‘extinguishers can both be used but CO: extinguishers have the edvantage of rot leaving behind a residue that might cause damage tothe equipment Which ype offre extinguer shouldbe se for fires involving Nammable liquids? Dry chemical (ABC) or carbon divide (BC) Which typeof fire shuld not be eninge with pressurized water? lectin fir’ or baring liquids What procedures shouldbe flowed when a fires discovered? © RACE: + Rescve patients and personnel Activate lam. ‘+ Conlin freby closing dors and windows. + Bningish ire ifposible, or evacuate.QUICK REVIEW Q& A'S 66. Describe the correct way to use a fie extingulsher, PASS + Pullthe pin, # Aimar the base of ire + Squeeze the handle + Sweep beck and forth | ‘SPECIMEN COLLECTION AND PROCESSING 1, What isthe difference between plasma and serum? Plasma contin fibrinogen and serum does no. Fibrinogen is converted t fibrin during coagulation. When plasm = required, blood is collected with an anticoagulant. 2, Name the anticoagulant in tubes with the following colored stoppers and explain the use of each~ green, grey lavender, light blue, STOPPER ANTICOAGULANT USE. | Green Sodium or lithium heparin Plasma determinations in Chemistry Gray Potassium oxalatefsodium | Glucose fluoride { Lavender K:EDTA | Hematology. Cen also be used for ABO, Rh, and antibody serening. Light blue Sodium ciate Coagulation i 3, What are pink top evacuated tubes? K,EDTA nes with a special crossmatch label for information required bythe American Association of Blood Banks (ABB). They can be used fr blood bank testing (ABO, Rh, and antibody screens) and for whole blood hematology detenminations, Although theadtive is the same a ina lavender top tube, hematology laboataris = not accept pink top tubes if the sizes not accepted by the automated analyzer. 4, When are royal blu top tubes used? For trace-element, toxicology, and nutritional chemistry determination, The tubes contain only low levels of trac: elements, ‘When are tan top tubes used? For lead determinations. The tube is certified to contain less than 0.01 pein. of lead 6 What s the purpose of the ge in serum separation tubes? ‘cforms a physical barier between the serum or plasma end the red blood cells during cetifugation. a10. , 13. GENERAL LABORATORY PRACTICE REVIEW ‘What additive can be used for stat serum determinations? ‘Thrombin, Clotting usually curs in less than S minutes. Thrombin tubes ae identified by orange or gray/yellow stoppers ‘Which type of serum tube is recommended for blood banking? Glass tubes without additives or gel (red top), Plastic tubes are not recommended for blood banking because they contain a clot activator, In what order should evacuated tubes be drawn? ‘The National Coramite for Clinical Laboratory Standards (NCCLS) issued new guidelines in 2004 that apy regardless of the type of tube being filed (glass or plastic) and regardless of whether the specimen is dravm using evacuated tubes or syringe. The new, simplified order of daw is: Sterile tubes 1. Sterile rubs for blood cultures fi 2. Sodium citrate tube (light blue top) light bic top 3 Serum tube (with or without lot activator or gel—re ed/lack, or god top) 4, Heparin tube (green top) 5. EDTA tube (lavender top) 6. OxalateMuoride tube (gray top) This oder prevents eroneous resus due to additive cary-over. Tubes with clot ectvatos are citi additive lubes and shoul be collected afer coagulation tubes since elt-actvator carry-over ean affet coagulation results, [NAACLS noted that glass nonaditve serum tubes canbe drawn before te citrate tube but, for simplicity, it grouped all serum tes together. How many times should evacuated blood collection tubes be inverted atthe time of collection? Most additive tubes should be inverted 8 times. The exceptions ar sodium citrate tubes (light blue top) which should be inverted 3-4 times and plastic serum tubes (with clot activators) which shouldbe inverted 5 times, Glas serum tubes do not require mixing since they contain no adtive, How do anticoagulants prevent coagulation? EDTA, citrate, and oxalate bind Ce™ Sit snot available for the coagulation cascade. Heparin inactivates thrombin, How does EDTA prevent coagulation? Itchelates Ca”. Chelation isthe combining of metalic ions with certain heterocyclic ring structures so that the ion is held by chemical bonds from each paicipating rng, When this structure is diagramed it appears thatthe metalic ion is being held by a claw. ‘Why is EDTA the preferred anticoagulant for differentials? Itprevents platelet clumping and causes minimal morphological changes in cells14, 15. 16, 17. 19, 20. 21. QUICK REVIEW Q& A'S ‘Why is sodium fluoride used when collecting blood for glucose determi ns? Sodium fluoride prevents glycolysis. Either sodium fluoride or lithium iodoacetate should be used when glucose analysis will not be performed for more than an hour after collection, Sodium fluoride preserves glucose for 24 hours al room temperature and 48 hours at 4°C. Why should sodium fluoride not be used when collecting blood for enzymes or for assays that use enzymes as reagents (ex: BUN by urease or uric acid by uricase)? Sodium Nuc is an “enzyme poison” that inhibits enzymatic reactions. A prothrombin time is ordered on a patient with polycythemia vera, What should be done prior to drawing hls blood? When the hematocrit is greater than 55%, the volume of sodium citrate should be reduced, otherwise over- anticoagulation will artfactually lengthen the PT. What is the proper ratio of sodium citrate to blood for accurate coagulation results? 1:9. Over-anticoagulation due to a short draw will artifactually lengthen the coagulation times. Why should blood not be drawn from above an IV site? The IV fluid may contaminate the specimen, increasing the concentration ofthe analytes contained in the IV fluid and decreasing the concentration of other analytes by dilution. Whenever possible, either the other arm should be used or, ifa large quantity of blood is not needed, a fingerstick could be performed, If neither of these options is possible, the blood should be drawn distal to the IV, with a tourniquet between the IV and the venipuncture site. If it is impossible to use a site distal to the IV, the IV should be shut off for at least 2 minutes and the first S mL. of blood discarded. The contents of the IV solution should be documented. What procedure should be followed when blood is collected from an indwelling line or heparin lock? The first 5 mL of blood should be discarded to avoid heparin contamination, Laboratory personnel usually do not draw from lines. ‘What is the most important step in collecting a blood specimen? Proper patient identification. Inpatients should only be identified by the identification bracelet, with special attention to two identifiers, usually name and identification number. Room number should never be used as an identifier. Ifa patient is not wearing an identification bracelet, the responsible nurse should be notified. Outpatients should be asked to state their name and provide some other identifying information such as social security number or address. The phlebotomist should not ask, “Are you Mrs, Jones?” Ifthe patient has difficulty hearing, she might respond affirmatively to someone else's name, leading to an improperly identified specimen. ‘When should blood collection tubes be labeled? Labels should always be applied at the bedside, never in advance, A 3-way check should always be performed, comparing patient identification information on the requisition, the patient's armband, and the tubes of blood 12n B. uM 25, 26. 2. 28. 29, 30, a GENERAL LABORATORY PRACTICE REVIEW ‘What are the advantages of the new systems that allow the phlebotomist to scan the patient's armband and generate labels at the bedside? Positive patient and specimen identification. Since multiple collection labels are not taken into the patient's room, there is no chance of accidentally applying a label with another patient's name. Why is important to follow the laboratory’s policy for placement of labels on blood collection tubes? Automated analyzers cannot read labels that are not properly placed ‘What type of gloves should phlebotomists wear? Because ofthe risk of hypersensitivity to latex proteins, phlebotomists should use only non-latex gloves for patient contact. ‘Which of the following needles has the largest bore: 18, 20,21) or 23 gauge? ‘The lower the number, the larger the bore, The most common needle size used for adult venipuncture is 21 gauge. ‘Why has OSHA prohibited the reuse of evacuated tube holders (adapters)? Because removal of the double-ended needle poses a risk to the employee, Also, studies showed that a high percentage of tube holders were contaminated with blood even after one use. Which phlebotomy products are associated with the highest rate of accidental needlesticks? Winged infusion sets (butterflies) and syringes. Recent design modifications have improved the safety of these devices. ‘When are winged blood collection sets (butterflies) used? For patients with small or fragile veins or those who are unable to move tothe arm-down position, Butterflies are expensive and have been associated with a higher rate of accidental needlesticks than the evacuated tube system. ‘They should be used judiously Why should the wings of the butterfly be held during insertion? For greater control and to ensure thatthe safety shield doesn’t slide over the needle when the needle makes contact with the skin, After insertion, the wings should never be touched again ‘Why shoulda’t the butterfly be withdrawn ig the wings? The safety shield cannot be pushed over the needle when the wings are being held. When using a blood collection set to collect omly a sodium citrate tube for PT and/or APTT, what additional step is required? A plain glass discard tube should be drawn first to purge the air from the tubing and allow a full draw into the citrate tube. 332. 33. 34. 35. 36. a. 38. 39 40. QUICK REVIEW Q & A'S Why i t best to purchase all venipuncture equipment from the same manufacturer? To ensure proper fit between components so that needles do not unscrew and tubes do not pop off. How do plastic serum tubes differ from glass serum tubes? Since plastic does not activate clotting like glass does, plastic tubes for serum testing (red top) must contain a cl- activator. If even a minute amount of the blood/ctot activator mixture carries over to the blue top tube, the physic = could adjust a patient's anticoagulant therapy based on erroneous results; therefore the plastic red top tube shou notification of critical values, result reporting, test interpretation by physician, follow-up patient care 24i, 3 2 14. GENERAL LABORATORY PRACTICE REVIEW In which phase of testing do most errors occur? In the preanalytical phase Define analytical sensitivity. The ability to detect small amounts of the analyte Define analytical specificity, ‘The ability to detect only the substance being measured, Freedom from false positives. Define accuracy. Closeness ofthe result to the trie value. Define precision. Reproducibility According to CAP, how often must quality control statistics be calculated? At least monthly. ‘What is the name of a sample which is chemically and physically simllar to the unknown and which is tested in the same manner as the unknown'@s monitor the precision of the test system? A conto How often should\controls be run for quantitative tests? A lest 2 levels of conta shouldbe run at ast once each dy of patient testing How often should controls be run for qualitative tests? A positive and negative contro shouldbe included with each run of patient specimens. What i the definition of an analytical run? The interval within which the accuracy and precision of the measuring system is expected to be stable, based upon manufacturer's recommendations and not to exceed 24 hours, Who should run controls? Personnel who routinely perform patient testing When should results of controls be verified for acceptability? Before patient results are reported. 2515. 16. A 18, 19. 20. ne. 2. 2. 24, 25, QUICK REVIEW Q & A’S How does one determine the mean of a series of test results? ‘The sum of the results divided by the number of tests performed, What is the minimum number of analyses needed to caleulate standard deviation? Twenty. In a normal Gaussi distribution, what percentage of the population falls between + 2 SD? 95.45%, ‘What is the coefficient of variation for a procedure whose mean is 100 mg/dL and whose standard deviatioz = 3 mg/dL? 3%, (CV = SD / mean x 100) ° What are the upper and lower limits of acceptable results for a control if the mean is 100 mg/dL and stands deviation is 3 mg/dL (95% confidence limits)? 94-106 mg/dL. (The mean +2 SD.) What is an outlier? A control value that falls outside of ¢ 2 SD. An outlier is due to random error. Random error is usually a one-tir: event that does not affect all results, for example, inaccurate pipetting of one sample. An outlier does not alway’ ‘mean that the procedure is out of control. How often is a control value expected to fall outside of 2 SD, due to normal variation? One time in twenty runs, ‘What is random error? —e ‘A foon-recirring er¥or due to factors such as diny glassware use ofthe wrong pipette, voltage fluctuation, or sampling érror. What is systematic error? Recurring error that affects all results. A dirty photometer, a faulty ISE, or evaporation or contamination of or reagents Would cause systematic error. Which type of error is inherent to some extent in all analyses? Random. ‘What is a trend? Control values increasing or decreasing for six consecutive runs. A trend is duc toa slowly developing syst error, for example, a feiling photocell or a deteriorating reagent or standard 2626. 1 28. 29. 30. 31 2. 3, GENERAL LABORATORY PRACTICE REVIEW What isa shift? Six consecutive control values on the same side ofthe mean. This isthe result ofa systematic eror. Improper calibration of an instrument or a change in lot numbers of reagents could lead toa shift in control values. Is it possible for a procedure to be out of control if all control values fall within the acceptable range? Yes, fa shift or trend is present. What isa false rejection? ‘A false rejection is when the analyst concludes that he procedure is out of control, when in fact the results were secure Explain the Westgard multirules. ‘Westgard multrules are applied across controls as well as across runs. RULE EXPLANATION 13s __ | One control exceeds the mean by more than 2 SD, but less than 3 SD. sg __| One control exceeds the mean by more than 3 SD. + ‘Two consecutive controls exceed the mean by more than 2 SD, but less than 3 SD, in the same direction. Res 4s, 10, __| Ten consecutive controls fall on one side ofthe’ mean, Which Westgard rule is a warning flag and not always a signal for rejection? J. The remaining rules must be tested to determine if here isa problem, ‘Which Westgard rule violations point to random error? Ig and Ras ‘Which Westgard rule violations point to systematic error? 2k 5, and 10, What is the advantage of using Westgard multirules? Westgard multirues ae used to reduce the number of false rejections and to facilitate the evaluation of two or three levels of control atthe same time. Computers aid inthe interpretation of these rules. 24, 35. 36. 37. 38. 39. QUICK REVIEW Q& A'S What steps are taken when a control value falls outside of the acceptable range? The laboratory's protocol should be followed. Following are typical steps. After each step is taken, the routine C pools are analyzed. If the results are within limits, the problem has been resolved. Ifthe results are still not acceptable, the next step is taken, 1, Do not release patient test results 2. Repeat the control. 3. Runa newly reconstituted control. Controls can be mishandled, resulting in changed analyte concentration: because of deterioration or evaporation, 4. Look for obvious problems--clots, reagent levels, mechanical problems. 5. Recalibrate the instrument forthe out-of-control analyte, then reassay all controls 6, Install a new bottle or new lot number for one or all of the reagents, recalibrate, and reassay all ofthe cont 7. Perform periodic maintenance, recalibrate, and reassay all ofthe controls 8. Ifcontrols are still ou, notify supervisor. 9. Document problem, investigation, and resolution in Out-of-Control Log Book. 10. Once the problem is resolved, rerun the patient specimens. 11, Patient test results obtained since the last acceptable test run should be re-evaluated to determing significant clinical difference in results, if there is = What is a primary standard? A standard prepared by weighing a highly purified chemical and dissolving itin a diluent tothe desired concentration. ‘What is a secondary standard? ‘A standard whose concentration is determined by a réference method using a primary standard for ‘What is the minimum number of standards required for construction of a standard curve? Three, Define calibration, Calibration refers to the operations that establish the relationship between instrument sesponse and the concer of afanalyte. Calibration is required when there isa change of reagents, when controls ae out of range, after instrurgent maintenance or service, when calibration verification fails, and when recommended by the manufe:~— Define calibration verification. Calibration verification is the process of confirming thatthe system will accurately measure the enalyte aver analytical measurement range. Materials with a matrix closely resembling the patient test samples and with k values are analyzed in the same manner as patient specimens. At least 3 levels must be tested~minimum, mic and maximum values for the analytical measurement range. 284 2. 4 44, 45, 46. a. GENERAL LABORATORY PRACTICE REVIEW ‘When should calibration verification be performed? © When QC fails to meet established eriteria + When ot numbers of reagents change + After major maintenance or service «When recommended by the manufecturer «Atleast every 6 months ‘What is matrix effect? ‘The matrix is all ofthe components ofa sample other than the analyte. The preparation of calibrators, quality control materials, and proficiency-testing samples may involve processes such as freezing or lyophilization or the additicn of substances such as preservatives that cause them to react differently than fresh patient specimens. Ths is known as matrix effect ‘What is linear range? Itis the range of analyte values that a method can directly measure on the specimen without any dilution, concentration, or other pretrealment not part ofthe usual assay process. It is also known as analytical range, analytical measurement range (AMR), or reportable range. The range must be revalidated at least every 6 months and following changes in ot numbers of reagents or major system components ‘What must be done when the patient value exceeds the linearity of the analyzer? The specimen must be diluted and run again. The answer obtained must be multiplied by the reciprocal ofthe dilution used. For example, 2 1:2 dilution would be tltiplied by 2. Meny analyzers are capable of doing this automatically What is autoverification? AA process by which the computer performs the initial verification of test results. Any data that fall outside of set parameters should be reviewed by the human operator. The autoverification process should be tested atleast annually and whenever there isa change tothe system. ‘What isa delta check? ‘A comparison of patient’ test results wit hisher previous results. When the change exceeds a predetermined limit, the cause must be investigated to rule out laboratory error. How are reference ranges established? ‘One hundred to 150 data points are gathered from a representative healthy client population and arranged in sequential order. The reference range is the values between the 2.5% position atthe low end and the 97.5% position at the high end. Since this represents the 95% confidence limit, one normal person in 20 will fall outside the reference range. Each laboratory should establish its own reference ranges or verify the use of published data, Reference ranges used to be called normal values. When should reference ranges be reevaluated? ‘When there i a change in methodology or patient population. 2948. 49, $0, 51. 32. 33, 54. 56. a QUICK REVIEW U6 AD ‘What must be done when a critical value is obtained on a specimen? ‘The physician or other responsible healthcare professional must be notified immediately. Ifthe results are delivered verbally or by phone, the receiver should read back the values to ensure accuracy. Critical value notification may be by computer or fax but delivery must be confirmed by telephone call. No read back is required. Critical value notification must be documented, This documentation should include the date, time, responsible laboratory individual, person notified and test results. The laboratory should document any failure of attempts to notify the appropriate person of critical results. What is turnaround time? The interval berween specimen receipt by laboratory personnel and results reporting, The laboratory must have defined tumsround times for each ofits tests and a policy for notifying the requester when testing i delayed, In general, 48 hours for routine tests and 1 hour for sats s usually considered acceptable, although there may be some exceptions. ‘When more than one instrument is used for a particular test, the lnboratory must verify that patient results produced are comparable. How often must this be done? i ‘Atleast twice a year. Fresh samples should be used rater than commercial controls, which maybe subject toa mat effect. What isa correlation study? A study to verify the accuracy of a new method, A minimum of 40 spit patient samples are analyzed by the existce ‘method and the new method. Values obtained by the existing method are plotted on the x-axis and those obtained t the new method on the y-axis. Perfect correlation is indicated by a straight line passing through the zero point at 8 45° angle. What is the correlation coefficient (r)? ‘A mathematically derived value that shows the degree of correlation between 2 methods, Values range from = 1 ‘+1, with +1 indicating a perfect direct relationship between the methods. A value of 0.95 or higher is considered excellent correlation How long should serum and body fluids be retained? At least 24 hours, except for blood bank specimens which should be retained for 7 days post-transfusion or 10 é: post-crossmatch How should erroneous laboratory reports be corrected? A corrected report should be generated and communicated to the physician as soon as possible. The original in result should be flagged as an error and remain in the medical record, Errors should not be deleted from the m== record What are proficiency tests? ‘Samples sent from an outside agency (ex: College of American Pathologists) that are tested as unknowns by & Taboratory. Results obtained are compared to those of other participating laboratories and reference labs 3056. 3. GENERAL LABORATORY PRACTICE REVIEW How should proficiency test samples be handled? Exactly like patient samples. They should not receive preferential treatment For exemple, a pathologist consult should nt be requested if one would not be requested for similar results ona patient specimen nor should the test be done by a special analyst unless that person routinely tess patient samples, Laboratories should not compere results with othe laboratories prior to submitting their sults to the proficiency testing agency. ‘When is replicate testing of proficiency samples allowed? (Only when patient specimens are routinely analyzed inthe same manner. ‘What should laboratory personnel do if they have concerns about a quality or safety issue? They should fist communicate their eoncems to ther supervisor or other laboratory manager. Ifthe concems are not addressed by laboratory management, they should communicate wit hospital administration. If the concems are still not addressed, they should communicate with the eppopriate accrediting egency or regulatory agency.QUICK REVIEW Q & A's 32Ss CLINICAL CHEMISTRY REVIEW % CARBOHYDRATES 1 Define glycolysis, elycogeness,elyeogenolyss and gluconeogenesis. «+ Glycoysis= Conversion af glucose to pyruvic acid rl ecid 1+ Glycogenesis = Production of glycogen from glucose + Glyeogenalysis = Production of glucose fem glyeogen 4 Gluconeogenesis = Production of glucose fom noncarbohy drat sources 2, Whatisthe end product of anaeroble glycolysis? Lactic acid 4, Which ug accounts fr ney al lod sugar andi the Body's major sure of cellular energy? : Glucose 1 4. Name$ hormone that regulate glucose level and tel wheter each ralses or lowers thelevel ee 2 Epinepin Insulin lowers glucose. Glucagon, cose, epinephrine and growth hormone all increse it, with gheagmn being for the greatest effet. 5, Whatisthe reference range fora fasting glucose in an adult? 70=110malal. 6 Compare the normal blood glucose level for newborns and adults -themean blood gcse eel for neonates is 38 mpl. Guests lower inthe newborn because ofthe small -giyeopen reserve inthe iver 1. Ho do ghsoe evel fern venous an capa specie? Following ingestion of ses gucos ves reali highrin copay bod In fasting specie, values are the same. 310. n 13. rr QUICK REVIEW Q & A'S What are critical values for glucose? Each laboratory establishes its own critical levels, but < 40 mg/dL and > 600.mg/dL are often used. When ac value is obtained and verified, the physician or other appropriate healthcare professional must be notified immediately so tat therapy can be initiate. What is glucosuri Glucose in the urine. Glycosuria is @ more general term, referring to the presence of any sugar in the urine Define renal threshold, The blood concentration of a substance that can be reabsorbed by the renal tubules. For glucose, the average r=~ threshold is 160-180 mg/dL. When the blood level exceeds this amount, glucose is excreted inthe urine. What is hyperglycemia? High blood suger. tis most often due to diabetes mellitus Compare Type 1 and Type 2 diabetes mellitus. TYPE | CAUSE, | CHARACTERISTICS Type! Pancreatic beta cell destruction Onset usually below age 40 Formerly known as juvenile | Absence of insulin Dependency on injected insuli= ‘onset diabetes.or insulin Autoimmune. Antibodies to insulin & | Prone to ketoacidosis and diabs: | dependent diabetes melfius | ise cells | complications (IDDM) Genetic predisposition (HLA DR.3 4) | Type 2 ‘Underproduction of insulin or insutin | Most common type Formerly known as adultonsei | resistance Onset usually after age 40 diabetes or non-insulin Associated with obesity Not dependent on exogenous i dependent diabetes mellitus Not prone to ketoacidosis (N'DDM) Which testis recommended by the American ion for screening for diabetes, except du pregnancy? ‘A fasting plasma glucose. What are the criteria of the American Diabetes Association for the diagnosis of diabetes mellitus? Any of the following on 2 occasions: a random plasma glucose 2 200 mg/dl, fasting plasma glucose 2 126 ‘or one oral glucose tolerance test (OGTT) value 2 200 mg/dL. The values were recently lowered so that diagne could be made earlier, It is thought that earlier treatment will reduce the risk of diabetic complications ‘What patient preparation is required for an oral glucose tolerance test (OGTT)? ‘An overnight fast. A fasting specimen is drawn and the patient is given a glucose oad (75 grams for an adult, 1~ grams per kg of body weight fora child), The 3-hour OGTT is falling out of favor because of its poor reproduc and the inconvenience to the patient, New guidelines from the American Diabetes Association recommend a and 2-hour specimen only, except during pregnancy. 34QUICK REVIEW Q& A'S. serum-separata tubes, following centrifugation, the gel forms @barier between the cells andthe serum, preventing alycolyss. 28 Whats glycated hemoglobin?) Hemoglobin A with glucose attached tothe beta chains (hemoglobin A). tis comprised of hemoglobin Ay Any and Are, Hemoglobin A isthe largest fraction, Some procedures measure tolal glycated hemoglobin; others measure | only hemoglobin Ay. Other names used are glycohemoglobin, glycosyated hemoglobin, an fest hemoglobin but the term “glycated hemoglobin” is prefered. Because of the confusing nomenclature, the term “Alc test” has been suggested Tits Textbook of Clinical Chemistry and Molecular Diograstics, 2096) Glycated hemoglobin is a useful indicator of fong-trm glucose contol 24, What specimen is required for glycated hemoglobin (hemoglobin A,.)? Whole blood, Fasting is not required. 25, What are the most common methods for glycated hemoglobin (hemoglobin Ay) in the U.S.? 12003 virtually all eboratris that participated in @ CAP survey used immunoassay or ion-exchange chromatography. Tiet Textbook of Clinical Chemistry and Molecular Diegnostics, 2006) 26, Whats the lineal signfeance ofan elevated glyated hemoglobin (hemoglobin A.) D> Iinctes por gucose conil ovr the past 68 weeks. Valuef2 12 ae pia of poor diabetic cont ‘Acordng tothe Ameccan Diabetes Assocaion’s (ADA) Clnkal Practice Recommendations of 2006, the hemoglobin A, ol for patent in general is than 7%, The ADA recommends Ale testing a east every 6 ‘months in patents who have stable lye corto. More fequent testing may be indicated in certain cnicl situations 27. Whyis the Ate test invalid in a patient with hemoglobin S or C? I Because of the shortened lifespan ofthe RBCS. | 28, A patient has a FBS of 10 mg/dL and a glycated hemoglobin (hemoglobin Ay) of 12%, What do these results indicate? Good short-term control, but poor long-term control. Fructosamine is glycated protein that can be used to determine glycemic contol over the past 2-3 weeks. The assay can be automated and is more precise and less expensive than glycated hemoglobin but there is curently no consensus on ils clinical value 30, What is microalbuminuria? Excretion of urinary albumin at a rate of 20-200 minute or 30-300 mg/24 hr. These low levels are below the sensitivity of routine urine screening methods, Mieroalbuinuriai highly predictive of diabetic nephropathy in type 1 : disbets. Early detection and tight plyeemie contol retard progression to nephropathy, Detection is by an | jmmunochemical measurement of te albumin excretion rate on 8 24-hour urine specimen using antibodies to human albumin 3618, 19. 20, 2, CLINICAL CHEMISTRY REVIEW ‘What is the screening test for'gestational diabetes? ‘The woman is given a $0-gram load of glucose at 24-28 weeks of gestation. The time of the last meal is not important. A 1-hour plasma glucose 2 140 mg/dl is abnormal and should be followed by an OGTT. Some » with gestational diabetes develop type 2 diabetes years later. How does the oral glucose tolerance test differ for a pregnant woman? ‘The standard testis the 100-gram OGTT pérformed after a 8-14 hour fast. A fasting, 1 hour, 2 hour, and 3 specimen are drawn, Gestational diabetes is diagnosed by two or more of the following venous plasma glucc values: fasting 2 95.mp/AL, | hour 2 180 mg/dL. 2 hour 2 155 mg/dL, and 3 hour 2 140 mg/dL. (Tietz Text: Clinical Chemistry and Molecular Diagnostics, 2006) ‘What should be done when # patient scheduled for a OGTT has a fasting glucose of 150 mg/dL? ‘The physician or pathologist should be consulted, Since the patient's glucose is already elevated, he/she shc be given a glucose load, If confirmed on another occasion, a fasting glucose of 150 mg/dL. is sufficient for = diagnosis of diabetes mellitus, An oral glucose tolerance test isnot required. [Name two enzymatic methods for glucose determinations. Glucose oxidase and hexdkinase. Glucose oxidase catalyzes the conversion of glucose to hydrogen pero Bluconic acid. The second step of the reaction is @ peroxidase reaction, which is much less specific than oxidase reaction, Hexokinase catalyzes the phosphorylation of glucose to glucose-6-phosphate, which is th: ‘oxidized by G-6-PD in the presence of NADP". The hexokinase method is more specific than the ghucose reaction. A 60-year-old diabetic has a blood glucose of 210 mg/dL. His urine glucose is negative, Assuming t! substances in his urine to inhibit the glucose reaction, what might account for the apparent diserepsr these test results? Diabetics may develop elevated renal thresholds, That is why urine testing is not a good screening test for = mellitus. — AA diabetic patient who performs home monitoring of his glucose is ordered by his physician to be te: local hospital laboratory. The patient is suspicious of the quality of care he receives from his HMO. immediately before going to the lab to have his blood drawn, he tests it himself, His result is 128 mg = laboratory result is 150 mg/dL. Assurning that both tests were performed correctly and that controls were within the ac: range, what might account for the discrepaney in values? ‘Home testing uses capillary whole blood; laboratory methods use venous plasma or serum. Whole blood : - 10-15% lower than plasma glucose, In addition, different methodologies re used. A specimen for a fasting glucose is drawn in a tube without anticoagulant at SAM in a nursing hor placed in a rack for pickup by the courier. The sample is picked up at 9 AM, delivered to the lab 2 and analyzed at 11:30 AM. Will the results be adversely affectéd by the handling of the specimen’ ‘Yes. Serum stiould be removed from cells within I hour of collection to prevent a decrease in glucose ¢ glycolysis. A preservative such as sodium fhuoride or lithium iodoacetate should be used when testing = delayed. Other options are to centrifuge the blood and remove the serum or to use serum-separator tbe: 3531 32. 3 Me 35, 36. 37. UPIDS ‘CLINICAL CHEMISTRY REVIEW ‘What are the most common causes of hypoglycemia? Inappropriate insulin production, insulin injection or ingestion of oral hypoglycemic agents Why isthe $ hour oral glucose tolerance test no longer recommended for the diagnosis of hypoglycemia? Because at east 10% of healthy individuals have glucose levels below 50 mg/dl. during this procedure Hypoglycemia is recognized by the presence of Whipple's trad plesma glucose less than 40 mg/dL, symptoms of hypoglycemia nervousness, anxiety, neurologic abnormalities), and relief of symptoms by administration of glucose. What happens when glucose levels drop below 20-30 mg/dL? Central nervous system dysfunction. ‘When are ketones present in the blood? ‘Whenever thee is impaired carbohydrate metabolism with breakdown of fatty acid, such as uncontrolled diabetes relitus, starvation, vomiting, or low carbohydrate diet. The Ketones are acetone, acetoacetic acid (diactic acid), and beta-hydroxybutyric acid (bela-hydroxybutyate) High levels lead to metabolic acidosis (ketoacidosis). ‘What reagent is used to detect ketones in blood and urine? \ Sodium nitroprusside, Actes tablets or Kets are frequently used, They are insensitive to beta-hyeroxybutyate, therfore, a negative nitroprusside est doesnot rule out ketoacidosis ‘What is the purpose ofthe lactose tolerance test? It aids inthe diagnosis of lactase deficiency. Lactase isthe enzyme that cleaves lactose into glucose and galactose. Following ingestion of mi or mil produc, affected individuals experience cramps and diarthea a lactose inthe tine i metabolized by bacteria. Inthe lctose tolerance test, glucose is measured inthe blood following oral administration of lactose. An increase in glucose of less than 20 mg/L. indicates that lactose was not broken down, and absorbed. Ithas been showa tat the most reliable method of determining lactose absorption isthe measurement ofthe amount of hydrogen in exheled breath after the ora administration of lactose. Levels ae above normal with lactase deficiency because hydrogen is one of he by-products of bacterial metabolism of lactose, The definitive diagnosis of lactase deficiency is made by tissue enzyme asstys on biopsies ofthe intestinal mucosa ‘What isthe purpose ofthe D-xylose absorption test? 1 itferentiates malabsorption of intestinal origin from malabsorpton:due to pancreatic insufficiency. D-xylose is pentose suger that is absorbed inthe small intestine without the action of pancreatic enzymes. Following oral atinisatin of D-xyose, blood or urine (olleted over a5 hour prio) is obained. Low levels of _ylose are suggestive ofan absorptive defect inthe jejunum. Describe the solubility properties of lipids. Lipids are insoluble in water and soluble inorganic solvent, Inblod, lipids are soluble due to their combination with protein (ipoproteins) 373. 1 10. nn 2 QUICK REVIEW Q & A'S What is the main form of lipid storage in the body? Triglycerides in adipose tissue [Name two substances that play a role in the digestion of lipids, Lipase and bile salts. Why is it recommended that a patient be seated for $ minutes prior to drawing blood for cholesterol? Values are higher when standing What is the major site of cholesterol synthesis? The liver. Which lipoprotein transports most cholesterol? Low density lipoprotein (LDL). List the risk factors for coronary heart disease (CHD) Smoking, hypertension, physical inactivity, obesity, type 2 diabetes, high total cholesterol and LDL-cholesterol, low HDL-cholesterol, male sex, family history of CHD. ‘What is the Natioual Cholesterol Education Project's (NCEP) recommended desirable level for total cholesterol for adults? fro eo Less than 200 mg/dl., Higher levels are a risk factor for coronary heart disease (CHD). Borderline high is 200- 239 mg/dL; high is 240 mg/dL and above. What specimen is required for total cholesterol? Serum or EDTA plasma. When EDTA is used, plasma should be cooled o 2-4°C immediately. Cholesterol ‘measurements made on EDTA plasma require correction by te facto of 1.03. Samples do not have tobe fasting unless triglycerides or LDL cholesterol will also be measured on the same specimen, What is the most common method for cholesterol? Enzymatic using cholesterol esterase and cholesterol oxidase. oe ‘What is the relationship between HDL cholesterol (HDL-C) and risk of coronary artery disease? Inverse-the higher the HDL cholesterol, the lower the risk. HDL-C less than 40 mg/dL is a major risk factor for CHD. Values over 60 mg/al. are protective. How is HDL cholesterol measured? Direct methods ae replacing the older methods that required pretreatment to precipitate LDL cholesterol and V cholesterol 383B. 14 15. 16. 18. 1». 20 a. ‘CLINICAL CHEMISTRY REVIEW ‘Whats the desirable level for HDL cholesterol? 240 md. ‘Which lipid i the major component of atherosclerotic plaques in arteries? LDL-cholesterol, Reduction of LDL-cholesterol has become the main gol of cholestero-lowering therapy. What isthe desirable level for LDL cholesterol (LDL-C)? wo Elevated LDL-C is recognized asa major cause of CHD. The Adult Treatment Pane! JH (ATPIII, issued in 2001, identifies the optimal level of LDL-C as less than 100 mg/d. It further identifies bordertne high s 130-159 mg/dL, high as 160-189 mg/L, and very high as 190 meld. or above. How is LDL cholesterol determined? can be calculated by the Fredewald formu: LDL. cholesterol = Total cholesterol HDL cholesterol - (Triglycerides), Triglycerides divided by 5 isan estimate of VLDL cholesterol. The formule isnot valid if triglycerides are greater than 400 mL. The disadvantage tothe calculated LDL. cholestrol is that itis dependent onthe accuracy of 3 other determinations, Several methods for dcect measurement of LDL cholesterol are now available and are recommended for improved precision and accuracy, ‘Calculate the LDL cholesterol and asses the patient's risk for coronary artery disease ifthe total cholesterol 1s 240 mg/dL, the HDL cholesterol is 64 mg/dL, and the triglycerides are 310 mg/dl. 1d maid ade re Both the total cholesterol andthe LDL cholesterol are above the desirable level but the HDL cholesterol is in he esiable level, Other rsk factors such as age, sex, family history, high blood pressure, diabetes mellitus, and smoking shouldbe taken nto account to assess the patiem’s overall risk, would also be important to ascertain if the specimen was fasting. Te elevated triglycerides could be due toa recent meal Discuss the structure oftrighcerides, ‘Triglyceride is made of one molecule of glycerol and three molecules of fatty aid What are exogenous trighyerides? ‘Triglycerides from the det. They are caried bythe chylomicrons. Endogenous triglycerides are synthesized inthe body. ‘What i usually measured in trigheeride assays? Glycerol, following cleavage of any acids by lipases and proteases What isthe desirable level for triglycerides? Triglycer follows sare now recognized as an independent risk actor for CHD. ATP Il clessifies serum triglycerides as 392, 2B. 24, 25. 26. 27. 28, 29, 30, QUICK REVIEW Q & A'S Normal Less than 150 mg/dL Borderline High 150-199 mg/dL. High 200-499 mg/dL Very High 500 mg/d L or higher What are chylomicrons? ‘The largest of the lipoproteins. They transport exogenous triglycerides and account forthe turbidity of serum following a meal Name the four classes of lipoproteins and the major lipid carried by each. BY ULTRACENTRIFUGATION MAJOR LIPID Chylomicrons Triglycerides (exogenous) Very Low Density Lipoproteins (VLDL) Triglycerides (endogenous) Low Density Lipoproteins (LDL) Cholesterol High Density Lipoproteins (HDL) Phospholipids ‘What tests should be Included in lipoprotein panel? Total cholesterol, LDL cholesterol, HDL cholesterol, and triglycerides, All adults over 20 years of age should be tested every 5 years, What specimen is required for a lipid profile? Serum collected after a 10-12 hour fast. Discuss patient preparation for a lipoprotein profile. ‘The patient should be in metabolic steady state and should maintain usual diet and weight for 2 weeks prior to collection. The patient should not exercise vigorously for 24 hours before the blood is drawn. The patient shoulé == fasting and should remain seated for $ minutes before being drawn, A non-fasting specimen is received for a lipoprotein panel, Which results, is any, will be useful? Only total cholesterol and HDL cholesterol. If the total cholesterol is 200 mg/dL or higher or the HDL choleste: Jess than 40 mg/dL, the tests should be repeated on a fasting specimen ‘What is the significance of turbid or lipemic serum? Iti indicative of increased chylomicrons and/or VLDL, both of which contain large amounts of triglycerides. ‘What causes a "cream layer" on top of plasma after ove refrigeration? High levels of triglycerides or chylomicrons. ‘What might lead you to suspect that a specimen was non-fasting? Lipemia or a chylomicron band on lipoprotein electrophoresis. 4031. ‘CLINICAL CHEMISTRY REVI ‘specimen fora lipid profile had elevated triglycerides but normal total cholesterel. What isthe most likely cause? ‘Anon-fastng specimen, Recent ingestion of fas wil elevate triglycerides but nt foal cholesterol. The tess should be repeated ona fasting specimen, PROTEINS What isthe most concentrated solute in the blood? Protein, The reference range is 6-8 gid. Most othe chemistry analytes are measured in maldL. ‘Which element is found in protein but not in carbohydrates oF ipks? [Nitrogen The protein molecule i 16% nitrogen ‘Where are most proteins synthesized? Inte five. ‘What is the wast product of protein catabolism? ‘res. I isencretd inthe urine. ‘What causes denaturation of protein? Heat pH, enzymes, and UV light all an disrupt the molecular structure of poten, es ing in loss of function. Why should repeated freeing and thawing ofa specimen be avoided? Repeated freezing and thawing may cause proteins to deteriorate What isthe most common method for measuring serum total protein? ‘iG ouret mehdCupousions eat with peptide bonds at an alain po rode colored comple. ‘Why Ite Daret method of protein analysis not sultable for urine or CSF protein? Tes ot sensitive enough othe small amount of protein inthe urne Trichloaesic acid can be used to precipitate ‘protein in rine and CSF. How would hemolysis affect the serum total protein level? ‘Hemoglobin inthe serum would neease the protein evel ‘What does low total protein use the patent? Edema, Water leaves the blood vessels and goes into the tissues. 41un. 13, 14 15, 16. 1. 18. 1. 20. QUICK REVIEW Q & A'S. ‘What is the main physiological function of albumin? 1 maintains the osmotic pressure and the distribution of water in the body. When albumin is low, edema results. Albumin also has an important role in binding and transporting various substances in the blood such as bilirubin and some drugs. What are the most common dyes used for the direct analysis of albumin? Bromeresol green (BCG) end bromeresol purple (BCP) ‘What happens to total protein and albumin levels in the nephrotic syndrome? ‘They decrease due to loss through the urine. How is the concentration of globulins determined in a chemistry profile? By subtracting albumin from total protein, ‘What is the normal A:G ratio? 1-1.8. A reversed A:G ratio is seen with monoclonal gammopathies and other increases in the globulin fraction Calculate the A:G ratio for a patient whose total protein is 9.2 g/dL and whose albumin is 4.2 g/dL.. 0.84, Globulins are determined by subtracting albumin from total protein (9.24.2 = 5.0). AG ratio is determined by dividing albumin by globulin (4.2 / 5.0 = 0.84). The normal ratio is 1-18. This patient has a reversed ratio, indicating increased globulins, possibly due to a gammopathy. What is amphoterism? | ‘The property of proteins to assume a positive, negative, or neutral charge, depending on the pH of the medium, With serum protein electrophoresis at pH 8.6, proteins carry a negative charge, Jn the body, what charge do most proteins carry? At pH 7.4 proteins are negatively charged (anions) Explain the principle of serum protein electrophoresis (SPE). When an electric current is applied to @ medium containing charged particles, the particles will migrate to the pole of the opposite charge. Negatively charged particles (anions) will migrate toward the positively charged pole (anode) and positively charged particles (cations) will migrate toward the negatively charged pole (cathode). The rate of. migration’ depends on the net charge of the particle, the size and shape of the molecule, the strength of the electric field, the properties of the support medium, and the temperature. ‘What support media are used for serum protein electrophoresis? Cellulose acetate or agarose are used to separate proteins on the basis of molecular charge. Starch and polyacrylamide gel can be used to separate proteins based on both size and charge. 42a 2, 2. 24, 25, 26, 2, 28. 30, 31. 32, CLINICAL CHEMISTRY REVIEW ‘What is electroendosmosis? ‘The flow of buffer toward the cathode due to the negative surface charge present on the support medium. On cellulose acetate, this results in gamma globulins being swept toward the cathode. Describe a normal serum protein electrophoretic pattern on cellulose acetate at pH 8.6. At pH 86, proteins are negatively charged and migrate toward the anode in the following order: albumin, alpha-1 globulin, alpha-2 globulin, beta globulin, and gamma globulin, What are the normal percentages of the five protein fractions? Albumin 53-65%, alpha-1 globulin 2.55%, alpha-2 globulin 713%, beta globulin 814%, and gamma globulin 12~ 22%, What is the significance of a sixth band migrating between beta and gamma on serum protein electrophoresis? Tt means thatthe specimen was plasma, not serum, The extra band is due to fibrinogen. ‘What stains are used in serum protein electrophoresis? ‘Amido black and Ponceau S were popular in the past, Coomassie brilliant blue (CBB) is more widely used today because itis more sensitive. ‘What is the name of the instrument used to quantitate protein fractions following serum protein, electrophoresis? A densitometer. Which protein fraction normally accounts for over half of the serum total protein? Albumin, In electrophoresis of serum at pH 8.6, which fraction is the fastest moving? Albumin ‘What eauses increased albumin? Dehydration What causes decreased alpha-I globulin? Alpha-I antitrypsin deficiency. This is seen with emphysema. ‘What causes a decreased gamma globulin fraction? Hypogammaglobulinemia Describe the acute phase reactant or inflammatory pattern, Allpha-1 and alpha-2 globul re increased. 4333. 34 38. 36. 3. 38, 39. 40. AL a 4B. QUICK REVIEW Q & A'S Describe the chronic response pattern. Albumin is decreased and alpha-1, alpha-2, and gamma globulins are increased Describe beta-gamma bridging. There is no valley between the beta and gamme globulin regions on SPE because of increased IgA. Beta-gamma bridging is seen with cirrhosis, Describe a polyclonal gammopathy. A diffuse increase in the gamma region Describe the electrophoretic pattern seen in the nephrotic syndrome, ‘Albumin is decreased and alpha-2 is increased. Wha 2 monoclonal gammopathy? AA sharp peak in the gamma region due to an increase in one immunoglobulin. It is known as an “M spike” because of its association with malignancy, such as multiple myeloma and Waldenstrom's macroglobulinemia. The name should not be misinterpreted to mean that the increased immunoglobulin is IgM. ‘What causes the hyperproteinemia seen in multiple myeloma? {An increase in one of the immunoglobulins, most often IgG Which immunoglobulin is increased in Waldenstrom's macroglobulinemia? IgM. This very high molecular weight protein causes the blood to have a high viscosity. A patient has a monaclonal gammopathy on serum protein electrophoresis. What test can be done to determine which immunoglobulin Is increased? Immunofixation has replaced immunoelectrophoresis as the method for identifying immunoglobulins. Explain the immunofixation procedure, Specimens are placed on separate tracks in an agarose gel or on cellulose acetate strips. The proteins are separated by electrophoresis. One track is treated with a fixative that fixes all proteins, creating a reference pattem. The other tracks are treated with monospecific antisera to IgG, IgM, IgA and kappa and lambda. Unprecipitated proteins are ‘washed away and the remaining proteins are stained. The locations of the stained proteins are compared to the reference pattern, How are immunoglobulins quantitated? Immunoglobulins are most often measured by immunoturbigimetry or immunonephelometry. ‘What must be done to the specimen prior to performing CSF or urine electrophoresis? Urine and CSF must be concentrated because of their low protein content. 444. 45, 46. 47. 48. 49. 50, CLINICAL CHEMISTRY REVIEW ‘Which band is normal urine electrophoresis? ‘Albumin, It isthe smallest protein, A small amount crosses the glomerular membrane and is excreted in the urine [A spike is observed in the gamma region on urine electrophoresis. What Is present? Bence Jones proteins. Bence Jones proteins are free immunaglobulin light chains (kappa or lambda) which are present in serum and/or urine of patients with muliple myeloma ‘What isthe name of the test that detects oligoclonal CSF bands associated with multiple sclerosis? High resolution electrophoresis. This technique allows separation of additonal proteins not usually detectable by serum protein electrophoresis. With polyacrylamide as the support, over 100 serum protein bands may be seen. [Name a band that fs seen in norma) CSF electrophoresis but not in normal serum protein electrophoresis. Prealbumin. It migrates ahead of albumin and accounts for approximately 4% of protein inthe CSF. ‘What is the clinical significance of prealbumin? Its an indicator of nutritional starus and can be used to assess the adequacy of a nutritional feeding plan. Low levels are seen with protein malnutrition. This protein was originally named prealbumin because it migrates ahead of, albumin on high resolution electrophoresis. I has been renamed transthyretin What are cryoglobulins and when are they present? Gamma plobulins that precipitate in the cold. They may be present with multiple myeloma, Waldenstrom’s macropiobulinemia, eukemia, systemic lupus erythematosus, sheumatoid arthritis, and polyeytheria. What are happens ifthe specimen for eryoglobulins is refrigerated? ‘The eryoglobulins precipitate out ofthe serum. A temperature of 37°C must be maintained during collection, processing, and storage What is ceruloplasmin? ‘The primary copper-containing protein in plasma, Levels are usually deceased in Wilgqn’s disease, gn inherited disorder of copper metabolism in which copper is deposited inthe skin, comeas, liver, and brain, Levels increase in inflammation and malignancy beceuse ceruloplasmin is an acute-phase reactant. ENZYMES AND CARDIAC MARKERS What isan enzyme? An organic catalyst All enzymes are proteins, The substance upon which they act is called the substrate. How are enzymes named by the International Union of Biochemistry (TUB)? systematic name is assigned, based on the substrate and the reaction catalyzed. For example, the systematic name for CK is ATP:creatine N-phophotransferase. Each enzyme is also assigned a numeric EC (Enzyme Commission) code consisting of 4 mumbers separated by periods. The fisst number assigns the enzyme to 1 of 6 categories of 485 6 10. UL. QUICK REVIEW Q.& A'S reaction-oxidoreductase, transferase, hydrolase, lyase, isomerase, or ligase, The second number denotes the subclass, based on the type of group that takes place in the reaction. The third number indicates the subsubclass of reaction, ‘ofien based on the acceptor group. The last number is the serial number of the particular enzyme. For example, the EC code for CK is 2.7.3.2. Most laboratories refer to enzymes by their trivial names What is a coenzyme? An organic cofactor required for an enzymatic reaction, Unlike the enzyme, which is not changed in the reaction, the coenzyme is changed. Common coenzymes are NAD (nicotinamide adenine dinucleotide), NADH, NADP, end NADPH, How is the change from NAD to NADH measured in automated enzyme analyses? By the change in absorbance at 340 nm. NADH absorbs light at 340 nm; NAD does not. ‘What are activators? Inorganic cofactors such as Ca” or Mg” that are required for some enzyme reactions. ‘What are isoenzymes? Slightly different forms of an enzyme that catalyze the same reaction. Because oftheir different molecular structure, isoenzymes can be separated by their electrophoretic mobility, heat stability or reaction with specific antibodies. How are enzymes measured? Based on their activity on the substrate, Measurements may be based on the decrease in the concentration of the substrate, the increase in the concentration of the end-product, the decrease inthe concentration ofthe coenzyme, or the increase in the concentration of the changed coenzyme. Define international unit (U). ‘The amount of enzyme that converts one micromole of substrate per minute under specified conditions. In the U.S,, what is the most common temperature used for enzyme determinations? The International Federation of Clinical Chemistry (1FCC) recommends 30°C, but 37°C is commonly used in the ULS. A tolerance of + 0.1°C is recommended, For many enzymes, an increase of 1°C produces @ 10% change in enzyme activity, Above 40°C, most enzymes are rapidly denatured and lose activity. What are zero-order kinetics? ‘When the rate of an enzyme reaction is dependent on the concentration of the enzyme only. This occurs when there is an excess of substrate. In first-order kinetics, the rate of the eaction is proportional to the substrate concentration, Enzyme assays should be based on zero-order kinetics, What specimen is required for most enzyme determinations? Serum is usually used because anticoagulants interfere with some assays. 4616. 1. 19. 20. 21 CLINICAL CHEMISTRY REVIEW ‘What is the clinical significance of an elevated lipase? Lipase is increased in acute pancreatitis. may aso be significantly increased in many other conditions (ex: opiate administration; pancreatic carcinoma; intestinal infarction, obstruction, or perforation; and pancreatic trauma). a ‘What is the substrate for lipase? Triglycerides. In turbidimetric assays, the rate of clearing of turbidity is related to the amount of lipase in the serum, What is the clinical significance of an elevated amylase? Amylase is increased in acute pancreatitis. It may also be significantly increased in many other conditions, such as opiate administration; pancreatic carcinoma; intestinal infarction, obstruction, or perforation; pancreatic trauma; ‘mumps; cholecystitis; hepatitis; citrhosis; ruptured ectopic pregnancy; and macroamylasemia, ‘What is the substrate for amylase? Starch, Itis broken down to sugars. Why shouldn’t EDTA plasma be used for an amylase determinatio Ca” is needed for te reaction and EDTA chelates C2~ How do amylase and lipase values compare in acute pancreatitis? The rise of both occurs at roughly the same time (within 5-8 hours of symptoms for amylase and within 4-8 hours for lipase). Amylase returns to normal by te third or fourth day whereas lipase doesn’t decrease for 8-14 days, (Older Iieratre described amytase as te earlier marker but new lipase assays with improved sensitivity have eliminated that advantage. Some now consider lipase the superior test because ofits higher specificity and longer elevation.) ‘Which enzyme is most specific for acute pancreatitis? Lipase. It is not found iss many tissues. ‘Are amylase and lipase elevated in chronie pancreatitis? Marginal elevation’ may be observed during atlacks When the disease is in its early stages, but as the disease progresses, severe destruction of acinar tissue eventually reduces the amount of enzymes released into the circulation. Then subnormal levels may be seen, ‘Why might a pleural uid amylase be ordered? ‘A normal pleural Quid amylase rul ut acute pancreatitis, ‘What is the major diagnostic significance of elevated alkaline phosphatase (ALP)? I is elevated with liver and bone disorders, 47n 23, 4. 25. 26. 21. 28. 29. . 30. 3 32, QUICK REVIEW Q & A'S In which disease do the highest elevations of alkaline phosphatase (ALP) occur? Paget's disease, a bone disease characterized by excessive bone destruction and unorganized bone repair, Values 10- 25 times the upper reference limit are not unusual. What is the optimum pH for alkaline phosphatase? 9.0-10.0 When is a physiological increas serum alkaline phosphatase seen? During pregnancy due to release of ALP from the placenta and during childhood due to rapid bone growth. ‘What is the clinical signifleance of acid phosphatase (ACP)? Itis elevated in prostatic cancer, but usually not until the cancer has metastasized. ACP in serum is rarely determined now because prostate specific antigen (PSA) is more sensitive. The presence of acid phosphatase in vaginal washings may be significant in rape cases, as it indicates the presence of semen, Vaginal swabs should be preserved by immersion in a protective broth. What reaction is catalyzed by CK in the body? Creatine phosphate + ADP ¢> Creatine + ATP With which clinical condition are the highest levels of CK seen? Duchenne’s muscular dystrophy. What are some physiologic causes for increased total CK? Intramuscular injections and vigorous exercise What reaction is catalyzed by LD in the body? Lactate + NAD © Pyruvate + NADH +H How should specimens for LD be stored and why? They should be stored at 25°C and analyzed within 48 hours. They should not be refrigerated because LD decreases more rapidly at 4°C than at 25°C. Which clinical condition results in the highest levels of LD? Pemicious anemia, Name the three isoenzymes of CK and the tissue with the highest concentration of each, CK-1 or CK-BB (brain), CK-2 or CK-MB (heart), and CK-3 or CK-MM (skeletal muscle). The Commission on Biochemical Nomenclature has recommended that isoenzymes be numbered on the basis of their electrophoretic mobility, with the most anodal form receiving the lowest number. Accordingly, CK-1, CK-2, and CK-3 is the preferred nomenclature, CK isoenzymes are not specific for any one tissue. Although CK-3 is primarily associated 483 38. 36. 31, 38 39, 40, a CLINICAL CHEMISTRY REVIEW with skeletal muscle, itis also found in cardiac tissue. CK-2 is primarily associated with cardiac tissue, but some is a present in skeletal muscle. Which cardiac enzyme is most specific? on CK-2isthe most specific cardiac enzyme, but not the mos specific cardiac marker. Cardiac iroponins are more specific ‘Which cardiac markers are currently used to diagnose acute myocardial infarction (AMI)? Cardiac troponin (Tor) and CK-MB, Most protocols measure these cardiae markers a admission and 6-9 hours late. Negative results on atleast two specimens cllected during ths interval effectively rules out AMI. AST, total CK, LD, and LD isoenzymes should no longer used to evaluate cardiac disease because they lack cardiac specificity. How is CK-2 measured? Immunoassays using monoclonal ani-CK.2 antibodies have replaced electrophoresis. How is ardiae troponin measured? y 4 Immunoassays using monoclonal antibodies. {A patient admitted tothe ER with chest pain of 6 hours duration has a negative myoglobin, What isthe WA Significance ofthis inding? Irules out acute myocardial infarction. Myoglobin isa sensitive, though nonspecific, marker for AMI. lis elevated ‘within 3-4 hours after onset of chest pain and remains elevated for about 24 hours. Because myoglobin is nonspecific, positive results must be followed up witha specific marker (cardiac troponins). Which cardige marker is most specific for cardiac damage? Cardiac troponins (TaT and Tol) Levels begin fo rise within 4 hours of myocardial damage and remain elevated for 10-14 days, so cardiaetroponins are useful for early diagnosis and for late diagnosis after cardiac enzymes have retuned to normal. Cardiae toponins ae sti to eliminate the need for LD isoenzymes inthe diagnosis of AMI Which cardiac marker is most likely tobe elevated in a patient who is admitted to the hospital 4 days after a suspected myocardial infaretion? Cardiac toponins. CK-2 would have retumed to normal What isthe clinical significance of B-type natriuretic peptide (BNP test)? BNP isa cardiac hormone that is produced by the heart ventricles in response to ventricular volume expansion and pressure overload. Iisa predictor of congestive heat flue in patients suffering from shortness of breath. BNP is, also to evaluate risk in persons who present with chest pain. It has been found that high BNP predicts an increased risk of death or subsequent hear atack n patients with acute coronary syndromes. The tests an immunoassay that can be performed at point of care ‘What isthe clinical significance of highly sensitive C-reactive protein (hs-CRP)? CRP isa marker of inflammation and appears to be associated with increased risk for coronary hear disease, sudden cleat, and peripheral artery disease. hsCRP is measured by turbidimetric immunoassays that use an antibody to an epitope onthe CRP molecule, These assays are sensitive t0 0.01 mg/dL. hs-CRP should not be confused with the 498. 4 45, 46. 41. 48 4 50, 51. QUICK REVIEW Q& A'S conventional flocculation assays for CRP, which only detect gross elevation. A this time, hs-CRP screening ofthe entire adult population is nt recommended. ‘Which enzymes are included ina hepatic function panel? ALT, AST, and alkaline phosphatase. The other tests are tot! protein, slbumin, and total and direct bilirubin Which enzyme is mast sensitive for al types of lve disease? GGT. Highest levels are seen with obstructive disorders, [ALT. AST is found in many tissues. ALT is found primarily in the lverand RBCs. What are the substrates for AST? Aspartate and acketoglutarate wi re the substrates for ALT? Alanine end a-ketoglutarate Which disease state has the highest elevation of AST and ALT? Acute hepatitis. Where is LD found? Inall cells ofthe body. Because of its wide distribution, serum LD elevations occu in a variety of elnical conditions including myocardial infarction, hemolysis, and disorders of the liver, kidneys lung, and muscle. Tn liver disease, LD does not increase as much as AST and ALT. At onetime, LD and LD isoenzymes were used in the disgnosis of myocardial infarction but they have been replaced by cardiac roponins. LD isoenzymes are rarely used in clinical practice anymore ‘What effect does hemolysis have on LD? Iris increased because LD is 100-150 times more concentrated inthe RBCS. ‘Which enzyme isa sensitive indicator of alcoholism? Gamma-gluamylianspeptidase (GGT) ‘Which enzyme would be helpful in establishing a diagnosis of bone disease? Alkaline phosphatase. Ww ‘enzymes would be helpful in establishing a diagnosis of muscle disorders? CK, AST, LD, and aldolase, ~~ S57 F ee Vee eS TSs53. 54, 55, CLINICAL CHEMISTRY REVIEW ‘What isthe clinical significance of a G-6-PD deficiency? 7 Inherited deficiencies of G-6-PD can lead to hemolytic anemia following exposure to certain drugs. G-6-PD is within the RBCs, so the specimen required is whole blood What is the clinical significance of low pscudocholinesterase levels? “ Low levels are seen following exposure to insecticides and nerve gases and in patients with hypersensitivity to the ‘muscle relaxant succinyleholine. ‘What is the advantage to using enzymes as reagents? Enzymatic assays are very specific. There are fewer false positives then with colorimetric assays. NON-PROTEIN NITROGEN COMPOUNDS Name three substances that are elevated in the blood with renal disease. BUN (blood urea nitrogen), creatinine, and uric acid What is urea? ‘The end product of protein metabolism, It is synthesized in the liver from ammonia and carbon dioxide, ‘What is the reference range for BUN? 7-18 mgldL (25-64 mm ). The term azotemia refers o elevated levels of urea in the blood. Why should tubes containing fluoride or citrate not be used when collecting blood for urea if analysis will be by the urease method? Fluoride and citrate inhibit urease What is the formula for converting BUN to urea? Urea = BUN x 2.14 ‘Where is 98% of the body's creatine located? In muscles, What is creatinine? The anhydride of creatine. Creatinine is formed from creatine by splitting out of water. ‘What reaction is used to measure creatinine? The Jaffe reaction using alkaline picrate. Its a nonspecific, but still clinially useful, method. 39. 10. Te 2. 13. MW 1s. “16. 11. 18, QUICK REVIEW Q.& A'S What is the reference range for creatinine? 0.5-1.2 mg/dL (44-196 mmol/L) ‘What is the significarice of the BUN:creatinine ratio? helps to determine the cause of an increased BUN, The normal ratio is approximately 10:1, Higher ratios are due to rnon-renal causes, such as a high protein dict, increased rate of protein catabolism, or decreased renal blood flow With these conditions the BUN increases more than the creatinine. With renal disease, the BUN and creatinine increase proportionately Calculate the BUN : creatinine ratio for a patient whose BUN is 45 mg/dL and whose creatinine is 2.1 mg/dL. 45214 21 ‘The normal ratio is 12-20. A ratio greater than 20 is suggestive of prerenal azotemia, ‘What is the least variable nitrogenous constituent of blood? Creatinine, because itis related to muscle mass and is not affected by diet ‘What is an estimated glomerular filtration rate (GFR)? ‘A calculation value based on serum creatinine and the patient's age, Sex, and race. The National Kidney Disease Education Program (NKDEP) encourages reporting of the estimated GFR along with the serum creatinine because it is a more sensitive indicator of kidney disease. At this time, the practice has not been widely adopted by clinical laboratories. What is urie acid? ‘The end product of purine metabolism. The purines are adenosine and guanine, components of nucleic acids. Uric acid is increased with gout, renal disease, and conditions where there is high cellular turnover, such as leukemia ‘Whit reagent is commonly used to measure uric acid? : Uricase, The preservative sodium fluoride must not be used to collec the blood sample because it destroys uricase What isthe reference range for uric acid? Females: 2.6-6.0 mg/dL (0.15-0.35 mmoVL), Males: 35-722 mg/l (0:21-0.42 mmoliL) What snay result from high levels of ure acid? rate crystals may precipitate in joims and tissue ‘Why must the pH of urine for a uric acid determination be adjusted to 7.5-82 To prevent precipitation of uric acid. Uie acid precipit cid pH 82| CLINICAL CHEMISTRY REVIEW | 19, Where ammonia formed? he ‘Mainly in the intestines from deamination of amino aids. It is converted to urea by the liver. 20, What isthe reference range for ammonia? ¢ 14-49 pill (11-35 pmol, 21. When is ammonia elevated? ‘With hepatic failure and Reye's syndrome. High levels are neurotoxic 22. What is Reye's syndrome? ‘An act, often fatal encephalopathy ané fatty degeneration ofthe liver, seen primarily in children, Is associated with the use of aspirin in children wth vital infections. 23. What are two technica difficulties in performing blood ammonia determinations? Levels increase rapidly after drawing, so the specimen must be placed on ice and the plasma separated from the cells 6 ‘rompily. Ammonia contamination from detergents, water, and smoke must be avoided, 24, Which amino acid is increased in the blood of patients with phenylketonuria (PKU)? Pa Phenylalanine. PKU is due toa deficiency of the enzyme phenylalanine hydrorylase which catalyzes the conversion of phenylalanine to tyrosine. If untreated, PKU leads to mental retardation. The Guthrie bacterial inhibition asay is used for screening, HPLC is the reference method 25. What may result if blood for PKU is drawn before 24 hours of age? False negatives. 26. Which amino acids are increased in maple syrup urine disease (MSUD)? Leucine, isoleucine, and valine. The urine has a bumt sugar odor. MSUD leads to mental retardation and sometimes death. Many states require newbom screening, A modified Guthrie test is commonly used. MSUD can be treated by dietary modification if diagnosed ex , ~ BILIRUBIN 1 What is btrubin? ‘The degradation product of heme. It is produced in the reticuloendothelial cells following breakdown of RBCs. 2 ‘Which protein transports bilirubin in the blood? Albumin, _ 536. 10, i. 12. 1B. 14, QUICK REVIEW Q.& A'S Name the two types of bilirubin, Direct and indirect. Indirect or unconjugated bilirubin is bilirubin en route to the liver. Once it goes through the liver it is known as direct bilirubin, conjugated bilirubin, or bilirubin diglucuronide. Explain what happens to bilirubin in the liver. Its conjugated with glucuronic acid by the enzyme uridyldiphosphate glucuronyl transferase (UDPG-T), Following conjugation, direct bilirubin is excreted into the intestine via the bile duct and is reduced by bacteria to urobilinogen Urobilinogen is oxidized to urobilin and gives the normal color to stools What is the significance of elay-colored or light stools? Iisa sign of obstruction of the bile duct. Urobilin is not being produced because bilirubin is not reaching the intestines. Which substances related to bilirubin metabolism are normally found in the urine? Only urobilinogen. Bilirubin should not be present in urine. What urine abnormality is seen with complete obstruction of the biliary tract? Decreased urobilinogen. Which bilirubin fractions are analyzed in the laboratory? Total and direct. The indirect level is calculated by subtracting direct from total. Compare the solubility of direct and indirect bilirubin. Direct bilirubin is soluble in water; indirect bilirubin is not. Both are soluble in alcohol. Which form of bilirubin can be excreted in the urine? Only direct bilirubin. What is a common method for determination of bilirubin levels? Diazotization with sulfanitic acid. Bilirubin reacts with diazotized sulfanilic acid to produce azobilirubin. Name several accelerators that are used in the total bilirubin reaction, Alcohol or caffeine-benzoate-acetate can be used to make the indirect bilirubin soluble. Name a source of error that can decrease the level of bilirubin in a specimen. Exposure to light. Hemolysis will also cause a decreased level by the Jendrassik-Grof method. What is the normal range for total bilirubin in an adult? 0.2-1.0 mg/dL. Conjugated (direct) bilirubin is < 0.2 mg/dL. 54CLINICAL CHEMISTRY REVIEW 15, How do normal values for bilirubin in a newborn compare to those in an adult? Levels are higher in the newborn. The total bilirubin in a 3-5 day old full-term infant is 4-6 mg/dL; for a premature. infant, 10-12 mg/dL. 16. What would cause an increase in total bilirubin with a normal concentration of direct bilirubi Prehepatic jaundice, for example, hemolytic transfusion reaction, hemolytic anemia, or hemolytic disease of the ewbom, on What causes physiologic jaundice of the newborn? Bilirubin metabolism is impaired beceuse the newbom's immature liver doesn't produce the enzyme required for bilirubin conjugation. Phototherapy is used to reduce the level of bilirubin. 18, In hemolytic disease of the newborn, which bilirubin fraction is elevated and why? Indirect due to excessive breakdown of RBCs by maternal antibody. 19, ‘What is the risk to the newborn from a high level of indirect bilirubin? Unconjugated bilirubin (indirect) has a high affinity for brain tissue and causes necrosis (kernicterus). Without appropriate treatment, mental retardation, hearing deficits, or cerebral palsy may result. 20, At what level of bilirubin would an exchange transfusion be indicated in a neonate? Each institution establishes its own criteria, but an exchange transfusion is usually performed when the unconjugated bilirubin reaches 20 mg/dL. 21. What method is used to determine neonatal bilirubin? Direct spectrophotometry at 454 nm, This method can’t be used for patients over one month of age because of interfering lipochromes, such a8 carotene. 22. ‘Name two conditions in which direct bilirubin is elevated. Hepatic and posthepatic jaun. 2B. What are the typical lab findings in posthepatic jaundice? Increased total bilirubin, increased direct bilirubin, decreased urine urobilinogen, and clay-colored stools 14, Which disorder results in the highest levels of conjugated bilirubin? Obstructive liver disease. HORMONES 1 What type of method is used for most hormone assays? Immusnoassays6 10. i. 1 QUICK REVIEW Q.& A'S ‘What is the precursor in the biosynthesis of all steroid hormones? Cholesterol List several steroid hormones. Contsol, aldosterone, estrogen, testosterone, progesterone, Which endocrine gland releases tropic hormones that regulate other endocrine glands? The anterior pituitary. ‘Where is follicle-stimulating hormone (FSH) produced and what i its main action? Itis produced in the anterior pituitary. It stimulates production of sperm and eggs. There isa sharp increase in FSH just before ovulation Where is growth hormone (GH) produced and what js its main action? Itis produced in the anterior pituitary. It stimulates protein symthesis and cell growth and division. Where is thyroid-stimulating hormone (TSH) produced and what is its main action? Its produced in the anterior pituitary. It stimulates the thyroid to produce T3 and 74, It is also known as thyrotropin. Where is adrenocorticotropic hormone (ACTH) produced and what is its main action? Its produced in the anterior pituitary. I stimulates the adrenal cortex to produce corticosteroids. diurnal variation. Levels are highest in early morning and lowest in late afternoon, Where is anti-diuretic hormone (ADH) produced and what is its main a Itis produced in the hypothalamus and stored in the posterior pituitary. I regulates reabsorption of water from the distal convoluted tubules. ADH is decreased in diabetes insipidus, leading to the excretion of an increased volume of dilute urine. ‘Where is cortisol produced and what is its main a Itis produced in the adrenal cortex. It regulates carbohydrate fat, and protein metabolism, water and electrolyte balance, and suppresses inflammatory and allergic reactions. Cortisol levels are regulated by ACTH and show diurnal variation, Levels in the evening are approximately 2/3 of moming levels. Cortisol is increased in Cushing's syndrome and decreased in Addison's disease. What is Addison's disease? ‘Adrenal insufficiency, characterized by decreased cortisol and increased ACTH. ‘What is Cushing's syndrome? ‘The signs and symptoms associated with elevated cortisol levels. Cushing’s syndrome may be due to tumors of the pituitary (Cushing's disease), tumors of the adrenal glands, ectopic ACTH-secreting tumors, or administration of glucocorticoids or ACTH. 563B. 14. 15. 16. 1. 19, 20, a. n CLINICAL CHEMISTRY REVIEW What do 17-ketosteroids and 17-hydroxycorticosteroids measure? Adrenal cortical function. Interpret this patient's plasma cortisol results: BAM: 30 pg/dL (reference range 5-23 gid) 4PM: 32 ug/AL. (reference range 3-16 g/dL) ‘The patient has hypercortisolism and no diurnal variation. Hypercortisolism is referred to as Cushing's syndrome. One cause of Cushing's syndrome is Cushing's disease, the result of an ACTH-producing pituitary adenoma. Other causes of Cushing's syndrome are tumors ofthe adrenal glands, ectopic ACTH-secreting tumors, and administration of glucocorticoids or ACTH. ‘Where is aldosterone produced and what is its main action? Itis produced in the adrenal cortex. It increases retention of Na" and excretion of K” and H. What are catecholamines? ‘The hormones secreted by the adrenal medulla~epinephrine, norepinephrine, and dopamine. Theit metabolite is vanillylmandetic acid (VMA), Wha pheochromocytoma? A tumor of the adrenal medulla that produces large amounts of catecholamines and causes hypertension. It can be diagnosed by urinary metanephrines, VMA, and plasma catecholamines. What substances can cause a false positive VMA? Bananas, vanilla, and some rugs. Because ofthe interfering factors in the VMA test, urinary melanephrines is considered the best screening test for pheochromocytoma ‘Where is progesterone produced and what is its main action? It is produced in the ovaries. It prepares the uterus for pregnancy and stimulates lactation, ‘What is the major estrogen produced by the ovaries? Estradiol (E2). Estrogens are involved in the development of the female reproductive organs and secondary sex characteristics, regulation of the menstrual cycle, and maintenance of pregnancy. ‘Which hormones are used to assess fetal well-being? Estriol, progesterone and its metabolite, pregnanediol ‘Which hormone is used to detect pregnancy? Human chorior gonadotropin (HCG). It is secreted by the placenta. HCG doubles approximately every 2 days during the first trimester, then slowly declines inthe second and third trimester. HCG can also be used to detect and monitor cancers of the ovaries, testes, and placenta. The befa subunit is measured to increase the specificity of the test. The alpha subunits are identical to those of LH and FSH. 8723. 24, 28. 26. 21. 28. 29. 30. a QUICK REVIEW Q & A'S Which hormone can be measured by a home testing kit to determine the time of ovulation? Luteinizing hormone (LIH), secreted by the anterior pituitary. There is a sharp peak just before ovulation. Why are estrogen and progesterone receptor assays performed? To establish a prognosis for patients with breast cancer, Where is thyroxine (4) produced and what is main action? It is produced in the thyroid and controls metabolic rate, growth and development, and sexual maturation, Triiodothyronine (T3) is formed primarily from deiodination of TS by tissues. ‘Which is the physiologically active form of T4? Free T4 (FT4). Most T4 is bound to thyroxine binding globulin (TBG), the main carrier protein for T4 and 73. What is the recommended screening test for thyroid function’ Recent improvements in sensitivity have made TSH the most important indicator of pritnary hyperthyroidism and hypothyroidism. With hypothyroidism, TSH may be increased before clinical symptoms. A normal TSH usually excludes a diagnosis of primary thyroid dysfunction. What further thyroid testing is recommended when the TSH is abnormal? Free T4. What further thyroid testing is recommended when the TSH is low and the free T¢ is low or normal? A total T3 to test for T3 thyrotoxicosis. T3 thyrotoxicosis is hyperthyroidism with elevated T3 and normal T4 and free T4 How can primary hypothyroidism be differentiated from secondary hypothyroi m? By TSH. Primary hypothyroidism is a disorder of the thyroid gland. TSH increases as the pituitary tries to stimulate the thyroid to produce more T3 and T4, Secondary hypothyroidism is a pituitary disorder. TSH levels are low. Discuss the use of the total T4 and T3 uptake. When free T4 testing was beyond the capabilities of most clinical labs, total T4 and T3 uptake were performed in order to calculate the free thyroxine index (FT«l) or T7, an estimate of free T4, Today, free T4 assays are readily available and provide a more accurate picture of the patient's thyroid status than total T and total T3, which are affected by abnormal concentrations of binding proteins, such as TBG. ‘What lab findings are typical of primary hypothyroidism? High TSH and low free T4. T3 levels are not routinely performed. Although T3 is decreased in patients who are severely hypothyroid, itis within the reference range in 15-30% of hypothyroid patients. 5833. 34 35. 36. 3. 38. CLINICAL CHEMISTRY REVIEW ‘What screening is done for neonatal hypothyroidism? All 50 states require newbom screening for hypothyroidism to eliminate severe mental retardation associated with thyroid hormone deficiency. Measurement of T4 or TSH or a combination of tests is performed using dry blood spots or cord blood. What lab findings are typical of hyperthyroidism? = GH ‘TSH is low and free TA in high. In over 90% of patients with hyperthyroidism, b serum T3 and TA levels are increased, with the increase in T3 great 2 the increase in T4: however, only free T4 is routinely measured, ‘What is Grave's Disease? An autoimmune disease that is the most common type of hyperthyroidism in the U.S. Thyroid stimulating hormone receptor antibodies (TRab) are present in the serum, Where is parathyroid hormone (PTH) produced and what is its main action? Itis produced in the parathyroid glands. It increases senfciyn and decreases phosphates\ PTH is increased in primary hyperparathyroidism and decreased in hypoparathytoidism, = ‘What is actually measured when serotonin is ordered? S-HIAA, the urinary metabolite of serotonin, Increased levels occur with argentaffinoma, a tumor of the GI tact, Bananas, walnuts, pineapples, plums, eggplants and avocados cause false positive S-HIAA, Where is glucagon produced and what is its main action? Itis produced in the alpha cells of the pancreas. It increases glucose levels. ELECTROLYTES 1 What are electrolytes? Substances that carry an electric curren when dissolved in water. Anions are negatively charged and cations are positively charged. ‘What are the major electrolytes? Sodium, potassium, chloride, and bicarbonate Wha the Law of Electric Neutrality? ‘The number of cations in the body must equal the number of anions. Ifa cation increases, either another cation must decrease or an anion must increase to maintain electric neutrality. ‘What is the SI unit for electrolytes? mmoUL. Some labs still report electrolytes in mi L. For monovalent electrolytes, the number remains the same. 599. 10. i 12. 13, 18, 16. mmol/L = mEq/L valence What is the major extracellular cation? Sodium. What is the major intracellular cation? Potassium What is the major intracellular anion? Phosphate, What is the major extracellular anion? Chloride ‘What are some sources of error in drawing blood for electrolytes? Drawing the specimen above an IV site. fist pumping during collection, prolonged tourniquet time (longer then one ‘minute), and “milking” the finger or heel during sample-collection by skin puncture. ‘What is the normal value for sodium? 135-145 mmol. Which hormone regulates the concentration of sodium? Aldosterone. Why should standards for sodium be stored in plastic containers instead of glass? Sodium can leech from glass containers and cause falsely elevated values. ‘What is the normal value for potassium? 3.45.0 mmoltt. How are sodium and potassium usually measured? By ion selective electrodes (ISE). ‘Which ISE has a liquid ion-exchange membrane incorporating the antibiotic valinomycin? The potassium-selective electrode. Because of is ability to bind potassium. valinomycin is used as the neutral carrier for the potassium-selective electrode, ‘What is the approximate ratio of potassium between RBCs and serum? 20:1. . 6018, 20. a. n, 2B. as. 26, 21, CLINICAL CHEMISTRY REVIEW What is the approximate ratio of sodium to potassium in serum? 30:1 List several factors that can cause artifactual elevations of potassium. Fist clenching, prolonged tourniquet time, contamination with KsEDTA or IV fluid, hemolysis, thrombocytosis, and Teukocytoss. why potassium slightly higher in serum than in plasma? Because potassium is released from platelets during clotting Before reporting an elevated potassium, what should be checked? ‘The specimen should be checked for hemolysis or excessive delay in separating the serum/plasma from the RBCs. Either could be responsible fora false elevation. ‘What clinical condition results from very high or very low potassium levels? Cardiac arthythmias. A specimen for potassium was drawn in a tube without anticoagulant at 6 AM from a patient in a nursing home. It was delivered to the lab at 11 AM and analyzed at 12 PM. The result was 6.0 mmol/L. What would the best course of action be? ‘A new specimen should be drawn. Potassium may be falsely elevated when the serum is in contact with the RBCs too long, What is the normal value for chloride? 98-106 mmol/L. What is chloride’s role in the body? 1k maintains hydration, osmotie pressure, and electrolyte balance. Which disease is characterized by a high concentration of sodium and chloride in sweat? Cystic fibrosis What is the most accepted test for the diagnosis of eystie fibrosis? ‘The sweat test, Sodium and chlotide are elevated with cystic fibrosis, Chloride is more commonly measured Which sweat testis recommended by the Cystic Fibrosis Foundation? ‘The Gibson-Cooke quantitative pilocarpine iontophoretic sweat test.28, 2, 30, 31. 3, 33 crs ‘What is pilocarpine? ° A topical drug used in the sweat test to stimulate sweat glands. In iontophoresis, an electric current delivers pilocarpine nitrate to the sweat glands. Sweat is collected and chloride or sodium is measured. Chloride i the preferred analyte, A sweat chloride greater than 60 mmol. is consistent with eystic fibrosis. What is the normal value for total C0? 29 mmol, What happens to CO; ifthe sample is exposed to ai In decreases. Which of the following results, if confirmed, would warrant an immediate call to the patient's physician? Sodium 130 mmol/L Potassium 6.8 mmoVL. Chloride 100 mmol/L. co; 28 mmol/L. ‘The potassiuim. Each institution establishes its own critical values, bt 6 mmol/L is often used as the upper limit for potassium. ‘What formulas are used as a quality control check of electrolyte results? (Na" + K") (CI + COz) = 16-20 mmobL or Na’ - (CI + CO.) = 8-16 mmoVL. Ifall values obtained are high or ow, it points to a possible effa¥ in one of the determinations. A negative number indicates an analytical error in at east one of the analytes, These are the same formulas used to calculate the anion gap. Interpret the following results: Sodium 130 mot. pte Potassium 3.5 mmol, Chloride 105 mmol/L, Bad > co; 27 mmol, , These findings are suggestive of a laboratory error since the difference between the cations and anions is only 1.5. The tests should be repeated ‘What does anion gap measure? ‘Unmeasured anions. The anion gap is increased with renal failure, diabetic acidosis, lactic a of drugs or toxins. sis, and the presence Calculate the anion gap for the following data: Sodium 132 mmov. end 2 Potassium 3.5 mmol. Chloride 93 mmol, CO; 25 mmol/L, 178 ~ 6236. 3. 38. 39. 40. 4 42. 43 44. 43. 46. 47. CLINICAL CHEMISTRY REVIEW (Na + K")- (CI + CO) (132 = 3,5) ~ (93 + 25) = 17.5, This is within the reference range of 10-20. What is the most abundant mineral in the body? Calcium, 99% is in the bones. ‘What is the normal value for total calcium? 8.7-10.2 mgidL (2.18 -2.55 mmol}. Which anticoagulants cause a false decrease in calcium? EDTA, citrate, and oxalate, All prevent coagulation by binding Ca". Heparin is the only acceptable anticoagulant for calcium determinations Which form of caleium is physiologically active? —, Tonized. Why is pH an important consideration in ionized calcium determinations? As pH decreases (acidosis), calcium dissociates from its complexed forms, increasing the amount of free ionized calcium in the serum. What is the reference method for total calcium? ‘Atomic absorption. How is ionized calcium measured? By ion selective electrodes What substances regulate calcium levels? PTH, calcitonin, and vitamin D. What is tetany? Musele spasms, cramps, and iritabilty due to decreased caleium or magnesium, What is the most common cause of hypercalcemia? Primary hyperparathyroidism, How does hyperparathyroidism affect the level of serum phosphorus? Inis decreased. What happens to calcium when phosphorus is increased? It decreases, There is a reciprocal relationship between calcium and phosphorus. 63 oeQUICK REVIEW Q & A'S A hospitalized patient exhibits signs ofteta ‘but her ionized calcium is normal, What other analyte should be checked? Magnesium. Low levels of magnesium also cause tetany 49. How does the reference range for phosphorus in growing children compare to that of adults? Wis higher in children. 50. What must be done to urine prior to performing a urine phosphorus analysis? i must be acidified to lt 6 to prevent precipitation of phosphates $1. How does hemolyss affect iron level? Because ofthe high concentration of iron in hemoglobin, even minimal hemolysis wll give falsely elevated result. ‘Tominimize this effect. serum/plasma should be separate from RBCs within one hour of collection and even slightly hemolyzed specimens should not be analyzed. 52. How are iron levels affected by the time of day when the specimen is drawn? Iron shows a marked diumal variation. Levels are approximately 30% higher in the moming 53. Which protein transports iron? Transferrin. It is normally 20-55% satureted with iron. 54, Where is most of the iron inthe body? In hemoglobin, 55, Name two storage forms of fron, _Feritin is the primary storage form. IL presen in mos cells and isa readily mobilized form of storage iron. A ‘Sal amount of iron is also stored as hemosiderin, 56. Howare the iron and total iron binding capacity (TIBC) affected in iron deficfency anemia? ‘Serum iron is decreased and TIBC is increased. TIBC is an indirect measurement of transferrin. TIBC is infrequens performed since the development of improved transferrin assays. 57. What is the most sensitive test for detection of iron deficiency anemia? i Serum ferritin, A decreased serum ferritin is almost always indicative of iron deficiency anemia, 58. What are the lab findings in hereditary hemochromatosis? Hereditary hemochromatosis isthe most common ofthe iron overload diseases. It causes the body to absorb and store too much icon. The iron panel consists of serum non (SI), ferritin total iron binding capacity (TIBC) and transferrin saturation (TS) TS determines how much iron is bound to transferrin, the protein that caries ion in he blood. After a 12-hour fast, the TIBC and SI are measured and the TS is calculated (SUTIBC ='TS). The serum ferritin test shows the level of ion inthe liver 6459. 60. 61. 02. 63. 64, 65. 66. 67. CLINICAL CHEMISTRY REVIEW Name two conditions in which free erythrocyte protoporphyrin (FEP) is increased, Iron deficiency anemia and anemia of chronic infection. FEP is protoporphyrin that is not complexed with Fe™. It is increased when there is decreased iron available to the developing RBCs. FEP is most commonly used as a screen for iron deficiency anemia. It was formerly used to screen for lead poisoning, but has been replaced by direct measurement of blood lead. ‘What is lactate? 1. Lactic acid, an intermediary in carbohydrate metabolism. There are 2 types of lactic acidosis: 2. Hypoxic, due to decreased oxygen delivery to the tissues 3, Metabolic, associated with disease, drugs/toxins, and inborn errors of metabolism ‘The mortality rate for lactic acidosis is greater than 60%. Name a reagent used to measure lactate, Lactate dehydrogenase (LD). Lactate is oxidized to pyruvate by LD in the presence of NAD”. The NADH formed is measured at 340 nm. What happens to lactate in the blood following collection? Itincreases due to glycolysis. What special handling is required for specimens for lactate? lodoacetate or sodium fluoride should be used to inhibit glycolysis. The specimen should be iced and the plasma separated from the RBCs within 15 minutes of collection. What is a colligative property? One that depends on the number of solute particles, regardless of size or molecular weight. The colligative properties are osmotic pressure, vapor pressure, boiling point, and freezing point. How is osmolality usually measured in the clinical Inb? By freezing point depression. Each milliosmote of solute lowers the freezing point by 0.00186° C. Osmolality is reported in milliosmoles of solute per kilogram of water (mOsm/kg). Normal serum osmolality is 275-295 mOsm/kg. Normal urine osmolality (24 hr. specimen) is 300-900 mOsmvkg. ‘What does the urine to serum osmolality ratio indicate? The degree to which the kidneys concentrate the glomerular filtrate, The normal urine:serum ratio is 1:1-3:1, What is the formula for calculated osmolality? 2a’ mmol/L + glucose mg/dL + BUN mg/dL. : 20 3 6568, 69. QUICK REVIEW Q & A'S Which substance contributes most to serum osmolality? Sodium accounts for almost ha. Name several clinical conditions that result in an increased serum osmolality. Dehydration, uremia, diabetes mellitus, alcohol intoxication, salicylate intoxication, excessive electrolyte IVs. What is osmolal gay The difference between measured osmolality and calculated osmolality. I is used to diagnose poisonings and to estimate blood alcohol levels. The reference range is 0-10 mOsrtvkg. Higher levels indicate an abnormal concentration of an unmeasured substance such as isopropanol, methanol, ethylene glycol, or acetone. ACID-BASE BALANCE 5. Define pH. Jog [H+] or log 1 (H+) ‘Write the Henderson-Hasselbalch equation for the determination of blood pH. pH = pK + log {HCO¥) or pH =6.1 + log [HCO3 (H:CO3) PCO; x 0.03 What is the normal pH of arterial blood? 7.35145, ‘What is acidosis? An arterial blood pH < 7.35. It is the result of a decreased bicarbonate:carbonic acid ratio, The normal ratio is 20:1 Acidosis is either metabolic (due to a decreased bicarbonate) or respiratory (due to an increased carbonic acid) ‘What is alkalosis? ‘An arterial blood pH > 7.45. Its the result of an increased bicarbonate;carbonic acid ratio, Alkslosis is either metabolic (due to an increased bicarbonate) or respiratory (due to a decreased carbonic acid) ‘What is a buffer? ‘A weak acid and its salt, Buffers minimize changes in pH. The most important blood buffer is the carbonic acid / bicarbonate buffer system, Phosphates, proteins, and hemoglobin are also blood buffers. What is PCO,? The partial pressure of CO». It is a measurement of dissolved CO3. PCO: is controlled by the lungs. The reference range for arterial blood is 35-45 mm Hg. 66a 12. 13, 14, 15, 16, Tm 18, CLINICAL CHEMISTRY REVIEW What is the normal total CO; of arterial blood? 23-27 mmoVL. Total C0: is primarily bicarbonate How do the lungs affect the blood pli? By regulating the PCO:, and subsequently, the concentration of carbonic acid. Hyperventilation d and carbonic acid concentration, which increases the pH. Hypoventilation increases the PCOs and ca concentration, which decreases the pli...) How do the kidneys affect the blood pH? By regulating the concentration of bicarbonate and hydrogen ions. What are the typical lab findings in respiratory acidosis? Decreased pli, Get 0s sGamaD bicarbonate. The kidneys wil attempt to compensate by retaining bicarbonate, thereby reestablishing the 20:1 Bicarbonate:carbonic acid ratio. ‘What are the typical lab findings in respiratory alkalosis? Increased pl, decreased PCO: and Fama? arbor The kidneys will attempt to compensate by excreting bicarbonate, thereby reestablishing thé 20:1 bicarbonale-carbone acid ratio. What are the typical lab findings in metabolic acidosis? Decreased pH and bicarbonate, and normal PCO; The lungs will attempt to compensate through hyperventilation to reduce carbonic acid, thereby reestablishing the 20:1 bicarbonate:carbonic acid ratio. What are the typical lab findings in metabolic alkalosis? Increased pH and bicarbonate and normal PCO: The lungs will attempt to compensate through hypoventilation to increase carbonic acid, thereby reestablishing the 20:1 bicarbonate:carbonic acid ratio. This compensatory ‘mechanism is only partially effective because breathing can only be slowed to a certain extent, ‘Whats the relationship between pH and H’ concentration? Inverse. As (H] increases, pH decreases and as (H'] decreases, pH increases. - Sp ‘Whats the relationship betweey pH and reas eee Inverse, As PCOz increases, pH deci®8Ses and as PCO; decreases, pH increases. sated? dial Direct, As bicarbonate increases, pH increases and as bicarbonate decreases, pH decreases, What is the relationship between; ‘What is the compensatory mechanism to reestablish pH in a patient with a high HCOs level? Hypoventilation, CO; is retained to reestablish the 20:1 ratio. 6719. 20. 2. 2. 23. 24. 28. 26. 27. QUILR REVIEW Wo A'D Interpret the following arterial blood gas results: pH 7.07 (Reference range: 7.35-7.45) PCco2 90 mm Hg, (Reference range: 35-45) & TotalCO2 27 mEq/L —_(Reference range: 23-27) {/ Respiratory acidosis. The low pH indicates acidosis and the high PCO: indicates a respiratory problem. Interpret the following arterial blood gas results: pH 135 PCO; 43 mm Hg TotalCO; 39 mEq/L. x Metabolic alkalosis. The high pH indicates alkalosis and the high total CO: (bicarbonate) indicates a metabolic problem. ‘What parameter is measured instead of bicarbonate? Total CO, This is an acceptable substinute because total CO; is primarily bicarbonate. Bicarbonate is approximately equal to total CO)-I. Total CO; can be calculated from pH and PCO; or it can be measured by colorimetric methods. ‘What is measured instead of carbonic acid? PCO;. Carbonic acid = PCO; x 0.03. A patient has a slightly decreased pH, a low HCO;", and low PCO}. What do these findings indicate? Compensated metabolic acidosis. Compensation for metabolic acidosis is hyperventilation to decrease PCO>. What Is base excess (BE) and how is it used clinically? BE js defined as the theoretical amount of acid or base needed to return the plasma pH to 7.40. 1t is used by some clinicians to assess the metabolic component of a patient's acid-base disorder. A positive value indicates an excess of bicarbonate or relative deficit on noncarbonic acids and suggests metabolic alkalosis. A negative value indicates a deficit of bicarbonate or relative excess of noncarbonic acids and suggests metabolic acidosis. BE is calculated by blood gas analyzers from pH, PCO;, and hemoglobin, How much oxygen can each gram of hemoglobin bind? 1.39 mb. What is PO;? The partial pressure of oxygen. Arterial PO: assesses the ability ofthe lungs to oxygenate the blood. It is used as an indirect measurement of the body's tissue oxygenation status. The normal PO; of arterial blood is 80-110 mmoVI. ‘What does oxygen saturation indicate? ‘The percentage of heme groups that are carrying oxygen. Oxygen saturation is used to assess a patient's oxygen status. Normal oxygen saturation of arterial blood is > 95%. 6828. 29, 30. 3. 32, 33. 34. 35. 36. a. CLINICAL CHEMISTRY REVIEW ‘What specimen is required for blood gases? Heparinized arterial blood. The specimen should be iced and should not contain air bubbles. Ifthe patient was hyperventilating when the sample was drawn, how would blood gases be affected? PCO; would be decreased, pH increased, and PO increased. If an arterial blood sample was held at room temperature for one hour before testing, how would the results be affected? Cells would continue to use oxygen and produce carbon dioxide so the PO; would decrease and the PCO; would increase. The pHl would decrease If an arterial blood sample was exposed to air before testing, how would results be affected? PO; would be increased, PCO: would be decreased, and the pH would be increased. How can ea ary blood be arterialized? By warming the site prior to collection, How is blood pH measured? By a potentiometric measurement with a pH electrode on a blood gas analyzer. A potentiometric measurement is one based on a change in voltage. The pH electrode is a H” sensitive glass electrode containing a silver/silver chloride wire in an electrolyte of known pH and a reference (calomel) electrode, How is PO; measured? By an amperometric measurement with a PO: electrode (Clark electrode) on a blood gas analyzer. An amperometric measurement is one based on the amount of current low. Thé PO; electrode has a silver/silver chloride anode and a platinum wire cathode. It is suspended in a KCI solution and is covered by a selectively permeable membrane. How is PCO; measured? By a potentiometric measurement with 2 PCO: electrode (Severinghaus electtade) on a blood gas analyzer. The PCO: electrode is modified pH electrode covered with a membrane permeable to COs. How is oxygen saturation measured? By an oximeter that measures absorbance or reflectance at isobestic and differential points. An isobestic point is a wavelength where reduced and oxyhemoglobin have the same absorbance or reflectance, for example 805 nm. A differential point is where they have different absorbance or reflectance, for example 650 nm, Some oximeters also measure carboxyhemoglobin, methemoglobin and sulfhemoglobin by using additional wavelengths. What is Ps? ‘The partial pressure of oxygen at which the oxygen saturation in $0%. A low value indicates increased oxygen affinity (shift to the left in the oxygen dissociation curve). A high value indicates decreased oxygen affinity (shift to the right) 6938, QUICK REVIEW Q.& A'S How is the oxygen dissociation curve affected by decreased 2,3-DPG? Itis shifted tothe lef. Increased affinity of hemoglobin for oxygen results in decreased release of oxygen to the tissues. Levels of 2,3-DPG decrease in stored blood TOXICOLOGY AND THERAPEUTIC DRUG MONITORING What are the most widely abused drugs in the U.S.? Amphetamines, cocaine, cannabinoids, opiates, barbiturtes, benzodiazepines, methadone, methaqualone, and phencyclidine (PCP). ‘What are other names for marijuana? Cannabinoids or THC (tetrhydrocannabino. ‘What is the most common method for screening for drugs of abuse? Immunoassays such as EIA and FPIA. Confirmation of presumptive positive is mandatory in forensic drug testing. The analytical method of choice for confirmation is gas chromatography with mass spectroscopy _ detection (GCIMS), How can an altered urine specimen for drug testing be detected? Dilution is recognized by a specific gravity less than 1.003 and a creatinine less than 20 mg/dL. Substitution is recognized when specific gravity is less than 1.001 or greater than 1.020 and creatinine is less than $ mg/dL. ‘Adulteration is present if nitrite is greater than 500 jgimL or if the sample contains a substance that is not ‘normally found in urine or is found at a concentration higher than physiologic. The temperature of urine immediately following collection should be 90.5*-98.9°F. Spot and strip tests are available to detect commercial adulterants that are not detected by routine specimen integrity tests Discuss the Substance Abuse and Mental Health Services Administration's (SAMHSA) recommended protocol for collection of urine for drug testing. ‘The water supply should be tumed off in the collection area. Bluing should be added tothe toilet water. Patents should not be permited to take coats, purses, etc. into the collection facility ‘Why should serum separator tubes not be used for most therapeutic drug monitoring assays? Some drug concentrations are affected by contact with some types of gel separator tubes, What isthe role ofthe medical review officer? To determine the cause of positive drug test results and provide counseling. What method is most often used to perform therapeutic drug monitoring? Immunoassay. 7012, 13, 14, 15, 16. 17, 18. 19. CLINICAL CHEMISTRY REVIEW Name the most common anticonvulsant used to control grand mal seizures. Phenytoin or dilantin. ‘What is the major metabolite of the anticonvulsant primidoni Phenobarbital. When a primidone assay is ordered, phenobarbital must also be assayed for complete result interpretation. Name a drug that relaxes the smooth muscles of the bronchial passages. Theophylline, It is used to treat asthmatics. What is the metabolite of theophylline? Caffeine, e When should blood be drawn for a peak drug level? Upon achievement of steady state. Steady state is reached when the amount of drug absorbed and distributed is equal to the amount of drug metabolized and excreted. This usually occurs after $—7 half-lives. The half-life of a drug is the time required for the concentration of the drug to be decreased by half. ‘A patient is to be given an orai dose of a drug every 8 hours, The drug has a half-life of 8 hours. After how many doses should the patient reach steady state concentration? 2) STA yy When should blood be drawn for a trough drug level? Just before the next scheduled dose Name three anti-arrhythmies. Lidocaine, digoxin, and digitoxin. 7 7 7 Which antibiotics are monitored by therapeutic drug monitoring? Aminoglycosides (amikacin, gentamicin, neomycin, netiimicin, kanamycin, streptomycin, tobramycin), chloramphenicol, and vancomycin, These antibiotics have toxic effects such as nephrotoxicity and ototoxicity outside of the therapeutic range. Which hemoglobin is elevated in a patient with carbon monoxide poisoning? Carboxyhemoglobin. Levels are determined on a co-oximeter. Carbon monoxide is toxic because the affinity of hemoglobin for carbon monoxide is 200 times that of oxygen. How is lithium measured? By flame photometry, ion selective electrode, or atomic absorption. Lithium is administered to treat manic- depressives, Levels must be monitored to avoid toxicity. 7exmmeenme PEELE eeeteereeee TUMOR MARKERS 1 QUICK REVIEW Q& A'S ‘What are tumor markers? ‘Tumor markers are constituents of healthy cells that are produced in large quamty by cancer cells How are tumor markers used clinically? “They ate used to assess the response to cancer reatment and to moniter for recurrence of the cancer, is changes in concentration over months or yeas thai significent, rather than a single value. Consecuively rising levels are an indicator thatthe cancer has returned, Tumor markers are nt used o screen fr cancer because their specifiy is low and thee would be many false postive resus. ‘What isthe clinical significance of earcinoembyronic antigen (CEA)? CCEA is substance normally found in fetal tissu. fs found inthe blood of patients with cancer of the colon and other types of cancer. CEA testing is not approved s screening method for detecting cancer. ls primary application if for monitoring patients who have been treated for colorectal cancer. A steadily rising CEA level may be the first sign thatthe cancer has retuned. CEA is measured by enzyme immunoassay. What is alpha-fetoprotein (AFP)? AFP isa protein produced bythe liver ofthe fetus that declines ro adult levels by 18 months of age. It is elevated in the serum of patients with cancer ofthe liver. testicles, or ovaries. Matemal serum AFP s one of the tests inthe maternal serum triple or quadruple screening test tht i done between 16 and 18 weeks of gestation to diagnose spina bifida, anencephaly, and Down's syndrome, The other tests in the triple screen are bete-HCG and unconjugated estriol. The quad screen also measures the hormone inhibin. A. ‘What isthe role of human chorionic gonadotropin (hCG) inthe management of cancer? It is used to monitor patients with testicular cancer and gestational trophoblastic disease (e.g molar pregnancy) What is CA-125? ‘Cancer antigen (CA) 125 is an oncofetal antigen thats sometimes present with ovarian and ater types of cancer, ‘The CA-125 testis used to monitor, not to diagnose, ovarian cancer. Decreasing levels indicate thatthe cancer is ‘esponding to therapy. Increasing levels indicate a return or continued growth of the cancer, Tere are no preseily no reliable sereening tests forthe diagnosis of ovarian cant What are CA 18.3 and CA 27.29? ‘They are cancer antigens tha are used to monitor therapy in patents with breast cancer and to evaluate for cancer, _ recurrence ‘What is the clinical utility of CA 19.97 In patients with pancreatic cancer, CA 19-9 levels comelate well with the stage ofthe disease. CA 19-9 is a Lewis, blood group antigen so it is of no rity in Lewis-negative individuals, 729. M. CLINICAL CHEMISTRY REVIEW ‘What is prostate-specific antigen (PSA)? ‘A matker for prostate cancer. PSA isthe only rumor marker approved for general screening, PSA has replaced acid phosphatase because of its increased sensitivity ‘What is the clinical utility of thyroglobulin (Te)? {tis useful in the management of patients with thyroid cancer. Following total thyroidectomy and/or radioablation, and in the absence of recurrent disease, Tg should be undetectable. AntithyFoglobulin antibody levels should also be measured because their presence can render Te results invalid ‘What method is used for testing for tumor-associated antigens? Immunoassays. CLINICAL CHEMISTRY INSTRUMENTATION 5. ‘Which methods used in clinical chemistry are photometric methods? Spectrophotometry, flame emission photometry, slomic absorption spectrophotometry, and fluoromety Is light of 340 nm is in the ultraviolet, visible, or infrared range? Ultraviolet. The visible range is from 400-700 nm. Infrared is from 750-2000 nm. What light source is used for work inthe ultraviolet range? Deuterium-discharge or mercury-are lamp. What light source is used for work inthe visible and nea Incandescent tungsten or rungsten-iodide In.a spectrophotometer, what isolates light ofa specific wavelength from white light? “The monochromator. Filters. prisms, of diffraction gratings are used. Diffraction gratings are most commonly used. ‘What is bandpass on a spectrophotometer? “The widih ofthe segment ofthe spectrum that will be isolated by a monochromator. The better the spectrophotometer, the narrower the bandpass. ‘What isthe purpose of a photodetector in a spectrophotometer? It converts light energy into electrical energy. What isthe advantage of a double beam spectrophotometer? Ithas a reference light beam and a measuring light beam, Variation in lamp intensity or detector sensitivity is clpensaed for by expressing the absorbance as arti between the reference light beam and the measuring ight ‘beam B% 10. un id 1B. 4 15. 1. QUICK REVIEW Q.& A'S, What isthe relationship between percent transmittance (%T) and absorbance? A= 2-log%T. Why should al the cuvettes used in a series of colorimetric readings be of the same diameter? ‘So that the light path through the solutions is the same. What type of cuvettes are required for UV and infrared work? Quartz, Borosilicate cuvettes are used in the visible range. What is the purpose of a blank in spectrophotometry? Ii subtracts out any absorbance due to the reagents alone. ‘According to Beer's law, wliat is directly proportional to'concentration? Absorbance, Ina colorimetric procedure that follows Beer's law ifthe 100 mg/dL standard has an absorbance of 25 and the absorbance of the unknown is .15, what is the concentration of the unknown? Concentration of the unknown = &.of the unknown x concentration of the standard A of the standard Concentration of the unknown = 1S x 100 25 Concentration of the unknown = 60 mg/dL. What is the concentration of an unknown, ifthe absorbance of a I: absorbance of the 100 mg/d standard is 0.18? dilution of the specimen is 0.3 and the Concentration of diluted specimen = A of the unknown x concentration ofthe standard : ‘A of the standard \ Concentration of diluted specimen = 0.3 x 100 = 200 mg/D! os Concentration of undiluted specimen = 200 x 2 = 400 mg/dl. “ ‘What is the main advantage of fluorometry over spectrophotometry”? Fluorometry is much more sensitive. In a fluorometer, how does the light emitted by the specimen differ from the light absorbed? “The emitted light is of a longer wavelength (lower energy). 7420. a 2 23, 26. CLINICAL CHEMISTRY REVIEW Why is the photodetector in the fluorometer placed at right angles to the light source? So that only light emitted from the sample is measured and not light from the light source. What isthe principle of flame emission photometry? A photon of light with a wavelength specific for a given element is emitted when orbital electrons excited by heat reum tothe ground state. Once used io measure sodium and potassium, today use ofthe flame photometer is limited to measuring lithium, ‘What is the purpose of the internal standard in flame photometry? To minimize the effect of variations due tothe rate of atomization, lame stability, viscosity of the solution, or fluctuations in gas/air flow rates, Lithium or cesium is added to al calibrators, blanks, and samples in equal concentrations, The flame photometer makes a comparison of the emission of the analyte withthe emission ofthe internal standard. What is the light source in atomic absorption spectrophotometry? A hollow cathode tube with cathode of the material tobe analyzed. Atomic absorption is used to measure trace metals, Its te reference method for calcium (but not routinely used) and is one of the recommended methods for measuring lead. ‘What does a nephelometer measure? Light scattering by antigen-antibody complexes in a Solution. This method is used to measure immunoglobulins, immune complexes, complement, and other proteins such as transferrin and prealbumin What is the difference between nephelometry and turbidimetry? ‘Turbidimeters and nephelometers both measure the intensity of light scattering. Turbidity decreases the intensity of the beam of light as it passes through a solution of particles. The measurement is at 180°, just as absorbance is measured in a spectrophotometer. Turbidity can be measured on most spectrophotometers and automated chemistry analyzers. Nephelometers measure scattered light at right angles to the incident light, much like a fluorometer Nephelometry is a more sensitive technique when measuring low-level antigen-antibody reactions. What isthe basis for separation of compounds in chromatographic procedures? ‘Chromatography separates solutes based on their differential attraction to the stationary phase as they ar¢ carried through in the mobile phase. In thin layer chromatography, what is the Rf value? The ratio ofthe distance moved by the compound tothe distance moved by the solvent. What type of substances can be analyzed by gas chromatography (GC)? Volatile substances or substances that can be made volatile, These compounds are identified by their retention time. Ethatiol can be measured by GC.27. 28. 29. 30. 3. 32. 33. 34. 38. 36. Uy QUICK REVIEW Q & A'S In high-performance liquid chromatography (HPLC), how is the concentration of the analyte determined? By the peak height ratio (height of the analyte peak / height of the internal standard peck) What is a discrete analyzer? One in which each sample and accompanying reagents are in 2 separate container. Discrete analyzers can run on est ‘on multiple samples or multiple tests on one sample. They are the most widely used analyzers today, having almost ‘completely replaced continuous flow and centrifugal analyzers ‘What is a random access analyzer? One which allows measurement of variable number and variety of tests on each specimen. Only discrete analyzers offer random access and stat capabilites. ‘What is the most common type of measurement used in today’s chemistry analyzers? Visible and ultraviolet light spectrophotometry. lon-selective electrodes and other electrochemical techniques are also widely used. What is the name of the component that produces a specific wavelength of light on analyzers that use spectrophotometry? - ‘A monochromator. Most new systems use diffraction gratings to separate light into its component colors. What does a photodetector do? It converts light into electrical energy. The electrical impulses are sent 10 a readout device, such as a printer or computer. What are some common types of measurement used in Immunochemistry analyzers? Fluorescence-polarization immunoassay, nephelometry, and chemiluminescence What types of substances are measured by immunochemistry analyzers? ‘Drugs, specific proteins, tumor markers, and hormones. Immunochemistry techniques are very sensitive. ‘What is the purpose of a bar code label? It provides patient identification and demographics and may also include tests ordered, What are some techniques being used to reduce the time required for specimen clotting, centrifugation, and transfer of the specimen to the sample cup? 4, Use of plasma separator tubes and primary tube sampling 5. Use of whole blood for analysis (primarily on point-of-care analyzers) 6. Use of robotics, or front-end automation, to centrifuge, decap, and aliquot the samples 7631. 38. 39. 40. 41. 2. 43. 44, 45, 46. 47. CLINICAL CHEMISTRY REVIEW ‘What are some mechanfsms used on automated analyzers to reduce exposure of the sample to air, evaporation, and loss of CO;? Lid covers for trays and individusl caps that can be pierced (including closed tube sarnpling from primary collection tubes). ‘What is the purpose of a liquid-sensing probe? |i controls the depth that the sample probe {20¢S to allow aspiration of the correct amount while avoiding clogging of ‘the probe with serum separator gel or clot ‘What are some conditions that can lead to sampling errors on an automated analyzer? Inadequate amount of specimen, fibrin clots or ar bubbles in the boitom of the cup can lead to falsely low values ‘What is carryover? ‘The unintended transfer of analyte or reage adequate cleaning of the probe, tubing, sample, ent from one specimen reaction to another. Carryover can be minimized by and cuvets between samples or by use of a separate probe or tip for each In automated analyses, what else is analyzed along with patient samples? Controls and standards fa laboratory has more than one instrument to measure a specific analyte or analytes, what must be done? ‘The instruments must be calibrated to give comparable results Before attempting to repair any electronic equipment, what should be done? The instrument should be tumed off and unplugged. What is relex testing? Testing that automatically occurs based on an abnormal result fora related test. For example, a high TSH can automatically trigger performance of a free TA. ‘What is the name of the computer software that enables a chemistry analyzer to "talk" to the laboratory information system to receive test orders and to transmit test results? Bidirectional interface. ‘What is point-of-care testing (POCT)? ‘Testing that takes place outside of the clinical laboratory. POCT is often performed by other healthcare workers. What is the advantage of point-of-care testing? ‘The theory is that faster tumaround time leads to better patient care and reduced healtheare costs because of shorter patient stays, 7QUICK REVIEW Q & A'S 48, What are the disadvantages of point-of-care testing? ‘The tests may be more expensive and less precise and accurate than those performed in the central laboratory. They are often performed by individuals with no clinical laboratory background or understanding of quality control. The quality of POCT is improved when it is overseen by the clinical laboratory. 49, Name several tests that are currently av: ble by point-of-care testing. Glucose. electrolytes. BUN, creatinine, lactate, ionized calcium, cardiac markers, BNP. blood gases, activated clotting time (ACT), prothrombin time (PT), hemoglobin, hematocrit, and others. The list is rapidly expanding, CASE STUDIES 1. A patient has an elevated total bilirubin, a slight increase in alkaline phosphatase, 2 marked increase in AST and ALT, and an increase in LD 4 and 5, What might cause these results? Acute viral hepatitis )) An elderly man admitted to the ER following an automobile accident has an elevated AST and CK. The CK- MB is within the reference range. What might account for these results? Skeletal muscle injury. 3. ‘An unconscious patient is admitted to the ER. The following test results were obtained: 138 mmoUL. 3.5 mmo’, Chloride 95 mmoVL 1 co, 20mmoVL + Glucose 90 mg/dL. BUN 20 mg/dl. _ Serum osmolality 300 mOsm/Re Calculate the anion gap and the osmolal gap for 1 patient and interpret the results. (Nal +K7)~(Cl'+CO3) (135 + 3,5) ~ (95 + 20) = 23.5 (Reference range = 10-20) Calculated osmolality = 2 Na” mmoVL. + glucose mg/dL + BUN me/dL. 20 3 = 2(135)+90+20 20 3 =2812 Osmotal gap = measured osmolality ~ calculated osmolality = 300- 281.2 18.8 (Reference range = -10 mOsnvkg) «sf The increased anion gap and osmolal gap indicate thatthe patient's serum has an abnormal concentration of an ‘unmeasured substance such as isopropanol, methanol, ethylene glycol, or acetoue. 784. CLINICAL CHEMISTRY REVIEW ‘An 18-year-old college student was found unconscious in his dorm room. He was taken to the emergency room where it was noted that his breathing was deep and rapid and that his breath had a “fruity” odor. Examine the results of his laboratory tests. What is the diagnosis? bah REFERENCE RANGE 135-145 35-50 cl 2 e 98-106 mol/L [coz 9 y [2329 mmol, Glucose 750 = 70-110 mg/dL. Creatinine 09 a 05-12 mg/dL BUN 22 f 10-20 me/dL. Total protein 65 64.83 yal Albumin 38 37-55 gil Cholesterol 190 <200 mydL Triglycerides 150 <200 mgdL Uric acid $2 J [2672 mpl Total bilirubin 03 a 0.210 meidL AST 3B $30 UL ALT 19 ; 10-30 UL ALP [29 v7 20.90 UA LD 153 a 80-200 UL Calcium as [85-105 mg/dL Phosphorus 32 u {25-40 Iron 89 : 50-150 pe/dl Serum ketones 3 x Neg Urine glucose ‘The patient has diabetic ketoacidosis, a type of metabolic acidosis. The lungs are trying to compensate by lowering the CO; Blood gases should be performed to determine the blood pH. This patient's serum osmolality and anion gap ‘would be increased, 795. QUICK REVIEW Q & A'S ‘The following lab results are from a 20-yeer-old woman with complaints of anorexia, mi ‘What is the most likely diagnosis? ise, and edema. TEST RESULT REFERENCE RANG UNITS, Na 130) 135-185 mmol/L K 53 35.50 rama ter 2 [98-106 mmol co? hie 23.29 [ moi. Glucose 100) 70-110 meidl Creatinine 23 [osi2 [ mera | BUN oF 10-20) meidL, Total protein 40 64.83 gal ‘Albumin ra 3155 gid. Cholesterol 400 ft) <200 mgidL, Triglycerides: 350 <200 mgfdL Unc acid 89 2612 mgldl, Total bilirubin 03 02-10 rgidl, AST 3 [30 vA. _| | ALT 19 10:30 vn. ALP, 9 20.90 Lo 153 80.200 | Calcium [33 85-105 [Phosphorus _ [46 25-40 Iron 34 [50.150 [gs This patient has signs of renal disease (increased BUN, creatinine, and uric acid). The decreased protein and increased lipids are suggestive of the nephrotic syndrome. The classic findings of the nephrotic syndrome are /erotinura igdura, byposlbyrinemia, hypelipidstia and ede, With nephvote syndrome ther is increased glomerular permeability and fnassive loss of protein inthe urine. The decreased plasma protein leads to edema as fluid moves fromthe blood vessels into the tissues. The patient should have a urinalysis and a quantitative urine protein, With nephrotic syndrome oval fat bodies and a variety of casts, including fatty casts, are seen inthe urine sediment, Serum protein electrophoresis would show decreased albumin and increased alpha 2-globulin, ‘A 31-year-old female was admitted to the ER with “kife-like” abdominal pain, nausea, and vomiting of two days duration, What additional testing should be performed? TEST [RESULT REFERENCE RANGE _| UNITS. Ne 143 135. amet. K a 35:50 ramoVL. ch 95 [98-106 mmoV. (coz 0 [329 mmol 80CLINICAL CHEMISTRY REVIEW (Glucose 250 med. [Creatinine Wo 05-12 [maja ay 16 1020 Teal otal protein 35 6483 yal Albumin 30 3755 eidl (Cholesterol 190 «200 mga Triglycerides 400 < 200 me/dL. Uric acid 52 2672 moa. Foil bilirubin 13 02-10 mya [Amylase 1000 FF 60-180 sua AST Bo 5330 un [ALT 19 1030 uA. ALP 9 20.90 UL LD. 13 80-200 ua (Calejum is. 85-1058 mea Phosphoras 32 25-40 mel ron 89 50-150 [ug/l “The symptoms and elevated amylase are suggestive of acute pancreatitis. Since amylase isnot spe pancreatitis, a lipase should be dane for confirmation, Laboratory findings in acute pancreatitis include increased 5, ligase, triglycerides, and hypoproteinemia, Caleium may be increased or decreased. ‘The following results are from a laboratory technician who has been feeling unusually tired for the ast month and reports a loss of appetite and frequent nausea, Suggest a diagnosis and further laboratory tests for acute TEST [RESULT | REFERENCE RANGE UNITS. [xe 0 135145 [molt | x 43 7 3580 nol cl ee 98-106 mmol con 5 Z [mmol Glucose 90 [mga Creatinine 09 mg/dl BUN 2 mg/d “Total proein 65 wil [Albumin [38 yal Cholesterol ae <200 afd Trilyeerides 150 <200 mal Uric aid 32 26.12 mul Toalbilinbin | 20 RK [oza | mel AST 650 a $30 ut pele OFQUICK REVIEW Q & A'S ALT 525 10-30 UL ALP 130 20.90 uA. LD 30 80-200 ui Calcium 85 a 85-105 mg/dL | Phosphorus 32 [2540 med ron 89 50-150 ngdL The elevation of bilirubjn and liver enzymes. particularly the very high levels of AST and ALT, suggest viral hepatitis. This patient is at risk for occupational exposure to the bloodbome pathogens hepatitis B and hepatitis C. Serologic tests for acute hepatitis, including anti-HAV, HBsAg, anti-HBe, and anti-HCV, should be performed. A patient suspected of overdosing on drugs is admitted to the ER. Interpret the results of his arterial blood gases. Which values afe expected to change with compensation? TEST RESULT REFERENCE RANGE ITS: pH ~ 735-745 PCO: sot 35-45 mm Hg HCO; 23 23.27 mmol. | PO; 72 80-110 mm He ‘The decreased pH and increased PCO, are indicative of respiratory acidosis’ The HCO; should begin to increase as the kidneys attempt to compensate. This will move the pH toward normal A 6Seyear-old man presents with long term back pain and chronic fatigue. Examine his lab results and suggest additional tests to establish a diagnosis. cE Sn) Na 140 : 135-145 mmol/L K 45 _7_|35-s0 mmol. Lc 99 Z [98-106 mmol/L co2 25 7 23.29 mmol. Glucose 90 70-110 meld Creatinine 24 05-12 mgd BUN 40 10-20 mydl | Toul protein 140 64-83 idl. | Albumin 27 3.155 gal . Cholesterol 190 i < 200 meal. “Triglycerides 150 : < 200 mya Uric acid 80 ¥ 26-12 mpd {Total bitirabin [os _[02-10 meld. 4 82i ea Be tal 10, CLINICAL CHEMISTRY REVIEW UL | ALT UL. i ALP UL wb UL Caleium mya. Phosphorus meal Iron upd ‘The patient's symptoms and his elevated total prove increased BUN, creatinine, and uric acid point io rene! = Hypercalcemia also occurs with multiple myeloma. be performed. Multiple myeloma is characterized by @ presence of Bence Jones protein. Other lab findings in mi anemia and elevated erythrocyte sedimentation rate, The bo Rouleaux may be seen on the blood smear and may interfere bulins are suggestive of multiple myeloma, The 2 common complication of multiple myeloma. resis and urine electrophoresis should hy. Urine electrophoresis may show the <"ude a normocytic, normochromic “cas sheets or clusters of plasma cells. tests ‘An 18-month old boy was taken to the ER after the mother found his: :0 the garage next to an open bottle of ethylene glycol. What tests should be ordered? Electrolytes, blood gases, and serum osmolality. With ethylene glycol p increased serum osmolality, and metabolic acidosis, ‘ll be an increased anion gap, Interpret the following results obtained on blood drawn from a middle-aged busines: employment physical. cutive during a pre~ f TEST RESULT REFERENCE RANGE | UNITS. Na 140 | 135-145 K _-| 3.5-5.0 mmobl, i a [9106 mmol. | coz 23-29 mmol/L i Glucose 70-110 mg/dl. { Creatinine 0512 mel, | BUN 10-20 mpd | Total protein | 64:83 gidl | Albumin 375.5 eid | Cholesterol <200 meld | Triglycerides <200 mgidl } Uric acid 20 ‘ 2.6.7.2 mg/dL. i Total bilirubin 15 f02.10 mgd. i AST 120 SX [s30 _[ut i ALT 60, t 10.30 UL, | [ave a) 4 20-90 UL | 83LD GGT mw 645 va Calcium 90 4 8.5-10.5 mg/dL. i Phosphorus Se 25-40 meidL Tron 9 ——~\ The elevation of liver enzymes_panicularly GGT. is suggestive of alcohol abuée, The elevated cholesterol and triglycerides suggest that the patient may be al risk for coronary antéry disease. HDL and LDL cholesterol should be ordered. 12, AS3-year-old runner experienced chest pain near the end of a marathon, He completed the race and when the pain persisted, went to the ER for evaluation. At hours after the onset of pain, total CK was elevated with CK-MB 1% of total CK. Did this patient have « myocardial infarction? What other tests could be done? ‘With MI, the CK-MB is elevated in 4-8 hours and is > 6% of total CK so it appears that this patient did rot have an MI. Myoglobin or cardiac troponins could be done to rule out Ml. The elevated total CK was probably due tothe intense exercise ofthe marathon 13. The following results were abtained on a patient admitted tothe hospital for treatment of renal calcul, Examine the results and suggest follow-up testing, [test ILT__|_ REFERENCE RANGE UNITS —] [Na M0 [i3saas mmol K 45 3550 mmol, | cl ira [ ramet co [a7 [23.29 [mmoit. Glucose on Ton med Creatinine oo 05-12 mgldl BUN 18 | mrt | Tol protein 65 6483 wl ‘Albumin Bo [375s im Cholesterol 90 <200 mg/dl ‘Triglycerides 150 <200 gil : Uric acid [27 26.12 mya. | i Total bitirabin Lo! {02.10 me/dl, i AST Is fy $30 UL ; ALT 2 10.30 un ‘ ALP 30 {20-90 UL. & 150 {80-200 UL | : ns 85-105 med. fh 19) i 254.0 mg/dL u A 84 |CLINICAL CHEMISTRY REVIEW ‘The patient has hypercalcemia and hypophosphatemia. These findings are typical of primary hyperparathyroidism, a disorder resulting from excessive secretion of PTH. High levels of serum calcium result in excess calcium excretion inthe urine and may result in calcium-containing renal stones. A PTH should be ordered to confirm the diagnosis Interpret the following results from a 14-year-old girl being treated for acute lymphoblast leukemia, RESULT REFERENCE RANGE Na 140, 135-145, mmol/L 45 35:50 mmol/L 99 98-106 mmol/L coz ae 23.29 mmol. Glucose 90 70-110 mg/d Creatinine 10 05-12 med BUN n= 10-20 me/dL Total protein 65 6483, eid. ‘Albumin 38 3155 wal. Cholesterol 190 <200 mg/dL. Triglycerides 150 < 200 mg/dl Urie acid 20 26-12 mgidL Tota bilirubin 10 02:10 | meat AST 15 5330 UL ALT 20 10-30 UL. ALP 30 20.90 UL Lb 150 80-200 jun Calcium 85 85-105 mg/d Phosphorus 29 25-40 mg/dL The patient hasan increased uric acidThe three major disease tates associated with elevated urie acid are ott, renal disease, and increased nlelear breakdown, Renal disease can be ruled out in this patient because ofthe normal BUN and creatinine. Gout is most common in men between the ages of 30 and 50. The increased uric acid in this patient is due to increased nuclear breakdown from the chemotherapy. CLINICAL CHEMISTRY CALCULATIONS jy. bot 3 Convert 76°F to C, 04 ee] hap Ad sc+n ll Ve No 76 = 18°C +32 ‘ ui Cm (1632) 18 = 244" ne We L 856. QUICK REVIEW Q & A’>, TY Convert 100 microliters to milliliters. 0.1 mL Convert 1 deciliter to milliliters. 100 mL. How would you prepare a 1:10 dilution of urine? One part of urine and nine pans of diluent A glucose exceeded the linearity of the analyzer so the technician diluted $00 microliters of serum with milliliters of saline and repeated the test. The concentration of the diluted sample was determined to be 1: ‘mg/dL. What value should be reported? 400 mg/dL. The dilution was 0.5:2 or 1:4 so the value obtained must be multiplied by 4. A calcium is reported as 10 mg/dL. What is the concentration in mEq/L? (Ator weight of calcium = 40. Valence of calcium = 2+) mEq/L = mgidl. x 10 ow 7 GEW mEq/L = 10x10 = 5 20 A calcium is reported as 10 mg/dL. What is the concentration in mmol/L? (Atomie weight of ealcium = 40. Valence of calcium = 2+) mmoVL = mgidL x 10 GMW mmoV/L = 10x10=2.5 40 A calcium is reported as S mEq/L. What is the concentration in mmol/L? (Atomic weight of calcium = 40, Valence of calcium = 2+) mmoVL = meg valence mmol/l. ‘What is the molarity ofa solution that contains 48 grams of NaCl per liter? (Atomic weights: Na = 23, Cl= 35.5) rk \ Molarity = grams per lite Guay = C yy aa 86CLINICAL CHEMISTRY REVIEW 10. What is the normality ofa solution that contains 98 grams of H;SO, per S00 ml.? (Atomic weights: H = 1, $= 32, 0= 16) Nomality= gts pe lier») Bett oy GEN. . N= 196=4 49 11, What is the percent concentration of a solution that contains 8.5 grams of NaCl per liter? Percent concentration = grams or milliliters per 100 ml») 85g = _x , - 1000 mL “100 mi 1000 x = (8.5) x 100 x= 0.85% 12, Whats the normality of a 3 M H,S0, solution? x valence) xTEO 13, Whatis the molarity of 0.3 N H;S0. solution? MeN valence M=03=0.15 14, How many mL of 95% alcohol are needed to prepare 100 mL. of 70% alcohol? ViCi= Vics Vo 2 Vp ee (x) (95) = (100) (70) Vy Uy . x= 73.7 mL. “ans 87CLINICAL MICROBIOLOGY REVIEW GENERAL BACTERIOLOGY ‘Name the two mechanisms by which bacteria produce disease. Itivasiveness and toxigenicity. Define virulence. ‘The degree of pathogenicity of a microorganism. Name several bacterial virulence factors. Capsules, toxins, enzymes, cell wall receptors, and pli How do capsules contribute to the virulence of certain bacteria? They enable the organism to resist phagocytosis. Compare endotoxins and exotoxins. EXOTOXIN ENDOTOXIN Produced by gram positives Produced by gram negatives Secreted by cell Component of cell wall. Released when cell multiplies/dies System effect [Local effect High toxicity and antigenicity | Low toxicity and antigenicity Stimulates enti-toxin production | Does not stimulate ant-toxin production Can be converted to toxoid ‘Cannot be converted to toxoid What is a toxoid? A toxin treated to decrease its toxicity but retain its antigenicity. I is given to stimulate ant-toxin production. The seanus toxoid is an example, What is an antictoxin? .= exvitody against a toxin. Antitoxins are used to treat diseases caused by toxins, for example, botulism, 89NM. 12, 13, 14. 15, 16, 1. 18, QUICK REVIEW Q & A'S What kind of immunizat n results when a person is injected with a toxoid? Aniificially acquired active immunity. ‘What is an opportunistic pathogen? ‘An organism that does not cause harm in a healthy host, but which can cause disease in a debilitated or immunocompromised host What are some factors that can render an individual susceptible to opportunistic infections? Very young or very old age, immune deficiencies, bums, dialysis, diabetes, chronic diseases, alcoholism. chemotherapy, organ transplants, invasive medical techniques, and antibiotics. ‘What is a commensal organism? One that lives on or in the host without causing harm, for example, the normal flora of the skin, mouth, and G' ‘What is a nosocomial infection? ‘An infection acquired while in the hospital. Define zoonosis. {An infectious disease contracted from direct or indirect animal contact How do bacteria reproduce? By binary fission, The cell divides into two genetically identical cells, ‘What is generation time? ‘The time it takes for a population to double. This varies from approximately 15 minutes for E. coli to 24 hours for Mycobacterium tuberculosis. ‘What is a facultative anaerobe? ‘An organism that can grow with or without oxygen, Most pathogens are facultative anaerobes. What isan obligate anaerobe? ‘An organism that can not grow in the presence of oxygen, for example, Clostridium and Bacteroides. Moderately obligate anaerobes can tolerate some exposure to oxygen but require an oxygen-free environment for growth, Strict ‘obligate anaerobes are killed by even a few minutes of exposure to oxygen, so they require special handling to protect them from oxygen. ‘What is the optimum atmosphere for strict anaerobes? 80-85% nitrogen, 10% hydrogen, 5-10% carbon dioxide, and less than 1% oxygen.F 7A TET Se 19. 20. 2. 2. 2B. 24, 25. 27. 28, 29, CLINICAL MICROBIOLOGY REVIEW What is a capnophilic organism? ‘An organism that requires increased carbon dioxide, for example, pathogenic neissera, ‘What concentration of CO; is achieved with a candle jar? ‘Approximately 3%, This concentration is not sufficient for some capnophilic organisms such as Campylobacter. A (CO; incubator can be used to provide a concentration of 510% and commercial bio-bags are available for incubation of small numbers of plates What type of candles should be used in « candle jar and why? White unscented. Others may emit substances toxic to bacteria, Which type of organisms will grow in a candle jar? ‘Acrobes, facultative anaerobes, and capnophilic organisms. The candle jar is not oxygen fre. What is a microaerophilic (microaerobic) organism? ‘An organism that requires a reduced level of oxygen in order to grow, for example, Campylobacter. ‘What is the usual temperature of incubation for bacterial cultures? 35.37°C. Most human pathogens are mesophiles and grow best close to body temperature, Name two organisms that grow at 42°C. Campylobacter and Pseudomonas aeruginosa ‘Name two bacteria that can grow below 35°C. Listeria monocytogenes and Yersinia enterocolitica can grow from 0°-40° C, Cold enrichment may be used to enhance the recovery of these organisms. ‘What is the usual incubation time for bacterial cultures? ‘Most bacteria routinely encountered grow in 24-48 hours. Mycobacteria and some anaerobes require longer incubation, How do automated blood culture systems detect bacterial growth? ‘They monitor production of CO; of consumption of O> within the blood culture bottle. What are molecular diagnostic techniques? Techniques in which specific sequences of DNA or RNA are targeted for analysis. These methods allow for detection of microorganisms directly in the clinical specimen, gt30. ce 32, 33. 34, 35, 36. QUICK REVIEW Q & A'S What is DNA? Deoxyribonucleic acid, the chemical unit of the genes. DNA is composed of structural units called nucleosides == nucleotide consists of deoxyribose (a $-carbon sugar), triphosphate group, and a nitrogenous base: adeni thymine (T), guanine (G), or eytosine (C). Adenine and guanine are purines. Cytosine and thymine are py Inthe nucleus, DNA exists in a double-stranded configuration that is produced when two single-stranded DS's ‘molecules undergo complementary base pairing of A to T and C to G, The unique series of nucteotides in e2c' codes for a sequence of amino acids that make a specific protein, Compare DNA and RNA. Ribonucleic acid (RNA) is similar to DNA except that it contains ribose sugar instead of deoxyribose suger, uraci instead of thymine, and it only exists in the single-stranded form, Define: amplicon, amplification, anneal, and hybridization, + Amplicon= A DNA fragment produced by amplification of a specific DNA sequence ‘+ Amplification = A process to produce multiple copies ofa specific DNA sequence ‘+ _Anneal = Base-pairing of complementary nucleotides to form a double-stranded nucleic acid molecule ‘+ Hybridization = Interaction between two single-stranded nucleic acid molecules to form a double-stranded molecule What is polymerase chain reaction (PCR)? A nucleic acid amplification technique in which a target sequence of DNA or RNA is identified and amplified ‘enough so that it can be detected by gene probe technology. What are nucleic acid probes? Probes are labeled single-strand sequences of DNA or RNA that are complementary to the nucleic acid sequence to be detected. What are the reagents in PCR? + The four nucleotides (A, C, G, 1), referted to as deoxynucleotide triphosphates (ANTPs) + Primers (short sequences of nucleotides that are complimentary to a unique region of the target and that start the amplification) + DNA polymerase (an enzyme that adds nucleotides to the primers) ‘+ Magnesium (required for proper function of the DNA polymerase) ’ ‘+A buffer (required for proper function of the DNA polymerase) Explain the three steps in PCR + Denaturation: A high temperature (94 or 95° C) is used to separate target (template) double-stranded DNA into single strands. © Primer annealing: Single-stranded oligonucleotides bind to the ends of the DNA strands. This is usually carried out at $0-60° C. 92| | aw. 38, 3. 40, 41. 2. 3, CLINICAL MICROBIOLOGY REVIEW ‘+ Primer extension: DNA polymerase adds individual DNA nucleotides to the primers to synthesize anew ‘complimentary strand of DNA. Ifthe template contains an A nucleotide, the polymerase adds a T nucleotide to the primer. Ifthe template contains a G nucleotide, the polymerase adds a C nucleotide to the primer. These three steps take less than two minutes and result in a doubling of the target DNA. The steps are repeated 25-40 times to synthesize a detectable amount of amplified target, The procedure is carried out in a thermal cycler What is Tag DNA polymerase? Inis an enzyme isolated from the thermophilic bacterium 7. aguaticus and isthe most commonly used DNA polymerase used in PCR. It does not lose function a the high temperature needed to denature double-stranded DNA. What isa major cause of false-positive results with PCR? Contamination with nuclei acids. Serupuious technique is required. Extracted DNA from specimens must be kept separate and must not be allowed to contaminate PCR components. Operator DNA must not contaminate specimen DNA ot PCR components List several precautions to prevent contamination in PCR. + DNA extraction, assembly of PCR components, adition of DNA template, and anal bbe done on separate benches or in different rooms, using separate equipment and supplies + Aerosol-resistant pipette tips should be used. ‘+ Sample tubes and reagent tu ould be capped a allies when they are not being handled + Lab coats and gloves should be wor during all phases ofthe procedure. Gloves should be changed between samples or if they become contaminated, +All benches and equipment should be cleaned with a decontaminating solution (e.g. freshly prepared 10% bleach) before and after proce: ‘+ UV light can be used to destroy contamina primers are not exposed, ig nucleic ac but care must be taken that PCR components and ‘What methods are used to detect PCR products? Agarose gel electrophoresis and real-time PCR. What is real-time PCR? {A variation of standard PCR in which the product is measured throughout the procedure through use of dyes or fluorescent-labeled probes, Real-time PCR is faster than standard PCR analyzed by agarose gel electrophoresis. What is reverse transeriptase PCR (RT-PCR)? ‘A molecular method that can be used to detect RNA viruses. The RNA target is converted to complementary DNA which is then amplified by standard PCR. What are some microorganisms that can be identified by PCR? ; Chlamydia trachomatis, Neisseria gonorrhoeae, he Nile virus, enteroviruses, and others, its A virus, hepatitis B virus, hepatitis C virus, HIV, West 93QUICK REVIEW Q & A'S 44, What is ligase chain reaction (LCR)? ‘A molecular amplification method that uses DNA ligase, DNA polymerase, and pairs of probes. LCR is performed in a thermal cycler, like PCR. LCR is used to detect Chlamydia trachomatis and Neisseria gonorrhoeae. 45. What is branched DNA (bDNA) detection? ‘A moleculat method that uses signal amplification instead of target amplification. bDNA assays are being used to measure viral load of HBV, HBC, HIV, and CMV. SAFETY IN THE MICROBIOLOGY LABORATORY 1 Discuss the CDC classification of infectious agents, * Biosafety Level 1 (BSL-1): Agents with no known potential for infecting healthy people + Biosafety Level 2 (BSL-2): Agents that are most commonly encountered in clinical specimens + Biosafety Level 3 (BSL-3): Agents with potential for aerosol transmission; disease may have serious or lethal consequences. Ex: Cultures growing Mycobacteria tuberculosis, mold stages of systemic Fungi + Biosafety Level 4 (BSL-4): Agents that pose high risk of life-threatening disease. Ex: Ebola virus, smallpox virus 2 What isthe biosafety level of most hospi I clinical microbiology laboratories? BSL-2or3. ‘What is the biosafety level of Bacillus anthracis and Yersinia pestis? B. anthracis is BSL-2 for processing of clinical specimens and BSL-3 for all culture manipulations that might produce aerosols. Yersinia pestis is BSL-2. 4. Name several biosafety-level 3 pathogens, Mycobacterium wberculosis, Francisella, Brucella, Rickettsia rckettsii, Coxiella burneti, West Nile viru, and the ‘mold stages of systemic fungi 5. Describe the 3 classes of biological safety cabinets (BSC). + Class I: Room air passes into the cabinet. Only the air being exhausted is sterilized. This type is rarely used today. ‘© Class Il: Air flowing into and out of the cabinet is sterilized. * Class Ill: Completely enclosed; work is done with rubber gloves that are attached and sealed to the cabinet. Air coming in and going out is sterilized. Used in BSL-4 facilities. 94Ege 6 Te 2 14. CLINICAL MICROBIOLOGY REVIEW State the biosafety level for which each biological safety cabinet class is appropriate, BSC TYPE BSL LEVEL Which class of biological safety cabinet is used for most clinical work? Class IL How is ar sterilized in a biological safety cabinet? Most commonly by HEPA filters. UV light or heat are also used. ‘What work should be conducted in a biological safety eabinet? Any manipulation of clinical specimens, for example, plating and direct smear preparation, and all mycobacteriology and mycology. In most cases, patient specimens pose a greater risk to laboratory workers than bacteria in culture, so most plate-reading and follow-up testing is performed outside of the BSC. The exception is BSL-3 pathogens, which should always be bandled in a BSC. ‘When should masks be worn in the microbiology lab? Whenever there is a risk of aerosol formation during specimen handling and when working with AFB, fungsl, and other BSL-3 cultures ‘What are the 5 infections that have been most frequently acquired in the clinical microbiology laboratory? Shigelloss, salmonellosis, tuberculosis, brucellosis, and hepatitis. What is the difference between a bacteriostatic and bacterioeidal agent? A bacteriostatic agent inhibits the growth of bacteria; a bacteriocidal agent kills bacteria How do disinfectants and antisepties differ? ‘Antiseptis are used on living tissue to prevent or arrest the growth of microorganisms. Disinfectants are used on inanimate objects to kill microorganisms. Disinfectants vary in ther effectiveness in killing spore-forming bacteria and Mycobacteria ‘What is the phenol coefficient? ‘Avvalue which compares the killing power ofa disinfectant to that of phenol. 9515. 18, wD. 20. QUICK REVIEW Q & A'S. What is the most practical and dependable method of sterilization? Steam under pressure (autoclave). What are the pressure, time, and temperature routinely used for autoclaving? 15 pounds of pressure, 15 minutes, 121°C. Which type of bacteria is most difficult to eliminate from the environment? Bacteria that produce spores Is boi an effective method of sterilization? No, because spores can survive. Name a gas used for sterilization. Ethylene oxide. tis used to sterilize hospital equipment that can't withstand steam, Explain how to package a biological agent for shipping. The specimen is placed in a watertight primary container. The primary container is placed ina secondary container ‘with enough absorbent material to absorb the contents inthe event of breakage. The secondary container is placed in ‘mailing container labeled with an etiologic agent label, biohazard symbol, and phone number of the Centers for Disease Control and Prevention, All personnel who package specimens for shipment must complete certified training every 2 years. ‘SPECIMENS FOR BACTERIOLOGY 5. List general guidelines for proper specimen collection from a suspected site of infection, ‘The specimen should be obtained during the acute phase, before antibiotics are given. The appropriate site should be sampled and a sufficient quantity obtained. Contamination with normal flora should be avoided. The specimen should be collected ina sterile, leak-proof container and delivered tothe lab within 30 minutes of collection Which type of swabs may contain fatty acids that inhibit some strains of fastidious bacterla? Cotton. What type of swabs are s ble for collection ofa specimen for culture of Netsseria gonorrhoeae? Dacron or rayon, Calcium alginate and cotton may contain inhibitory substances. ‘What can be used as a preservative for stool specimens for culture? Cary-Blair, Stuarts, Amies, and phosphate buffered saline ‘What can be used as a preservative for urine specimens? Boric aca. 96fi REE 6 9. 10. 12. 13, 4, CLINICAL MICROBIOLOGY REVIEW How should specimens for microbiology be transported? Ina sealed secondary container such asa plastic zip-lock bag, The bag should be marked with biohazard symbol. ‘What information is required on microbiology requisitions? The patient's name, identification number, age or date of birth, gender, and diagnosis; the name of the ordering physician; the source ofthe specimen; information about antibiotic therapy, the tests requested; the date and time of | collection, and the date and time the specimen was received in lab List several causes for specimen rejection in the microbiology laboratory. ‘An unlabeled or improperly labeled specimen, prolonged transit time (over 2 hours without preservation), improper temperature during transport or storage, a leaking specimen, an improper transport medium, a dried out swab, a specimen received on a dried out or expired agar plate, wood ot calcium alginate swab for viruses or Chlamydia, a specimen submitted in an unsterle container, insufficient quantity, or an inappropriate specimen forthe culture requested. Asa rule, in what situation should every effort be made not to reject a specimen? For invasive specimens like tissue and CSF or other specimens that are difficult to recollect. The physician should be consulted, Whenever a specimen is rejected, the caregiver must be notified and the notification must be documented. Which specimens may be refrigerated? Sputum (up to 2 hours), urine (up to 24 hours), and specimens for viral culture (f the delay will not exceed 24 hours) Which specimens should never be refrigerated? Cerebrospinal fluid (CSF) and other body fluids, blood, cultures for. gonorrhoeae, and specimens for anaerobic culture. Some labs don’t refrigerate stool specimens either because Shigella may be killed at low temperatures. It is best to process stoal and respiratory specimens without delay. Ideally, CSF should be held at 37°C. Which microorganisms are particularly susceptible to adverse environmental condi temperature, and pH? ns such as atmosphere, Anaerobes, Chlamydia, Haemophilus influenzae, Neisseria gonorrhoeae, Neisseria meningitidis, Salmonella, Shigella, Sreptacoccus pneumoniae, viruses, and parasites. Specimens likely to harbor these pathogens require special consideration with regard to transit ime, preservation, and storage temperature ‘When is the ideal time to obtain a blood culture? ‘As soon as possible after a fever spike. How many blood cultures should be drawn? Usually 2-3 ses, each consisting of 2 bottles. At leat 2 venipunctures should be performed, bu all specimens can be collected at one time, Most authorities now believe that there's ltl to gain By dolletting specitieds at different times. (Koneman’s Color Atlas and Textbook of Diagnostic Microbiology, 2006) 7QUICK REVIEW Q & A'S 15, What volume of blood should be drawn for blood cultures? ‘The total volume of blood cultured should be approximately 30 mL in an adult. The manufacturer's instructions should be followed as to the optimum volume of blood for each bottle, the bottles hold 10 ml. of blood, two sets of cultures (2 bottles each) will satisfy the volume requirement. The total volume collected is more important than the ‘number of specimens obtained. (Koneman's Color Atlas and Textbook of Diagnostic Microbiology, 2006) 16, Which anticoagulant Is used for blood cultures and why? Sodium polyanethol sulfonate (SPS). I prevents clotting ofthe blood, has anli-complementary and anti-phagocytic properties, and inactivates some antibiotics. Many blood culture bottles also contain antibiotic-removing resins. 17. What ratio of blood to broth is recommended for blood cultures? is. 18. Which tube of CSF should be sent to the microbiology lab for culture and sensitivity and why? Tube #2. Its less likely to have skin contaminants than tube #1 BACTERIOLOGICAL STAINS 1 List, in order, the reagents used in the Gram stain and tell the purpose of exch step. REAGENT FUNCTION | Crystal violet Primary stain| Stains all cells purple Grams iodine Mordant Fixes crystal violet to cells Alcohol, acetone, or combination | Decolorizer Removes crystal violet from gram negatives Safranin Counterstain Stains gram negatives pink 2 What accounts for the different staining characteristics of gram-positive and gram-negative cells? The different cell wall compositions. Teichoic acid cross-links inthe thick peptidoglycan layer of the gram-positive cell wall enable it to resist decolorization. Large amount of lipopolysaccharide in the gram-negative cell wall allows decolorizer to enter 3 A technician performs a Gram stain of a colony growing on a blood agar plate. After careful examination, no bacteria can be located on the slide. What error might have been made? “The technician might have forgoten to fix the slide, Without fixation the specimen may be washed off during the staining process, Heat or alcohol (methanol or ethanol) ean be used for fixation, A direct smear of a specimen reveals many gram-negative cocci in chains, A control slide run at the same time reveals gram-negative cocci and gram-negative bacilli. What should be done? Since the Gram stain control slide does not give the expected results (gram-positive cocci and gram-negative bacilli), the procedure should be repeated. The slides were probably overdecolorized. The decolorizer should be ‘added drop by drop just until it begins to run clear. 9810. M4 CLINICAL MICROBIOLOGY REVIEW ‘What is the term to describe bacterial cells of one species that vary in size and shape? Pleomorphie. Name two things that might cause gram-positive bacteria to stain gram negative, A culture that is too old or overdecolorization during the staining process. All of the polys on a Gram stain appear blue, What does this mean? The slide was underdecolorized. ‘What is the significance of many polys on the Gram stain of a stool? It indicates an infection due to an invasive organism such as enteroinvasive B. coli, Salmonella, Shigella, Campylobacter, or Clostridium dificie. A colony growing on chocolate agar is Gram stained. The organisms stain pink and vary in size and shape. Some appear as very short rods, while others are longer. How should these findings be reported? Pleomorphic gram-negative rods ‘Which enteric pathogens aré associated with RBCs on a Gram stain of the stool? Enterohemorthagic E. coli (EHEC. Serotype 0157:H7 is most common) and Shigella How do yeast stain on a Gram stain? Gram-positive How can the Gram stain be used to assess the acceptability of a sputum specimen? I helps to differentiate sputum from saliva. A good sputum specimen will have 25 or more polys and less then 10 epithelial cells per low power field (unless the patient is neutropenic). Saliva will have more than 25 epithelial cells per low power field. ‘What Gram stain findings are indicative of bacteriuria? One or more bacteria per oil immersion field on a drop of uncentrifuged urine. This correlates 1 a colony count of 10° CFUImL. How is a Gram stain of CSF prepared? From the sediment. The CSF should be centrifuged for at least 15 minutes at 1500x g, Many labs use a cytocentrifuge preparation for increased Sensitivity. How isthe acridine orange sain used in microbiology? Acridine orange is fluorochrome dye that stains both gram-positive and gram-negative bacteria, living or dead. It is used to locate bacteria in blood cultures and other specimens where they are difficult to see withthe Gram stain. 99MEDIA 1 4, 9. 10, QUICK REVIEW Q & A'S. ‘What are enriched media? Media with supplements added to support the growth of fastidious organisms. Blood agar and chocolate gar are examples of enriched media, ‘What are selective media? Media with substances such as bile salts, dyes, or antibiotics added to inhibit certain bacteria. Examples of selective = media are CNA, PEA, EMB, MacConkey, XLD, Hektoen Enteric, Modified Thayer Martin, and Martin Lewis. What are differential media? Media formulated to provide distinct colonial appearance based on certain biochemical reactions such as lactose fermentation or hydrogen sulfide production. MacConkey, EMB, XLD, and Hekioen Enteric are differential media, What might cause blood agar plates to appear brown? Lysis of the RBCs. This can result from exposure to temperature extremes or from age of the medium. ‘What can be added to media to make it selective for gram negatives? Crystal violet, bile sats, eosin, or methylene blue. ‘What can be added to media to make it selective for gram posi Colistin, nalidixic acid, or phenylethy! alcohol ‘What type of bacteria grow on CNA and PEA? Gram positives. ‘When growth is observed on blood agar and MacConkey agar but not on CNA or PEA, what type of organism — has been isolated? ‘A gram-negative rod, ‘Why is chocolate agar used? To grow fastidious organisms such as Neisseria and Haemophilus. (Most other organisms also grow on chocolate agar.) ‘When js xylose lysine deoxycholate agar (XLD) used? For the recovery of Salmonella and Shigella from stool cultures, Hektoen enteric agar or SS ager may be used instead. 100E k b 12: 13, 14, 15, 16, 18, 19, 20. CLINICAL MICROBIOLOGY REVIEW ‘A pure growth of colorless colonies is observed on a MacConkey plate. What type of colonies would be expected on the XLD plate set up from the same specimen? Colorless or red colonies which may have black centers ifthe organism produces HS. Name four media specifically used for the isolation of anaerobes. CDC anaerobic blood agar, Bacteroides bile esculin ager (BBE), laked kanamycin-vancomycin blood agar (LKV), phenylethyl alcohol sheep blood agar (PEA), thioglycolate broth, and chopped meat broth ‘Will anything other than anaerobes grow in thioglycolate broth? Yes, Most aerobes grow as well. The location of the growth indicates the organism's oxygen requirements. Aerobic ‘organisms grow at the top of the tube, strict anaerobes at the bottom, and facultative anaerobeé throughout. In the Gulf states, where one might expect to isolate Vibrio, what medium should be added to those routinely used for stool cultures? Thiosulfate citrate bile salts sucrose (TCBS). Alkaline peptone water may be used as an enrichment broth. Which medium is best for recovery of Legionella pneumophila from clinical specimens? fered charcoal yeast extract agar (BCYE). Which medium should be used to recover Bordetella pertussis from a nasopharyngeal specimen? Bordet Gengou, Regan Lowe, or Jones Kendrick charcoal agar. What media are used to isolate mycobacteria? Lowenstein Jensen, Middlebrook 7H10 and 7H1 1, and broth medi ‘When are Thayer Martin or Martin Lewis agar used? To isolate pathogenic Neisseria from sites with normal flora. Antibiotics in these media suppress the normal flora E. coli was isolated on the Thayer Martin plates from several different patients in the same day. What might account for this unexpected finding? The antibiotics were left out of the medium or had deteriorated. This illustrates the importance of performing quality control on media, Which media are used for fecal / rectal cultures? A supportive medium (blood agar), a medium selective for gram-negative rods (MacConkey or EMB), a medium selective for Salmonella and Shigella (X1.D or Hektoen enteric), and Campylobacter blood agar. Some labs also use an enrichment broth for Salmonella and Shigella (GN, tetrathionate, or selenite broth). Many include MacConkey sorbitol agar (SMAC) for £. coli 0157:H7. In certain geographic areas, CIN agar for Yersinia enterocolitica and/or TCBS for Vibrio may be included 103QUICK REVIEW Q & A'S ry In some laboratories, MacConkey plates inoculated with stool specimens are incubated at room temperature when which fecal pathogen is suspected? Yersinia enterocolitica, The optimum temperature for Y. enterocolitica is 28-30°C. 22, When is bismuth sulfite agar used? When Salmonella is suspected. S. npphi produces black colonies surrounded by ¢ metallic sheen 23. Name a medium that is selective for Clostridium difficile. Cycloserine cefoxitin fructose agar (CCFA). C. difficile produces yellow ground glass colonies. 24, What media are inoculated for urine cultures? ‘Usually blood agar and MacConkey. Some laboratories prefer EMB to MacConkey and some also use a medium selective for gram positives, such as CNA or PEA. 25. What is added to holding media to absorb fatty acids present in the specimen that could kill fragile organisms such as Neisseria gonorrhoeae? Charcoal 26. Which cultures require incubation in increased CO;? Blood, CSF, genital, respiratory, wound, and cultures for AFB and Campylobacter, 27, Which plates are routinely incubated in increased CO, at 35-37°C? Chocolate, modified Thayer Martin or Manin Lewi , Human Blood Tween (HBT) and Vaginalis (V) ager. 28. Which plates are routinely incubated in increased CO, at 42°C? Campylobacter blood agar plates. 29, Why is media heated before autoclaving? To dissolve the agar. 30, How is most media sterilized? By autoclaving for 15 minutes at 1$ pounds of pressure (121°C.) 31. How should urea broth be sterilized and why? By filtration, Urea cannot withstand high temperatures, 32. How should most agar plates be stored? Plates are stored upside down to prevent condensation water from falling on the surface of the agar. Most media is stored under refrigeration. 102CLINICAL MICROBIOLOGY REVIEW GRAM-POSITIVE COCCI A swab of superficial skin lesion is received for culture. A direct smear is prepared and gram-positive cocci are seen. The culture shows a pure growth of moderate-sized, white, nonhemolytic colonies on blood agar, chocolate agar, and CNA, No growth is seen on MacConkey. What is the first biochemical test that should be performed? Catalase, to determine ifthe isolate is Staphylococcus ot Streptococcus. Staphylococcus is catalase positive. Streptococcus is catalase negative. ‘What is the reagent for the catalase test and how should it be stored? 3% hydrogen peroxide. It should be stored in the reffigerator in a dark bottle, Deseribe a positive catalase reaction. Immediate and rapid bubbling. Which bacteria grow on mannitol salt agar (MSA)? S. aureus, coagulase negative staphylococci, and micrococei will grow. The high salt concentration inhibits most others, S. aureus ferments mannitol and causes a color change in the agar from pink to yellow. S. saprophyticus may also. MSA is not used much today since more rapid tests are available for the identification of S. aureus. ‘What does the tube coagulase test detect? Free coagulase secreted by S. aureus. It reacts with fibrinogen in plasma to form fibrin (clot formation). The tube ‘coagulase testis still the reference method for the identification ofS. aureus. What does the slide coagulise test detect? Cell-bound coagutase (clumping factor) on the surface of the S. aureus the cell wall A gram-positive coccus resembling 5, aureus is isolated. A slide test for coagulase is negative, What should be done next? A tube coagulase, The slide testis only a screening test. ‘What type of plasma is best for the coagulase test? Rabbit plasma. ‘What types of tests may be used instead of the congulase test to identifly S. aureus? utination and passive hemagglutination. Inthe latex agglutination test, latex beads are coated with plasma, gen detects clumping factor and the immunoglobulin molecules detect protein A, a staphylococcal cell- a, In the hemagglutination test, sheep red blood cells are sensitized with fibrinogen, which detects tor. 103un. 4 15, 16 18. Interpret the following results of a latex agglutination test for S. aureus. ¥! QUICK REVIEW Q & A'S i ' | The testis invalid, Agelutination in the negative control indicates nonspecific agglutination, Material was received from an abscess. The smear showed many gram-positive coee in clusters with polys. There was growth on the blood agar plates, but nat on MacConkey. The colonies on blood azar ‘nouhemolytic and white, Catalase was positive and coagulase was negative. How should this organism be identified? Coagulase negative staphylococci Which Staphylococeus species causes urinary tract infections in sexually active young women? S. saprophyticus. In this population, S. sapraphyticus is the second most common cause ofeysttis, after E.col can also cause urinary tract infections in other populations and, in some cases, bacteremia, ‘A urine culture from a 20-year-old female grows 100 medium sized, round, white, nonhemolytic colonies on BAP. There is no growth on MacConkey Agar. A Gram stain of the culture shows gram-positive cocci in clusters. The catalase testis positive. The latex test for S. aureus is negative. What is the next step? ‘A novobiocin disk to nue out S. saprophyvicus. S. saprophyticus is resistant to novobiocin, other coagulase negative- staphylococci are sensitive, When should coagulase-negative staphylococci be identified to the species level? ‘When isolated in urine, blood and other normal ly sterile body Muids, and su cal specimens. ‘Some species of Staphylococcus produce an enterotoxin. What does it eause? Food poisoning, A gram-positive coccus is isolated on blood agar. It is catalase positive, modified oxidase (microdase) positive, susceptible to bacitracin, and resistant to furazolidone and lysostaphin. What is it? Micrococcus. Tis organism is rarely associated with infection. It is usually a contaminant that must be differentiated from Staphylococcus. Compare the appearance of colonies of S. aureus and group A streptococci. S. aureus colonies are larger, may be golden, and may produce a nartow zone of beta hemolysis compared to the diameter ofthe colony. Group A streptococci colonies are pinpoint, translucent, and produce a wide zone of beta hemolysis compared to the diameter of the colony. Lancefild's serological grouping ofthe streptococct is based on what cellular component? The cell wall C carbohydrate. 104: 20. a. 2. 2B. 24. 28. 29. 30. 's TAL MICROBIOLOGY REVIEW To which Laneefield group does S. p) = Group A To which Lancefield group does S. agaloctice belong? Group B. To which Lancefield group do enterococci belong? Group D What are the most common species of enterococci? E faecalis and E. faecium Name two streptococci that do not belong to any Lanceffeld group. Streptococcus pneumoniae and viridans streptococci Which Lancefield group causes most streptococeal infections? Group A, What is the only type of blood agar that should be used to determine hemolysis of streptococci? Sheep blood (5%), Describe alpha hemolysis, beta hemoty and gamma hemolysis on sheep blood agar. Alpha hemolysis is partial hemolysis; the colony is surrounded by an olive-green zone, Beta hemolysis is complete hemolysis; the colony is surrounded by a clear zone. Gamma hemolysis means no hemolysis; there is no change in the agar around colony Which Lancefield groups are usually beta-hemolytic? Groups A, B, C, F, and G. Which streptococci are usually alpha-hemolytie? S. pneumoniae and viridans streptococci Which streptococci are usually gamma-hemolytic? oup D. Enterococci are also usually gamma-hemolytic. A sputum culture grows small glistening colonies with depressed centers and raised edges, surrounded by greenish zones of hemolysis on a blood agar plate, What would probably be seen on a Gram stain of these colonies? The colonies resemble S. pneumoniae, so gram-positive lance-sheped diplococei would be expected. 105a 36. 3. 38, 39. 7 CK REVIEW Q& A'S + the previous question was mixed with a drop of 3% hydrogen peroxide, +, #2 {rere would be no bubbles, So ps Sus costar ssers.cve 10 optochin and positive for bile solubility? 4n alpha-hemols tic colony is isolated on blood agar. It is catalase negative. The Gram stain shows gram- positive cocei, An optochin test is performed and after overnight incubation, there is no inhibition of growth. Bile esculin is negative, What has been isolated? sidans streptococci, Identification to species level is not required unless isolated from a critical specimen such as ‘What is the advantage of “stab streaking” or anaerobi hemolytic streptococei? cubation of blood agar plates when looking for beta sure detection of beta-hemolysis by those streptococci that only produce streptolysin O. Hemolysis cocci is due to two enzymes, streptolysin O and streptolysin S, Most streptococci produce both hemolysins, sm only produces streptolysin O, hemolysis will not be seen on surface colonies because streptolysin abile, Stab streaking or anaerobic incubation preserves streptolysin O. Which beta-hemolytic streptococci are sensitive to bacitracin? Group A. Bacitracin susceptibility is an unreliable indicator for group A strep. Why is the bacitracin disk alone not recommended for the identi ation of group A streptococei? Itis not specific. Groups C and G streptococci may also be sensitive to bacitracin. Which beta-hemolytic streptococei are resistant to SXT (sulfamethoxazole and trimethoprim)? Groups A and B. Groups C, F, and G are sensitive Name a test preferred over bacitracin for the identification of group A streptococci. The PYR test because it is more specific than bacitracin for S. pyogenes. S. pyogenes is the only species of beta- hemolytic streptococci that will give a positive PYR reaction. (Enterococci are also PYR positive, but are usually nonhemolytic.) The enzyme L-pyrroghutamyl-aminopeptidase can be detected using commercially available PYR disks. The tests a rapid test. Identification may also be made with streptococcal grouping reagents A child complaining of fever and sore throat is examined in the physician's office. A rapid group A streptococcal antigen testis performed in the office and the result is negative. Control results were acceptable, What should be done and why? A culture because, although specific, rapid antigen tests are not sensitive. 10640. 41. 43. 44. 46. 48, 49. CLINICAL MICROBIOLOGY REVIEW Which streptococc! are CAMP positive? Group B. A positive result isan area of enhanced hemolysis (arrowhead formation) atthe junction of the Streptococcus and beta hemolytic S. aureus streaks. The CAMP test is rarely done now that more rapid identification methods are available. Which gram-positive cocci are most resistant to antibiotics and always requires sensitivity testing? Enterococci In what part of the body are the enterococci considered normal fora? In the’GI tract, vagina, and male urethra, Prenatal anogenital cultures are recommended in order to identify which group of streptococci? Group B streptococci (GBS). GBS is the leading bacterial cause of neonatal illness and death. It asymptomatically colonizes the vaginal or rectal areas of 10-30% of pregnant women. Ifa pregnant woman is colonized with GBS, antibiotics are administered during delivery to prevent infection in the neonate. Some laboratories are now using PCR to rapidly sercen for GBS at the time of delivery A vaccine is available to confer immunity to which species of Streptococcus? 5. pneumoniae, Immunization is recommended for the elderly, thos: have had a splenectomy. vith chronic respiratory disease, and those who Which species of Streprococcus causes dental caries and endocarditis? S. mutans, Name two diseases that follow untreated or partially treated streptococcal pharyngitis or skia infections. Rheumatic fever and glomerulonephiitis, The antistreptolysin O titer is increased with these diseases. What is the most common cause of subacute bacterial endocarditis? idans strepto Which organisms may cause bacteremia following dental procedures? Viridans streptocoeei A throat specimen is inoculated on a selective streptocaceal blood agar plate and a bacitracin disk is applied. After overnight incubation, beta-hemolytic colonies are observed but there is no growth next to the disk. Which Lancefield group has been isolated? Group A. Both group A and group B streptococci grow on selective streptococcal blood agar containing SXT but only group A is sensitive to bacitracin 10750. sl. 52, 33. 56. QUICK REVIEW Q & A'S Gram-positive cocct in chains are isolated on blood agar. The colonies are beta-hemolytic and catalase negative, What test(s) should be done next? Tests for beta-hemolytic streptococci include bacitracin or PYR to identify group A streptococci, CAM? ci hippurate hydrolysis to identify group B streptococci, ora slide agglutination test which identifies grc_zs = = 2 7 and G strepiococci. PYR and slide agglutination are more commonly performed today because the. =: 7 ihe classic tests. Which gram-positive cocei hydrolyze eseulin? Group D streptococci and enterococci Which gram-positive cocei hydrolyze esculin, grow in 6.5% NaCl broth, and are PYR positive? Enterococci atalase Gram-positive cocei in chains are isolated on blood agar. The colonies are gamma-hemols negative, What test(s) should be done next? Bile esculin, PYR, and 6.5% NaCl to differentiate group D streptococci, enterococci ar GROUP D STREP ENTEROCOCC! NSSTREP | Bile eseulin + I + | 0 [ : | o ‘ ‘ | ‘The bacitracin testis only performed on colonies of which heniolytic type? Bou. The optochin test is only performed on colonies of which hemolytic type? Alpha. ‘The CAMP test is only performed on colonies of which hemolytic type? Beta, A blood culture is received from a patient suspected of having endocarditis, The blood culture instrument flags the culture as positive. A Gram stain is prepared and gram-positive cocci in chains are observed, The blood culture bottle is subcultured to aerobic and anaerobic blood ager and chocolate agar. No growth is observed on any of the plates at 24 or 48 hours. What should be done and why? ‘The broth should be subcultured again adding a pyridoxal disk to detect 4biotrophia spp., formerly known as nutritionally-variant streptococei. These opportunistic pathogens, which are normal flora in the mouth, are gram- positive cocci that do not grow on blood or chocolate agars unless pyridoxal (vitamin B,) i8 added, ether by a pyridoxal disk or by cross-streaking with Staphylococcus 10858. 39. 60. 61. CLINICAL MICROBIOLOGY REVIEW Which Streptococcus has a distinctive Gram stain morphology that differentiates S. pneumoniae is the only strep that appears as lancet-shaped diplococci (ovoid with 22 x'#2 = The Gram stain of a sputum specimen shows many gram-positive cocei in chains an hemolytic colonies are seen on the primary sheep blood agar plate. Which tests could the organisms are S. pneumoniae or alpha streptococci? small alpha- 9 determine if ‘A rapid slide agglutination test for S. pneumoniae, an optochin test, or a bile-solub sensitive to optochin (P disk) and bile soluble. 5. pneumoniae is What is the principle of the bile-solubility test? Bile salts (e.g, sodium deoxycholate) activate the autolysins and accelerate t pneumoniae, The bile-solubility test can be performed on a broth culture or solubility is indicated by clearing of the broth suspension or disappears: 2! lytic property of S. ies growing on agar. Bile jonies after addition of bile salts What is the Quellung reaction? A method for identifying S. pneumoniae and determining its capsular tyre W'hen S, pneumoniae is mixed with capsular antiserum, the capsule becomes more refractile and appears to be swollen. The testis seldom used in labs nical GRAM-POSITIVE BACILLI A Gram-stained smear from growth on an aerobic blood agar plate shows small gram-positive bacilli that are pleomorphic and club shaped. They are arranged parallel to each other and in V and Y formation. To which genus do these organisms most likely belong? um species pathogeni No, Coryne classical pathogen in this genus. Other corynebacteria (Corynebacterium spp., sometimes re 28 art of the normal flora of the skin and upper respiratory t Some Coryne’ 1 may cause disease, especially in compromised hosts, Name a rapid test to differentiate nonhemols tie streptococci from Corynebacterium spp. (diphtheroids). tives cocci are catalase negative. Name the organism that causes diphtheria. Corynebacterium diphtheriae. Diphtheria is uncominon in developed countries because of vaccination (DPT) but it causes significant mortality and morbidity in developing countries What media are used to isolate C. diphtheriae? Cystine tellurite or modified Tinsdale is used for development of characteristic colonial morphology. C. diphtheriae produces black colonies with dark brown halos on modified Tinsdale agar, and black or gray colonies on cystine tellurite. Loeffler medium is used for development of characteristic microscopic morphology. It stimulates production of metachromatic granules. 109QUICK REVIEW Q & A'S. Which testis re : sation of C diphtheriae? Tonigenicity siscg sey: 2155.5 78 pathogen, C diphtheriae, from the nonpathogenic corynet “iy 42 TE Sg8r':7742 0 reference labs What is the clinics) sg° Scarce of C+ +ssa.seriam jeikelum (CDC Group JK)? wikeiums is -7 J5H: #5 758 commonly isolated from human infections. I colonizes the ~ of hospualzed pasiemis er + ~ s:unocompromised hosts and in patients with catheters & matic devices, It is the most se-r ~2> 2. valve endocarditis in adults and i is very resistant 19 Pasties gram-positive sporeformi 2:mboo on a Gram stain, and prod. ced from handling contaminated wool or hides, looks like enhemolytic "Medusa head” colonies on blood agar? us anthracis Which organism causes anthrax? Bacillus anthracis. Anthrax is pr cases due to occupational expos of renewed concern with the advent o Ithas been rare in the U.S., with only occasional ¢ hices, wool, goat or camel hair, or cashmere, Anthrax is What is the most useful microbiologic test fo> © sgn esis ef anthrax? The standard blood culture Describe the Gram stain morphology of B. ath ‘oad, gram-positive rods 1-1.5 x 3-5 jt, Oval, the cell. (Spores are usually not seen in clinica! specs 1x 1.5 {with no significant swelling of Describe B. anthracis in direct smears from clinical specimens. Encapsulated, broad, gram-positive rods in shor chains of 2-4 ceils, An Ind the capsule. Spores are usually not present in clinical specimens a ink stain should be used to visualize Deseribe the colonial characteristics of B. anthracis. Blood ager: + Colonies 2-5 mm in diameter + Gray-white with ground glass appearance + Flat or slightly convex Irregularly round with edges that slightly undulate, often with comma-shaped protrusions from the edge (Medusa head colonies) Tenacious consistency (When teased with a loop, the growth will stand up like a beaten egg white.) + Nonhemolytic MacConkey agar: no growth 110