EPID 525 Lecture 1

1. EPID 525 Lecture 1 General Lab Issues

2. UMHS Pathology Anatomic Clinical Surgical Pathology Blood Bank Autopsy Hematology Cytopathology Chemistry Immunohistochemistry Cytogenetics Molecular Microbiology

3. Role of the clinical microbiology laboratory • Diagnostic functions • Direct examination of the specimen • Grow/cultivate/DETECT organisms present • Analyze cultivated/DETECTED organisms • Communication of findings • present info in such a way as to avoid or minimize confusion • add interpretive comments where appropriate • provide educational updates

5. Expedited result reporting • computerized through laboratory information systems • Review various sources of technical guidelines • Manual of Clinical Microbiology • Clinical Microbiology Procedures Handbook • Clinical Laboratory Standards Institute • College of American Pathologists

6. Specimen collection and handling • Collection techniques • laboratory provides instructions • collect during acute phase, prior to administration of antibiotics • prepare site to minimize contamination with normal flora • sputum vs. saliva http://www.pathology.med.umich.edu/handbook/

9. Specimen preservation, storage, labeling, requisition • transport to the lab ASAP • oxygen  anaerobes • temperature Neisseria • pH Shigella • use preservatives, or anticoagulants if delays

11. Specimen rejection • mislabeled/unlabeled • improper transport temp. or container/medium • quantity not sufficient (QNS) • leaking • delay in transport (> 2 hrs unpreserved) • inappropriately received in fixative, or received dried up • MUST COMMUNICATE WITH CARE TEAM

12. Specimen processing • Direct microscopic examination • can assess specimen quality (e.g. sputum) • can assess inflammation (e.g. WBCs) • compare direct smear with culture • different stains: • Gram stain: bacteria, WBC, RBC, epithelial cells • Fungi: KOH or calcofluor white (fluorescent) • AFB: Kinyoun, Ziehl-Neelsen, or auramine-rhodamine (fluorescent)

13. Specimen processing • Selection of culture media • nutritive: support the growth of wide range of (most) organisms • differential: allow for distinguishing between organisms because of different growth characteristics • selective: support the growth of one group of organisms but not another because of the addition of inhibitors (antibiotics, dyes, alcohol)

14. Specimen processing • Specimen preparation • homogenization (tissue) • concentration (CSF) • decontamination (respiratory) • Inoculation of solid media • quantitative cultures • streaking for isolation • Nucleic acid extraction/detection

15. Laboratory Safety • Sterilization and Disinfection • Methods of sterilization: all forms of microbial life (including spores) are killed • physical methods: • incineration: flame • moist heat: autoclave; 121ºC, 15 psi • dry heat: 160-180ºC • filtration: 0.2 μm • gamma irradiation: microwaves, X-rays • chemical method: • ethylene oxide: gas for heat sensitive material

16. Sterilization and Disinfection • Methods of disinfection: only pathogenic microorganisms are destroyed • physical methods: • boiling: 100ºC, 15 min • pasteurization: 63ºC, 30 min or 72ºC, 15 sec • UV light • chemical methods: • alcohol, bleach, phenol • impacted by organism load, concentration, environmental conditions

17. Biosafety & Exposure Control Plan • Employee education and orientation • Disposal of hazardous waste • Standard precautions • No eating, drinking, smoking, applying cosmetics • Treat every specimen as if it is HIV+ • Wash hands • Avoid needlesticks, sharps exposures • Engineering controls • Biological safety cabinets (BSC) • Personal protective equipment (PPE) • Post-exposure control

21. Classification of Biologic Agents • Biosafety level 1 agents: no potential to cause disease in healthy people • Standard precautions • Biosafety level 2 agents: most common agents of infectious diseases • Standard precautions, limit access to lab, special training and supervision, BSCs for aerosols

22. Classification of Biologic Agents • Biosafety level 3 agents: unusual pathogens not routinely encountered. • Mycobacterium tuberculosis, mould forms of dimorphic fungi, Francisella, Brucella TRANSMITTED BY AEROSOL • BSL 2, plus engineering controls, additional PPE • Biosafety level 4 agents: rarely encountered hemorrhagic fever viruses and arboviruses • BSL 3, plus special containment and PPE • Mailing Biohazardous Materials • Regulated by the International Air Transport Association (IATA) • Dangerous goods regulations

23. CLIA • Clinical Laboratory Improvement Act of 1988 • Resulted from public and Congressional concerns about the quality of clinical laboratory testing in the U.S. • Basic set of guidelines to apply to all labs, regardless of size, complexity, or location. • Implementation and development of working guideline was assigned to HCFA (Health Care Finance Agency), now known as CMS (Center for Medicare and Medicaid Services).

24. Labs exempted from CLIA 88 • Labs accredited by state agencies (NY, WA) • Law enforcement agencies • Forensic testing and SAMSHA accredited labs • Patient self-testing • Research testing of human samples when there is no report of patient specific results. • VA system

25. CLIA • The intent of CLIA is to promote the development, implementation, delivery, monitoring, and improvement of high quality laboratory services.

26. CLIA Original consisted of 4 sets of rules describing: • Laboratory standards • Personnel standards • Quality control requirements • Test complexity model • Quality assessment of the complete testing process • Application process and user fees • Enforcement procedures • Approval of accreditation programs

27. Total Testing Process Pre Analytic Analytic Post-Analytic Physician order Sample prep Test report Patient preparation Analyzer setup Transmittal of report Specimen acquisition Test calibration Receipt of report Specimen handling Quality Control Review of test results Sample transport Sample analysis Action on test results

28. Quality Assurance Program designed to monitor and evaluate the ongoing and overall quality of the total testing process (preanalytic, analytic, and postanalytic)

29. Quality Control Activities designed to monitor and evaluate the performance of instruments and reagents used in the testing process Is a component of a QA program

30. Quality Assurance activities Patient test management assessment - specimen collection, labeling, transport - test requisition - specimen rejection - test report format and reporting systems Quality control assessment - calibrations and controls - patient data ranges - reporting errors

31. Quality Assurance activities (cont.) Proficiency testing assessment - regulated and unregulated analytes Comparison of test results - different assays or instruments used for same test - accuracy and reproducibility

32. Quality Assurance activities (cont.) Relationship of patient info. to test results - results consistent with patient info. - age, sex, diagnosis, other results Personnel assessment - education; competency

33. Quality Assurance activities (cont.) Communications and complaint investigations - communications log QA review with staff - review during regular meetings

34. Quality Assurance activities (cont.) QA records - retention for 2 years Verification of methods - accuracy, precision - analytical sensitivity and specificity - reportable range - reference range(s) (normal values)

35. Quality Assurance activities (cont.) Quality monitors - TAT - smear/culture correlation - contamination rates

36. Assessment of compliance College of American Pathologists (CAP) - Profession pathology organization - Been granted “deemed status” by CMS - Groups of peers conduct bi-annual site inspections - Publish checklists for laboratories to document compliance

39. The next driver of changes? • Maryland General Hospital in Baltimore • Over a period of 14 months, the lab reported almost 500 HIV and hepatitis serologies when quality control was out. • Lab and hospital passed CAP and JCAHO inspections during this time period. • Result  “Unannounced” inspections

40. How do we assess the performance of our tests?

41. Verification • Background • CLIA requirement to check (verify) the manufacturer’s performance specifications provided in package insert • Assures that the test is performing as intended by the manufacturer • Your testing personnel • Your patient population • Your laboratory setting • One time process performed prior to implementation

42. Verification • Accuracy • Are your test results correct? • Assures that the test is performing as intended by the manufacturer • Use QC materials, PT materials, or previously tested patient specimens

43. Verification • Precision • Can you obtain the same test result time after time? • Same samples on same/different days (reproducible) • Tested by different lab personnel (operator variance)

44. Verification • Reportable Range • How high and how low can test values be and still be accurate (qualitative)? • Choose samples with known values at high and low end of range claimed by manufacturer • What is the range where the test is linear (quantitative)? • Test samples across the range

45. Verification • Reference ranges/intervals (normal values) • Do the reference ranges provided by the test system’s manufacturer fit your patient population? • Start with manufacturer’s suggested ranges • Use published ranges • Can vary based on type of patient • May need to adjust over time • Normal patients should be within range, abnormal patients should be outside range

46. Verification • Number of samples to test • Depends on the test system and laboratory testing volume • FDA-approved: 20 positive and negatives • Non-FDA approved: 50 positive and negatives • The number used for each part of the verification will vary • Laboratory director must review and approve results before reporting patient results

47. Sensitivity • The probability of a positive test result given the presence of disease • How good is the test at detecting infection in those who have the disease? • A sensitive test will rarely miss people who have the disease (few false negatives).

48. Specificity • The probability of a negative test result given the absence of disease. • How good is the test at calling uninfected people negative? • A specific test will rarely misclassify people without the disease as infected (few false positives).

49. Sensitivity and Specificity DISEASE TEST Sensitivity = TP/TP+FN Specificity = TN/TN+FP

50. Predictive Value • The probability of the presence or absence of disease given the results of a test • PVP is the probability of disease in a patient with a positive test result. • PVN is the probability of not having disease when the test result is negative.