Quality Control in The Clinical Microbiology Laboratory Mohammad

Quality Control in The Clinical Microbiology Laboratory Mohammad Rahbar (Ph. D) Professor of clinical Microbiology Department of Microbiology Iranian Reference Health Laboratory , Tehran Iran

QC program The laboratory director is primarily responsible for QC and QA programs. However all laboratory personnel must actively participate in both program

Positive Patient Outcome in the Microbiology laboratory Reduced length of stay Reduced cost of stay Reduced turn-around time for diagnosis of infection Change to appropriate antimicrobial therapy Customer ( physician or patients )satisfaction

The basic elements of QC Specimen collation Standard operating procedures (SOPM). Personnel Proficiency testing Antimicrobial Susceptibility tests Eminence of QC procedures Maintenance of QC stocks Patients reports

SPECIMEN COLLECTION AND TRANSPORT The laboratory is responsible for providing instructions for the proper collection and transport of specimens. These instructions should be available to the clinical staff for use when specimens collected.

Written collection instruction Test selection criteria Patients selection criteria Timing of specimen collection (e. g. antimicrobial are administration) Optimal specimen collection site Approved specimen collection method Specimen transport medium before

Continue… Specimen transport time and temperature Specimen holding instructions if it cannot be transported immediately (e. g. hold at 4 C for 24 hours) Availability of test (on –site or sent to reference laboratory ) Hours test performed (daily or batch) Turn-around time Result reporting procedures

Information should be filled Patient name Hospital or laboratory number Ordering physician Whether the patient receiving antimicrobial therapy Suspect agent or syndrome

Criteria for Unacceptable Specimens Unlabeled or mislabeled specimens Use of improper transport medium Excessive transport time Improper temperature during transport or storage Improper collection site for test request Specimen leakage out of transport container Sera that are excessively hemolyzed , lipemic, or contaminated with bacteria

Standard Operating Procedure Manuel (SOPM) The SOPM is considered part of QC program. The SOPM should define test performance , tolerance limits, reagent preparation, required quality control , result reporting and references. The SOPM should be written in CLSI format and must be reviewed and signed annually by the microbiology director.

Continue. . The SPOM should be available in the work area. It is the definitive laboratory reference and is used often for questions relating to individual test. Any obsolete procedures should be dated when removed from SPOM and retained for at least 2 years.

Personnel It is laboratory director's responsibility to employ sufficient qualified personnel for the volume and complexity of the work performed. Document competency and training twice a year Continuing education program should be provided All documentation should maintained in personnel file.

Proficiency Testing Laboratories are required to participate in an external proficiency testing (PT) The laboratory must maintain an average score of 80% to maintain licensure in any subspecialty area. The laboratory's procedures, reagents, equipments and personnel are all checked in the process of proficiency testing.

PT or External quality control Provide laboratory management with an insight into their performance Improve both local and national standards Reveals unsuspected area of difficulty Provides an educational stimulate for improvements Acts as a check on the efficacy of internal quality control procedures Demonstrates to colleagues and customers a commitment to quality

Performance Checks Instrument Equipment logs should contain the following information Instrument name, serial number, and date put use Procedure and periodicity( daily, weekly, monthly) for routine function check)

Continue… Acceptable performance ranges Instrument function failure , including specific details of steps taken to correct the problems (corrective action) Date and time of services requests and response. Date of routine preventive maintenance (PM) which should follow manufactures recommendations.

Continue. . Maintenance records should be retained in the laboratory for the life of instrument. Specific guidelines regarding periodicity of testing for autoclaves, biological safety cabinets , centrifuges , incubators, microscope, refrigerators , freezers, water bathes , heat blocks and other microbiology laboratory can be found in reference books.

Quality control of Culture Meda Commercially prepared culture media In house prepared culture media (User-Prepared)

Commercially Prepared Culture Media The CLSI subcommittee on media quality control collected data over several` years regarding the incidence of QC failure of commonly used microbiology media Based on its finding the subcommittee published a list of media that did not require retesting in the user's laboratory if purchased from a manufactures who follow CLSI guidelines

Continue. . The laboratory must inspect each shipment for Cracked media Excessive bubbles Clarity Hemolysis Freezing Unequal filling Visible contamination

User-Prepared and , commercially prepared Media QC forms for user-prepared media should contain: The amount of prepared The source of each ingredient The lot number Sterilization methods

Continue. . The preparation date The expiration date (Usually 1 month for agar plate and 6 month for tube media) The name of prepare All user prepared colures media also should checked for:

Continue. Proper color Depth Smoothness Hemolysis Excessive bubbles Contamination

Sterility Check A representative sample of the lot should be test for sterility; 5%of any lot is tested when a batch of 100 or fewer unit is received and maximum of 10 units are tested in large batches. Sterility is routinely checked by incubating the medium for 48 hours at the temperature at which it will be used.

Performance testing When medium dose need to quality controlled because it was prepared “in house (in the laboratory) or because it is complex, several basic rules must be followed : All media must be tested before use Each medium must be tested with organisms expected to give positive reaction as well as with organism expected either not to grow or produce a negative reaction

Continue. . The medium should be tested for sterility and p. H The organisms selected for QC should represent the most fastidious organisms for which the medium was designed Testing technique should be different for primary plating media that for biochemical or subculture media. Primary plating media should be tested with dilute suspensions of organisms, whereas biochemical media can be tested with undiluted organisms QC testing should be performed according to CLSI recommendation Expiration date must be established

Media failure log Date 2/14/2014 Media Urea Agar Lot# In house preparation 2/13/2014 Expiration date 6 month from preparation Quantity 2 racks Failure failure to give proper reaction with P. mirabilis Action taken Technologist QC repeated with P. mirabilis failed All tubes discarded. New Urea agar prepared MAR

Use of Stock Cultures To operate a quality control program in Microbiology lab. stock culture must be maintained by all laboratories. They are available from many sources. Commercial sources Proficiency testing source Patients isolates American Type Culture Collection (ATCC)

Continue… When quality control testing appears have failed, it is usually the stock culture rather than the test itself that has failed. Organisms may mutate with repeated sub culturing. for best results , a stock culture should be grown in a large volume of broth , then divided among enough small freezer vials to last a year.

Continue. . With this technique a new vial can be removed from the freezer weekly so that organism do not have to be continually subcultured. An organism may need to be subcultured twice after thawing to return it to a healthy state. Media selection for freezing is at the discretion of individual laboratories but should not contain sugar. If organism utilize sugar while being maintained , the acid products that result may kill organism with time.

Popular Media for Stock Cultures Schaeler broth wit glycerol Chopped meat (anaerobes) Tryptic Soy agar deeps (at room temperature) Tryptic Soy Broth (with 15 % Glycerol ) at -70 Cystein-tryptic agar (CTA) without carbohydrates

Continue… Nonfastidious (rapidly growing), aerobic bacterial organisms can be saved up to 1 years on TSA slants. Long –term storage of aerobes or anaerobes can be accomplished either by lyophilizartion (freeze drying) or freezing , at -70 C. Frozen , no fastidious organism should be thawed , reisolated and refrozen every 5 years; fastidious organisms should be thawed reisolated , and refrozen every 3 years. Stock isolated may be maintained by freezing them in 10% skim milk , Trypticase Soy Broth (TSB) with 15% glycerol.

Stain and Reagents Containers of stains and reagents should be labeled as to contents, concentration , storage requirements, date prepared (or received) date placed in service , expiration date, source (commercial manufacture or user prepared) and lot number. All stains and reagents should be stored according manufacture's recommendations and tested with positive and negative controls before use.

Continue. All stain Bacitracin B-lactamase CAMP Catalase Coagulase Fe. Cl 3 Gelatin Germ tube Hippurate Instrument failure Nitrate inoculum Optochin Temperature PYR Moisture Typing se Difficulty in VP determining X and V strips endpoint Cation content Thymidin

Antisera The lot number, date received, condition received , and expiration date must be recorded for all shipments of antisera. In addition , the antisera should be dated when opened. New lots must be tested concurrently with previous lots, and testing must include positive and negative controls.

Antimicrobial Susceptibility Testing For antimicrobial susceptibility testing, control organisms must be tested with each new lot or batch of antimicrobials or media, and daily thereafter. However, the frequency of test monitoring may be reduced to weekly (including the testing of new lots or batches of antimicrobials or media) if the laboratory can document satisfactory performance with daily control tests as suggested by CLSI guidelines.

Continued. . Single isolates or pure cultures only should be used for final performance of antimicrobial susceptibility testing (i. e. , no mixed susceptibilities). There must be stated criteria to determine the presence of an endpoint or zone size in the antimicrobial susceptibility testing system. The laboratory may use CLSI (NCCLS) criteria, but the use of other validated criteria is acceptable.

Continued. . The inoculum used for antimicrobial susceptibility testing (i. e. , inoculum size) must be controlled using a turbidity standard or other acceptable method. Antibiotic susceptibility may be substantially affected by inoculum size. The microbiology department should consult with the medical staff and pharmacy to develop a list of antibiotics to be reported for organisms isolated from different sites. (number and type of antibiotics)

Continued. . The goals of a quality control program are to monitor the precision (repeatability) and accuracy of susceptibility test procedures. Multiple parameters are monitored by following quality control recommendations such as : Reagents & equipments used in the tests Persons who carry out the tests and read the result

Continued. . Tools of QC: Reference strains Reagents & Equipments Chart of acceptble limits for quality control strains Quality control chart

Continued. . Corrective Action: Out-Of Control Result due to an obvious error Use of the wrong disk Use of wrong control strain Obvious contamination of strain Wrong procedure

Continued. . Reference Strains for Quality Control: n n n n Enterococcus Faecalis Escherichia Coli Staphylococcus aureus Strep. Pneumoniae P. aeruginosa Kleb. Pneumonia Haem. influenzae • • ATCC 29212 ATCC 25922 ATCC 35218 ATCC 25923 ATCC 49619 ATCC 27853 ATCC 700603 ATCC 49247

Maintence of QC records All QC results should be recorded on an appropriate QC form. Corrective action should be noted on this form. If temperature is adjusted or a biochemical test repeated, the new reading within the tolerance limits should be listed. In many laboratories the supervisor reviews and initials all forms weekly and the director then reviews each one monthly. QC records should be maintained for at least 2 years except those on equipment , which must be saved for the life of instrument

Patient Report The laboratory should established a system for supervisory of all laboratory repots. This review should involve checking the specimens workup to verify that the correct conclusion were drawn and no clerical errors were made in reporting results. Reports should be given only authorized by law to receive them.

Continue. . Clinician should be notified about ‘’panic values’’ immediately. Panic values are such as : potential-threatening results, for example positive Gram stain for CSF or AFB and positive blood culture. All patients records should be maintained for least 2 years.