MICROBIOLOGY - pathology.med.umich.edu€¦ · Web viewIn the specific case of photomicrographs,...

Revised: 10/06/2005

MICROBIOLOGY

This College of American Pathologists (CAP) Laboratory Accreditation Program (LAP) Checklist is provided as a Microsoft® Word 2000 electronic file for convenience and for educational purposes. It represents the fully-approved version for use in the LAP as of the date given in the header.

Newer approved versions of this Checklist may be found via the Internet at the CAP Web site (http://www.cap.org/apps/docs/laboratory_accreditation/checklists/checklistftp.html) for both viewing and download to your computer.

If you are currently enrolled in the CAP LAP and are preparing for an inspection, please note:

The Checklists undergo frequent revision, and the contents may have changed after you receive your inspection packet. If a Checklist has been updated since receiving your packet, you will be inspected based upon the Checklists that were mailed to you in your application or reapplication packet.

For questions about the use of Checklists in the inspection process, please e-mail the CAP at [email protected], or call (800) 323-4040, ext. 6065. Suggestions for content improvement should be sent by e-mail to LAP at [email protected].

All checklists are © 2005 College of American Pathologists. All rights reserved.

http://www.cap.org/apps/docs/laboratory_accreditation/checklists/checklistftp.html

College of American Pathologists Revised: 10/06/2005

OUTLINE

SUMMARY OF CHANGESINSPECTION TECHNIQUES – KEY POINTS

GENERAL MICROBIOLOGYPROFICIENCY TESTINGQUALITY CONTROL AND QUALITY MANAGEMENT

SUPERVISIONPROCEDURE MANUALSPECIMEN COLLECTION AND HANDLINGREAGENTS - GENERALREPORTING OF RESULTSINSTRUMENTS AND EQUIPMENT

Temperature-Dependent EquipmentPERSONNELPHYSICAL FACILITIESBIOSAFETY

BACTERIOLOGYQUALITY CONTROL

MEDIASTAINSREAGENTSANTIMICROBIAL SUSCEPTIBILITY TESTING, QC REQUIREMENTS, AND RESULTS REPORTING

PROCEDURES AND TESTSRESPIRATORY CULTURESURINE CULTURESGYNECOLOGICAL CULTURESSTOOL CULTURESCEREBROSPINAL FLUID CULTURESBLOOD CULTURESWOUND CULTURES

LABORATORY SAFETYMYCOBACTERIOLOGY

QUALITY CONTROLSPECIMEN HANDLINGREPORTING OF RESULTSMEDIACONTROLS AND STANDARDS

PROCEDURES AND TESTSRAPID METHODSCONCENTRATION, INOCULATION, INCUBATIONCULTURESDIFFERENTIAL BIOCHEMICAL PROCEDURES

LABORATORY SAFETYMYCOLOGY

QUALITY CONTROLMEDIACONTROLS AND STANDARDS:

PROCEDURES AND TESTSLABORATORY SAFETY

PARASITOLOGYQUALITY CONTROL

REAGENTSINSTRUMENTS AND EQUIPMENT

MICROBIOLOGY Page 2 of 105


PROCEDURES AND TESTSSTOOLS FOR OVA AND PARASITESBLOOD FILMS FOR MALARIA AND OTHER PARASITES

LABORATORY SAFETYVIROLOGY

QUALITY CONTROLREAGENTSCONTROLS AND STANDARDS

LABORATORY SAFETYMOLECULAR MICROBIOLOGY

QUALITY CONTROLPROCEDURES AND TESTSLABORATORY SAFETY



SUMMARY OF CHANGESMICROBIOLOGY Checklist

10/6/2005 Edition

The following questions have been added, revised, or deleted in this edition of the checklist, or in the two editions immediately previous to this one.

If this checklist was created for a reapplication, on-site inspection or self-evaluation it has been customized based on the laboratory's activity menu. The listing below is comprehensive; therefore some of the questions included may not appear in the customized checklist. Such questions are not applicable to the testing performed by the laboratory.

Note: For revised checklist questions, a comparison of the previous and current text may be found on the CAP website. Click on Laboratory Accreditation, Checklists, and then click the column marked Changes for the particular checklist of interest.

NEW Checklist Questions

Question Effective Date MIC.13800 09/30/2004MIC.21626 09/30/2004MIC.21813 09/30/2004MIC.63350 09/30/2004MIC.63450 09/30/2004MIC.63550 09/30/2004MIC.63650 09/30/2004MIC.63750 09/30/2004MIC.63850 09/30/2004MIC.63950 09/30/2004MIC.64050 09/30/2004MIC.64150 09/30/2004MIC.64250 09/30/2004MIC.64350 09/30/2004MIC.64450 09/30/2004MIC.64550 09/30/2004MIC.64650 09/30/2004

REVISED Checklist Questions

Question Effective Date MIC.21930 10/06/2005MIC.21626 04/28/2005MIC.13100 09/30/2004MIC.22700 09/30/2004MIC.31680 09/30/2004



MIC.53050 09/30/2004

DELETED Checklist Questions

Question Effective Date MIC.21690 10/06/2005MIC.13050 09/30/2004MIC.22120 09/30/2004MIC.22720 09/30/2004MIC.23000 09/30/2004MIC.33000 09/30/2004MIC.43000 09/30/2004MIC.53000 09/30/2004MIC.63000 09/30/2004MIC.63300 09/30/2004



The checklists used in connection with the inspection of laboratories by the Commission on Laboratory Accreditation (“CLA”) of the College of American Pathologists have been created by the College and are copyrighted works of the College. The College has authorized copying and use of the checklists by College inspectors in conducting laboratory inspections for the CLA and by laboratories that are preparing for such inspections. Except as permitted by section 107 of the Copyright Act, 17 U.S.C. sec. 107, any other use of the checklists constitutes infringement of the College’s copyrights in the checklists. The College will take appropriate legal action to protect these copyrights.

NOTE ON MOLECULAR TESTING IN MICROBIOLOGY

This checklist covers molecular testing for unmodified, FDA-approved molecular methods only. Microbiology laboratories that have modified FDA-approved methods, or that use molecular methods that are not FDA-approved, must also be inspected with the Molecular Pathology checklist. Note: This requirement applies to all CAP-accredited laboratories, including non-U.S. laboratories.

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INSPECTION TECHNIQUES – KEY POINTS

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I. READ – OBSERVE – ASK – the three methods of eliciting information during the inspection process. These three methods may be used throughout the day in no particular order. Plan the inspection in a way that allows adequate time for all three components.

READ = Review of Records and DocumentsDocument review verifies that procedures and manuals are complete, current, available to staff, accurate and reviewed, and describe good laboratory practice. Make notes of any questions you may have, or processes you would like to observe as you read the documentation.

OBSERVE – ASK = Direct Observation and Asking QuestionsObserving and asking questions accomplish the following:

1. Verifies that the actual practice matches the written policy or procedure2. Ensures that the laboratory processes are appropriate for the testing performed3. Ensures that outcomes for any problem areas, such as PT failures and issues/problems

identified through the quality management process, have been adequately investigated and resolved

4. Ensures that previously cited deficiencies have been corrected

Use the following techniques: Observe laboratory practices – look at what the laboratory is actually doing. Compare the

written policy/procedure to what you actually observe in the laboratory to ensure the written



policy/procedure accurately reflects laboratory practice. Note if practice deviates from the documented policies/procedures.

Ask open ended, probing questions – these are starting points that will allow you to obtain large amounts of information, and help you clarify your understanding of the documentation you’ve seen and observations you’ve made. This eliminates the need to ask every single checklist question, as the dialogue between you and the laboratory may address multiple checklist questions.

Ask open-ended questions that start with phrases such as “show me how…” or “tell me about …” or “what would you do if…”. By asking questions that are open-ended, or by posing a hypothetical problem, you will avoid “cookbook” answers. For example, ask “Could you show me the specimen transport policy and show me how you ensure optimum specimen quality?” This will help you to determine how well the technical staff is trained, whether or not they are adhering to the lab’s procedures and policies, and give you a feel for the general level of performance of the laboratory.

Ask follow-up questions for clarification. Generally, it is best not to ask the checklist questions verbatim. For example, instead of asking the checklist question “Is there documentation of corrective action when control results exceed defined tolerance limits?” ask, “What would you do if the SD or CV doubles one month?” A follow-up probing question could be, “What would you do if you were unable to find a cause for the change in SD or CV?”

II. Evaluate Selected Specimens and Tests in Detail

For the Laboratory General Checklist: Follow a specimen through the laboratory. By following a specimen from collection to test result, you can cover multiple checklist questions in the Laboratory General checklist: questions on the specimen collection manual; phlebotomy; verbal orders; identification of patients and specimens; accessioning; and result reporting, including appropriate reference ranges, retention of test records, maintaining confidentiality of patient data, and proper handling of critical values and revisions to reports.

For the individual laboratory sections: Consult the laboratory’s activity menu and focus on tests that potentially have the greatest impact on patient care. Examples of such tests include HIV antibodies, hepatitis B surface antigen, urine drugs of abuse, quantitative beta-hCG, cultures of blood or CSF, acid-fast cultures, prothrombin time and INR reporting, and compatibility testing and unexpected antibody detection. Other potentially high-impact tests may be identified by looking at very high or low volume tests in the particular laboratory, or problems identified by reviewing the Variant Proficiency Testing Performance Report.

To evaluate preanalytic and postanalytic issues: Choose a representative specimen and “follow" the specimen through the laboratory or section of the laboratory, reviewing appropriate records in the preanalytic and postanalytic categories.

To evaluate analytic processes: Choose 2 or 3 analytes and perform a comprehensive review of records, including procedure manuals, quality control and proficiency testing records, instrument maintenance records and method performance validations for the last 2 years, selecting timeframes at



the beginning, mid-point, and end of this timeframe. Compare instrument print-outs to patient reports and proficiency testing results to ensure accurate data entry. If problems are identified, choose additional tests or months to review.

III. Verify that proficiency testing problem have been resolved: From the inspector’s packet, review the Variant PT Performance Report that identifies, by analyte, all of the PT scores below 100%. Correlate any PT problems to QC or maintenance records from the same time period. Be thorough when reviewing these representative records, selecting data from the beginning, middle and end of the period since the last on-site inspection.

IV. Review correction of previous deficiencies: Review the list of deficiencies from the previous on-site inspection provided in the inspector’s packet. Ensure that they have been appropriately addressed.

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GENERAL MICROBIOLOGY

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Questions in this section apply to ALL of the subsections in the microbiology laboratory (bacteriology, mycobacteriology, mycology, parasitology, and virology). When the microbiology department is inspected by a team, each member of the team must survey individual subsections for compliance with questions in this General Microbiology section. The team leader is then responsible for completing this section at the conclusion of the inspection.

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PROFICIENCY TESTING

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MIC.00010 Phase II N/A YES NO

Is the laboratory enrolled in the appropriate required CAP Surveys or CAP-approved alternative proficiency testing (PT) program for the patient testing performed?

NOTE: The list of analytes for which CAP requires proficiency testing is available on the CAP website [http://www.cap.org/apps/docs/laboratory_accreditation/ptgraded.html] or by phoning 800-323-4040 (or 847-832-7000), option 1. The laboratory’s participation in proficiency testing must include all analytes on this list for which it performs patient testing. Participation in proficiency testing may be through CAP Surveys or a CAP-approved proficiency testing provider. Laboratories will not be penalized if they are unable to enroll in an oversubscribed program. If unable to enroll, however, the laboratory must implement an alternative assessment procedure for the affected analytes.


http://www.cap.org/apps/docs/laboratory_accreditation/ptgraded.html


For regulated analytes, if the CAP and CAP-approved alternative PT programs are oversubscribed, CMS requires the laboratory to attempt to enroll in another CMS-approved PT program.

COMMENTARY:

N/A

REFERENCES: 1) Woods GL, Witebsky FG. College of American Pathologists mycobacteriology E proficiency testing survey; summary of participant performance, 1979-1992. Arch Pathol Lab Med. 1995;119:17-22; 2) Woods GL, Witebsky FG. Susceptibility testing of Mycobacterium avium complex in clinical laboratories. Results of a questionnaire and proficiency test performance by participants in the College of American Pathologists mycobacteriology E survey. Arch Pathol Lab Med. 1996;120:436-439; 3) Richardson H, et al. Quality improvement of diagnostic microbiology through a peer-group proficiency assessment program. A 20-year experience in Ontario. Arch Pathol Lab Med. 1996;120:445-455; 4) NCCLS. Continuous quality improvement: essential management approaches; approved guideline GP22-A. Wayne, PA: NCCLS, 1999; 5) College of American Pathologists, Commission on Laboratory Accreditation. Standards for laboratory accreditation; standard III. Northfield, IL: CAP, 1998; 6) Thomson S, et al. External quality assessment in the examination of blood films for malarial parasites within Ontario, Canada. Arch Pathol Lab Med. 2000;124:57-60; 7) Church DL, et al. Effects of restructuring on the performance of microbiology laboratories in Alberta. Arch Pathol Lab Med. 2000;124:357-361; 8) Method Preferences and Test Accuracy of Antimicrobial Susceptibility Testing: Updates from the CAP Microbiology Surveys Program. Arch Path Lab Med. 2001;125:(10)1285-1289.


For tests for which CAP does not require PT, does the laboratory at least semiannually 1) participate in external PT, or 2) exercise an alternative performance assessment system for determining the reliability of analytic testing?

NOTE: Appropriate alternative performance assessment procedures may include: split sample analysis with reference or other laboratories, split samples with an established in-house method, assayed material, regional pools, clinical validation by chart review, or other suitable and documented means. It is the responsibility of the laboratory director to define such alternative performance assessment procedures, as applicable, in accordance with good clinical and scientific laboratory practice. Participation in ungraded/educational proficiency testing programs also satisfies this checklist question.

COMMENTARY:

For areas where graded proficiency testing is not available, performance must be checked at least semi-annually with appropriate procedures such as: participation in ungraded proficiency surveys, split sample analysis with reference or other laboratories, split samples with an established in-house method, assayed material, regional pools, clinical validation by chart review, or other suitable and



documented means. It is the responsibility of the laboratory director to define such procedures, as applicable, in accordance with good clinical and scientific laboratory practice.

REFERENCES: 1) Department of Health and Human Services, Centers for Medicare and Medicaid Services. Clinical laboratory improvement amendments of 1988; final rule. Fed Register. 1992(Feb 28):7184 [42CFR493.1709]; 2) Shahangian S, et al. A system to monitor a portion of the total testing process in medical clinics and laboratories. Feasibility of a split-specimen design. Arch Pathol Lab Med. 1998;122:503-511; 3) NCCLS. Assessment of Laboratory Tests When Proficiency Testing is Not Available; Approved Guideline. NCCLS document GP29-A [ISBN 1-56238-479-1]. NCCLS, 940 West Valley Road, Suite 1400, Wayne, PA 19087-1898 USA, 2002.


Does the laboratory integrate all proficiency testing samples within the routine laboratory workload, and are those samples analyzed by personnel who routinely test patient samples, using the same methods as for patient samples?

NOTE: Replicate analysis of proficiency samples is acceptable only if patient specimens are routinely analyzed in the same manner. If the laboratory uses multiple methods for an analyte, proficiency samples should be analyzed by the primary method. There must not be any interlaboratory or instrument/reagent manufacturer communication on proficiency testing data before results reporting. The educational purposes of proficiency testing are best served by a rotation that allows all technologists to be involved in the proficiency testing program. Records of these studies must be kept and can be an important part of the competency and continuing education documentation in the personnel files of the individuals. In the specific case of photomicrographs, reported identifications must be made by a single individual who normally performs such identifications in patient samples. Responsibility for identifications should be rotated over time among all staff who render morphologic assessments in clinical samples. Group review and consensus identifications are permitted only for those unknown samples that would ordinarily be reviewed by more than one person in an actual patient sample. When external proficiency testing materials are not available, the semi-annual alternative performance assessment process should also be integrated within the routine workload.

COMMENTARY:

N/A

REFERENCES: 1) Department of Health and Human Services, Centers for Medicare and Medicaid Services. Clinical laboratory improvement amendments of 1988; final rule. Fed Register. 1992(Feb 28):7146 [42CFR493.801(b)]; 2) Shahangian S, et al. Toward optimal PT use. Med Lab Observ. 2000;32(4):32-43.




Are organisms in proficiency testing specimens identified to the same level as those from patient samples?

NOTE: If the laboratory's proficiency testing reports include incomplete identifications (e.g., "Gram positive cocci" or "Mycobacterium species, not tuberculosis"), it must document that this matches the information produced by the laboratory's internal capabilities in patient reports. In other words, patient reports cannot be more specific than the identification level reporting in proficiency testing, unless the former contain more specific information provided by reference laboratories.

COMMENTARY:

The microbiology laboratory must match its proficiency testing organism reporting level to the same degree used in patient reports. If the laboratory's proficiency testing reports include incomplete identifications (e.g., "Gram positive cocci" or "Mycobacterium species, not tuberculosis"), it must document that this matches the information produced by the laboratory's internal capabilities in patient reports. In other words, patient reports cannot be more specific than the identification level reporting in proficiency testing, unless the former contain more specific information provided by reference laboratories.


Is there evidence of evaluation and, if indicated, prompt corrective action in response to "unacceptable" results on the proficiency testing reports and results of the alternative performance assessment system?

NOTE: The evaluation must document the specific reason(s) for the "unacceptable" result(s) and actions taken to reduce the likelihood of recurrence. This must be done within one month after the laboratory receives its proficiency testing evaluation, unless one is dealing with repeat testing of samples requiring longer time intervals (e.g., mycobacterial cultures). In addition, each ungraded challenge, each educational challenge, and each episode of nonparticipation must be reviewed and corrective action instituted as appropriate.

COMMENTARY:

There must be evidence of evaluation and, if indicated, corrective action in response to each "unacceptable" result on the proficiency testing reports and results of the alternative performance assessment system. The evaluation must document the specific reason(s) for the "unacceptable" results and actions taken to reduce the likelihood of recurrence. This must be done within one month after the laboratory receives its evaluation, unless one is dealing with repeat testing of samples requiring longer time intervals (e.g., mycobacterial cultures).

REFERENCES: 1) NCCLS. Using proficiency testing (PT) to improve the clinical laboratory; approved guideline GP27-A. Wayne, PA: NCCLS,1999; 2) Novak RW. Do proficiency testing



participants learn from their mistakes. Experience from the EXCEL throat culture module. Arch Pathol Lab Med. 2002;126:147-149.


Is there documented evidence of ongoing evaluation by the laboratory director or designee of the proficiency testing and alternative performance assessment results?

COMMENTARY:

There must be evidence of ongoing evaluation by the laboratory director or designee of proficiency testing and alternative performance assessment results.

REFERENCES: 1) NCCLS. Using proficiency testing (PT) to improve the clinical laboratory; approved guideline GP27-A. Wayne, PA: NCCLS, 1999; 2) Somoskovi A, et al. Lessons from a proficiency testing event for acid-fast microscopy. Chest. 2001;120:250-257; 3) Novak RW. Do proficiency testing participants learn from their mistakes. Experience from the EXCEL throat culture module. Arch Pathol Lab Med. 2002;126:147-149.

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QUALITY CONTROL AND QUALITY MANAGEMENT

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SUPERVISION

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Before patient results are reported, QC data must be judged acceptable. The laboratory director or designee must review QC data at least monthly. Beyond these specific requirements, a laboratory may (optionally) perform more frequent review at intervals that it determines appropriate. Because of the many variables across laboratories, the CAP makes no specific recommendations on the frequency of any additional review of QC data.

All quality management (QM) questions in the Laboratory General Checklist pertain to the microbiology laboratory.


Does the microbiology laboratory have a written QC/QM program?



NOTE: The QM/QC program in the microbiology laboratory must be clearly defined and documented. The program must ensure quality throughout the preanalytic, analytic, and post-analytic (reporting) phases of testing, including patient identification and preparation; specimen collection, identification, preservation, transportation, and processing; and accurate, timely result reporting. The program must be capable of detecting problems in the laboratory’s systems, and identifying opportunities for system improvement. The laboratory must be able to develop plans of corrective/preventive action based on data from its QM system.

Appropriate laboratory personnel must judge QC data acceptable before patient results are reported. The laboratory director or designee must review QC data at least monthly. Beyond these specific requirements, a laboratory may (optionally) perform more frequent review at intervals that it determines appropriate. Because of the many variables across laboratories, the CAP makes no specific recommendations on the frequency of any additional review of QC data.

COMMENTARY:

N/A


Is there a documented procedure describing methods for patient identification, patient preparation, specimen collection and labeling, specimen preservation, and conditions for transportation, and storage before testing, consistent with good laboratory practice?

COMMENTARY:

The laboratory must have a completely documented procedure describing methods for patient identification, patient preparation, specimen collection and labeling, specimen preservation, conditions for transportation, and storage before testing. Such protocols must be consistent with good laboratory practice.


Is there evidence of ongoing evaluation of instrument maintenance and function, temperature, etc.?

COMMENTARY:

N/A




Is there a documented system in operation to detect and correct significant clerical and analytical errors, and unusual laboratory results that could affect patient management?

COMMENTARY:

The laboratory must have a documented system in operation to detect and correct significant clerical errors, significant analytical errors, and unusual laboratory results that could affect patient management. One common method is review of results by a qualified person (technologist, supervisor, pathologist), but there is no requirement for supervisory review of all reported data. The selective use of delta checks also may be useful in detecting clerical errors in consecutive samples from the same patient. In computerized laboratories, there should be automatic "traps" for improbable results.


Does the system provide for the timely correction of errors?

COMMENTARY:

The system for detecting significant clerical errors, significant analytical errors, and unusual laboratory results must provide for timely correction of errors, i.e., before results become available for clinical decision making. For suspected errors detected by the end user after reporting, corrections must be promptly made if such errors are confirmed by the laboratory.


In the absence of on-site supervisors, are results of tests performed by personnel reviewed by the laboratory director or general supervisor within 24 hours?

NOTE: The CAP does NOT require supervisory review of all test results before or after reporting to patient records. Rather, this question is intended to address only that situation defined under CLIA-88 for "high complexity testing" performed by trained high school graduates qualified under 42CFR493.1489(b)(5) when a qualified general supervisor is not present.

COMMENTARY:

In the absence of on-site supervisors, the results of tests performed by personnel must be reviewed by the laboratory director or general supervisor within 24 hours. The CAP does not require supervisory review of all test results before or after reporting to patient records. Rather, this question is intended to address only that situation defined under CLIA-88 for "high complexity testing" performed by trained high school graduates qualified under 42CFR493.1489(b)(5) when a qualified general supervisor is not present.



REFERENCE: Department of Health and Human Services, Centers for Medicare and Medicaid Services. Clinical laboratory improvement amendments of 1988; final rule. Fed Register. 1992(Feb 28):7182 [42CFR493.1463(a)(3) and 42CFR493.1463(c)]: 7183 [42CFR493.1489(b)(1) and 42CFR493.1489(b)(5)].

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PROCEDURE MANUAL

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The procedure manual should be used by personnel at the workbench and should include: test principle, clinical significance, specimen type, required reagents, test calibration, quality control, procedural steps, calculations, reference intervals, and interpretation of results. The manual should address relevant pre-analytic and post-analytic considerations, as well as the analytic activities of the laboratory. The specific style and format of procedure manuals are at the discretion of the laboratory director.

The inspection team should review the procedure manual in detail to understand the laboratory's standard operating procedures, ensure that all significant information and instructions are included, and that actual practice matches the contents of the procedure manuals.


Is a complete procedure manual available at the workbench or in the work area?

NOTE 1: The use of inserts provided by manufacturers is not acceptable in place of a procedure manual. However, such inserts may be used as part of a procedure description if the insert accurately and precisely describes the procedure as performed in the laboratory. Any variation from this printed procedure must be detailed in the procedure manual. In all cases, appropriate reviews must occur.

NOTE 2: A manufacturer's procedure manual for an instrument reagent system may be acceptable as a component of procedures. Any modification to, or deviation from, the procedure manual must be clearly documented.

NOTE 3: Card files or similar systems that summarize key information are acceptable for use as quick reference at the workbench provided that:

a. A complete manual is available for referenceb. The card file or similar system corresponds to the complete manual and is subject to

document control

NOTE 4: Electronic (computerized) manuals are fully acceptable. There is no requirement for paper copies to be available for the routine operation of the laboratory, so long as the



electronic versions are readily available to all personnel. Such electronic versions must be subjected to proper document control (i.e., only authorized persons may make changes, changes are dated/signed (manual or electronic), and there is documentation of periodic review). Current paper copies of electronically stored procedures should be available at the time of the CAP inspection, or rapidly generated at the request of the Inspector.

COMMENTARY:

A documented procedure manual must be developed for the microbiology section of the laboratory and be available at the workbench. Its elements should include: test principle, clinical significance, specimen type(s), required reagents, calibration, quality control, procedural steps, calculations, reference intervals, and interpretation, as applicable.

NOTE 1: The use of inserts provided by manufacturers is not acceptable in place of a procedure manual. However, such inserts may be used as part of a procedure description, if the insert accurately and precisely describes the procedure as performed in the laboratory. Any variation from this printed procedure must be detailed in the procedure manual. In all cases appropriate reviews must occur.

NOTE 2: A manufacturer's procedure manual for an instrument/reagent system may be acceptable as a component of the overall departmental procedures. Any modification to or deviation from the procedure manual must be clearly documented.

NOTE 3: Card files or similar systems that summarize key information are acceptable for use as quick reference at the workbench provided that:

a. A complete manual is available for reference,b. The card file or similar system corresponds to the complete manual and is subject to

document control.

NOTE 4: Electronic (computerized) manuals are fully acceptable. There is no requirement for paper copies to be available for the routine operation of the laboratory, so long as the electronic versions are readily available to all personnel. Such electronic versions must be subjected to proper document control (i.e., only authorized persons may make changes, changes are dated/signed (manual or electronic), and there is documentation of periodic review). Current paper copies of electronically stored procedures should be available at the time of the CAP inspection, or rapidly generated at the request of the Inspector.

REFERENCES: 1) NCCLS. Clinical Laboratory Technical Procedure Manuals; Approved Guideline—Fourth Edition. NCCLS document GP2-A4 (ISBN 1-56238-458-9). NCCLS, 940 West Valley Road, Suite 1400, Wayne, PA 19087-1898, USA 2002; 2) van Leeuwen AM. 6 Steps to building an efficiency tool. Advance/Laboratory. 1999:8(6):88-91; 3) Borkowski A, et al. Intranet-based quality improvement documentation at the Veterans Affairs Maryland health care system. Mod. Pathol. 2001;14:1-5.




Is there documentation of at least annual review of all policies and procedures in the entire microbiology laboratory by the current laboratory director or designee?

NOTE The director must ensure that the collection of policies and technical protocols is complete, current, and has been thoroughly reviewed by a knowledgeable person. Technical approaches must be scientifically valid and clinically relevant. To minimize the burden on the laboratory and reviewer(s), it is suggested that a schedule be developed whereby roughly 1/12 of all procedures are reviewed monthly. Paper/electronic signature review must be at the level of each procedure, or as multiple signatures on a listing of named procedures. A single signature on a Title Page or Index of all procedures is not sufficient documentation that each procedure has been carefully reviewed. Signature or initials on each page of a procedure is not required.

COMMENTARY:

There must be documentation of at least annual review of all policies and procedures in the microbiology laboratory section by the current laboratory director or designee. The director is responsible for ensuring that the collection of technical protocols is complete, current, and has been thoroughly reviewed by a knowledgeable person. Technical approaches must be scientifically valid and clinically relevant. To minimize the burden on the laboratory and reviewer(s), it is suggested that a schedule be developed whereby roughly 1/12 of all procedures are reviewed monthly. Paper/electronic signature review must be at the level of each procedure, or as multiple signatures on a listing of named procedures. A single signature on a title page or index of all procedures is not sufficient documentation that each procedure has been carefully reviewed. Signature or initials on each page of a procedure is not required.

REFERENCES: 1) Department of Health and Human Services, Centers for Medicare and Medicaid Services. Clinical laboratory improvement amendments of 1988; final rule. Fed Register. 1992(Feb 28):7173 [42CFR493.1407(e)(13)]; 2) Borkowski A, et al. Intranet-based quality improvement documentation at the Veterans Affairs Maryland health care system. Mod. Pathol. 2001;14:1-5.


Does the director or designee review and approve all new policies and procedures, as well as substantial changes to existing documents, before implementation?

NOTE: Current practice must match the policy and procedure documents.

COMMENTARY:

The director or designee must review and approve all new policies and procedures, as well as substantial changes to existing documents before implementation. Current practice must match these documents.



REFERENCE: Department of Health and Human Services, Centers for Medicare and Medicaid Services. Clinical laboratory improvement amendments of 1988; final rule. Fed Register. 2003(Jan 24):7164 [42CFR493.1251 (d)].


If there is a change in directorship, does the new director ensure (over a reasonable period of time) that laboratory procedures are well-documented and undergo at least annual review?

COMMENTARY:

If there is a change in directorship of the laboratory, the new director must ensure (over a reasonable period of time) that all microbiology laboratory procedures are well-documented and undergo at least annual review.

REFERENCE: Department of Health and Human Services, Centers for Medicare and Medicaid Services. Clinical laboratory improvement amendments of 1988; final rule. Fed Register. 2003(Jan 24):7164 [42CFR493.1251 (d)(e)].


When a procedure is discontinued, is a paper or electronic copy maintained for at least 2 years, recording initial date of use and retirement date?

COMMENTARY:

A paper or electronic copy of a discontinued procedure must be maintained for at least 2 years, recording initial date of use and retirement date.

REFERENCE: Department of Health and Human Services, Centers for Medicare and Medicaid Services. Clinical laboratory improvement amendments of 1988; final rule. Fed Register. 2003(Jan 24):7164 [42CFR493.1105 (a)(2); 493.1251 (e)].


Does the laboratory have a system documenting that all personnel are knowledgeable about the contents of procedure manuals relevant to the scope of their testing activities?

NOTE This does not specifically require annual procedure sign-off by testing personnel. The form of this system is at the discretion of the laboratory director.

COMMENTARY:



The laboratory must have a system documenting that all personnel are knowledgeable about the contents of procedure manuals relevant to the scope of their testing activities. This does not specifically require annual procedure sign-off by testing personnel. The form of this system is at the discretion of the laboratory director.

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SPECIMEN COLLECTION AND HANDLING

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Culture specimens are often collected by nurses or others outside the laboratory. An important aspect of quality control is the provision of adequate instructions to ensure proper collection and handling of specimens before they are received by the laboratory.

**REVISED** 09/30/2004


Are there criteria for establishing specimen acceptability?

NOTE: This could include important issues such as absence of gross external contamination, adequate specimen type/quantity, suitable preservation, prevention of dried swabs, and correct use of transport media when required.

COMMENTARY:

N/A


Are all specimens recorded in an accession book, day book, worksheet, computer or other comparable document?

COMMENTARY:

All specimens must be recorded in an accession book, day book, computer, or other comparable document.

MIC.13200 Phase I N/A YES NO

Do requests for isolation include source of specimen and, when appropriate, type of infection and/or organism expected?



COMMENTARY:

Requisitions for microbiology studies should require information regarding source of specimen and, when appropriate, type of infection and/or organism expected.


Are there documented instructions for microbiology specimen collection and handling that include all of the following?

1. Method for proper collection of culture specimens from different sources2. Need for prompt delivery of specimens to ensure minimum delay and prompt

processing (CSF, wound cultures, anaerobes)3. Use of transport media when necessary4. Method for preservation of specimens if processing is delayed (e.g., refrigeration of

urines)5. Procedures for safe handling of specimens (tightly sealed containers, no external

spillage)6. Proper labeling of culture specimens

NOTE: The Inspector must provide specific details of any deficiencies in Part B (Deficiency Summary) of the Inspector's Summation Report.

COMMENTARY:

Instructions for microbiology specimen collection and handling must include proper collection procedures for different types of cultures (such as sputum, urine, urethral, or wounds), and must include all of the following elements, as applicable:

1. Method for proper collection of culture specimens from different sources2. Need for prompt delivery of specimens to ensure minimum delay and prompt

processing (CSF, wound cultures, anaerobes)3. Use of transport media when necessary4. Method for preservation of specimens if processing is delayed (e.g., refrigeration of

urines)5. Procedures for safe handling of specimens (tightly sealed containers, no external

spillage)6. Proper labeling of culture specimens

Specific details of non-compliance are identified in part B (deficiency summary) of the Inspector's Summation Report.

REFERENCE: Miller JM, et al. Specimen collection, transport, and storage. In: Murrary PR, et al, ed. Manual of Clinical Microbiology, 8th ed. Washington, DC: ASM Press; 2003:55-66.



**NEW** 09/30/2004


For post-vasectomy checks for reproductive sterility, is a concentrating technique employed on seminal fluid?

NOTE: For post-vasectomy checks for reproductive sterility, a concentrating technique must be employed on the seminal fluid sample. Without such an approach, the presence of both motile and non-motile sperm may not be detected.

COMMENTARY:

N/A

REFERENCES: 1) Jones CD, Cornbleet PJ. Wright-Giemsa cytology of body fluids. Techniques for optimal cytocentrifuge slide preparation. Lab Med. 1997;28:713-716; 2) Jaffe TM, et al. Sperm pellet analysis: a technique to detect the presence of sperm in men considered to have azoospermia by routine semen analysis. J Urol. 1998;159:1548-1550.

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REAGENTS - GENERAL

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The following generic questions apply to all subsections of the Microbiology Laboratory.


Are reagents and solutions properly labeled, as applicable and appropriate, with the following elements?

1. Content and quantity, concentration or titer2. Storage requirements3. Date prepared or reconstituted by laboratory4. Expiration date

NOTE: The above elements may be recorded in a log (paper or electronic), rather than on the containers themselves, providing that all containers are identified so as to be traceable to the appropriate data in the log. While useful for inventory management, labeling with "date received" is not routinely required. There is no requirement to routinely label individual containers with "date opened"; however, a new expiration date must be recorded if opening the container changes the



expiration date, storage requirement, etc. The inspector will describe specific issues of non-compliance in the Inspector's Summation Report.

COMMENTARY:

All reagents must be properly labeled, as applicable and appropriate, with the following elements:

1. Content and quantity, concentration or titer2. Storage requirements3. Date prepared or reconstituted by laboratory4. Expiration date

The above elements may be recorded in a log (paper or electronic), rather than on the containers themselves, providing that all containers are identified so as to be traceable to the appropriate data in the log. While perhaps useful for inventory management, labeling with "date received" is not routinely required. There is no requirement to routinely label individual containers with "date opened"; however, a new expiration date must be recorded if opening the container changes the expiration date, storage requirement, etc. One or more of the above elements were absent during the on-site inspection. Details are provided in the Inspector's Summation Report.

REFERENCES: 1) Department of Health and Human Services, Centers for Medicare and Medicaid Services. Clinical laboratory improvement amendments of 1988; final rule. Fed Register. 2003(Jan 24):7164 [42CFR493.1252(c)]; 2) NCCLS. Clinical laboratory technical procedure manuals - fourth edition; approved guideline GP2-A4. Wayne, PA: NCCLS, 2002.


Are all reagents stored as recommended by the manufacturer?

COMMENTARY:

Reagents must be stored as recommended by the manufacturer in order to prevent environmentally-induced alterations that could affect test performance. If ambient temperature is indicated, there must be documentation that the defined ambient temperature is maintained and corrective action is taken when tolerance limits are exceeded.


Are all reagents used within their indicated expiration date?

COMMENTARY:

Reagents must not be used beyond their stated or assigned expiration date.



REFERENCE: Department of Health and Human Services, Centers for Medicare and Medicaid Services. Clinical laboratory improvement amendments of 1988; final rule. Fed Register. 2003(Jan 24):7164 [42CFR493.1252 (d); 493.1271 (b)].


For direct antigen tests on patient specimens that DO include internal controls, is a positive and negative external control tested and documented with each new kit lot number or separate shipments of a given lot number?

NOTE: Manufacturers' recommendations must be followed. For panels or batteries, controls must be employed for each antigen sought. For tests classified as "high complexity" under CLIA-88 that include an extraction phase, the system must be checked each day of use with a positive organism.

COMMENTARY:

For direct antigen tests on patient specimens that include internal controls, a positive and negative external control (organism or antigen extract) must be tested and documented with each new kit lot number or separate shipments of a given lot number. Internal quality control tests can be used for subsequent testing, based upon manufacturers' recommendations. For panels or batteries, controls must be employed for each bacterial antigen sought. For tests classified as "high complexity" under CLIA-88 that include an extraction phase, the system must be checked each day of use with a positive organism.


For direct antigen tests on patient specimens that do NOT include internal controls, is a positive and negative control tested and documented each day of patient testing?

NOTE: For each test system that requires an antigen extraction phase, the system must be checked with a positive control (e.g., a whole positive organism) that will detect problems in the extraction process.

COMMENTARY:

For direct antigen tests on patient specimens that do NOT include internal controls, a positive and negative control (organism or antigen extract) must be tested each day of patient testing. For panels or batteries, controls must be employed for each bacterial antigen sought in patient specimens. For each test system that requires an antigen extraction phase, the system must be checked with a positive control (e.g., a whole positive organism) that will detect problems in the extraction process.

REFERENCE: Department of Health and Human Services, Centers for Medicare and Medicaid Services. Fed Register. 2003(Jan 24):3708 [42CFR493.1256(d)].




Are results of reagent quality control testing recorded?

COMMENTARY:

Reagent performance and adequacy must be verified before placing the material in service or concurrent with use. Various methods, such as direct analysis, use of reference materials, or parallel testing of old versus new reagents, are acceptable. The results of verification checks must be recorded.

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REPORTING OF RESULTS

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When indicated, are preliminary reports promptly generated?

COMMENTARY:

Results of cultures should be reported promptly to provide clinically useful information. The quality of service should be improved by submitting timely preliminary reports based on the initial reading of direct smears or wet mounts of infectious materials or the first reading of culture plates.


Are documented criteria established for immediate notification of a physician or other clinical personnel responsible for patient care when results of certain tests exceed alert limits important for prompt patient management decisions?

NOTE: May be indicated either in the procedure manual and/or in a separate manual. The bench technologists must be familiar with alert limits for procedures that they perform.

COMMENTARY:

The microbiology laboratory must have documented criteria for the immediate notification of a physician or other clinical personnel when results of certain tests exceed alert limits. While sometimes informally known as "panic values," these are better described as "alert," "critical" or "life-threatening" data that usually require prompt patient management action by a clinician. These are best developed by the laboratory in consultation with user clinicians, and must be published. These may be indicated in the procedure manual and/or in a separate manual or policy. The bench technologists must be familiar with alert limits for procedures that they perform.



REFERENCES: 1) Department of Health and Human Services, Centers for Medicare and Medicaid Services. Clinical laboratory improvement amendments of 1988; final rule. Fed Register. 2003(Jan 24):[42CFR493. 1251(b)(13); 493.1291(g)]; 2) Steindel SJ, Heard NV. Critical values: data analysis and critique. Q-Probes 92-04. Northfield, IL: College of American Pathologists, 1992; 3) Kost GJ. Using critical limits to improve patient outcome. Med Lab Observ. 1993;25(3):22-27; 4) Tate KE, Gardner RM. Computers, quality, and the clinical laboratory: a look at critical values. Proc Annu Symp Comput Appl Med Care. 1993;193-197; 5) Kaufman HW, Collins C. Notifying clients of life-threatening results. Med Lab Observ. 1994;26(8):44-45; 6) Lum G. Evaluation of a laboratory critical limit (alert value) policy for hypercalcemia. Arch Pathol Lab Med. 1996;120:633-636; 7) Emancipator K. Critical values. ASCP practice parameter. Am J Clin Pathol. 1997:108:247-253; 8) Dalton-Beninato K. Critical value notifications are never welcome news. Lab Med. 2000;31:319-323; 9) Howanitz PJ, et al. Laboratory critical values policies and procedures. A College of American Pathologists Q-probes study in 623 institutions. Arch Pathol Lab Med. 20002;126:663-669.


Is there documentation of prompt notification of the physician (or other clinical personnel responsible for patient care) of results of all alert values?

NOTE: In addition, the laboratory should document any failure of attempts to notify the appropriate person of alert results, and document the action taken to prevent recurrence of this problem.

COMMENTARY:

Records must be maintained indicating the notification of the appropriate clinical individual promptly after observing results in an alert range. These records should include: date, time, responsible laboratory individual, person notified and test results. In addition, the laboratory should document any failure of attempts to notify the appropriate person of alert results, and document the action taken to prevent recurrence of this problem.

REFERENCES: 1) Kost GJ. Critical limits for urgent clinician notification at US medical centers. JAMA. 1990;263:704-707; 2) Department of Health and Human Services, Centers for Medicare and Medicaid Services. Clinical laboratory improvement amendments of 1988; final rule. Fed Register. 2003(Jan 24): [42CFR493.1251(b)(13); 493.1291(g)]; 3) Steindel SJ, Heard NV. Critical values: data analysis and critique. Q-probes 92-04. Northfield, IL: College of American Pathologists, 1992; 4) Kost GJ. Using critical limits to improve patient outcome. Med Lab Observ. 1993;25(3):22-27; 5) Dalton-Beninato K. Critical value notifications are never welcome news. Lab Med. 2000;31:319-323. ----------------------------------------------------

INSTRUMENTS AND EQUIPMENT

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All instruments and equipment should be properly operated, maintained, serviced, and monitored to ensure that malfunctions of these instruments and equipment do not adversely affect the analytical results. The inspection team should review the procedures for instrument/equipment operations, maintenance and monitoring records to ensure that these devices are properly used.


Are instruments (microscopes, centrifuges, etc.) on a regular instrument maintenance schedule?

COMMENTARY:

Instruments (microscopes, centrifuges, etc.) must be on a regular maintenance schedule.


Are records of instrument function checks maintained?

COMMENTARY:

Records of instrument function checks (incubators, refrigerators, water baths and autoclaves, etc.) must be maintained and available.


Are instrument manuals and maintenance, service and repair records (or copies) promptly available to, and usable by, the technical staff operating the equipment?

NOTE: Effective utilization of instruments by the technical staff depends upon the prompt availability of maintenance, repair, and service documentation (copies are acceptable). Laboratory personnel are responsible for the reliability and proper function of their instruments and must have access to this information. Off-site storage, such as with centralized medical maintenance or computer files, is not precluded if the inspector is satisfied that the records can be promptly retrieved.

COMMENTARY:

N/A


Are pipettes, microtiter diluters or automatic dispensers that are used for quantitative dispensing of material checked for accuracy and reproducibility at specified intervals, and results documented?



NOTE: This question is “not applicable” for precalibrated inoculation loops that are used in the direct plating of clinical specimens such as urine cultures.

COMMENTARY:

Pipettes, microtiter diluters and automatic dispensers that are used for quantitative dispensing of material must be checked for accuracy and reproducibility at specified intervals, and results documented.

REFERENCES: 1) Curtis RH. Performance verification of manual action pipets. Part I. Am Clin Lab. 1994;12(7):8-9; 2) Curtis RH. Performance verification of manual action pipets. Part II. Am Clin Lab. 1994;12(9):16-17; 3) Perrier S, et al. Micro-pipette calibration using a ratiometric photometer-reagent system as compared to the gravimetric method. Clin Chem. 1995;41:S183; 4) Bray W. Software for the gravimetric calibration testing of pipets. Am Clin Lab. Oct 1995 (available on the Internet at http://www.labtronics.com/pt_art.htm); 5) Kroll MH, et al (eds). Laboratory instrument evaluation, verification & maintenance manual, 5th edition. Northfield, IL: College of American Pathologists, 1999:126-127; 6) Johnson B. Calibration to dye for: Artel's new pipette calibration system. Scientist. 1999;13(12):14; 7) Connors M, Curtis R. Pipetting error: a real problem with a simple solution. Parts I and II. Am Lab News. 1999;31(13):20-22; 8) Skeen GA, Ashwwod ER. Using spectrophotometry to evaluate volumetric devices. Lab Med. 2000;31:478-479.


Is an appropriate thermometric standard device of known accuracy available (NIST certified or guaranteed by manufacturer to meet NIST standards)?

COMMENTARY:

The laboratory must have an appropriate thermometric standard device of known accuracy. Thermometers meeting NIST standards are available from commercial sources and one must be obtained.


Are all non-certified thermometers in use checked against an appropriate thermometric standard device before being placed in service?

COMMENTARY:

All non-certified thermometers must be checked against an appropriate thermometric standard device (reference thermometer) before being placed in service.



.................................................................

Temperature-Dependent Equipment

.................................................................


Are thermometers placed in, or integrated in all of the following equipment?

1. Refrigerators2. Incubators3. Water baths4. Heating blocks


COMMENTARY:

Thermometers must be placed in, or integrated in all of the following equipment:

1. Refrigerators2. Incubators3. Water baths4. Heating blocks

Specific details of non-compliance are identified in part B (Deficiency Summary) of the Inspector's Summation Report.


Are temperatures checked and recorded appropriately?

NOTE: Temperature-dependent equipment containing reagents and patient specimens must be monitored daily, as equipment failures could affect accuracy of patient test results. Items such as water baths and heat blocks used for procedures need only be checked on days of patient testing. The Inspector must provide specific details of any deficiencies in Part B (Deficiency Summary) of the Inspector's Summation Report.

COMMENTARY:

Temperatures must be checked and recorded appropriately for all instruments. Temperature-dependent equipment containing reagents and patient specimens must be monitored daily, as equipment failures



could affect accuracy of patient test results. Items such as water baths and heat blocks used for procedures need only be checked on days of patient testing.


Are there sufficient, clean, and well-maintained incubators available at specified temperature ranges?

COMMENTARY:

The laboratory should have adequate numbers and types of incubators for the extent of diagnostic services offered. There is insufficient incubator space for the number and types of cultures handled.

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PERSONNEL

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Does the person(s) in charge of technical operations in microbiology have education and experience in microbiology equivalent to an MT(ASCP) and at least 4 years experience (one of which is in microbiology) under a qualified laboratory director?

COMMENTARY:

N/A

REFERENCE: Church DL, et al. Effects of restructuring on the performance of microbiology laboratories in Alberta. Arch Pathol Lab Med. 2000;124:357-361.


Are personnel working in microbiology checked for visual color discrimination?

NOTE: Testing is not required for personnel who do not perform laboratory tests requiring color discrimination. This does not mean that visually color-impaired technical personnel cannot be employed, only that they be tested, with job assignments and responsibilities evaluated accordingly.

COMMENTARY:



Personnel working in microbiology should be checked for difficulty with visual color discrimination. Testing is not required for personnel who do not perform laboratory tests requiring color discrimination. This does not mean that visually color-impaired technical personnel cannot be employed, only that they be tested, with job assignments and responsibilities evaluated accordingly.

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PHYSICAL FACILITIES

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Sufficient space and utilities need to be provided for the overall workload of all microbiology sections, and to meet all safety requirements.


Is there adequate space for administrative functions?

COMMENTARY:

Additional space should be provided for administrative functions.


Is there adequate space for clerical work?

COMMENTARY:

Additional space should be provided for clerical work.


Is there adequate space for technical work (bench space)?

COMMENTARY:

Additional space should be provided for technical work (bench space).

REFERENCES: 1) https://www.irnoise.com/absa/respubs.html; 2) Richmond J. "Perspectives on Laboratory Design", Anthology of Biosafety I: American Biological Safety Association, Mundelein, IL January 1999; 3) Richmond J. "Facility Design Considerations", Anthology of Biosafety II: American Biological Safety Association, Mundelein, IL April 2000; 4) Richmond J. "Application of Principles", Anthology of Biosafety III: American Biological Safety Association, Mundelein, IL May 2000; 5)


https://www.irnoise.com/absa/respubs.html


Richmond J. "Issues In Public Health", Anthology of Biosafety IV: American Biological Safety Association, Mundelein, IL September 2001.


Is there adequate space for media preparation?

COMMENTARY:

Additional space should be provided for media preparation.


Is there adequate space for instruments and equipment?

COMMENTARY:

Additional space should be provided for instruments and equipment.


Is there adequate space for shelf storage?

COMMENTARY:

Additional space should be provided for storage.


Is there adequate refrigerator/freezer storage?

COMMENTARY:

Additional space should be provided for refrigerator/freezer storage.


Is there adequate incubator space?

COMMENTARY:



Additional space should be provided for incubators.


Is the space available efficiently utilized?

COMMENTARY:

Existing space should be utilized more efficiently.


Is the space available such that there is no compromise of the quality of work, safety of personnel, or limitation of quality control activities?

NOTE: The Inspector must provide specific details of which section(s) of the microbiology laboratory has serious space deficiencies in Part B (Deficiency Summary) of the Inspector's Summation Report.

COMMENTARY:

Existing space limitations in one or more sections of the microbiology laboratory were so severe as to interfere with the quality of work, the safety of personnel, and/or ability of personnel to carry out adequate quality control procedures with appropriate documentation. Specific details of non-compliance are identified in part B (Deficiency Summary) of the Inspector's Summation Report.


Are floors and benches clean, free of clutter and well-maintained?

COMMENTARY:

Floors and benches appeared cluttered or otherwise deteriorated. Additional cleaning and maintenance procedures need to be implemented.


Are water taps, sinks and drains adequate?

COMMENTARY:

Water taps, sinks and drains should be improved to support the types of procedures and workload of the laboratory.




Is lighting adequate?

NOTE: Direct sunlight should be avoided because of its extreme variability and the need for low light levels necessary to observe various computer consoles, etc. Lighting control should be sectionalized so general levels of illumination can be controlled in areas of the room if desired.

COMMENTARY:

N/A

REFERENCE: College of American Pathologists. Medical laboratory planning and design. Northfield, IL: CAP, 1986.


Are gas supplies adequate?

COMMENTARY:

Gas supplies should be improved to support the types of procedures and workload of the laboratory.


Are electrical outlets adequate?

COMMENTARY:

Electrical outlets should be improved to support the types of procedures and workload of the laboratory.


Is ventilation adequate?

COMMENTARY:

Ventilation should be improved to support the types of procedures and workload of the laboratory.




Is temperature/humidity control adequate?

COMMENTARY:

Temperature/humidity control should be improved to support the types of procedures and workload of the laboratory. This is partly for the comfort of personnel, but primarily to provide proper conditions for organisms incubated at room temperature.


Are telephones conveniently located, and are calls easily transferred?

COMMENTARY:

Telephones (number and/or location) should be improved to support the types of procedures and workload of the laboratory.

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BIOSAFETY

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Items in this section apply to ALL areas of the microbiology laboratory. Additional items for specific subsections (bacteriology, mycobacteriology, mycology, parasitology, and virology) are found under the Laboratory Safety subsections for each of those areas. If any of these safety deficiencies apply only to specific subsections of the laboratory, the Inspector must so specify in Part B of the Inspector's Summation Report.


Does the microbiology laboratory have policies and procedures for the recognition of isolates that may be used as agents of bioterrorism?

NOTE: Microorganisms likely to be utilized as biological weapons include Bacillus anthracis (anthrax), Brucella species (brucellosis), Clostridium botulium (botulism), Francisella tularensis (tularemia), Yersinia pestis (plague) and variola major (smallpox).

As part of an institution-wide plan to prepare and respond to a bioterrorism event, the hospital-based microbiology laboratory should have policies and procedures for the recognition of isolates that may be used as agents of bioterrorism.



COMMENTARY:

N/A

REFERENCES: 1) Snyder JW. Role of the hospital-based microbiology laboratory in preparation and response to a bioterrorism event. J Clin Microbiol. January, 2003; 2) Gilchrist MJR. Laboratory Safety, Management, and Diagnosis of Biological Agents Associated with Bioterrorism; 3) Robinson-Dunn B. The microbiology laboratory’s role in response to bioterrorism. Arch Pathol Lab Med. March 2002; 126; 4) Morse SA. Bioterrorism: Laboratory Security. Lab Med. June 2001; 5) Sewell, DL. Laboratory safety practices associated with potential agents of biocrime or bioterrorism. J. Clin. Microbiology. July 2003;41(7):2801-2809.


Does the laboratory participate in the institution’s bioterrorism response plan?

COMMENTARY:

N/A

REFERENCES: 1) Snyder JW. Role of the hospital-based microbiology laboratory in preparation and response to a bioterrorism event. J Clin Microbiol. January, 2003; 2) Gilchrist MJR. Laboratory Safety, Management, and Diagram of Biological Agents Associated with Bioterrorism; 3) Robinson-Dunn B. The microbiology laboratory’s role in response to bioterrorism. Arch Pathol Lab Med. March 2002; 126; 4) Morse SA. Bioterrorism: Laboratory Security. Lab Med. June 2001.


Are there documented policies for handling spills of contaminated materials?

COMMENTARY:

Safety policies and procedures must be improved by having documented policies for handling spills of contaminated materials.

REFERENCE: Department of Health and Human Services. Biosafety in Microbiological and Biomedical Laboratories, 4th ed. Washington, DC: Superintendent of Documents, Document 017-040-00523-7, May 1993.


Is there documentation of daily decontamination of bench tops?



COMMENTARY:

There must be documentation of daily decontamination of bench tops.


Are there documented policies and procedures for the safe handling and processing of specimens?

NOTE: Suggested topics to be considered in the policies and procedures for the safe handling and processing of specimens include the need for tight sealing of containers, avoiding spills of hazardous materials, requirements for wearing gloves, the need for respirator protection, availability and use of vaccinations, and the potential hazards of sniffing plates.

COMMENTARY:

There must be documented procedures that are consistently adhered to for the safe handling of specimens such as tight sealing of containers and avoiding spills, etc.

REFERENCES: 1) Jamison R, et al. Laboratory Safety in Clinical Microbiology, Cumitech 29, July 1996, ASM Press; Washington DC; 2) Fleming DO, Hunt DL. Biological Safety, Principles and Practices, 3rd ed. ASM Press; Washington DC.


Have policies and procedures been developed to minimize the occupational risk of exposure to infectious agents handled in the microbiology laboratory, in accordance with current recommendations regarding the biosafety levels for working with different organisms?

NOTE: The four biosafety levels (B.S.L.) for working with infectious agents are described in the CDC-NIH guidelines (Biosafety in Microbiological and Biomedical Laboratories. U.S. Dept. of Health and Human Services, fourth edition, 1999). Each B.S.L. consists of combinations of equipment, procedures and techniques, and laboratory design that are appropriate for the type of laboratory and infectious agent handled. The intent is for the laboratory director to know what manipulations are performed with pathogens (e.g., M. tuberculosis) and that appropriate precautions are in place to minimize the risk of laboratory personnel infection.

COMMENTARY:

Policies and procedures must be developed to minimize the occupational risk of exposure to infectious agents handled in the microbiology laboratory, in accordance with current recommendations regarding the biosafety levels (B.S.L.) for working with different organisms. The four levels of biosafety for working with infectious agents are described in the CDC-NIH guidelines. Each B.S.L. consists of



combinations of equipment, procedures and techniques, and laboratory design that are appropriate for the type of laboratory and infectious agent handled. The intent is for the laboratory director know what manipulations are performed when handling pathogens (e.g., M. tuberculosis) and that appropriate precautions are in place to minimize the risk of laboratory personnel infection.

REFERENCES: 1) Biosafety in microbiological and biomedical laboratories, fourth edition. Washington, DC: U.S. Dept Health and Human Services, 1999; 2) Richmond J. "Arthropod Borne Diseases", Anthology of Biosafety VI: American Biological Safety Association, Mundelein, IL April 2003.


Are engineering and work practice controls appropriate to the Biosafety level of the laboratory defined and implemented?

NOTE: Each increasing B.S.L. number (1 to 4) implies increased occupational risk from exposure to an agent or performance of a procedure, and therefore is associated with more stringent control and containment practices.

COMMENTARY:

Engineering and work practice controls appropriate to the biosafety level of the laboratory must be defined and implemented. Each increasing B.S.L. Number (1 to 4) implies increased occupational risk from exposure to an agent or performance of a procedure, and therefore is associated with more stringent control and containment practices.

REFERENCES: 1) Biosafety in microbiological and biomedical laboratories, fourth edition. Washington, DC: U.S. Dept Health and Human Services, 1999; 2) Richmond J. BSL-4 Laboratories. Anthology of Biosafety V: American Biological Safety Association, Mundelein, IL January 2002; 3) Richmond J. Biosafety Level 3. Anthology of Biosafety VII: American Biological Safety Association, Mundelein, IL December 2003.


Is a biological safety cabinet available and properly used for handling specimens or organisms considered highly contagious by airborne routes?

COMMENTARY:

A biological safety cabinet must be provided for handling specimens or organisms considered highly contagious by airborne routes.



REFERENCE: Department of Health and Human Services. Biosafety in microbiological and biomedical laboratories, 4th edition. Washington, DC: Superintendent of Documents, Document 017-040-00523-7, May 1993.


Is the biologic safety cabinet certified at least annually to ensure that filters are functioning properly and that airflow rates meet specifications?

COMMENTARY:

The biologic safety cabinet must be certified at least annually to ensure that filters are functioning properly and that airflow rates meet specifications.

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BACTERIOLOGY

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QUALITY CONTROL

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MEDIA

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The laboratory has the responsibility for ensuring that all media used, whether purchased or prepared by the laboratory, are sterile, able to support growth appropriately and are appropriately reactive biochemically. This will ordinarily require that the laboratory maintain a stock of reference organisms and test the media before or concurrent with use. Explicit documentation of such testing is essential.

For prepared, purchased media the laboratory must have explicit documentation that each lot of purchased medium has been tested for sterility, ability to support growth of appropriate organisms and biochemical reactivity at the time of preparation or concurrent with use in the laboratory. The recipient laboratory must have a copy of National Committee for Clinical Laboratory Standards (NCCLS) Document Number M-22-A3 (Quality Assurance for Commercially Prepared Microbiological Culture Media) as a reference source. The manufacturer or preparer must document to the user that their quality control activities meet the NCCLS guidelines, or are otherwise equivalent.



The laboratory director may wish to have a signed contractual arrangement with his/her selected manufacturer to cover all expected quality control and documentation. For each lot the preparer will certify that quality control performance was acceptable and maintain a record of test results and the lot numbers for ALL media for at least 2 years. The user laboratory may record that fact in place of the more detailed documentation of media performance. The user must visually examine each shipment for breakage, contamination, appearance, or evidence of freezing or overheating. Transportation of media/reagents under unfavorable environmental conditions may adversely affect product performance.

The user laboratory must continue to test each lot of media except those listed as being exempt from such testing in the tables in M22-A3, using quality control methods employed for media manufactured in-house (which are also listed in the M22 tables). In addition, each shipment of a commercial identification system must be tested for appropriate performance. If more than one lot number is received per shipment, each lot number must be tested.

The user must control media for critical reactions that are not tested by the preparer, even though the media may be exempted from testing for other purposes, as specified in M22-A3 tables. The director is responsible for the quality and performance of media and must document all media failures and the resultant corrective action taken.


Does the laboratory have documentation that its media supplier carries out the quality assurance guidelines enumerated in NCCLS Document M22-A3?

COMMENTARY:

The manufacturer or preparer must document to the user that their quality control activities meet the NCCLS guidelines.

The laboratory has the responsibility for ensuring that all media used, whether purchased or prepared by the laboratory, are sterile, able to support growth appropriately and are appropriately reactive biochemically. This will ordinarily require that the laboratory maintain a stock of reference organisms and test the media before or concurrent with use. Explicit documentation of such testing is essential.

For prepared, purchased media, the laboratory must have explicit documentation that each lot of purchased medium has been tested for sterility, ability to support growth of appropriate organisms and biochemical reactivity at the time of preparation or concurrent with use in the laboratory. The recipient laboratory should have a copy of the NCCLS document number M22-A3 (Quality assurance for commercially prepared microbiological culture media) as a reference source. The manufacturer or preparer must document to the user that their quality control activities meet the NCCLS guidelines, or are otherwise equivalent. The laboratory director may wish to have a signed contractual arrangement with his/her selected manufacturer to cover all expected quality control and documentation thereof. For each lot, the preparer will certify that quality control performance was acceptable, and maintain a record of test results and the lot numbers for all media for at least 2 years. The user laboratory may



record that fact in place of the more detailed documentation of media performance. The user must visually examine each shipment for breakage, contamination, appearance, or evidence of freezing or overheating. Transportation of media/reagents under unfavorable environmental conditions may adversely affect product performance.

The user laboratory must continue to test each lot of media except those listed as being exempt from such testing in the tables in M22-A3, using quality control methods that are used for media manufactured in-house. In addition, each shipment or lot, if more than one lot number is received per shipment of a commercial identification system must be tested for appropriate performance.

The user must control media for critical reactions that are not tested by the preparer, even though the media may be exempted from testing for other purposes, as specified in M22-A3 tables. The director is responsible for the quality and performance of media, and must document all media failures and the resultant corrective action taken.

REFERENCE: NCCLS. Quality assurance for commercially prepared microbiological culture media - third edition; approved standard M22-A3. Wayne, PA: NCCLS, 2004.


Does the laboratory have documentation that each shipment of purchased media is examined for breakage, contamination, appearance, and evidence of freezing or overheating?

COMMENTARY:

The laboratory should have documentation that each shipment of purchased media is examined for breakage, contamination, appearance, and evidence of freezing or overheating.


Does the laboratory have documentation that an appropriate sample of each purchased medium that is not listed in M22-A3 as exempt from testing is checked for each of the following?

1. Ability to support growth (where applicable) by means of stock cultures or by parallel testing with previous batches

2. Biochemical reactivity, where appropriate


COMMENTARY:

The laboratory must have documentation that an appropriate sample of each purchased medium that is not listed in NCCLS M22-A3 as exempt from testing is checked for each of the following:



1. Ability to support growth (where applicable) by means of stock cultures or by parallel testing with previous batches

2. Biochemical reactivity, where appropriate

Specific details of non-compliance are identified in Part B (Deficiency Summary) of the Inspector's Summation Report.

REFERENCE: NCCLS. Quality assurance for commercially prepared microbiological culture media - third edition; approved standard M22-A3. Wayne, PA: NCCLS, 2004.


For microbiology media prepared in-house, is there documentation that an appropriate sample of each medium prepared by the laboratory is checked for each of the following?

1. Sterility (following introduction of additives after sterilization)2. Ability to support growth (where applicable) by means of stock cultures or by

parallel testing with previous batches3. Biochemical reactivity (where appropriate)


COMMENTARY:

There must be documentation that an appropriate sample of each medium prepared by the laboratory is checked for each of the following:

1. Sterility (following introduction of additives after sterilization)2. Ability to support growth (where applicable) by means of stock cultures or by parallel

testing with previous batches3. Biochemical reactivity (where appropriate)



Are all media in visibly satisfactory condition (with expiration date, plates smooth, adequately hydrated, uncontaminated, appropriate color and thickness, tubed media not dried or loose from sides)?

COMMENTARY:



Media were observed that were unsatisfactory for use, such as dried plates or tubes. Deteriorated media must be discarded.


Are reference organisms used to check stains, reagents and susceptibility test methods?

COMMENTARY:

Reference organisms must be used to check stains, reagents and susceptibility discs.

REFERENCE: Jones RN, et al. Method preferences and test accuracy of antimicrobial susceptibility testing. Updates from the College of American Pathologists microbiology surveys program (2000). Arch Pathol Lab Med. 2001;125:1285-1289.


Are control specimens tested in the same manner and by the same personnel as patient samples?

COMMENTARY:

It is implicit in quality control (QC) that control specimens are tested in the same manner as patient specimens. Moreover, QC specimens must be analyzed by personnel who routinely perform patient testing. This does not imply that each operator must perform QC daily, so long as each instrument and/or test system has QC performed at required frequencies, and all analysts participate in QC on a regular basis. To the extent possible, all steps of the testing process must be controlled, recognizing that preanalytic and postanalytic variables may differ from those encountered with patients.

REFERENCE: Department of Health and Human Services, Centers for Medicare and Medicaid Services. Clinical laboratory improvement amendments of 1988; final rule. Fed Register. 2003(Jan 24):7166 [42CFR493.1256(d)(8)]. 1256(f)].


Are the results of controls reviewed for acceptability before reporting patient results?

COMMENTARY:

The results of controls must be reviewed before reporting patient results. It is implicit in quality control that patient test results will not be reported when controls are unacceptable.



REFERENCE: Department of Health and Human Services, Centers for Medicare and Medicaid Services. Clinical laboratory improvement amendments of 1988; final rule. Fed Register. 2003(Jan 24)1992(Feb 28):7166 [42CFR493.1256(f)].

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STAINS

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All staining procedures (gram stains, special stains, fluorescent stains) should be checked and results recorded for each new batch and at least weekly with known positive and negative (when appropriate) control organisms.


Is the gram staining procedure checked and recorded for each new batch of stains and at least weekly against known gram-positive and gram-negative control organisms?

COMMENTARY:

Gram stains must be checked and results recorded for each new batch, and at least weekly, with known positive and negative (when appropriate) control organisms.

REFERENCES: 1) August, Hindler, Huber, Sewell. Quality control and quality assurance practices. In: Clinical microbiology, Cumitech 3A. Washington, DC: American Society for Microbiology, 1990; 2) Department of Health and Human Services, Centers for Medicare and Medicaid Services. Clinical laboratory improvement amendments of 1988, final rule. Fed Register. 2003(Jan 24):3708 [42CFR493.1261(a)(2)].


Are all non-immunofluorescent, non-immunologic-based stains (other than Gram stains) checked with a positive control and negative control for intended reactivity each day of use, and for each new batch, lot number and shipment?

COMMENTARY:

N/A




Does the microbiology section of the laboratory have a defined, documented system to ensure consistency of microscopic observations/interpretations among all personnel performing gram and other organism stains?

NOTE: Suggested methods to accomplish this include:

1. Circulation of organisms with defined staining characteristics, and/or2. Multi-headed microscopy, and/or3. Use of photomicrographs with referee and participant identifications (e.g., former CAP

microbiology Surveys or other photomicrographs from teaching collections)

COMMENTARY:

The microbiology laboratory should have a documented system to ensure that all personnel report microscopic observations and interpretations on patient samples in a similar fashion. For initial accuracy, as well as consistency in serial samples from the same patient, the laboratory should be able to document that all of its staff are consistent with respect to morphologic identifications with gram and other microbiology stains. Suggested methods to accomplish this include:

1. Circulation of organisms with defined staining characteristics, and/or2. Multi-headed microscopy, and/or3. Use of photomicrographs with referee and participant identifications (e.g., former CAP

microbiology Surveys or other photomicrographs from teaching collections)

REFERENCE: Flournoy DJ. Interpreting the sputum gram stain report. Lab Med. 1998;29:763-768.


Are fluorescent stains checked for positive and negative reactivity each time of use?

COMMENTARY:

Positive and negative controls must be tested each time of use with all fluorescent stains to ensure appropriate reactivity.

REFERENCE: Department of Health and Human Services, Centers for Medicare and Medicaid Services. Clinical laboratory improvement amendments of 1988; final rule. Fed Register. 2003(Jan 24):7146 [42CFR493.1256(e)(3); 493.1273(a)].



----------------------------------------------------

REAGENTS

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The laboratory has the responsibility for ensuring that all reagents used, whether purchased or prepared by the laboratory, are appropriately reactive. The verification of reagent performance is required and must be documented. Any of several methods may be appropriate, such as direct analysis with reference materials, parallel testing of old vs. new reagents, and checking against routine controls. The intent of the questions is for new reagents to be checked by an appropriate method and the results recorded before patient results are reported. Where individually packaged reagents/kits are used, there should be criteria established for monitoring reagent quality and stability, based on volume of usage and storage requirements. Processing of periodic "wet controls" to validate reagent quality and operator technique is a typical component of such a system.


Are new reagent lots and/or shipments checked against old reagent lots or with suitable reference material before or concurrently with being placed in service?

NOTE: For qualitative tests, minimum cross-checking includes testing at least one known positive and one known negative sample with the new reagent lot or shipment. It is often preferable to do these validations with patient samples that have been tested previously, or can be tested simultaneously with the old reagent lot. Good clinical laboratory science includes patient-based comparisons in many situations, since it is patient results that are “controlled”. However, comparison with the old reagent lot is neither practical nor preferred in all cases, as in the case of a direct test for Shigella in stool, where the specimen is unstable and positives are infrequent. Another example where comparison with an old reagent lot may not be preferred would be when most patient specimens or the organisms derived from such specimens react very strongly in the test of interest. In this case, it might be preferable to use a well-characterized, weakly positive control to validate sensitivity of the new lot of reagents.

COMMENTARY:

New reagents must be tested in parallel or validated with old reagents or checked against other reference material to ensure appropriate reactivity before or concurrently with being placed in service. For qualitative tests (as applicable), minimum cross-checking should include testing one known positive and one known negative sample with the new reagent lot. It is often preferable to do these validations with patient samples that have been tested previously, or can be tested simultaneously with the old reagent lot. However, comparison with the old reagent lot is neither practical nor preferred in all cases, as in the case of a direct test for Shigella in stool, where the specimen is unstable and positives are infrequent. Another example where comparison with an old reagent lot may not be preferred would be when most patient specimens or the organisms derived from such specimens react very strongly in the test of interest. In this case, it might be preferable to use a well-characterized, weakly positive control to validate sensitivity of the new lot of reagents.




Are positive and negative controls tested and results recorded for each new batch, lot number, and shipment of reagents, disks and stains?

NOTE: Reagents subject to this requirement include (but are not limited to) catalase, coagulase, oxidase and indole reagents; bacitracin, optochin, ONPG, X, V, and XV disks.

COMMENTARY:

N/A

REFERENCE: Department of Health and Human Services, Centers for Medicare and Medicaid Services. Clinical laboratory improvement amendments of 1988; final rule. Fed Register. 2003(Jan 24): 3708 [42CFR493.1256 (e) (1) and (2)].

**NEW** 09/30/2004**REVISED** 04/28/2005


Is each new lot number and shipment of reagents used in bacterial identification systems tested with a positive and negative organism?

NOTE: All biochemical tests in each new lot number and shipment should be evaluated with a known positive and negative organism, to assure appropriate reactivity.

COMMENTARY:

N/A

REFERENCE: Department of Health and Human Services, Centers for Medicare and Medicaid Services. Clinical laboratory improvement amendments of 1988; final rule. Fed Register. 2003(Jan 24):3708 [42CFR493.1256(e)(1)].


Are positive and negative controls tested and results recorded for each new batch, lot number and shipment of antisera when prepared or opened and once every 6 months thereafter?

COMMENTARY:



Positive and negative controls must be tested and results recorded for each new batch, lot number and shipment of antisera when prepared or opened and once every 6 months thereafter.

REFERENCE: Department of Health and Human Services, Centers for Medicare and Medicaid Services. Clinical laboratory improvement amendments of 1988; final rule. Fed Register. 2003 (January 24):3708 [42CFR493.1261(a)(3)].


Are positive and negative controls tested and results recorded for beta-lactamase (other than commercial chromogenic cephalosporin reagents) on each day of use?

NOTE: Beta lactamase tests using commercial chromogenic cephalosporin reagents need be checked only with each batch, or lot number.

COMMENTARY:

N/A

REFERENCE: Department of Health and Human Services, Centers for Medicare and Medicaid Services. Clinical laboratory improvement amendments of 1988; final rule. Fed Register. 2003 (January 24):3708 [42CFR493.1261 (a) (1)].


Are results of serologic control tests recorded?

COMMENTARY:

Results of all serological control procedures must be recorded.


If there are multiple components of a reagent kit, does the laboratory use components of reagent kits only within the kit lot, unless otherwise specified by the manufacturer?

COMMENTARY:

If there are multiple components of a reagent kit, the laboratory must use components of reagent kits only with other kits that are in the same lot number, unless otherwise specified by the manufacturer.



REFERENCE: Department of Health and Human Services, Centers for Medicare and Medicaid Services. Clinical laboratory improvement amendments of 1988; final rule. Fed Register. 2003 (Jan 24):7164 [42CFR493.1252(d)].


Are anaerobic systems (e.g., jars, chambers, bags) checked for adequate anaerobic conditions with methylene blue strips, fastidious anaerobic organisms or other appropriate procedures?

COMMENTARY:

Methylene blue strips, cultures of fastidious anaerobic organisms, or other appropriate procedures must be used in the anaerobic system (e.g., jars, chambers, bags) to verify anaerobiosis.

**NEW** 09/30/2004


Are CO2 incubators checked daily for adequate CO2 levels, with recording of results?

NOTE: Some organisms require CO2 to grow sufficiently to form visible colonies. CO2 monitoring is required in all CO2 incubators, including those that adjust gas flow to maintain a set CO2 level.

COMMENTARY:

N/A


Are out of control results reported to the supervisor or laboratory director, and is there documented evidence of corrective actions taken?

COMMENTARY:

Results that are out of control must be reported immediately to the supervisor or laboratory director and corrective action taken must be documented.


If the laboratory performs test procedures for which calibration and control materials are not available, have procedures been established to verify the accuracy of patient test results?



COMMENTARY:

Procedures must be established to verify accuracy of patient test results if calibration and control materials are not available.

REFERENCE: Elder BL, et al. Verification and Validation of Procedures in the Clinical Microbiology Laboratory. Cumitech 31, February 1997. ASM Press; Washington DC.

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ANTIMICROBIAL SUSCEPTIBILITY TESTING, QC REQUIREMENTS, AND RESULTS REPORTING

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Are single isolates or pure cultures only used for final performance of antimicrobial susceptibility testing (i.e., no mixed susceptibilities)?

COMMENTARY:

Antimicrobial susceptibility testing must be performed only on pure cultures or single isolates.


Is each new lot of susceptibility disks checked for activity before use?

COMMENTARY:

The potency of the antimicrobial susceptibility disks must be checked before use with reference organisms having known susceptibility patterns.

REFERENCES: 1) Department of Health and Human Services, Centers for Medicare and Medicaid Services. Clinical laboratory improvement amendments of 1988; final rule. Fed Register. 2003(Jan 24):7167 [42CFR493.1261(b)(1); 2) NCCLS. Performance standards for antimicrobial disk susceptibility tests - eighth edition; approved standard M2-A8. Wayne, PA: NCCLS, 2003; 3) NCCLS. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically - sixth edition; approved standard M7-A6. Wayne, PA: NCCLS, 2003; 4) NCCLS. Anaerobic dilution supplemental tables; thirteenth informational supplement M100-S13. Wayne, PA: NCCLS, 2003.




For antimicrobial susceptibility testing of either disk or dilution type, are control organisms tested with each new lot or batch of antimicrobials or media, and each day the test is performed thereafter?

NOTE: 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 NCCLS guidelines. For this purpose, satisfactory performance is defined as follows:

1. There is documentation that all reference strains were tested for 20 or 30 consecutive test days, and

2. For each drug/microorganism combination, no more than 1 of 20 or 3 of the 30 values (zone diameter or MICs) may be outside the accuracy ranges. These limits may be established by the laboratory or NCCLS Guidelines M2-A8 or M7-A6

COMMENTARY:

N/A

REFERENCES: 1) Department of Health and Human Services, Centers for Medicare and Medicaid Services. Clinical laboratory improvement amendments of 1988; final rule. Fed Register. 2003(Jan 24):7167 [42CFR493.1261(b)(1)]; 2) NCCLS. Methods for antimicrobial susceptibility testing of anaerobic bacteria - fifth edition; approved standard M11-A5. Wayne, PA: NCCLS, 2001; 3) NCCLS. Performance standards for antimicrobial disk susceptibility - eighth edition; approved standard M2-A8 . Wayne, PA: NCCLS, 2003; 4) NCCLS. Methods for dilution antimicrobial susceptibility tests for bacteria that grow - sixth edition; approved standard M7-A6. Wayne, PA: NCCLS, 2003; 5) NCCLS. Anaerobic dilution supplemental tables; thirteenth informational supplement M100-S13. Wayne, PA: NCCLS, 2003.


Are tolerance limits for potency of antimicrobials established (criteria for "out of control")?

COMMENTARY:

Tolerance limits for potency of antimicrobials (criteria for "out of control") must be established.

REFERENCES: 1) NCCLS. Performance standards for antimicrobial disk susceptibility - eighth edition; approved standard M2-A8. Wayne, PA: NCCLS, 2003; 2) NCCLS. Anaerobic dilution supplemental tables; thirteenth informational supplement M100-S13. Wayne, PA: NCCLS, 2003.



**REVISED** 10/06/2005


For antimicrobial susceptibility testing systems, are there documented criteria for interpretation of the endpoint or zone size?

NOTE: 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.

COMMENTARY:

N/A

REFERENCES: 1) NCCLS. Performance standards for antimicrobial disk susceptibility tests - eighth edition; approved standard M2-A8. Wayne, PA: NCCLS, 2003; 2) NCCLS. Anaerobic dilution supplemental tables; thirteenth informational supplement M100-S13. Wayne, PA: NCCLS, 2003.


Is the inoculum used for antimicrobial susceptibility testing (i.e., inoculum size) controlled using a turbidity standard or other acceptable method?

COMMENTARY:

The susceptibility testing inoculum size should be controlled. Antibiotic susceptibility may be substantially affected by inoculum size.

REFERENCES: 1) NCCLS. Performance standards for antimicrobial disk susceptibility tests - eighth edition; approved standard M2-A8. Wayne, PA: NCCLS, 2003; 2) NCCLS. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically - sixth edition; approved standard M7-A6. Wayne, PA: NCCLS, 2003; 3) NCCLS. Anaerobic dilution supplemental tables; thirteenth informational supplement M100-S13. Wayne, PA: NCCLS, 2003.


Are guidelines established for the number and type of antibiotics reported for organisms isolated from different sites of infection?

COMMENTARY:



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. These lists may be based on the guidelines developed by the NCCLS table to report routinely (Group A) and which might be reported only selectively (Group B). Limiting the number of antimicrobial agents reported, referred to as “cascade reporting”, usually means reporting no more than four potential agents to which in at least one instance the isolate in question is susceptible. Selective reporting should help improve the clinical relevance of test reporting and help minimize the selection of multi resistant nosocomial strains by overuse of broad-spectrum agents. Laboratories should also only report those antimicrobial agents that are effective at the site from which the organism was isolated. Documentation of agreed upon policies should be available in the laboratory.

REFERENCE: Poulter MD, Hindler JF. Challenges in Antimicrobial Susceptibility Testing and Reporting. Lab Med, November, 2002:11:33.


For hospital based Microbiology Laboratories, is cumulative antimicrobial susceptibility test data maintained and reported to the medical staff at least yearly?

COMMENTARY:

For hospital based Microbiology Laboratories, cumulative antimicrobial susceptibility test data should be maintained and reported to the medical staff at least yearly.

REFERENCE: NCCLS. Analysis and presentation of cumulative antimicrobial susceptibility test data; approved guideline M39-A. Wayne, PA: NCCLS, 2002.


Does the procedure manual address unusual or inconsistent antimicrobial testing results?

NOTE: Acceptable results derived from testing QC strains does not guarantee accurate results with all patient isolates. When unusual or inconsistent results are encountered with patient isolates, the results should be investigated further to ensure accuracy. For example, repeat testing and/or repeat identification procedures using an alternative method (if possible) should be performed in an effort to ensure accurate results. Some examples include:

1. Escherichia coli that appears resistant to imipenem2. Klebsiella spp. susceptible to ampicillin3. Proteus mirabilis resistant to ampicillin4. Staphylococcus aureus resistant to vancomycin

COMMENTARY:



Acceptable results derived from testing QC strains does not guarantee accurate results with all patient isolates. When unusual or inconsistent results are encountered with patient isolates, the results should be investigated further to ensure accuracy. For example, repeat testing and/or repeat identification procedures using an alternative method (if possible) should be performed in an effort to ensure accurate results.

1. Escherichia coli that appears resistant to imipenem2. Klebsiella spp. susceptible to ampicillin3. Proteus mirabilis resistant to ampicillin4. Staphylococcus aureus resistant to vancomycin

REFERENCES: 1) NCCLS. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically - sixth edition; approved standard M7-A6. Wayne, PA: NCCLS, 2003; 2) NCCLS. Performance standards for antimicrobial disk susceptibility tests - eighth edition; approved standard M2-A8. Wayne, PA: NCCLS, 2003; 3) NCCLS. Analysis and presentation of cumulative antimicrobial susceptibility test data; approved guideline M39-A. Wayne PA: NCCLS, 2002; 4) NCCLS. Aerobic dilution supplemental tables; thirteenth informational supplement M100-S13. Wayne, PA: NCCLS, 2003.

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PROCEDURES AND TESTS

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ROUTINE PROCEDURES: The following questions define minimum standards for evaluation of routine cultures. This does not preclude the use of screening cultures (limited studies) and should not be construed to mean that all routine cultures require special media. Special media should be available if needed.

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RESPIRATORY CULTURES

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Potential pathogens may be part of the oral flora of patients and may not require full identification or susceptibility testing, if there is evidence of heavy contamination of the sputum with saliva. Attempts should be made to correlate culture results (e.g., the presence of a single species in large numbers) with gram-stained smear results (e.g., the presence of single morphotype in large numbers) and whenever possible with clinical information obtained from the physician concerning objective evidence of pneumonia.

Routine procedures from acceptable sputum cultures should allow the isolation of pneumococci, Staphylococcus aureus, Pseudomonas aeruginosa, and Enterobacteriaceae. Isolation identification, and reporting of Hemophilus species, Neisseria meningitidis, and Moraxella (Branhamella)



catarrhalis should be reserved for those instances in which there is a predominance of morphotypes resembling these organisms on the gram-stained smears.


Is a gram-stained smear performed routinely on all expectorated sputa to determine acceptability of a specimen for bacterial culture or the extent of culture workup?

COMMENTARY:

A gram-stained smear should be routinely performed on all expectorated sputa to determine acceptability of a specimen for bacterial culture or the extent of culture workup.

REFERENCES: 1) Valenstein PN. Semiquantitation of bacteria in sputum Gram stains. J Clin Microbiol. 1988;26:1791-1794; 2) Miller JM, et al. Specimen collection, transport, and storage. In: Murrary PR, et al, ed. Manual of Clinical Microbiology, 8th ed. Washington, DC: ASM Press; 2003:55-66; 3) Baron, et al. Infectious disease physicians rate microbiology services and practices. J Clin Microbiol. 1996;34:496-500; 4) Wilson ML. Clinically relevant, cost-effective clinical microbiology. Strategies to decrease unnecessary testing. Am J Clin Pathol. 1997;107:154-165; 5) Merrick ST, et al. Comparison of induced versus expectorated sputum for diagnosis of pulmonary tuberculosis by acid-fast smear. Am J Infect Control. 1997;25:463-433; 6) Namias N, et al. A reappraisal of the role of gram's stain of tracheal aspirates in guiding antibiotic selection in the surgical intensive care unit. J Trauma. 1998;44:102-106; 7) Al-Moamary M. et al. The significance of the persistent presence of acid-fast bacilli in sputum smears in pulmonary tuberculosis. Chest. 1999;116:726-731; 8) Church DL. Are there published standards for Gram stain results for sputa to establish specimen quality for culture? College of American Pathologists CAP Today. 2000;14(4):97.


Are unacceptable sputum samples not cultured (or cultured only by special request) and is the clinician or nursing station notified so another specimen can be collected without delay?

NOTE: It is suggested that the laboratory notify an appropriate caregiver about an inadequate specimen even in an outpatient setting, such as a reference laboratory. Notification can be by phone or computer report.

COMMENTARY:

Unacceptable sputum specimens should not be cultured, and/or the clinician or nursing station should be notified of unacceptable specimens so another sample can be collected without delay.

REFERENCES: 1) Bartlett RC. Medical microbiology: quality cost and clinical relevance. New York, NY: Wiley, 1974:24-31; 2) Carroll KC. Laboratory Diagnosis of Lower Respiratory Tract Infection: Controversy and Conundrums. J Clin Microbiol. 2002;3115-3120.



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URINE CULTURES

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Are quantitative cultures (colony counts) performed?

COMMENTARY:

The minimal standards for evaluation of urine cultures should include an estimate of number of organisms and identification of gram-positive and gram-negative organisms. Quantitative cultures (colony counts) must be done routinely.


Do the media and procedures used permit the isolation and identification of both gram-positive and gram-negative bacteria?

COMMENTARY:

Media and procedures must be used to ensure isolation and identification of both gram-positive and gram-negative bacteria in urine.

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GYNECOLOGICAL CULTURES

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Are group B streptococcus screens from pregnant women collected and cultured in accordance with the current guidelines?

NOTE: In 2002, the CDC released revised guidelines for the prevention of perinatal Group B streptococcal disease. The new guidelines recommend universal prenatal screening for vaginal and rectal Group B streptococcal (GBS) colonization of all pregnant women at 35-37 weeks gestation. Procedures for collecting and processing clinical specimens for GBS culture and performing susceptibility testing to clindamycin and erythromycin are also included in the guidelines



COMMENTARY:

Group B streptococcus screens from pregnant women should be collected and cultured in accordance with current guidelines.

REFERENCES: 1) Center for Disease Control and Prevention, 2002. Prevention of perinatal Group B streptococcal disease. MMWR 51(RR-11);1-22; 2) Baron, EJ. Laboratory support for prevention of perinatal Group B streptococcal disease: Commentary on the guidelines on screening for Group B streptococci during pregnancy. Clin Micro Newsletter. Vol 25, No 9, May, 2003; 3) Schrag, SJ, et al. A population-based comparison of strategies to prevent early-onset Group B streptococcal disease in neonates. N Engl J Med 2002;347:233-9.

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STOOL CULTURES

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Does the final report for routine bacterial stool cultures list the organisms for which the specimen was cultured (e.g., Salmonella, Shigella, Vibrio, etc.)?

NOTE: It is inappropriate to report “No enteric pathogens isolated.” The report should list the organisms whose presence was sought by culture (e.g., No Salmonella, Shigella, or Campylobacter, etc., isolated).

COMMENTARY:

With the increasing number of bacterial agents associated with diarrhea, it is inappropriate to issue a report “No enteric pathogen isolated,” if the stool was only cultured for Salmonella and Shigella. The report should state, “No Salmonella or Shigella isolated.” The laboratory should list the organisms which were sought.

REFERENCE: Gilligan PH, et al. Laboratory diagnosis of bacterial diarrhea. Cumitech 12A, 1992. ASM Press; Washington DC.


Do routine procedures permit isolation and identification of enteric pathogens in patients with diarrhea?

COMMENTARY:



The routine procedure for processing specimens for enteric pathogens must be designed to permit the isolation and identification of enteric pathogens in patients with diarrhea.


Do routine procedures permit recovery of small numbers of enteric pathogens in asymptomatic carriers (enrichment or selective media)?

COMMENTARY:

Enrichment procedures or selective media must be used routinely to allow recovery and identification of small numbers of pathogens in asymptomatic carriers.


Does the laboratory have guidelines (developed with clinicians) for the number and/or timing of collection of stool specimens submitted for routine bacterial testing?

COMMENTARY:

The laboratory should consider developing guidelines with its clinicians for the number and/or timing of collection of stool specimens submitted for routine bacterial testing. Suggestions made by the authors of a 1996 CAP Q-Probes study (Valenstein et al) include:

1. Accept no more than 2 specimens/patient without prior consultation with an individual who can explain the limited yield provided by additional specimens

2. Do not accept specimens from inpatients after the third hospital day, without prior consultation

3. Test stool for Clostridium difficile toxin for all patients over 6 months of age with clinically significant diarrhea and a history of antibiotic exposure. Consider C. difficile testing as an alternative to routine microbiologic studies for inpatients over 6 months of age who have test requests for routine enteric pathogens

These recommendations are for diagnostic testing. Different guidelines may apply to tests ordered for follow-up.

REFERENCES: 1) Yannelli B, et al. Yield of stool cultures, ova and parasite tests, and Clostridium difficile determinations in nosocomial diarrhea. Am J Infect Control. 1988;16:246-249; 2) Siegel DL, et al. Inappropriate testing for diarrheal diseases in the hospital. JAMA. 1990;263:979-982; 3) Asnis DS, et al. Cost-effective approach to evaluation of diarrheal illness in hospitals. J Clin Microbiol. 1993;31:1675; 4) Fan K, et al. Application of rejection criteria for stool cultures for bacterial enteric pathogens. J Clin Microbiol. 1993;31:2233-2235; 5) Valenstein P, et al. The use and abuse of routine



stool microbiology. A College of American Pathologists Q-probes study of 601 institutions. Arch Pathol Lab Med. 1996;120:206-211; 6) Wilson ML. Clinically relevant, cost-effective clinical microbiology. Strategies to decrease unnecessary testing. Am J Clin Pathol. 1997;107:154-165; 7) Blackman E, et al. Cryptosporidiosis in HIV-infected patients: diagnostic sensitivity of stool examination, based on number of specimens submitted. Am J Gastroenterol. 1997;92:451-453; 8) Wood M. When stool cultures from adult inpatients are not appropriate. Lancet. 2001;357:901-902; 9) Bauer, TM, et al. Derivation and validation of guidelines for stool cultures for enteropathogenic bacteria other than Clostridium difficile in hospitalized patients. JAMA. 2001;285:313-319.

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CEREBROSPINAL FLUID CULTURES

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Are CSF samples for culture processed immediately on receipt?

COMMENTARY:

Cerebrospinal fluid samples for culture must be processed without delay when received by the laboratory.


Are gram stains done routinely, and positive results reported in accordance with laboratory policy for alert values?

COMMENTARY:

Smears must be prepared routinely, gram stains done and positive results reported in accordance with laboratory policy for alert values.

REFERENCE: Dunbar SA, et al. Microscopic examination and broth culture of cerebrospinal fluid in diagnosis of meningitis. J Clin Microbiol. 1998;1617-1620.


Does the procedure (media and incubation conditions) permit recovery of fastidious bacteria expected in this type of specimen (N. meningitidis, H. influenzae, etc.)?

COMMENTARY:



The types of media inoculated and method of incubation must be selected to ensure recovery of common pathogens with fastidious requirements, such as Neisseria meningitidis and Hemophilus influenzae.

REFERENCE: Sturgis CD, et al. Cerebrospinal fluid broth culture isolates. Their significance for antibiotic treatment. Am J Clin Pathol. 1997;108:217-221.


If antigen-detection methods are used, are back-up cultures performed on both positive and negative CSF specimens?

NOTE: Total dependence on a bacterial antigen test for the diagnosis of bacterial meningitis does NOT meet accreditational requirements. The sensitivity and, especially in urine, the specificity of such tests are not adequate. In addition, meningitis may be caused by bacteria not covered by the antigen tests. Thus, culture is essential for evaluation of bacterial meningitis, and must be performed on the patient specimen - if not performed by the present laboratory, the Inspector must seek evidence that culture has been performed elsewhere.

COMMENTARY:

If antigen-detection methods are used, back-up cultures must be performed on both positive and negative CSF specimens. Total dependence on a bacterial antigen test for the diagnosis of bacterial meningitis does not meet CAP accreditational requirements. The sensitivity and, especially in urine, the specificity of such tests are not adequate. In addition, meningitis may be caused by bacteria not covered by the antigen tests. Thus, culture is essential for evaluation of bacterial meningitis, and must be performed on the patient specimen - if not performed by the present laboratory, the Inspector must seek evidence that culture has been performed elsewhere.

REFERENCES: 1) Forward KR. Prospective evaluation of bacterial antigen detection in cerebral spinal fluid in the diagnosis of bacterial meningitis in a predominantly adult hospital. Diagn Micro Infect Dis. 1988;11:61-63; 2) Maxson S, et al. Clinical usefulness of cerebrospinal fluid bacterial antigen studies. J Pediat. 1994; 125:235-238; 3) Finlay FO, et al. Latex agglutination testing in bacterial meningitis. Arch Dis Child. 1995;73:160-161; 4) Rathore MH, et al. Latex particle agglutination tests on the cerebrospinal fluid. A reappraisal. J Florida Med Assoc. 1995;82:21-23; 5) Kiska DL, et al. Quality assurance study of bacterial antigen testing of cerebrospinal fluid. J Clin Micro. 1995;33:1141-1144; 6) Perkins MD, et al. Rapid bacterial antigen detection is not clinically useful. J Clin Micro. 1995;33:1486-1491.

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BLOOD CULTURES

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Is the blood culture system in use designed to recover both aerobic and, when indicated or if intended to be part of the routine procedure, anaerobic organisms?

COMMENTARY:

The routine system for blood cultures must be improved to ensure recovery of both aerobic and when appropriate, anaerobic organisms. The minimum standards for the evaluation of blood cultures require adequate procedures for the recovery and identification of both aerobic and, when appropriate, anaerobic organisms. All macroscopically negative blood cultures should be checked by stain and/or aerobic subculture at some point before discarding as negative; however, negative blood cultures processed on automated systems need not be subcultured or stained before discarding as negative if they have been monitored for at least 5 days.

REFERENCES: 1) Wilson SJ, et al. Diagnostic utility of postmortem blood cultures. Arch Pathol Lab Med. 1993;117:986-988; 2) Henke PK, Polk HC. Efficacy of blood cultures in the critically ill surgical patient. Surgery. 1996;120:752-759; 3) Sturmann KM, et al. Blood cultures in adult patients released from an urban emergency department: a 15 month experience. Acad Emerg Med. 1996;3:768-775; 4) Delaflor-Weiss E, Al-Haddadin D. Routine use of anaerobic blood culture bottles in a hospital setting. Am J Clin Pathol. 1997;108:351; 5) Swisher ED, et al. Blood cultures in febrile patients after hysterectomy: cost-effectiveness. J Reprod Med. 1997;42:547-550; 6) Grinsztejy B, et al. Mycobacteremia in patients with acquired immunodeficiency syndrome. Arch Intern Med. 1997;157:2359-2363; 7) Peacock SJ et al. Positive intravenous line tip cultures as predictors of bacteremia. J Hosp Infect. 1998;40:35-38; 8) DesJardin JA, et al. Clinical utility of blood cultures drawn from indwelling central venous catheters in hospitalized patients with cancer. Ann Int Med. 1999;131:641-647.


Are macroscopically negative aerobic blood cultures stained and/or subcultured at some point before discarding?

NOTE: Subcultures and/or stains need not be done on blood cultures performed by automated methods if bottles are monitored for at least 5 days.

COMMENTARY:

Macroscopically negative aerobic blood cultures must be stained and/or subcultured at some point before discarding. Studies have shown that subcultures or stains are not necessary on blood cultures performed by automated methods if bottles are monitored for 5 days.

REFERENCES: 1) Courcol RJ, et al. Routine evaluation of the nonradiometric Bactec NR 660 system. J Clin Microbiol. 1986;24:26-29; 2) Jungkind D, et al. Clinical comparison of new automated infrared blood culture system with the Bactec 460 system. J Clin Microbiol. 1986;23:262-266; 3) Wilson ML,



et al. Recovery of clinically important microorganisms from the BacT/Alert blood culture system does not require 7 day testing. Diag Microbiol Infect Dis. 1993;16:31-34; 4) Doern GV, et al. Four-day incubation period for blood culture bottles processed with the Difco ESP blood culture system. J Clin Microbiol. 1997;35:1290-2.


Are initial positive cultures reported in accordance with laboratory policy for alert values?

COMMENTARY:

An initial positive blood culture must be reported in accordance with laboratory policy for alert values.


Are sterile techniques for drawing and handling of blood cultures defined, made available to individuals responsible for specimen collection, and practiced?

COMMENTARY:

Sterile techniques for drawing of blood cultures must be defined, made available to individuals responsible for specimen collection, and practiced. It is recommended that blood culture statistics, including number of contaminated cultures, be maintained and reviewed regularly by the laboratory director. The laboratory should establish a threshold for an acceptable rate of contamination. Tracking the contamination rate and providing feedback to phlebotomists or other persons drawing cultures has been shown to reduce contamination rates. Other measures to monitor include types of skin disinfection, volume of blood drawn, number of culture sets drawn, number of single cultures and line draws.

REFERENCES: 1) Bates DW, et al. Contaminant blood cultures and resource utilization: the true consequences of false-positive results. JAMA. 1991;265:365-369; 2) Schifman RB, Pindur A. The effect of skin disinfection materials on reducing blood culture contamination. Am J Clin Pathol. 1991;99:536-538; 3) Strand CL, et al. Effect of iodophor versus iodine skin preparation on blood culture contamination rate. JAMA. 1993;269:1004-1006; 4) Spitalnic SJ, et al. The significance of changing needles when inoculating blood cultures; a meta-analysis. Clin Infect Dis. 1995;21:1103-1106; 5) Gibb AP, et al. Reduction in blood culture contamination rate by feedback to phlebotomists. Arch Pathol Lab Med. 1997;121:50-507; 6) Schifman RB, et al. Blood culture contamination. A College of American Pathologists Q-probes study involving 640 institutions and 497 134 specimens from adult patients. Arch Pathol Lab Med. 1998;122:216-221; 7) DesJardin JA, et al. Clinical utility of blood cultures drawn from indwelling central venous catheters in hospitalized patients with cancer. Ann Int Med. 1999;131:641-647; 8) Ernst DJ. Controlling blood culture contamination rates. Med Lab Observ. 2000;32(5):36-47; 9) Ruge DG, et al. Reduction of blood culture contamination rates by establishment of policy for central intravenous catheters. Lab Med. 2002;33:797-800.




Are adequate volumes of blood collected for detection of sepsis?

NOTE: In adults, at least 20 mL of blood per culture set (2 bottles) should be collected for culture. Automated blood culture systems approved or cleared by the FDA may use smaller volumes per culture set and are acceptable.

COMMENTARY:

Adequate volumes of blood should be drawn for blood cultures. In adults, at least 20 mL of blood per septic episode should be collected for culture. Larger volumes of blood increase the yield of true positive cultures. Automated blood culture systems approved or cleared by the FDA may use smaller volumes per culture set and are acceptable.

REFERENCES: 1) Kellogg JA, et al. Justification and implementation of a policy requiring two blood cultures when one is ordered. Lab Med. 1994;25:323-330; 2) Li J, et al. Effects of volume and periodicity on blood cultures. J Clin Microbiol. 1994;32:2829-2831; 3) Washington J, Ilstrup. Blood cultures: issues and controversies. Rev Infect Dis. 1986;8:792-802; 4) Schiffman RB, et al. Blood culture quality improvement. A College of American Pathologists Q-probes study involving 909 institutions and 289 572 blood culture sets. Arch Pathol Lab Med. 1996;120:999-1002; 5) Arendup M, et al. Diagnosing bacteremia at a Danish hospital using one early large blood volume for culture. Scand J Infect Dis. 1996;28:609-614; 6) Wilson ML. Clinically relevant, cost-effective clinical microbiology. Strategies to decrease unnecessary testing. Am J Clin Pathol. 1997;107:154-165; 7) Goonewardene S. Evaluating the efficiency of the workup of multiple positive blood cultures for individual patients in a university hospital setting. Am J Clin Pathol. 1998;110:542; 8) Novis DA, et al. Solitary blood cultures. A College of American Pathologists Q-Probes study of 132778 blood culture sets in 333 small hospitals. Arch Pathol Lab Med. 2001;125:1290-1294.

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WOUND CULTURES

----------------------------------------------------

**REVISED** 09/30/2004


Are special procedures defined to culture anaerobic organisms when indicated?

NOTE: The minimum standards for the evaluation of deep wound cultures require adequate procedures for the collection, recovery and identification of aerobic organisms, as well as moderately anaerobic organisms when indicated. Usual media for anaerobes include an anaerobic blood agar



plate, a medium that inhibits gram-positive and facultative gram-negative bacilli such as KV blood agar, a differential or selective medium such as BBE (Bacteroides bile-esculin), and a gram-positive selective medium (colistin-nalidixic acid blood agar or phenylethyl alcohol blood agar). Provisions for adequate anaerobic incubation, with monitoring of the anaerobic environment, must be available. If the laboratory is not equipped to handle anaerobic incubation, there must be a procedure to refer the specimen to a reference laboratory in an expeditious fashion using a satisfactory transport system.

COMMENTARY:

N/A


Are gram stains of direct smears examined and results routinely reported, when indicated?

NOTE: Gram stains are used to evaluate specimen quality and guide the work-up of the specimen. Examination of the smear may reveal morphotypes of the organisms present, acute inflammatory cells and squamous epithelial cells.

COMMENTARY:

N/A

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LABORATORY SAFETY

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NOTE TO THE INSPECTOR: The inspector should review relevant questions from the Safety section of the Laboratory General checklist, to assure that the bacteriology laboratory is in compliance. Please elaborate upon the location and the details of each deficiency in the Inspector's Summation Report.

The following question pertains specifically to the bacteriology laboratory.


Are microbiology specimen residuals and contaminated media disposed of in a manner to minimize hazards to all personnel handling the material?

NOTE: Sterilization or decontamination within the microbiology section before disposal is preferred. If such material is transported before treatment, it must be placed into a leak-resistant rigid container, and appropriately labeled.



COMMENTARY:

N/A

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MYCOBACTERIOLOGY

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QUALITY CONTROL

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SPECIMEN HANDLING

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Are specimens for mycobacterial culture collected appropriately and transported to the laboratory without delay?

COMMENTARY:

The laboratory should recommend collecting 3 first-morning sputum specimens for acid-fast smears and culture in patients with clinical and chest x-ray findings compatible with tuberculosis (preferably on 3 separate days). Specimens must be delivered to the laboratory promptly, ideally within 30 minutes, but at least within 1 day of collection. Specimens should be refrigerated within 1 hour of collection.

REFERENCES: 1) Tenover FC, et al. The resurgence of tuberculosis: is your laboratory ready? J Clin Microbiol. 1993;31:767-770; 2) McCarter YS, Robinson A. Quality evaluation of sputum specimens for mycobacterial culture. Am J Clin Pathol. 1996;105:769-773; 3) Bhattacharya M, et al. Cross-contamination of specimens with Mycobacterium tuberculosis. Clinical significance, causes, and prevention. Am J Clin Pathol. 1998;109:324-330; 4) Merrick ST, et al. Comparison of induced versus expectorated sputum for diagnosis of pulmonary tuberculosis by acid-fast smear. Am J Infect Control. 1997;25:463-433; 5) Divinagracia RM, et al. Screening by specialists to reduce unnecessary test ordering in patients evaluated for tuberculosis. Chest. 1998;114:681-684.



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REPORTING OF RESULTS

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When clinically indicated, are results of acid-fast stains reported within 24 hours of specimen receipt by the testing laboratory?

COMMENTARY:

When clinically indicated, the results of acid-fast smears should be reported within 24 hours of specimen receipt in the testing laboratory.

REFERENCES: 1) Huebner RE, et al. Current practices in mycobacteriology: results of a survey of state public health laboratories. J Clin Microbiol. 1993;31:771-775; 2) Tenover FC, et al. The resurgence of tuberculosis: is your laboratory ready? J Clin Microbiol. 1993;31:767-770; 3) Woods GL, et al. Mycobacterial testing in clinical laboratories that participate in the College of American Pathologists mycobacteriology surveys. Changes in practices based on responses to 1992, 1993, and 1995 questionnaires. Arch Pathol Lab Med. 1996;120:429-435.


Are susceptibility test results for M. tuberculosis available in a timely manner?

COMMENTARY:

The rapid recognition of drug-resistant organisms is essential to the control of multidrug-resistant tuberculosis. For isolates of M. tuberculosis complex, the CDC and Prevention Laboratory work group recommends that laboratories use methods that may allow susceptibility test results to be available within 28 days of specimen receipt. From a CAP accreditation perspective, 28 days is a goal, not a requirement.

REFERENCES: 1) Tenover FC, et al. The resurgence of tuberculosis: is your laboratory ready? J Clin Microbiol. 1993;31:767-770; 2) College of American Pathologists position statement regarding rapid detection of Mycobacterium tuberculosis. Arch Pathol Lab Med. 1993;117:873; 3) Woods GL, Witebsky FG. Current status of mycobacterial testing in clinical laboratories. Arch Pathol Lab Med. 1993;117:876-884; 4) Huebner RE, et al. Current practices in mycobacteriology: results of a survey of state public health laboratories. J Clin Microbiol. 1993;31:771-775; 5) NCCLS. Susceptibility testing for mycobacteria, nocardia, and other aerobic actinomycetes; approved standard M24-A. Wayne, PA: NCCLS, 2003; 6) Woods GL, et al. Mycobacterial testing in clinical laboratories that participate in the College of American Pathologists mycobacteriology surveys. Changes in practices based on responses to 1992, 1993, and 1995 questionnaires. Arch Pathol Lab Med. 1996;120:429-435; 7)



Woods GL, Witebsky FG. Susceptibility testing of Mycobacterium avium complex in clinical laboratories. Results of a questionnaire and proficiency test performance by participants in the College of American Pathologists mycobacteriology E survey. Arch Pathol Lab Med. 1996;120:436-439; 8) Howanitz JH, Howanitz PJ. Timeliness as a quality attribute and strategy. Am J Clin Pathol. 2001;116:311-315.

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MEDIA

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Is an appropriate sample of each medium and additive prepared by the laboratory checked for all of the following elements?

1. Sterility (if additives are introduced after initial sterilization)2. Ability to support growth (when applicable) by means of stock cultures or by

parallel testing with previous batches3. Biochemical reactivity (where appropriate)


COMMENTARY:

An appropriate sample of each medium and additive prepared by the laboratory must be checked for all of the following elements:

1. Sterility (if additives are introduced after initial sterilization)2. Ability to support growth (when applicable) by means of stock cultures or by parallel

testing with previous batches3. Biochemical reactivity (where appropriate)


REFERENCE: Sharp SE, et al. Lowenstein-Jensen media. No longer necessary for mycobacterial isolation. Am J Clin Pathol. 2000:113:770-773.


Are all media in satisfactory condition (adequately hydrated, tubed media not dried or loose from sides)?



COMMENTARY:

Deteriorated mycobacteriology media must be discarded.

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CONTROLS AND STANDARDS

----------------------------------------------------



COMMENTARY:

In mycobacteriology, procedures must be established to verify accuracy of patient test results if calibration and control materials are not available.



Are control specimens tested in the same manner as patient samples?

COMMENTARY:

It is implicit in quality control that control specimens are tested in the same manner as patient specimens. Moreover, QC specimens must be analyzed by personnel who routinely perform patient testing - this does not imply that each operator must perform QC daily, so long as each instrument and/or test system has QC performed at required frequencies, and all analysts participate in QC on a regular basis. To the extent possible, all steps of the testing process must be controlled, recognizing that pre-analytic and post-analytic variables may differ from those encountered with patients.

REFERENCE: Department of Health and Human Services, Centers for Medicare and Medicaid Services. Clinical laboratory improvement amendments of 1988; final rule. Fed Register. 2003(Jan 24):7146 [42CFR493.1256(d)(8)].





COMMENTARY:

The results of controls must be reviewed before reporting patient results. It is implicit in quality control that patient test results will not be reported when controls are unacceptable.

REFERENCE: Department of Health and Human Services, Centers for Medicare and Medicaid Services. Clinical laboratory improvement amendments of 1988; final rule. Fed Register. 2003(Jan 24):7166 [42CFR493.1256(f)].


Are AFB stains checked each day of use with appropriate positive and negative controls, and results documented?

COMMENTARY:

Acid fast bacterial stains must be each day of use with appropriate controls and the results recorded.



Are fluorescent stains checked with positive and negative controls each time of use and results documented?

COMMENTARY:

Fluorescent stains must be checked with positive and negative controls each time of use, and results documented.



Is a known strain of M. tuberculosis tested whenever the NAP test is performed?

COMMENTARY:



A known strain of Mycobacterium tuberculosis should be tested whenever the NAP test is performed.


If nucleic acid probes are used for identification of mycobacteria grown in culture, are appropriate positive and negative controls tested on each day of use?

COMMENTARY:

Appropriate positive and negative controls must be tested on each day of use of nucleic acid probes for identification of mycobacteria grown in culture.

REFERENCE: Woods GL. Molecular methods in the detection and identification of mycobacterial infections. Arch Pathol Lab Med. 1999;123:1002-1006.

**REVISED** 09/30/2004


If the laboratory performs susceptibility testing of M. tuberculosis, is a control strain sensitive to all antimycobacterial agents run each week of patient testing, and with each new batch/lot number of media and antimicrobial agents?

COMMENTARY:

N/A

REFERENCE: Department of Health and Human Services, Centers for Medicare and Medicaid Services. Clinical laboratory improvement amendments of 1988; final rule. Fed Register. 2003(Jan 24):3708 [42CFR493.1262(b)].

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RAPID METHODS

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The College of American Pathologists encourages laboratories in areas of the country where the incidence of tuberculosis has increased over the past several years and laboratories in other parts of



the country that have experienced an increased rate of recovery of mycobacteria to utilize the most rapid and reliable methods available for detection and identification of mycobacteria, especially M. tuberculosis, and the most rapid and reliable methods available for susceptibility testing of isolates of M. tuberculosis.


Is fluorochrome staining performed on mycobacterial smears prepared from primary specimens, either in the laboratory or by the reference laboratory?

NOTE: Such smears are easier to read than those stained with a conventional carbol-fuchsin based stain. Fluorescing organisms stand out prominently against the background of the smear, and the smears can be examined at a lower power than conventionally-stained smears, so that a larger amount of material can be examined in a given period of time. As with the interpretation of Ziehl-Neelsen- and Kinyoun-stained smears, expertise is needed for interpretation of smears stained with a fluorescent stain; not everything that fluoresces in such a stain is necessarily a mycobacterium. Particularly when only a few organism-like structures are seen, it is important to pay careful attention to their morphology before interpreting them as Mycobacteria.

COMMENTARY:

N/A

REFERENCES: 1) Narain R, et al. Microscopy positive and microscopy negative cases of pulmonary tuberculosis. Am Rev Resp Dis. 1971;103:761-773; 2) Lipsky BA, et al. Factors affecting the clinical value of microscopy for acid-fast bacilli. Rev Infect Dis. 1984;6:214-222; 3) Witebsky FG. Q&A. College of American Pathologists CAP TODAY, 1999;13(1):72; 4) Somoskovi A, et al. Lessons from a proficiency testing event for acid-fast microscopy. Chest. 2001;120:250-257.


Are nucleic acid probes, chromatography the NAP test, or other rapid method (e.g., nucleic acid amplification or sequencing) employed for identification of mycobacterial isolates?

COMMENTARY:

Nucleic acid probes, chromatography, and/or the NAP test should be used for identification of mycobacterial isolates.

REFERENCES: 1) Smith MB, et al. Evaluation of the enhanced amplified Mycobacterium tuberculosis direct test for direct detection of Mycobacterium tuberculosis complex in respiratory specimens. Arch Pathol Lab Med. 1999;123:1101-1103; 2) Driscoll JR, et al. How and why we fingerprint tuberculosis. RT J Resp Care Pract. 2001:Feb/Mar.



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CONCENTRATION, INOCULATION, INCUBATION

----------------------------------------------------


Are certain specimens (e.g., sputum) concentrated before AFB smear examination and culture?

COMMENTARY:

Certain specimens (e.g., sputum) submitted for culture of mycobacteria must routinely be concentrated and AFB smears prepared from the concentrate in addition to culture.


Are specimens (other than blood) routinely inoculated on at least 2 types of media or agar, including either liquid or thin agar plates with microscopic examination, when a sufficient amount of specimen is obtained?

COMMENTARY:

When a sufficient amount of specimen is obtained, the laboratory should inoculate specimens (other than blood) to at least 2 types of media or agar. This should include either liquid or thin-prep plates.

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CULTURES

----------------------------------------------------

Laboratories providing complete identification must provide a sufficient variety of differential tests to accurately identify and differentiate the different types of mycobacteria, including temperature growth requirements and photoreactivity studies. Laboratories not providing complete identification are encouraged to at least provide photoreactivity studies.


Are mycobacterial cultures maintained at 35-37C?

NOTE: The optimal incubation temperature for most mycobacterial specimens is 35 to 37 degrees C. Exceptions to this include specimens obtained from skin or soft tissue suspected to contain M. marinum



(incubate at 30-32 degrees C) or M. xenopi (incubate at 42 degrees C). These specimens should be held at 35 – 37 degrees in addition to the lower or higher temperature.

COMMENTARY:

Mycobacterial cultures must be incubated at 35-37C, and at 30 - 32 degrees Celsius or at 42 degrees Celsius when clinically indicated.

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DIFFERENTIAL BIOCHEMICAL PROCEDURES

----------------------------------------------------


Are differential biochemical tests appropriate for the extent and manner of mycobacterial identification?

NOTE: The number and types of biochemical tests needed depend upon (a) the extent to which mycobacteria are identified (e.g., "Mycobacterium kansasii" or "photochromogen"), (b) the particular species which a laboratory attempts to identify (e.g., does it attempt to identify Mycobacterium terrae complex, or the species and subspecies of the Mycobacterium chelonae-Mycobacterium fortuitum complex), and (c) the degree to which biochemical testing is ancillary to other methods such as nucleic acid probes and HPLC. Useful biochemical tests include, but are not limited to, arylsulfatase, 68C catalase, semiquantitative catalase, iron uptake, MacConkey agar, 5% NaCl, niacin accumulation, nitrate reductase, Tween 80 hydrolysis, and urease. These tests are particularly useful for the following identifications and discriminations:

TEST UTILITYArylsulfatase Helps distinguish pathogenic from non-pathogenic rapid growers; also useful

for M. marinum, M. szulgai, M. xenopi, M. triviale.68C catalase Helpful for identification of M. tuberculosisSemiquantitativecatalase

Helpful in certain circumstances. M.tuberculosis complex, MAC, M. xenopi, and a few other species produce <45 mm of bubbles.

Iron uptake Helps distinguish M. chelonae from M. fortuitum.MacConkey agar Helps with identification of rapid growers.5% NaCl Helps with identification of rapid growers and M. triviale.Niacin accumulation Helps with identification of M. tuberculosis, M. simiae, some strains of M.

bovis.Nitrate reductase Helpful in identifying many mycobacterial species.Tween 80 hydrolysis Helps distinguish some usually pathogenic from some usually non-pathogenic

mycobacterial species.Urease Helpful in identifying many mycobacterial species.



COMMENTARY:

N/A

REFERENCE: Kent PT, Kubica GP. Public health mycobacteriology: a guide for the level III laboratory. Atlanta, GA: US Department of Health and Human Services, Centers for Disease Control, 1985.


Are all biochemical tests employed checked each day of use with appropriate positive and negative controls and results recorded?

COMMENTARY:

Appropriate positive and negative controls must be tested and results recorded with all biochemical procedures.

REFERENCE: Department of Health and Human Services, Centers for Medicare and Medicaid Services. Clinical laboratory improvement amendments of 1988; final rule. Fed Register. 2003(Jan 24):3708 [42CFR493.1262(a)].

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LABORATORY SAFETY

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NOTE TO THE INSPECTOR: The inspector should review relevant questions from the Safety section of the Laboratory General checklist, to assure that the mycobacteriology laboratory is in compliance. Please elaborate upon the location and the details of each deficiency in the Inspector's Summation Report.

The following questions pertain specifically to the mycobacteriology laboratory.


Are all specimens for mycobacterial culture collected and/or received in sealed leak-proof containers?

COMMENTARY:

Leak-proof containers must be provided for specimens for mycobacteria cultures.




In centrifuging specimens, are sealed screw capped tubes enclosed in sealed safety centrifuge carriers (i.e., a double closure system) used to minimize aerosol hazards?

COMMENTARY:

In centrifuging specimens, a double closure system using a screw capped centrifuge tube inside a screw capped leak-proof centrifuge cup must be used.

REFERENCE: NCCLS. Clinical laboratory safety; approved guideline GP17-A. Wayne, PA: NCCLS, 1996.


Does the biologic safety cabinet meet minimum requirements for mycobacteriologic work?

NOTE: Exhaust air from a class I or class II biological safety cabinet must be filtered through HEPA filters. Air from Class I and IIB cabinets is hard-ducted to the outside. Air from Class IIA cabinets may be recirculated within the laboratory if the cabinet is tested and certified at least every 12 months. It may be exhausted through a dedicated stack that protects against backflow of air from adverse weather conditions or through the building exhaust air system in a manner (e.g., thimble convection) that avoids any interference with the air balance of the biological safety cabinet or building exhaust system.

COMMENTARY:

The biologic safety cabinet must meet minimum requirements for mycobacteriologic work. Exhaust air from a class I or class II biological safety cabinet must be filtered through HEPA filters. Air from class I and IIB cabinets is hard-ducted to the outside. Air from class IIA cabinets may be recirculated within the laboratory if the cabinet is tested and certified at least every 12 months. It may be exhausted through a dedicated stack that protects against backflow of air from adverse weather conditions or through the building exhaust air system in a manner (e.g., thimble convection) that avoids any interference with the air balance of the biological safety cabinet or building exhaust system.

REFERENCES: 1) Department of Health and Human Services. Biosafety in microbiological and biomedical laboratories, 3rd edition. Washington, DC: Superintendent of Documents, Document 017-040-00523-7, May 1993; 2) NCCLS. Protection of laboratory workers from occupationally acquired infections - second edition; approved guideline M29-A2. Wayne, PA: NCCLS, 2001.



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MYCOLOGY

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QUALITY CONTROL

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MEDIA

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NOTE: See QUALITY CONTROL text preceding Question MIC.21200 concerning requirements for commercially prepared media.


Is an appropriate sample of each medium prepared by the laboratory or purchased but not excluded from testing in NCCLS M22-A3 checked for each of the following?

1. Sterility (following introduction of additives after sterilization)2. Ability to support growth and biochemical reactivity (where applicable) by means

of stock cultures or by parallel testing with previous batches

COMMENTARY:

All media must be checked for sterility (following introduction of additives after sterilization); and ability to support growth and biochemical reactivity (where applicable) by means of stock cultures or by parallel testing with previous batches.

REFERENCE: NCCLS. Quality assurance for commercially prepared microbiological culture media – third edition; approved standard M22-A3. Wayne, PA: NCCLS, 2004.

----------------------------------------------------

CONTROLS AND STANDARDS:

----------------------------------------------------



Good laboratory practice includes checking all media either at the time of receipt or concurrently with use. This applies to purchased media as well as media prepared by the laboratory. See text preceding MIC.21200 concerning requirements for commercially prepared media.


Are reference cultures maintained?

COMMENTARY:

Reference cultures must be maintained by the laboratory for proper quality control of media, reagents, antimicrobial susceptibility materials, and serologic tests.


Are reference cultures used to check stains and reagents at appropriate intervals?

COMMENTARY:

Reference cultures must be used to check stains and reagents at appropriate intervals.



If nucleic acid probes or exo-antigen tests are used for identification of fungi isolated from culture, are appropriate positive and negative controls tested on each day of use?

COMMENTARY:

If nucleic acid probes or exo-antigen tests are used for identification of fungi isolated from culture, appropriate positive and negative controls must be tested on each day of use.



COMMENTARY:







COMMENTARY:





COMMENTARY:

Controls must be reviewed before reporting patient results. It is implicit in quality control that patient test results will not be reported when controls are unacceptable.



Are direct patient specimen stains (e.g., acid fast, PAS, Giemsa, Gomori's methenamine silver, Calcofluor white, India ink) checked with positive and negative controls on each day of patient sample testing?



NOTE: For certain stains such as GMS and Giemsa, the slide itself serves as the negative control. Controls for KOH preparations are not required.

COMMENTARY:

N/A

REFERENCES: 1) August, Hindler, Huber, Sewell. Quality control and quality assurance practices. In: Clinical Microbiology, Cumitech 3A. Washington, DC: American Society for Microbiology, 1990; 2) Department of Health and Human Services, Centers for Medicare and Medicaid Services. Clinical laboratory improvement amendments of 1988; final rule. Fed Register. 2003(Jan 24):7166 [42CFR493. 1256(e)(2); 493.1256(e)(3); 493.1273(a)]; 3) Department of Health and Human Services, Centers for Medicare and Medicaid Services. Clinical laboratory improvement amendments of 1988; final rule. Fed Register. 2003(Jan 24):3709 [42CFR493.1263(a)].


Are out of control results reported to the supervisor or laboratory director and is there documented evidence of corrective actions taken?

COMMENTARY:

Results that are out of control must be reported immediately to the supervisor or laboratory director and corrective action must be documented.

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The intent of this series of questions is to ensure the use of an appropriate variety of media and growth conditions to isolate the significant pathogens with minimal interference from contaminants.


Are preliminary screening procedures, such as direct wet mount preparations and stains performed when indicated (e.g., 10% KOH, India ink, Giemsa)?

COMMENTARY:

Preliminary screening procedures (such as 10% KOH, India ink, Giemsa stains) must be available and performed when indicated. Isolation and identification procedures should include preliminary



screening with direct or stained preparations, use of selective media for dermatophytes and systemic fungi and use of media with antimicrobial agents.


Are suitable selective media used for the growth and isolation of dermatophytes and/or systemic fungi?

COMMENTARY:

Additional or different media must be used for culture of dermatophytes and/or systemic fungi.


Are media with antimicrobial agents used to suppress the growth of contaminants?

NOTE: Antimicrobial agents may inhibit some yeasts and the yeast phase of dimorphic organisms. Both types of media (with and without antimicrobials) should be available and used when indicated.

COMMENTARY:

Media with antimicrobial agents must be available and used, when indicated, to suppress overgrowth of bacteria. Antimicrobial agents may inhibit some yeasts and the yeast phase of dimorphic organisms. Both types of media (with and without antimicrobials) must be available and used when indicated.


Are incubation temperatures for the growth and isolation of dermatophytes and systemic fungi defined and followed under culture conditions?

COMMENTARY:

Incubation temperatures for the growth and isolation of dermatophytes and systemic fungi must be defined and adhered to under culture conditions.


If cultures are incubated at room temperatures, is actual ambient temperature (22-26C) checked daily to determine if proper growth conditions are being maintained?

COMMENTARY:



If cultures are left at room temperature, the ambient temperature (22-26C) must be checked and recorded daily.


Are procedures for the differentiation and identification of fungi (differential tests) adequate for the needs of the laboratory?

NOTE: Laboratories offering full identification must have sufficient procedures to do so. Smaller laboratories with limited services should have an arrangement with an approved reference laboratory for back-up and complete identification of mycology specimens.

COMMENTARY:

If the laboratory is providing full identification services, adequate procedures for differentiation and identification of fungi must be provided. Individual judgment will be required. Laboratories offering full identification must have sufficient procedures to do so. Smaller laboratories with limited services must have an arrangement with an approved reference laboratory for back-up and complete identification of mycology specimens.

REFERENCE: Riddle DL, et al. Clinical comparison of the Baxter Microscan yeast identification panel and the Vitek yeast biochemical card. Am J Clin Pathol. 1994;101:438-442.


Do differential tests include biochemical tests (e.g., urease, carbohydrate assimilation and/or fermentation)?

COMMENTARY:

Biochemical tests (e.g., urease, carbohydrate assimilation and/or fermentation) must be used routinely.


Do differential tests include slide cultures (when appropriate)?

COMMENTARY:

Slide cultures should be performed when needed, but are not required for the identification of dermatophytes.




Do differential tests include nutritional studies for dermatophytes?

COMMENTARY:

Nutritional studies should be performed for the differential identification of dermatophytes.


Are exo-antigen tests, DNA probes for fungi, or yeast phase conversion tests with conversion-negative tested in the laboratory or sent to a reference laboratory for confirmation by exoantigen tests or DNA probes for dimorphic fungi?

COMMENTARY:

Exo-antigen tests or DNA probes are recommended for dimorphic fungi. If these are not performed in the laboratory, then yeast phase conversion-negative isolates should be sent to a reference laboratory for confirmation by these methods.

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LABORATORY SAFETY

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NOTE TO THE INSPECTOR: The inspector should review relevant questions from the Safety section of the Laboratory General checklist, to assure that the mycology laboratory is in compliance. Please elaborate upon the location and the details of each deficiency in the Inspector's Summation Report.

The following questions pertain specifically to the mycology laboratory.


If plate culture media is used in mycology, are appropriate safety precautions taken (such as taping lid to plate on both sides when not in use or other appropriate measures) to prevent the accidental opening of a plate?

COMMENTARY:

Appropriate safety precautions must be taken if culture media for mycology are used in Petri plates. Taping the lids of plate media on both sides reduces the chance of accidental opening and exposure of infectious spores to laboratory personnel. Furthermore, some authorities recommend the transfer of growing colonies from plate to tubed media, if the former is routinely used for initial inoculation.




When working with a colony exhibiting mycelial growth, are transfers performed within a biological safety cabinet?

COMMENTARY:

Upon observing growth of aerial mycelia on culture media, it must always be handled in a functional biological safety cabinet.


Is the use of slide culture techniques limited, where possible, to work with low virulence organisms; or if used for dimorphic fungi, are special safety precautions defined and rigidly adhered to?

COMMENTARY:

When working with highly infectious dimorphic fungi, slide cultures must only be performed when strict and appropriate safety measures are followed. This technique must not be used if there is any question of it being a dimorphic fungus.


When preparing teased preparations or "scotch" tape preps, are mycelia always submerged in some liquid medium (such as lactophenol cotton blue)?

COMMENTARY:

When dealing with teased or "scotch" tape preparations, mycelia must always be submerged in some liquid medium such as lactophenol cotton blue.


Is a biological safety cabinet (BSC) or hood available for handling organisms considered to be highly contagious by airborne routes?

COMMENTARY:

A biologic safety cabinet or hood must be provided for handling cultures containing organisms considered to be highly contagious by airborne routes.



REFERENCE: Department of Health and Human Services. Biosafety in microbiological and biomedical laboratories, 3rd edition. Washington, DC: Superintendent of Documents, Document 017-040-00523-7, May 1993.


Is the BSC certified annually to ensure that filters are functioning properly and that airflow rates meet specifications?

COMMENTARY:

The biologic safety cabinet must be certified annually to ensure that filters are functioning properly and that airflow rates meet specifications.


Does the BSC meet minimum requirements for mycologic work?

NOTE: Exhaust air from a class I or class II BSC must be filtered through HEPA filters. It may be recirculated within the laboratory if the BSC is tested and certified at least every 12 months. It may be exhausted through a dedicated stack that protects against backflow of air from adverse weather conditions or through the building exhaust air system in a manner (e.g., thimble convection) that avoids any interference with the air balance of the BSC or building exhaust system.

COMMENTARY:

The biologic safety cabinet (BSC) must meet minimum requirements for microbiologic work. Exhaust air from a class I or class II BSC must be filtered through HEPA filters. It may be recirculated within the laboratory if the cabinet is tested and certified at least every 12 months. It may be exhausted through a dedicated stack that protects against backflow of air from adverse weather conditions or through the building exhaust air system in a manner (e.g., thimble convection) that avoids any interference with the air balance of the BSC or building exhaust system.


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PARASITOLOGY

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QUALITY CONTROL

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Are reference materials, such as permanent mounts, photomicrographs, NCCLS documents M15-A and M28-A, or printed atlases available at the work bench to assist with identifications?

COMMENTARY:

Reference materials, such as permanent mounts, photomicrographs, NCCLS documents M15-A or M28-A or printed atlases must be available at the work bench to assist with identifications.

REFERENCES: 1) NCCLS. Laboratory diagnosis of blood-borne parasitic diseases; approved guideline M15-A. Wayne, PA: NCCLS, 2000; 2) Garcia LS, Shimizu R. Diagnostic parasitology: parasitic infections and the compromised host. Lab Med. 1993;24:205-215; 3) Ash R, Orihel TC. Atlas of human parasitology, 4th ed. Chicago, IL: American Society of Clinical Pathology, 1996; 4) NCCLS. Procedures for the recovery and identification of parasites from the intestinal tract; approved guideline M28-A. Wayne, PA: NCCLS, 1997; 5) Garcia LS,. Diagnostic medical parasitology. 4th ed. Washington, DC: ASM Press, 2001; 6) Colmer-Hamood JA, Fecal microscopy. Artifacts mimicking ova and parasites. Lab Med. 2001;32:80-84.

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REAGENTS

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If zinc sulfate is used, is the solution checked for specific gravity (1.18 for fresh specimens and 1.20 for formalin-fixed specimens) with a hydrometer whose scale is large enough to differentiate the two values?

COMMENTARY:

If zinc sulfate is used, the solution must be checked periodically for specific gravity (1.18 for fresh specimens and 1.20 for formalin-fixed specimens) with a hydrometer whose scale is large enough to differentiate the two values.




Is the zinc sulfate flotation solution stored in a tightly-stoppered bottle?

COMMENTARY:

Zinc sulfate solution should be stored in a tightly-stoppered bottle.


Are all permanent parasitology stains checked for intended reactivity at least monthly (or with each test if performed less frequently than every month)?

COMMENTARY:

All permanent parasitology stains must be checked for intended staining characteristics at least monthly (or with each test if performed less frequently than every month) with controls or reference materials. PVA fixative solutions thoroughly mixed with fresh fecal material that has been seeded with buffy coat leukocytes usually provides reliable controls for permanent stains.

REFERENCES: 1) Department of Health and Human Services, Centers for Medicare and Medicaid Services. Clinical laboratory improvement amendments of 1988; final rule. Fed Register. 2003(Jan 24):7167 [42CFR493.1264(c)]; 2) Garcia LS, Diagnositic medical parasitology, 4th ed. Washington, DC: ASM Press, 2001.


Are stains that are used to detect specific parasites (e.g., acid fast, fluorescent) checked with appropriate control organisms each time that stain is used?

COMMENTARY:

Stains that are used to detect specific parasites (e.g., acid fast, fluorescent) must be checked with appropriate control organisms each time that stain is used.

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INSTRUMENTS AND EQUIPMENT

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Is an ocular micrometer available for determining the size of eggs, larvae, cysts or trophozoites?



COMMENTARY:

An ocular micrometer is required in the parasitology section.



Has the ocular micrometer been calibrated for the microscope(s) in which it is used and is it recalibrated each time the eyepieces or objectives are changed?

NOTE: Calibrations can be checked against a micrometer or other objects of known dimensions. If there are no changes to a particular microscope's optical components, there is no need for checking calibration on some fixed calendar schedule.

COMMENTARY:

Ocular micrometers must be calibrated for the microscope(s) in which they are used. Recalibration is needed any time eyepieces or objectives are changed. If there are no changes to a particular microscope's optical components, there is no need for checking calibration on some fixed calendar schedule.


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STOOLS FOR OVA AND PARASITES

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Does the microscopic examination of all stools submitted for an ova and parasite (O&P) examination include a concentration procedure and a permanent stain?



NOTE: When a stool specimen is submitted fresh, the usual approach would be to perform a direct wet preparation (looking for motility), a concentration (helminth eggs/larvae/protozoan cysts), and the permanent stained smear (identification of protozoa missed by concentration and confirmation of suspect organisms). As a minimum (and certainly if the stool is submitted in preservatives), the standard O&P examination would include the concentration procedure and a permanent stained smear. The main point is to ensure that the permanent stained smear is performed on all stool specimens, regardless of what was or was not seen in the concentration wet preparation. Often, intestinal protozoa will be seen in the permanent stained smear, but may be missed in the concentration examination.

COMMENTARY:

The microscopic examination of all stools submitted for an ova and parasite (O&P) examination must include a concentration procedure and a permanent stain. When a stool specimen is submitted fresh, the usual approach would be to perform a direct wet preparation (looking for motility), a concentration (helminth eggs/larvae/protozoan cysts), and the permanent stained smear (identification of protozoa missed by concentration and confirmation of suspect organisms). As a minimum (and certainly if the stool is submitted in preservatives), the standard O&P examination would include the concentration procedure and a permanent stained smear. The main point is to ensure that the permanent stained smear is performed on all stool specimens, regardless of what was or was not seen in the concentration wet preparation. Often, intestinal protozoa will be seen in the permanent stained smear, but may be missed in the concentration examination.

REFERENCES: 1) Garcia LS, Diagnostic medical parasitology, 4th edition. Washington, DC: ASM Press, 2001; 2) NCCLS. Procedures for the recovery and identification of parasites from the intestinal tract; approved guideline M28-A. Wayne, PA: NCCLS, 1997.


Does the microscopic examination of liquid stools include a direct wet mount if submitted fresh?

NOTE: Liquid stools contain the motile form of the intestinal protozoa (the trophozoite). Thus, in fresh stool, the examination of a direct saline preparation might allow one to see motile organisms. However, there are often significant delays between the time of stool passage and when the laboratory receives the specimen. Thus, the ability to see motile organisms may or may not be possible, depending on the condition of the specimen. The number of times motile protozoa are seen will vary tremendously.

COMMENTARY:

The microscopic examination of fresh (unpreserved) liquid stools should include a wet mount of fresh specimen by direct technique. Liquid stools contain the motile form of the intestinal protozoa (the trophozoite). Thus, in fresh stool, the examination of a direct saline preparation might allow one to see motile organisms. However, there are often significant delays between the time of stool passage and



when the laboratory receives the specimen. Thus, the ability to see motile organisms may or may not be possible, depending on the condition of the specimen. The number of times motile protozoa are seen will vary tremendously.

REFERENCES: 1) Ash LR, Orihel TC. Parasites: a guide to laboratory procedures and identification. Chicago, IL: American Society of Clinical Pathology, 1987; 2) NCCLS. Procedures for the recovery and identification of parasites from the intestinal tract; approved guideline M28-A. Wayne, PA: NCCLS, 1997; 3) Garcia LS, Diagnostic medical parasitology. 4th ed. Washington, DC: ASM Press, 2001.


Does the laboratory have guidelines (developed with clinicians) for the number and/or timing of collection of stool specimens submitted for routine parasitology testing?

COMMENTARY:

The laboratory should consider developing guidelines with its clinicians for the number and/or timing of stool specimens submitted for routine "ova and parasite" testing. Suggestions made by the authors of a 1996 CAP Q-Probes study (Valenstein et al) include:

1. Accept no more than 2 or 3 specimens/patient without prior consultation with an individual who can explain the limited yield provided by additional specimens

2. Do not accept specimens from inpatients after the fourth hospital day, without prior consultation

These recommendations are for diagnostic testing. Different guidelines may apply to tests ordered for follow-up

REFERENCES: 1) Yannelli B, et al. Yield of stool cultures, ova and parasite tests, and Clostridium difficile determinations in nosocomial diarrhea. Am J Infect Control. 1988;16:246-249; 2) Morris AJ, et al. Application of rejection criteria for stool ovum and parasite examinations. J Clin Microbiol. 1992;30:3213-3216; 3) Valenstein P, et al. The use and abuse of routine stool microbiology. A College of American Pathologists Q-probes study of 601 institutions. Arch Pathol Lab Med. 1996;120:206-211; 4) Cartwright CP. Utility of multiple-stool-specimen ova and parasite examinations in high-prevalence setting. J Clin Microbiol. 1999;37:2408-2411.

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BLOOD FILMS FOR MALARIA AND OTHER PARASITES

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When blood films are positive for malaria parasites (Plasmodium spp.), is the level of parasitemia reported along with the organism identification?

NOTE: It is important to report the level of parasitemia when blood films are reviewed and found to be positive for malaria parasites. Because of the potential for drug resistance in some of the Plasmodium species, particularly P. falciparum, it is important that every positive smear be assessed and the parasitemia reported exactly the same way on follow-up specimens as on the initial specimen. This allows the parasitemia to be followed after therapy has been initiated. The parasitemia will usually drop very quickly within the first 24 hours; however, in cases of drug resistance, the level may not decrease, but actually increase over time.

COMMENTARY:

N/A

REFERENCES: 1) NCCLS. Laboratory diagnosis of blood-borne parasitic diseases; approved guideline M-15-A. Wayne, PA: NCCLS, 2000; 2) Garcia LS, Diagnostic Medical Parasitology. Washington, DC, ASM Press, 2001.


Are both thick and thin films (routine blood films and/or buffy coat films) made to provide thorough examination for blood parasites?

COMMENTARY:

N/A

REFERENCES: 1) NCCLS. Laboratory diagnosis of blood-borne parasitic diseases; approved guideline M15-A. Wayne, PA: NCCLS, 2000; 2) Thomson S, et al. External quality assessment in the examination of blood films for malarial parasites within Ontario, Canada. Arch Pathol Lab Med. 2000;124:57-60.


Is there documentation that malaria stains are washed with a buffer of a pH appropriate for the stain used (e.g. pH 6.8-7.2 for Giemsa)?

COMMENTARY:

N/A



REFERENCES: 1) NCCLS. Laboratory diagnosis of blood-borne parasitic diseases; approved guideline M15-A. Wayne, PA: NCCLS, 2000; 2) Garcia LS, Diagnostic medical parasitology. 4th ed. Washington, DC: ASM Press, 2001.


Are an adequate number of fields examined under oil immersion using the 100X oil immersion objective (e.g., 300 fields)?

COMMENTARY:

N/A

REFERENCE: NCCLS. Laboratory diagnosis of blood-borne parasitic diseases; approved guideline M15-A. Wayne, PA: NCCLS, 2000.


Are the results of positive blood parasite films reported in accordance with laboratory policy for alert (critical) values?

COMMENTARY:

N/A

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LABORATORY SAFETY

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NOTE TO THE INSPECTOR: The inspector should review relevant questions from the Safety section of the Laboratory General checklist, to assure that the parasitology laboratory is in compliance. Please elaborate upon the location and the details of each deficiency in the Inspector's Summation Report.

The following questions pertain specifically to the parasitology laboratory.

**REVISED** 09/30/2004


If a procedure uses formalin, are formaldehyde vapor concentrations maintained below the following maxima, expressed as parts per million?



8 hr Time-Weighted Exposure Limit Action Level ( 8 hr Time-Weighted

Exposure)

15 min Short-Term Exposure Limit (STEL)

0.75 0.5 2.0

NOTE: Formaldehyde vapor concentrations must be monitored in all areas where formalin is used. Once an initial monitoring procedure has been performed, further periodic formaldehyde monitoring is mandated if the initial monitoring result equals or exceeds 0.5 ppm (8 hr time-weighted exposure, the “action level”) or 2.0 ppm (STEL). The laboratory may discontinue periodic formaldehyde monitoring if results from 2 consecutive sampling periods taken at least 7 days apart show that employee exposure is below the action level and the short-term exposure limit, and 1) no change has occurred in production, equipment, process or personnel or control measures that may result in new or additional exposure to formaldehyde, and 2) there have been no reports of conditions that may be associated with formaldehyde exposure.

Formaldehyde monitoring must be repeated any time there is a change in production, equipment, process, personnel, or control measures which may result in new or additional exposure to formaldehyde. If any personnel report signs or symptoms of respiratory or dermal conditions associated with formaldehyde exposure, the laboratory must promptly monitor the affected person’s exposure.

COMMENTARY:

N/A

REFERENCES: 1) Montanaro A. Formaldehyde in the workplace and in the home. Exploring its clinical toxicology. Lab Med. 1996;27:752-757; 2) Goris JA. Minimizing the toxic effects of formaldehyde. Lab Med. 1997;29:39-42 ; 3) Occupational Safety and Health Administration, 1998(Jul 1) [29CFR1910.1048].


If a procedure uses ether, is the diethylether stored on open shelves in a well-ventilated room using the smallest can feasible (as shipped by manufacturer)?

NOTE: The use of concentration techniques other than those requiring the use of ether is recommended.

COMMENTARY:

N/A



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VIROLOGY

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QUALITY CONTROL

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REAGENTS

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Are culture media tested for sterility (if additives are introduced after initial sterilization)?

NOTE: Entering the media to remove aliquots for refeeding, etc. does not generate the need for repeat sterility testing.

COMMENTARY:

Media to which additives are introduced after initial sterilization must be rechecked for sterility. Entering the media to remove aliquots for refeeding, etc., does not generate the need for repeat sterility testing.


Are continuous cell lines checked for mycoplasma contamination?

COMMENTARY:

Continuous cell lines must be checked periodically for mycoplasma contamination. An alternative method, rather than culture for mycoplasma, could be the monitoring of a negative, uninoculated control.




Are animal sera used for cell growth media checked for absence of toxicity to cells?

COMMENTARY:

Animal sera used for growth cell media must be checked for toxicity to cells.


Does the laboratory have the appropriate minimal cell line(s) available for all virology testing performed?

NOTE: The following is a suggested list of cell lines for the intended purpose:

ORGANISM CELL LINEChlamydia McCoy or Buffalo Green Monkey KidneyHerpes simplex HDF, Primary or First Pass Rabbit Kidney, MRC-5, or A549Varicella zoster HDF, Primary Monkey KidneyInfluenza Primary Monkey Kidney or MDCKParainfluenza Primary Monkey KidneyRSV HEp-2 or Primary Monkey KidneyEnteroviruses Primary Monkey KidneyAdenoviruses HEp-2, Human Embryonic Kidney, or A549CMV HDF

COMMENTARY:

The laboratory must have the appropriate minimal cell lines available for all of the virology services that are performed in the laboratory. The following is a suggested list for the intended purpose:

ORGANISM CELL LINEChlamydia McCoy or Buffalo Green Monkey KidneyHerpes simplex HDF, Primary or First Pass Rabbit Kidney, MRC-5, or A549Varicella zoster HDF, Primary Monkey KidneyInfluenza Primary Monkey Kidney or MDCKParainfluenza Primary Monkey KidneyRSV HEp-2 or Primary Monkey KidneyEnteroviruses Primary Monkey KidneyAdenoviruses HEp-2, Human Embryonic Kidney, or A549CMV HDF

REFERENCES: 1) Department of Health and Human Services, Centers for Medicare and Medicaid Services. Clinical laboratory improvement amendments of 1988; final rule. Fed Register. 2003(Jan 24):7168 [42CFR493.1101(b); 493.1252(a)]; 2) McCarter YS, Robinson A. Comparison of MRC-5



and primary rabbit kidney cells for the detection of Herpes simplex virus. Arch Pathol Lab Med. 1997;121:122-124; 3) McCarter YS, Ratkiewicz IN. Comparison of virus culture and direct immunofluorescent staining of cytocentrifuged virus transport medium for detection of Varicella-Zoster virus in skin lesions. Am J Clin Pathol. 1998;109:631-633.


Are tube monolayer cultures incubated for a sufficient time to recover the viruses for which service is offered?

NOTE: The following is a suggested list of minimum incubation times for the intended purpose:

ORGANISM INCUBATION TIMEHerpes simplex (genital) 5 daysHerpes simplex (other) 7 daysRespiratory viruses 10 daysOther viruses 14 days

Spin-amplified shell vials are not tube monolayer cultures. Most spin amplification cultures (shell vials) are completed after 24-48 hours; laboratories incubating their shell vials for this shorter period should have in-house data to support that practice.

COMMENTARY:

Tube monolayer cultures must be incubated for a sufficient time to recover the viruses for which service is offered. The following is a suggested list of minimum incubation times for the intended purpose:

ORGANISM INCUBATION TIMEHerpes simplex (genital) 5 daysHerpes simplex (other) 7 daysRespiratory viruses 10 daysOther viruses 14 days

Spin-amplified shell vials are not tube monolayer cultures. Most spin amplification cultures (shell vials) are completed after 24-48 hours; laboratories incubating their shell vials for this shorter period should have in-house data to support that practice.

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CONTROLS AND STANDARDS

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Are records kept of cell types, passage number, source, and media used for their growth and maintenance?

COMMENTARY:

Records must be maintained of cell types, passages, sources and media used for growth and maintenance.


Are inoculated cultures checked for cytopathic effect in a fashion that optimizes the time to detection of viral pathogens?

NOTE: If tube cultures are the only means for detection of virus, primary cultures must be checked at least every other working day for cytopathic effect during the first 2 weeks of incubation. If additional diagnostic methods are used (e.g., shell vials, antisera), the observation schedule may be modified as appropriate.

COMMENTARY:

If tube cultures are the only means for detection of virus, Primary cultures must be checked at least every other working day for cytopathic effect during the first 2 weeks of incubation. If additional diagnostic methods are used (e.g., shell vials, antisera), the observation schedule may be modified as appropriate.


Are uninoculated cell monolayers or monolayers that have been inoculated with sterile material available for comparison with cultures of clinical material?

NOTE: Uninoculated cell culture controls should be included for each lot of cell culture tubes in order to detect non-specific degeneration; or to detect extraneous infection of the cell culture with endogenous viral agents capable of producing cytopathic effects.

COMMENTARY:

N/A


Are media and diluents checked for sterility and pH?



COMMENTARY:

Media and diluents must be checked for sterility and pH. It is satisfactory to test either the individual components or the final product.


Are red cell suspensions that are used for quantitative serologic procedures standardized (photometrically or with some other equivalent procedure)?

COMMENTARY:

Red cell suspensions used for quantitative serologic procedures should be standardized and checked by photometric or some other equivalent procedure.


Are criteria for degrees of agglutination and lysis defined for quantitative serologic procedures?

COMMENTARY:

Criteria should be defined for degrees of agglutination and lysis for quantitative serologic procedures.


Do worksheets and/or records indicate actual titers, when known, of reagents and control sera?

COMMENTARY:

Worksheets and/or records must indicate results of titers, when known, of reagents and control sera.


Are reactive and non-reactive controls processed in serologic reactions for detection of antibodies or antigens?

COMMENTARY:

Reactive and nonreactive controls must be tested in all serologic procedures.





COMMENTARY:





COMMENTARY:





COMMENTARY:

Controls must be reviewed before reporting patient results. It is implicit in quality control that patient test results will not be reported when controls are unacceptable.




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LABORATORY SAFETY

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NOTE TO THE INSPECTOR: The inspector should review relevant questions from the Safety section of the Laboratory General checklist, to assure that the virology laboratory is in compliance. Please elaborate upon the location and the details of each deficiency in the Inspector's Summation Report.

The following questions pertain specifically to the virology laboratory.


Is a biological safety cabinet (BSC) or hood available for handling specimens or organisms considered to be highly contagious by air borne routes?

COMMENTARY:

A biologic safety cabinet or hood must be provided for handling specimens or cultures containing organisms considered to be highly contagious by airborne routes.


Is the BSC certified annually to ensure that filters are functioning properly and that airflow rates meet specifications?

COMMENTARY:

The biologic safety cabinet must be certified annually to ensure that filters are functioning properly and that airflow rates meet specifications.


Does the BSC meet minimum requirements for virology work?

NOTE: Exhaust air from a class I or class II BSC must be filtered through HEPA filters. It may be recirculated within the laboratory if the BSC is tested and certified at least every 12 months. It may be exhausted through a dedicated stack that protects against backflow of air from adverse weather conditions or through the building exhaust air system in a manner (e.g., thimble convection) that avoids any interference with the air balance of the BSC or building exhaust system.

COMMENTARY:



The biologic safety cabinet (BSC) must meet minimum requirements for virology work. Exhaust air from a Class I or Class II BSC must be filtered through HEPA filters. It may be recirculated within the laboratory if the cabinet is tested and certified at least every 12 months. It may be exhausted through a dedicated stack that protects against backflow of air from adverse weather conditions or through the building exhaust air system in a manner (e.g., thimble convection) that avoids any interference with the air balance of the BSC or building exhaust system.



Are there documented policies for the safe handling and processing of virology specimens?

COMMENTARY:

Documented policies for the safe handling and processing of virology specimens must be available to all personnel.


Are specimens and used media disinfected, sterilized, or contained in a manner to minimize the hazard of an accident during transportation to a remote autoclave or incinerator?

COMMENTARY:

All virology specimens and contaminated media must be handled in a manner so as to minimize the hazard of an accident during storage or transportation; this may include decontamination within the virology laboratory.

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MOLECULAR MICROBIOLOGY

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This section covers molecular testing for unmodified, FDA-approved molecular methods only. Microbiology laboratories that have modified FDA-approved methods, or that use molecular methods that are not FDA-approved, must also be inspected with the Molecular Pathology checklist.

Laboratories should consult the list of currently FDA-approved tests published on-line by the Association for Molecular Pathology (AMP) at www.ampweb.org



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QUALITY CONTROL

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**NEW** 09/30/2004


Are all test reagents including buffers and water stored properly and in a manner which minimizes target DNA/RNA contamination?

NOTE: Pre- and post-amplification reagents and water should be stored under appropriate temperature and other conditions in designated pre- and post-amplification areas.

COMMENTARY:

N/A

REFERENCES: 1) Borst A, et al. Eur J Clin Microbiol Infect Dis 2004 epub ahead of print; 2) Neumaier M et al. (1998) Clin. Chem. 44:1;12-26.

**NEW** 09/30/2004


Are acceptable positive controls containing the target sequence and negative controls containing nucleic acid but lacking the target sequence properly stored in the laboratory?

NOTE: Storage conditions should prevent degradation of the target of interest in positive controls and prevent contamination of negative controls.

COMMENTARY:

N/A



**NEW** 09/30/2004


Are appropriate positive and negative controls run in parallel with patient specimens on each day of patient testing?

COMMENTARY:

N/A

**NEW** 09/30/2004


Are results of positive and negative controls reviewed for acceptability prior to reporting patient results?

NOTE: Patient results must not be reported unless the results of controls are within acceptable limits. Conditions causing unacceptable control results must be corrected prior to reporting patient results. Corrective action must be documented.

COMMENTARY:

N/A

**NEW** 09/30/2004


On each day of use, are aliquots of patient specimens spiked with target nucleic acid in order to assess inhibition of nucleic acid amplification/detection?

NOTE: If the laboratory can document absence of nucleic acid inhibition in particular specimen types, an inhibition control is not required (see checklist question MIC.63850, below).

This requirement does not apply to probe-based solution hybridization methods (e.g., Gen-Probe AccuProbe) performed without nucleic acid amplification.

COMMENTARY:

N/A



REFERENCE: Ballagi-Pordany A, Belek S. Mol Cell Probes 1996 Jun 10(3):159-64.

**NEW** 09/30/2004


If spiked inhibition controls are not included for each specimen, is the absence of inhibition with particular specimen types documented by past experience?

NOTE: Documentation may include reference to parallel cultures, chart reviews, and/or daily spiked samples showing no evidence of inhibition over time.

This requirement does not apply to probe-based solution hybridization methods (e.g., Gen-Probe AccuProbe) performed without nucleic acid amplification.

COMMENTARY:

N/A

**NEW** 09/30/2004


If results of negative controls are positive or equivocal for the presence of nucleic acid, are nucleic acid surveillance (wipe tests) performed in the laboratory (on benchtops, microfuges, waterbaths, etc.)?

NOTE: Wipe tests should be documented and corrective action taken to clean contaminated areas. After a contamination event, nucleic acid surveillance should be performed at least semiannually for at least one year.

This checklist question does not apply to probe-based solution hybridization methods (e.g., Gen-Probe AccuProbe) performed without nucleic acid amplification.

COMMENTARY:

N/A

REFERENCES: 1) Borst A, et al. Eur J Clin Microbiol and Inf Dis 2004 epub ahead of print; 2) Cone RW, et al. Lancet 336:686-7, 1990.



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**NEW** 09/30/2004


Are reagent preparation, specimen preparation, and nucleic acid amplification/detection areas physically separated to minimize contamination risks?

NOTE: This requirement does not apply to probe-based solution hybridization methods (e.g., Gen-Probe AccuProbe) performed without nucleic acid amplification.

COMMENTARY:

N/A

REFERENCE: Neumaier M et al. (1998) Clin Chem 44:1;12-26.

**NEW** 09/30/2004


Is adequacy of nucleic acid isolation/preparation procedures evaluated with performance of each assay?

NOTE: Adequacy of nucleic acid isolation/preparation procedures (manual or automated) must be evaluated with each assay by the use of positive and negative controls that are run in parallel with patient samples.

COMMENTARY:

N/A

**NEW** 09/30/2004


Are tests performed and results reported as specified in package inserts without substitution of essential reagents/probes?



COMMENTARY:

N/A

**NEW** 09/30/2004


Are all incubation (reaction) temperatures defined and documented with the performance of each assay?

COMMENTARY:

N/A

**NEW** 09/30/2004


Are incubations (reactions) performed in appropriate baths/blocks/instruments?

COMMENTARY:

N/A

**NEW** 09/30/2004


If any incubation temperature is out of range, is the deviation reported to the supervisor and corrective action taken prior to reporting assay results?

COMMENTARY:

N/A

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LABORATORY SAFETY

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The inspector should review relevant questions from the Safety section of the Laboratory General checklist, to assure that the molecular testing section is in compliance. Please elaborate upon the location and the details of each deficiency in the Inspector's Summation Report.

The following question pertains specifically to the molecular microbiology section.

**NEW** 09/30/2004


Are biological safety cabinets (or hoods) certified annually to ensure that filters are functioning properly and that airflow rates meet specifications?

COMMENTARY:

N/A


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