Clinical Laboratory Regulatory and Accrediting Organizations

The U.S. Congress enacted the Clinical Laboratory Improvement Amendments of 1988 (CLIA ’88) in response to the concerns about laboratory testing errors. The final CLIA rule, Laboratory Requirements Relating to Quality Systems and Certain Personnel Qualification, was published in the Federal Register in January 2003. Enactment of the CLIA established a minimum threshold for all aspects of clinical laboratory testing.

Voluntary standards have been set by The Joint Commission (TJC), the Commission on Office Laboratory Accreditation (COLA), and the College of American Pathologists (CAP).

A more recent development in voluntary accreditation aimed at improving quality was the introduction of ISO 15189. The International Organization for Standardization (ISO) is the world’s largest nongovernmental developer and publisher of international standards. ISO standards and certification are widely used by industry, but ISO 15189 has now been formulated for clinical laboratories. ISO 15189 has gained some standing abroad as a mandatory accreditation, such as in Australia, Ontario, and many European countries. In the United States, ISO 15189 accreditation remains optional. Requirements for quality and competence in ISO 15189 are unique because it takes into consideration the specific requirements of the medical environment and the importance of the medical laboratory to patient care. CAP 15189 is a voluntary nonregulated accreditation to the ISO 15189:2007 standard as published by ISO. CAP 15189 does not replace CAP’s CLIA-based Laboratory Accreditation Program, but complements CAP accreditation and other quality systems by optimizing processes to improve patient care, strengthen the deployment of quality standard, reduce errors and risk, and control costs.

Nonanalytical Factors Related To Testing Accuracy

Qualified Personnel

The competence of personnel is an important determinant of the quality of the laboratory result. Only properly certified personnel can perform nonwaived assays (see Chapter 9 for levels of laboratory testing).

Established Laboratory Policies

Laboratory policies should be included in a laboratory reference manual that is available to all hospital personnel. Each laboratory must have an up-to-date safety manual. This manual contains a comprehensive listing of approved policies, acceptable practices, and precautions, including Standard Blood and Body Fluid Precautions. Specific regulations that conform to current state and general requirement, such as Occupational Safety and Health Administration (OSHA) regulations, must be included in the manual.

Laboratory Procedure Manual

A complete laboratory procedure manual for all analytical procedures performed in the laboratory must be provided. The manual must be reviewed regularly, in some cases annually, by the supervisory staff and updated as needed.

A complete laboratory procedure manual for all procedures performed in the laboratory must be provided. The Clinical and Laboratory Standards Institute (CLSI) recommends that these manuals follow a specific pattern for how procedures are organized (Box 7-1).

Box 7-1   Written Procedural Protocol

• Procedure name

• Name of the test method

• Principle and purpose of the test

• Specimen collection and storage

• Quality control

• Reagents, supplies, and equipment

• Procedural protocol

• Expected or normal (reference) values

• Procedural notes:

Sources of error

Limitations

Clinical applications

Adapted from Clinical and Laboratory Standards Institute: Clinical laboratory technical procedure manual: approved guideline, ed 4, Wayne, Pa, 2002, CLSI Document GP2-A4.

Test Requisitioning

A laboratory test request must include the following: (1) patient identification data; (2) time and date of specimen collection; (3) source of the specimen; and (4) analyses to be performed. The information on the accompanying specimen container must exactly match the patient identification on the test request.

Patient Identification, Specimen Procurement, and Labeling

Patients must be carefully identified. For outpatients, identification may be validated with two forms of identification. Using established specimen-processing information, the clinical specimens must be properly labeled or identified once obtained from the patient. An important rule is that the analytical result can only be as good as the specimen. Specimens must be efficiently transported to the laboratory.

For elimination of the most frequent source of pretesting error, a patient must be positively identified when a blood specimen is obtained. This specimen must be properly collected and labeled. In general, hemolyzed specimens should not be used for serologic testing.

Preventive Maintenance of Equipment

Microscopes, centrifuges, and other pieces of equipment need to be cleaned and checked for accuracy. A preventive maintenance schedule should be followed for all automated equipment. Failure to monitor equipment regularly can produce inaccurate test results and lead to expensive repairs.

Appropriate Testing Methods

Each laboratory must have an assessment routine for all procedures, performed on a daily, weekly, or monthly basis, to detect problems. When such problems are indicated, they must be corrected as soon as possible.

Another part of a quality control program concerns the way new procedures are validated before they are included in the methods routinely used by the laboratory. Each laboratory must determine the reproducibility (or confidence limits) for each procedure used and establish acceptable limits of variation for control specimens.

Inaccurate Results

Inaccuracies in testing can be systematic or sporadic. Systematic errors can be eliminated by a program that monitors equipment, reagents, and other supplies. Sporadic or isolated errors in technique can produce false-positive and false-negative results, depending on the technique used for testing (Box 7-2).

Box 7-2   Possible Causes of Technical Errors

False-Positive Errors

Overcentrifugation of serum cell mixture

Dirty glassware

Hemolyzed patient serum

Inadequate dispersal of centrifuged serum cell mixture

Extended incubation

False-Negative Errors

Omitting patient serum from test mixture

Omitting reagent from test mixture

Undercentrifugation of serum cell mixture

Vigorous shaking of centrifuged serum cell mixture

False-Positive or False-Negative Errors

Incorrect labeling of test tubes

Addition of wrong reagent to test tube

Erroneously reading or interpreting results

Inaccurately recording results

Expired or improperly stored reagents

An important aspect of quality is documentation of results. CLIA regulations mandate that any problem or situation that might affect the outcome of a test result be recorded and reported. These incidents can involve specimens that are improperly collected, labeled, or transported to the laboratory or problems concerning prolonged turnaround times for test results. There must be a reasonable attempt to correct the problems or situation and all steps in this process must be documented.

Errors Related to Phase of Testing

The Institute for Quality Laboratory Medicine has developed measures to evaluate quality in the laboratory based on the preanalytical, analytical, and postanalytical phases of testing.

Errors occurring during the analytical phase of testing in clinical laboratories are now relatively rare. Currently, most laboratory errors are related to the preanalytical and postanalytical phases of testing. To guarantee the highest quality laboratory results and to comply with CLIA regulations, various preanalytical factors need to be considered (Boxes 7-3 and 7-4).

Quality Descriptors

Quality control activities include monitoring the performance of laboratory instruments, reagents, other testing products, and equipment. A written record of QC activities for each procedure or function should include details of deviation from the usual results, problems, or failures in functioning or in the analytical procedure and any corrective action taken in response to these problems. All solutions and kits used in testing must be carefully checked before actually being used for testing patient samples.

Definitions

Quality control consists of procedures used to detect errors that result from test system failure, adverse environmental conditions, and differences between technologists, as well as the monitoring of the accuracy and precision of test performance over time. Accrediting agencies require monitoring and documentation of quality assessment records. Documentation of QC includes preventive maintenance records, temperature charts, and QC charts for specific assays.

Quality control monitors the accuracy and reproducibility of results through the use of control specimens. The diagnostic usefulness of a test and its procedure is assessed by using statistical evaluations, such as descriptions of the accuracy and reliability of the test and its methodology.

The terms accuracy and precision are often used to describe quality. Accuracy describes how close a test result is to the true value. Precision describes how close the test results are to one another when repeated analyses of the same specimen are performed. It is possible to achieve great precision, with all laboratory personnel who perform the same procedure arriving at the same answer, but without accuracy if the answer does not represent the actual value being tested. Accuracy can be improved by the following:

• Use of properly standardized procedures

• Statistically valid comparisons of new methods with established reference methods

• Use of samples of known values (controls)

• Participation in proficiency testing programs

The precision of a test, its reproducibility, may be expressed as the standard deviation (SD) or derived coefficient of variation (CV). A procedure may be extremely accurate, yet so difficult to perform that individual laboratory personnel are unable to arrive at values that are close enough to be clinically meaningful.

Precision can be ensured by the proper inclusion of standards, reference samples, and/or control solutions, statistically valid, replicate determinations of a single sample, or duplicate determinations of sufficient numbers of unknown samples. Day-to-day and between-run precision are measured by the inclusion of blind samples and control specimens.

Coefficient of Variation

The CV can be used to compare the standard deviations of two samples. Standard deviations cannot be compared directly without considering the mean. The CV can be used to compare a day’s work with that of a similar day or to compare test results from one laboratory with the same type of test results from another laboratory. The coefficient of variation (%) is equal to the standard deviation divided by the mean, as follows:

< ?xml:namespace prefix = "mml" />CV(%)=SDMean×100

Sensitivity and Specificity

Laboratory results should provide medically useful information, including the specificity and sensitivity of the tests being ordered and reported. Both specificity and sensitivity are desirable characteristics for a test, but in different clinical situations, one is generally preferred over the other.

Assessing the sensitivity and specificity of a test requires four factors: tests positive, tests negative, disease present (positive), and disease absent (negative). True positives are subjects who have a positive test result and who also have the disease in question. True negatives represent those who have a negative test result but do not have the disease. False positives are those who have a positive test result but do not have the disease. False negatives are those who have a negative test result but do have the disease.

Sensitivity

The sensitivity of a test is defined as the proportion of subjects with the specific disease or condition who have a positive test result (i.e., assay correctly predicts with a positive result):

Sensitivity(%)=True positivesTrue positives+False negatives×100

Practically, sensitivity represents how much of a given substance is measured; the more sensitive the test, the smaller the amount of assayed substance that is measured.

Specificity

The specificity of a test is defined as the proportion of subjects without the specific disease or condition who have a negative test result (i.e., assay correctly excludes with a negative result):

Sensitivity(%)=True negativesFalse positives+True negatives×100

Practically, specificity represents what is being measured. A highly specific test measures only the assay substance in question; it does not measure interfering or similar substances.

Predictive Values

To assess the predictive value (PV) for a test, the sensitivity, specificity, and prevalence of the disease in the population being studied must be known. The prevalence of a disease is the proportion of a population who have the disease. The incidence is the number of subjects found to have the disease within a defined period, such as 1 year, in a population of 100,000.

A positive predictive value for a test indicates the number of patients with an abnormal test result who have the disease compared with all patients with an abnormal result:

Positive PV=Number of patients with disease   and with abnormal test resultsTotal number of patients with        abnormal test results

Positive PV=True positivesTrue positives+False positives

A negative predictive value for a test indicates the number of patients with a normal test result who do not have the disease compared with all patients with a normal (negative) result:

Negative PV=True negativesTrue negatives+False negatives

Monitoring Quality

Proficiency Testing

Proficiency testing (PT) is incorporated into the CLIA requirements. In addition to the use of internal QC programs, each laboratory should participate in an external PT program as a means of verification of laboratory accuracy. Periodically, a laboratory tests a specimen that has been provided by a government agency, professional society, or commercial company. Identical samples are sent to a group of laboratories participating in the PT program. Each laboratory analyzes the specimen, reports the results to the agency, and is evaluated and graded on those results compared with results from other laboratories. In this way, quality control between laboratories is monitored.

Control Specimens

A QC program for the laboratory uses a control specimen, a specimen with a known value that is similar in composition to the patient’s blood. A control specimen must be carried through the entire test procedure and treated in exactly the same way as any unknown specimen; it must be affected by all the variables that affect the unknown specimen. Control specimens are used because repeated determinations on the same or different portions (or aliquots) of the same sample will not give identical values. Many factors can produce variations in laboratory analyses. With a properly designed control system, it is possible to monitor testing variables.

If the control value in a determination is out of the acceptable range (out of control), one or more of the following factors may be responsible:

1. Deterioration of reagents or standards

2. Faulty instrument or equipment

3. Dirty glassware

4. Lack of attention to timing or incubation temperature

5. Use of a method not suited to the needs and facilities of the laboratory

6. Use of poor technique by the technologist doing the test

7. Statistics: a certain percentage of all determinations will be statistically out of control.

Reference Range Statistics

In analytical immunology and serology testing using methods such as enzyme immunoassay, quantitative reference range statistics can be used. Statistically, the reference range for a particular measurement is usually related to a normal, bell-shaped curve (Fig. 7-1). This Gaussian curve has been shown to be correct for almost all types of biological, chemical, and physical measurements. A statistically valid series of individuals who are thought to represent a normal healthy group are measured and the average value is calculated. This mathematical average is defined as the mean (X¯, called the X-bar). The distribution of all values around the average for the particular group measured is described statistically by SD.

Use of the mean, median, and mode is explained in the following example:

A series of results reported for a laboratory test on seven different specimens = 7, 2, 3, 6, 5, 4, and 2

The mean is the mathematical average and is calculated by taking the sum of the values (29) and dividing by the number of values (7) in the list. The mean is 4.1 (rounded off to 4).

The median equals the middle value. To find the median, the list of numbers must first be ranked according to magnitude: 2, 2, 3, 4, 5, 6, 7. There are seven values in the list, and the median is the middle value, 4.

The mode is the value most frequently occurring value, or 2 in this example.

The standard deviation is the square root of the variance of the values in any one observation or in a series of test results. In a normal population, 68% of the values will be clustered above and below the average and defined statistically as falling within the first standard deviation (±1 SD; see Fig. 8-1). The second standard deviation (±2 SD) represents 95% of the values falling equally above and below the average, and 99.7% is included within the third standard deviation (±3 SD). (Again, variations occur equally above and below the average value [or mean] for any measurement.) Thus, in determining reference values for a particular measurement, a statistically valid series of people are chosen and assumed to represent a healthy population. These people are then tested and the results are averaged.

The reference range is the range of values that includes 95% of the test results for a healthy reference population. The term replaces normal values, or normal range. The limits (or range) of normal are defined in terms of the standard deviation from the average value. Thus, normal or reference values are stated as a range of values in terms of SD units.

Testing Outcomes

Before physicians can determine whether a patient has a disease, they must know what is acceptable for a representative population of similar patients (e.g., same age, same gender, same ethnicity), as well as the analytical method used for an assay. Furthermore, an individual may show daily, circadian, and physiologic variations.

Biometrics, the science of statistics applied to biologic observations, has been a rapidly expanding field that attempts to describe these variations. The selection of a group on whom to base reference groups is another problem confronting the individual laboratory. To develop reference values (normal values), the proper statistical tools of sampling, selection of the comparison group, and analysis of data must be used by the manufacturer of testing kits or individual laboratories.

Although generally accepted values are published, reference values will vary, especially between laboratories and between geographic locations. Each laboratory must give the physician information concerning the range of reference values for that particular laboratory.

Validating New Procedures

The QC program also determines how new procedures are validated before being included as one of the methods routinely used by the laboratory. Each laboratory must determine the reproducibility (or confidence limits) for each procedure used and establish acceptable limits of variation for control specimens. The QC program includes calculation of the mean (or average value) and standard deviation and the preparation of control charts for each procedure.

Parallel Testing of Test Kits

The requirements for the parallel testing of test kits differ depending on your accreditation agency. For example, the CAP and Clinical Laboratory Improvement Acts (CLIAs) have slightly different requirements. There is also a difference in the requirements, depending on the circumstances. Are you changing manufacturers and tests kits, or are you only changing lot numbers for the same kit?

The CAP asserts that CLIA-waived assays are not recognized and the laboratory must treat all tests the same way. It is best to check the immunology checklist at www.cap.org for the latest revisions to questions related to kits. Currently, CAP checklist question IMM.33150 (phase II) is “Are new reagent lots checked against old reagent lots, or with suitable reference material before, or concurrently with, being placed in service?”

A CLIA inspection focuses on the following:

• If the test is moderate or of high complexity and the change is to a new kit manufacturer, the new test kit must be validated for accuracy and precision. This can be done with controls, other known samples, or comparison with an old kit. If the laboratory is receiving a new lot shipment of the same test kit, only controls need to be done, or whatever the manufacturer requires.

• If the test is a waived test, a laboratory only needs to follow the manufacturer’s directions if a new test is put into use or if there is a lot change of a current test. This rule is also applicable if the waived test is being performed in a moderate- or high-complexity laboratory. If an assay is waived anywhere, it is performed under CLIA requirements.

CASE STUDY

A new employee was asked to exam a CLSI procedural protocol worksheet and rate the write up. She noted the following entries:

Title	Entry
Title	Test for Staphylococcus
Quality Control	No positive or negative controls available

Questions

1. Is the title acceptable as written?

a. Yes

b. No

2. Is the quality control requirement acceptable?

a. Yes

b. No

See Appendix A for the answers to these questions.

Critical Thinking Group Discussion Question

1. Why are positive and negative control essential to the accuracy of a test result?

See instructor site for the discussion of these questions.

Validation of a New Procedure Write-Up

Each student should develop a procedure checklist following the CLSI procedural format. A manufacturer’s package insert or book should be used as a source of information. After completing the CLSI procedural protocol, a fellow student should rate the write-up.

Are the steps in the procedure understandable?
Can the procedure be performed as described?   Reporting Results Positive reaction A titer >1:56 is considered to be a positive presumptive test.     Negative reaction The antigens on sheep erythrocytes are associated with infectious mononucleosis, serum sickness, and the Forssman antigen.     Procedural notes   Are the criteria for acceptable results clearly defined?   Sources of error False-positive reactions have been observed in conditions such as hepatitis infection and Hodgkin’s disease. An improperly inactivated serum will produce hemolysis.     Limitations The test is only indicative of the presence or absence of heterophil antibodies.
Demonstrating agglutination by using sheep erythrocytes does not make a distinction between antibodies associated with infectious mononucleosis, serum sickness, or the Forssman antigen.
Heterophil antibody assay lacks sensitivity as a diagnostic criterion for infectious mononucleosis.
Sheep erythrocytes are less sensitive than erythrocytes from other species such as the horse.
A patient may take as long as 3 mo to develop a detectable heterophil titer.     Clinical applications The Paul-Bunnell test is a useful screening test for the presence of heterophil antibodies because it is simple and inexpensive. Although the specificity of the heterophil assay is rated as good, negative results are demonstrated in individuals who do not produce infectious mononucleosis heterophil antibody. If negative results are displayed, however, Epstein-Barr virus (EBV) serology may be indicated.     General question: Are all necessary fields of the CLSI format addressed?
Additional general comments:
Evaluation of write-up validation by: _____________________________ Date _________
Supervisory reviewer: _________________________________________ Date _________

Chapter Highlights