Calibration and Calibration Verification

Each section must perform and document the number and concentration of calibrators used for calibration procedures following the manufacturer’s instructions or use the criteria established when establishing or verifying performance specifications according to CAP/CMS standards. Established calibration procedures must be performed and documented at the established frequency, but no less than once every 6 months, and each time any of the following occur:

• A complete change of reagents for a procedure is introduced, unless the lab can demonstrate that changing lot numbers does not affect the range used to report patient test results, and control values are not adversely affected by reagent lot changes.

• There is a major preventive maintenance or replacement of critical parts that may influence test performance.

• Control materials reflect an unusual trend or shift, or are outside of the lab’s acceptable limits, and other means of assessing and correcting unacceptable control values fail to identify and correct problems.

• The lab’s established schedule for verifying the reportable range for patient test results requires more frequent calibration verification.

Quality Control

Each clinical section must have a clearly defined and documented Quality Control policy that meets CAP/CMS standards, including the establishment of number, type, and frequency of testing control materials. For each test system, the section is responsible for having control procedures that monitor the accuracy and precision of the complete analytical process. The control procedures must:

• Detect immediate errors that occur due to test system failure, adverse environmental conditions, and operator performance.

• Monitor over time the accuracy and precision of test performance that may be influenced by changes in test system performance and environmental conditions, and variance in operator performance.

• At least once each day (when patient specimens are assayed) perform the following for:
  • Each quantitative procedure – include two control materials of different concentrations.
  • Each qualitative procedure – include a negative and positive control material.
  • Test procedures producing graded or titered results – include a negative control and a control material with graded or titered reactivity, respectively.
  • Each test system that has an extraction phase – include two control materials, including one that is capable of detecting errors in the extraction process.
  • Each molecular amplification procedure – include two control materials and, if reaction inhibition is a significant source of false negative results, a control material capable of detecting inhibition.

• Incorporate the following:
  • For each electrophoresis procedure, test at least one control material containing the substances being identified or measured, concurrent with patient specimens.
  • Perform control material testing as specified before resuming patient testing when a complete change of reagents is introduced, major preventive maintenance is performed, or any critical part that may influence test performance is replaced.
  • Rotate control testing material over time among all employees who perform the test, and test them in the same manner as patient samples.
  • Test control materials in the same manner as patient specimens.
  • When using calibration material as a control material, use calibration material from a different lot number than that used to establish a cut-off value or to calibrate the test system.

• Establish or verify the criteria for acceptability of all control materials.
  • When control materials providing quantitative results are used, statistical parameters (e.g., mean and standard deviation) for each batch and lot number of control materials must be defined and available.
  • The lab may use the stated value of commercially assayed control material provided the stated value is for the methodology and instrumentation employed by the laboratory and is verified by the laboratory.
  • Statistical parameters for unassayed control materials must be established over time by the lab through concurrent testing of control materials having previously determined statistical parameters.

• Incorporate steps for reagent, media, and supply checks, to perform and document the following:
  • Checking each batch, lot number, and shipment when prepared or opened for appropriate reactivity.
  • Testing staining materials each day of use, for intended reactivity using both positive and negative controls.
  • Checking fluorescent and immunohistochemical stains for positive and negative reactivity each time of use.

Results of control materials must meet laboratory and, as applicable, manufacturer’s criteria for acceptability before reporting patient test results. The laboratory must document all control procedures performed.

The Quality Control records are maintained in each laboratory and reviewed regularly by each laboratory supervisor or technical quality control coordinator. The Quality Control records are maintained for a minimum of two (2) years. The Quality Control programs have clearly defined goals for monitoring analytic performance, procedures, policies, tolerance limits, corrective action and related information in accord with CAP standards.

When neither calibration nor control materials are available for a particular test, each section must establish procedures to verify the reliability of patient test results.

As part of the department-wide Quality Control program, interim CAP self-inspections are performed, in which deficiencies as well as corrective actions taken are documented.

Special Stains and In-Situ Hybridization (ISH) Quality Control

For special stains, including histochemical stains, and studies using immunologic and ISH methodology, positive and negative controls are verified and recorded as acceptable prior to or concurrent with the reporting of patient results and records maintained.

• Controls must be verified and recorded as acceptable by a pathologist or designee (provided the designee meets high complexity testing qualifications).
• Positive tissue controls must contain the component specific to the special stain that is being applied to the specimen.

For immunofluorescence microscopy, appropriate positive and negative controls must be performed.

For Immunohistochemistry antibodies, a positive and negative control must be performed for each antibody. Immunohistochemical tests using polymer-based detection systems (biotin-free) are sufficiently free of background reactivity to obviate the need for a negative reagent control and such controls may be omitted at the discretion of the laboratory director following appropriate validation. 

• Positive controls assess the performance of the primary antibody. They are performed on sections of tissue known to contain the target antigen, using the same epitope retrieval and immunostaining protocols as the patient tissue. 
• Negative controls must assess the presence of nonspecific staining in patient tissue as well as the specificity of each antibody with the exception listed below. A negative tissue control must be processed for each antibody in a given run. Any of the following can serve as a negative tissue control:
  • Multitissue blocks. These can provide simultaneous positive and negative tissue controls, and are considered best practice.
  • The positive control slide or patient test slides, if these slides contain tissue elements that should not react with the antibody.
  • A separate negative tissue control slide. 
• The type of negative tissue control used (i.e. separate sections, internal controls or multitissue blocks) must be specified.

In situ hybridization controls depend on the specific assay, signal pattern present, and sample type. For example, assays designed to detect deletions may use internal controls that include both the probe of interest and a control locus probe, both of which map to the same chromosome. In this situation, there are two internal controls, the signal for the probe of interest on the normal homolog and the control locus signals on both the normal and deleted homolog. For a dual fusion assay, the probe signals on each of the normal homologs function as internal controls. If a probe is used that does not produce an internal control signal (e.g. a Y chromosome probe in a female), another sample that is known to have the probe target must be run in parallel as an external control with the patient sample. In addition, many ISH assays use an external control(s). For FDA-cleared or approved ISH assays, laboratories must follow manufacturer's instructions for quality control at minimum.

Results of controls must be recorded, either in internal laboratory records, or in the patient report. A statement in the report such as, "All controls show appropriate reactivity" is sufficient.