Clinical Neurochemistry Laboratory

CLIA # 21D0955652 LAB DIRECTOR: Graeme Eisenhofer, Ph.D.

Clinical Neurochemistry Laboratory
Quality Assurance Plan

____________________________________________________________________________

QUALITY CONTROL

All tests are validated before reporting patient results. Reagents are monitored for labeling, lot number (where appropriate), expiration date, and receipt or formulation date (where appropriate) to ensure quality testing. All reagents used for assays of plasma metanephrines are identified by a bright orange circular label. All reagents used for assays of plasma catechols are identified by a red circular label. Reagents are dated when received and again when opened. Relevant details about the key reagents used in each run (e.g., reagent formulation or expiry dates; lot or P/N numbers) are recorded for each run in a data sheet ("Reagent Data Sheet") that is appended to the data sheet detailing the assay results ("Assay Data Sheet") for each run. Reagents that do not have an expiration date are assumed to be expired one year from the date opened. Reagents, solutions, control materials, calibration materials, and other dated supplies will not be used when they have exceeded the expiration date, deteriorated, or are of substandard quality.

Two levels of control (a high and a normal level) are tested with each HPLC assay run. The controls are tested in the same manner as patient samples and the results are recorded on a data sheet ("Quality Control Data Sheet") that documents the values obtained for each assay, the recoveries of the internal standards run with each quality control sample, and the coefficients of variation of assay results. Any problems detected during evaluation of control data or errors in reported results are investigated and the action taken is recorded. Quality control records are kept for at least two years.

Quality Control Data Sheets are stored for reference along with this QA plan in the "Quality Assurance Log Folder". The individual Assay Data Sheets and Reagent Data Sheets are stored in the "Assay Results Folders", where they can be accessed by the date of each assay. (See folders labeled METANEPHRINES). Assay Results Folders are available in room 6N258 for plasma catechols assayed in room 6N258 (See folders labeled "CATECHOLS) and in room 6N250 for those assayed in Room 6N250 (see folders labeled "Catecholamine Assays"). Assay Results Folders for plasma metanephrines are available in Room 6N258

___________________________________________________

Quality Control Measures

• At least two Quality Control (QC) samples (1 low QC sample and 1 high QC sample) are included with each set of extracted samples and with each HPLC assay run.

• A water control ("water blank," HB or H2O BLK) is included with each set of extracted samples and with each HPLC assay run, to detect contamination.

• At least one internal standard is included with each sample taken through the extraction procedure and HPLC assay. The recovery of the internal standard is used to correct recoveries of analytes in each sample so that results can be presented as concentrations of analyte per ml of plasma.

• In the HPLC assay procedure for measurement of catechols, at lease one set of standards are taken through each extraction procedure and assay run. The recoveries of the standards (DHPG, NE, DOPA, EPI, DA, DOPAC) relative to the internal standard (3,4-dihydroxybenzylamine, DHBA) are used to correct for any discrepancies in systematic recoveries of analytes relative to the internal standard.

___________________________________________________

Procedure Manual

The Procedure Manual contains the procedures for all techniques used in the laboratory for HPLC assays of catechols and metanephrines. Also included are reagent source lists, the instructions for collection and transportation of blood and plasma samples, blank patient information sheets, and other documents relevant to the handling of specimens, analytical techniques, and production of data and patient reports. All testing procedures are available as written documents and include any modifications to the published methods. The procedures are reviewed annually by the Laboratory Director. Approval of laboratory procedures is indicated by the date and signature of the Laboratory Director on the cover sheet immediately inside the procedure folder.

The procedures for extraction and HPLC assay of catechols and metanephrines are documented separately in the Procedure Manual, in the two Sections entitled:

1. LIQUID CHROMATOGRAPHIC-ELECTROCHEMICAL DETERMINATION OF PLASMA CATECHOLS

and

2. LIQUID CHROMATOGRAPHIC-ELECTROCHEMICAL DETERMINATION OF PLASMA METANEPHRINES

Within each of these two sections are sub-sections that detail all the procedures necessary for liquid chromatographic-electrochemical detection of plasma catechols and metanephrines:

• Test name

• Principle of the liquid-chromatographic electrochemical detection (LC-EC) method

• Equipment for LC-EC

• Operation of LC-EC apparatus

• Principle of extraction procedures prior to LC-EC

• Equipment for extractions

• Step-by-step directions for performing the extractions prior to LC-EC

• Step-by-step directions for data acquisition and calibration

• Directions for chromatogram reprocessing and building of data and patient reports

• List of reagent sources, storage directions and expiration times

• Directions for preparation of standards and reagents

•.Directions for specimen collection and handling

•.Directions for specimen shipping to and transport within the NIH

• Patient information required with each sample

• Reference ranges

• References

___________________________________________________

Equipment Monitoring

The Laboratory follows the manufacturer's instructions for the operation and maintenance of all instruments used in the test procedures. Procedures for the operation of the equipment are available in the Procedure Manual for the assays of plasma metanephrines and catechols and in the specific manuals for each item of equipment (equipment manuals are available in rooms 6N250 and 6N258). Prior to delivery, the manufacturer calibrates instruments. Equipment that requires calibration will be calibrated at least every six months according to the manufacturer's instructions. If calibration fails, the calibration is repeated, or the manufacturer is contacted for service or replacement.

One liquid chromatographic-electrochemical detection system in Room 6N258 is used for assays of plasma metanephrines. Use of three different liquid chromatographic-electrochemical detection systems for assays of plasma catechols, two in Room 6N250 and one in Room 6N258, is validated by running the same quality control samples on all three systems. The same criteria for acceptable variation of one system are used for acceptable variation among the three systems. Problems or concerns are addressed in the form of a service call or repeat of maintenance or calibration. All actions are documented and kept for a least two years.

Items of equipment that require daily or periodic monitoring and bi-yearly calibration include:

The -70oC freezer (room 6N248/250) used for storage of samples prior to assays is monitored electronically by alarm system and temperature recorded continuously using an electronic temperature recording system. The temperature of the -70oC freezer should be no higher than -60o C. The temperatures of the refigerator (6N250), used for storage of standards, and the oven (6N250), used for storage of alumina, are recorded on each work day in a log stored in Room 6N248/250, with completed records stored in the Quality Assurance Manual Folder. This log also details daily records of the temperature of the -70oC freezer.

Water circulators and ESA electrochemical detector background currents are monitored manually on each day of use, with the records maintained in Reagent Data Sheets appended with Assay Data Sheets.

The centrifuge used for spinning blood samples, the balance used for weighing reagents and standards, and all pipettes used in assays are calibrated every 6 months. Documentation is recorded in the Quality Assurance Manual Folder.

Ambient room temperature between 60o and 86oF is appropriate for testing procedures. Where ambient room temperature varies outside this range, NIH maintenance personnel are to be contacted to correct any severe changes in room temperature.

___________________________________________________

Quality Control Recording, Review, and Remedial Action

The normal and high value quality controls, water blanks, and records of analyte recoveries and standard peak areas, heights, and retention times of chromatographed analytes provide a constant check on the catechol and metanephrine assay procedures. Additional monitoring of chromatography (e.g. baseline noise), analytical cell potentials and changes in retention times between and within chromatographic runs, as well as all equipment critical to the two assay procedures, provide additional quality control checks.

 HPLC Column Performance

HPLC column performance is checked from assay run to assay run, by visual inspection of chromatograms and by review of chromatographic characteristics from run to run (e.g., retention times and peak height : area ratios of standards).

Peak height : area drop offs. A drop off in peak height : area ratios at a particular retention time for a specific analyte can be expected as the HPLC analytical column ages. This is accompanied by visual evidence of rounder, less sharp chromatographic peaks and sometimes by peak tailing. Reduced sensitivity and increased interference are the potential problems resulting from this drop in analyical column performance (i.e., reduction in theoretical plates). Where peak height : area ratios fall by more than 30% compared with the first run when the column was introduced, the analytical column should be replaced.

Analyte retention times. A slow decrease in analyte retention times can be expected as the analytical column ages. To maintain reasonably constant retention times from run to run, the mobile phase concentration of acetonitrile can be reduced. On occasion, analytical columns may exhibit a sharp decrease in retention time. This may result from a number of possible causes: 1. the mobile phase is incorrectly made up; 2. the silica column saver is exhausted; or 3. the column is defective. The appropriate remedial action to a rapid decrease in retention time is first to make up new mobile phase. If that fails to rectify the situation, a new silica column saver is installed. If that fails, the analytical column is replaced. On occasion, retention times may fluctuate within a run. The most likely reason is a change in column temperature, resulting from impaired water flow around the column jacket or a breakdown in the water circulator cooler. The situation should be rectified by removal of the obstruction or repair or replacement of the water circulator cooler. For any problem in analyte retention times, the flow rate of the HPLC pump and the mobile phase composition must always be checked first.

 Peak Detection

Problems with peak detection may include development of noisy chromatograms and decreases in peak response, both of which may lead to unacceptable reduction in sensitivity (i.e., an increase in minimum amount of analyte detectable defined as the mass amount of analyte that provides a peak response 3 times higher than the baseline noise). Chromatographic contaminants may also result in unacceptable chromatography and poor peak detection.

Noise in chromatograms. Noise in chromatograms may result from: 1. pump oscillations; 2. dirty mobile phase; 3. dirty electrode or air in electrode; 4. electronic interference from other equipment; 5. an electronic fault in the ESA Coulochem. Appropriate remedial action involves exclusion of pump oscillations, by reducing and increasing pump speed/flow rate. If the noise shows periodicity that correlates with pump speed, then the pump requires maintenance or replacement. If the noise is independent of pump speed, then the mobile phase should be replaced and the background current checked. If the noise persists, then the cell can be purged to remove possible air and cleaned according to the manufacturer's operating manual. If the noise persists, then the electrode should be replaced. If the noise persists, it most likely reflects electronic interference. Remedial action at this stage includes the sequential turning off of all equipment, use of grounding wires and sequential replacement of parts of the HPLC apparatus, starting with replacement of the ESA Coulochem module.

Peak response. A decrease or increase in peak response (area under the curve) for the complete set of standards may indicate either that the standards were made up incorrectly or that there has been a change in the sensitivity of the detection system. Unless there has been considerable length of time between assays or there has been a change in the electrochemical cell, chromatographic peak areas for standards should not vary by more than 5% from the previous assay. Where chromatographic peak areas for standards vary by more than 5% from the previous assay, an additional set of standards should be made up and run at the time of calibration. If the additional set of standards remains out of range of the previous assay, but within range of the first set, then these standards are acceptable. If the additional set of standards is within range of the previous assay, but out of range of the first set, then the second set of standards is acceptable, and results of the first set should be ignored. Where peak areas of new standards appear to differ from the standards of a previous assay run, a notation is made on the Reagent and Quality Control Data Sheets, and the checklist (see below) of possible reasons is considered and remedial action taken if necessary.

Checklist of possible reasons for alterations in standard peak area response and remedial action

1. Different analytical electrochemical cell.
   No action unless sensitivity is decreased by over 30%, in which case the cell should be cleaned or a new analytical cell installed.

2. Different potentials of guard and analytical cells.
   Adjust potentials to appropriate settings.

3. Different mobile phase pH.
   This is only a potential problem with epinephrine or metanephrine at pH>3.4. If pH is above 3.4, adjust mobile phase pH appropriately.

4. Different lot numbers of standards.
   New standards made up and compared with original standards.

5. None of the above.
   No action required.

Chromatographic contaminants. Problems with chromatographic contaminants are manifest by anomalous peaks on chromatograms that may or may not interfere with peaks corresponding to the analytes of interest. Such interference may be due to contaminants within samples or may enter during sample processing and chromatography.

Sources of contamination during sample processing and chromatography are identified by inclusion of water blanks with every assay run. Contaminants appearing in water blanks that do not directly interfere with the analytes of interest do not justify exclusion of results. Contaminants appearing in water blanks that have the same retention times as analytes of interest may or may not justify exclusion of results for the specific analyte of interest. If the contamination appears to lead to erroneous results for the normal QC sample, then the results for tests of the specific analyte throughout that chromatographic run should be excluded. The samples should be rerun. If the erroneous results are not apparent for the normal QC sample, then results for that analyte are acceptable. If repeat assays fail to reproduce a contaminating peak, then no further action is necessary. If a repeat assay shows the same contaminant in the water blank, then all reagents used in the extraction procedure should be discarded and new reagents made up. If use of new reagents fails to remove the contaminant, then repeat assays should be carried out on another chromatographic system, to establish whether the interference is from within the HPLC apparatus (e.g., contaminated injection system on autosampler). If contamination appears to be due to the chromatographic system, then each component of the system should be replaced systematically until the problem component is found. Alternatively, it may be necessary to clean the chromatographic hardware according to the manufacturer's directions in the equipment manuals.

Sources of contamination within the patient material that lead do not lead to interference with analyte peaks of interest can be ignored. However, where contaminants cause direct, overwhelming interference with peaks of interest, then results for those peaks of interest should be excluded. If additional sample remains, then the assay should be repeated, with attention paid to reducing the interference. This may be achieved by one or more of several changes to the assay: 1. in catechol assays, elution from alumina with a lower strength acid; 2. additional washing of alumina; 3. alteration of chromatographic conditions. If the above remedial action fails to avoid the interference, or if there is no additional sample, then a repeat sample should be requested, with attention paid to potential dietary and pharmacological sources of interference. In assays of plasma free metanephrines, the presence of acetaminophen in samples is an established source of interference to measurements of normetanephrine. Therefore, for assays of plasma free metanephrines, all patients and physicians are to be explicitly informed that patients must not have taken acetaminophen in any form for 5 days before samples are taken. Analogously, a-methyl-para-tyrosine, a-methyldopa, isoproterenol, dobutamine, and dihydrocaffeic acid all are catechols and can interfere with the analytical results. Dihydrocaffeic acid occurs in the plasma of coffee drinkers and results from metabolism of caffeic acid, a catechol present in decaffeinated as well as caffeinated coffee (Goldstein et al., 1984).

 Analyte recovery

Recoveries of analytes are checked by both of two procedures in assays of both plasma metanephrines and catechols: 1. Recovery of internal standard; and 2. Recovery of analyte standards (in assays of plasma catechols) or analytes in spiked samples of plasma (in assays of plasma metanephrines).

Recoveries of internal itandard. Recoveries of internal standard for each sample are recorded in Assay Data Sheets, while recoveries of internal standard in quality control samples are additionally recorded in the Quality Control Data Sheet. Recoveries of the internal standard, dihydroxybenzylamine (DHBA) used in assays of catechols, and of 3-methoxy-4-hydroxybenzylamine (MHBA) and 3-ethoxy-4-hydroxyphenylethylamine (EHPEA), used in assays of plasma metanephrines, should not vary outside the range 50-95%.

In assays of plasma metanephrines, either of the internal standards may be used to correct for recoveries of analytes in the sample. In cases of isolated interference to one internal standard, the other internal standard may be used for correction of recoveries of analytes.

Unless a decreased recovery for a specific sample can be traced to loss of sample during extraction or reduced sample volume on injection, results from samples with less than 50% recovery should be excluded. Where recoveries of internal standard are less than 40% throughout an assay run, results of the entire run should be excluded and the samples assayed again after appropriate steps have been taken to rectify the cause of the decreased recoveries. Such steps may include discarding and replacing some or all of the reagents and consumables used in the extraction procedures.

In assays where recoveries exceed 95%, a check should be made that calibration of the HPLC system is correct and that the correct amount of internal standard was used. Results where recoveries are above 95% should be excluded, unless it can be determined that the erroneously high recoveries are secondary to incorrect calibration, use of an incorrect but known higher amount of internal standard in samples, or presence of a contaminating peak co-eluting with the internal standard. In the latter case, recovery of the internal standard in the QC samples or in the extracted mixture of external standards for that assay may be used to estimate analytical recoveries.

Recoveries of Analytes. In the HPLC assay procedure for measurement of catechols, at least one set of standards are taken through each extraction procedure and assay run (the "extracted mixture"). The recoveries of the standards (DHPG, NE, DOPA, EPI, DA, DOPAC) relative to the internal standard (3,4-dihydroxybenzylamine) are used to correct for systematic differences in recoveries of analytes relative to the internal standard. Recoveries of all analytes in the extracted mixture are recorded in the catechol Assay Data Sheet

In the HPLC assay procedure for measurement metanephrines, recoveries of analyte relative to internal standard should be similar and should not require correction. However, a check of this is provided by the use of the high QC sample. This QC sample is spiked with a given and known amount of normetanephrine, metanephrine, and methoxytyramine (500 pg/mL).

Quality control samples

Results for quality control samples are recorded for each assay in the Assay Data Sheets. A complete record of QC sample results for all assays is also recorded in the Quality Control Data Sheets for low and high QC samples, which are stored in the QA Log Folder. This Quality Control Data Sheet updates for each assay the mean values recorded for analytes for all assays until the latest assay. Also updated on the Quality Control Data Sheet are ranges in results and coefficients of variation for each analyte. The Quality Control Data Sheet also includes the dates of each assay, the initials of the operator responsible for each assay and the recoveries of internal standards for each QC sample.

Acceptable limits for the results of quality control samples are generally ± 15% of the mean value determined up until the current assay. For samples where concentrations of analytes are below 50 pg/mL the acceptable limits of ±15% are relaxed according to the guidelines detailed below:

Where the control values are out of range, an investigation is done and documented. If it can be determined that out of range control values are also associated with and explained by out of range standard peak areas, then corrective action, involving adjusting for the perceived error in standard peak heights, may be considered. In assays of catechols, if out of range values are associated with out of range recoveries of analytes for the extracted mixture, then corrective action can include appropriate correction of out of range extracted standard values, according to previous assay results. Out of range quality control values may be ignored, if the recoveries of the internal standard for the specific out of range QC sample and only that out of range QC sample are also out of range (e.g., low recovery of the internal standard for the QC sample alone). No patient results are reported until the controls are within acceptable ranges, and corrective action is documented.

 Recording of Remedial Actions

The results and date of all remedial action will be entered as soon as possible into the QA Log Manual Folder by the operator responsible for the assay, with review by the Laboratory Director.

 Periodic Meetings

The Laboratory Director and testing personel meet regularly after Clinical Neurochemistry Section meetings on Monday mornings. These meetings allow results and problems to be discussed and remedial action to be implemented. When necessary, others associated with the assays and interpretation of results may be called in for consultation at the Monday morning meetings. A record of meeting notes will be kept.

 Equipment

If the -70oC freezer temperature increases to above the acceptable value (-60 oC), the door will be kept closed for 30 minutes and the temperature rechecked. If it remains above acceptable, the contractor employed under the service plan for the freezer will be called for immediate corrective action. If there is any chance of thawing of samples, dry ice may be used to ensure samples remain frozen, or all samples may be transferred to an alternative freezer. If the water circulators show any indication of poor temperature control or strain due to dust build-up or electric or mechanical breakdown, they must be replaced immediately with a backup unit and serviced. Any evidence of malfunction in the liquid chromatography-electrochemical detection system equipment must be dealt with immediately, according to the directions available in this QA plan and the information in the equipment manuals. If necessary the manufacturer will be contacted for a service call, according to the service plan for the equipment components.

___________________________________________________

Proficiency Tests and Competency Checks

 Proficiency

Twice a year, a blinded assay of a previously tested specimen is repeated to ensure that similar results are obtained. Acceptable limits for agreement of results are generally ± 30% from the values recorded for the first result (i.e., both test results must be within 15% of the median value, for each analyte). For samples where concentrations of analytes are below 50 pg/mL the acceptable limits of ±15% from the median value are relaxed according to the guidelines detailed below: