This procedure is valid for the following chemistry analyzers


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Procedure:
Phencyclidine
OSR9J229

This procedure is valid for the following chemistry analyzers:

  • AU400/AU400e

  • AU640/AU640e

  • AU480

  • AU680

  • AU600

  • AU2700/AU5400



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PRINCIPLE:


Phencyclidine, also known as PCP and “angel dust," is a synthetic drug that was originally developed for its anesthetic properties but is now a drug of abuse used solely for its potent hallucinogenic effects. It may be self-administered in a variety of ways, including ingestion, inhalation, and intravenous injection. Phencyclidine is absorbed well and quickly and concen­trates in the brain and fatty tissues2. Excretion patterns vary widely ranging from several hours to a couple of weeks. Phencyclidine is excreted in the urine unchanged, as conjugated metaboIites, and primarily as unidentified compounds.

The Emit II Plus Phencyclidine Assay tests for phencyclidine in human urine. It also detects the analog 1-[1-(2-thienyl)-cyclohexyl]piperidine (TCP). High concentrations of several phencyclidine metabolites and analogs can also produce positive results in the assay. Positive results for samples containing other compounds structurally unrelated to phencyclidine usually have not been observed.

Methods historically used for detecting phencyclidine in biological fluids include thin-layer chromatograph,3 gas chromatography,4 ultraviolet spectroscopy, enzyme immunoassay, and radioimmunoassay.5, 6

INTENDED USE:


The Emit II Plus Phencyclidine Assay is intended for use in the qualitative and semi-quantitative analysis of phencyclidine in human urine. Emit II assays are designed for use with multiple Beckman Coulter AU analyzers.

METHODOLOGY:


The Emit II Plus Phencyclidine Assay is a homogeneous enzyme immunoassay technique used for the analysis of specific compounds in human urine6. The assay is based on competition between drug in the sample and drug labeled with the enzyme glucose-6-phosphate dehydrogenase (G6PDH) for antibody binding sites. Enzyme activity decreases upon binding to the antibody, so the drug concentration in the sample can be measured in terms of enzyme activity. Active enzyme converts nicotinamide adenine dinucleotide (NAD) to NADH, resulting in an absorbance change that is measured spectrophotometrically. Endogenous serum G6PDH does not interfere because the coenzyme NAD functions only with the bacterial (Leuconostoc mesenteroides) enzyme employed in the assay.

The Emit II Plus Phencyclidine Assay provides only a preliminary analytical test result. A more specific alternative chemical method must be used to obtain a confirmed analytical result. Gas chromatography/ mass spectrometry (GC/MS) is the preferred confirmatory method1. Other chemical confirmation methods are available. Clinical consideration and professional judgment should be applied to any drug of abuse test result, particularly when preliminary positive results are used.

SPECIMEN:

Patient/Sample Preparation:


None required.

Additional instructions for preparation as designated by this laboratory:


Type:


Urine samples are the recommended specimen type.

Additional type conditions as designated by this laboratory:

Handling Conditions:


Urine specimens may be collected in plastic (i.e., polypropylene, polycarbonate, polyethylene) or glass containers. Some plastics, other than those listed, can adsorb certain drugs.

If not analyzed immediately, specimens may be stored at room temperature (15-25C) for up to 7 days following collection. After 7 days, specimens should be stored frozen (< -20C). Frozen specimens must be completely thawed and mixed thoroughly prior to analysis.

Specimens with high turbidity should be centrifuged before analysis.

The recommended pH range for urine specimens is 3.0-10.0.

Adulteration of the urine specimen may cause erroneous results. If adulteration is suspected, obtain another specimen.

Human urine specimens should be handled and treated as if they are potentially infectious.

Additional handling conditions as designated by this laboratory:

EQUIPMENT AND MATERIALS:

Equipment:


Beckman Coulter AU400/AU400e, AU480, AU600, AU640/AU640e, AU680, AU2700, and AU5400 analyzers.

Materials:


Emit II Plus Phencyclidine Assay

Antibody/Substrate Reagent -- Sheep polyclonal antibodies reactive to phencyclidine, glucose-6-phosphate, nicotinamide adenine dinucleotide, bovine serum albumin, preservatives, and stabilizers.

Enzyme Reagent 2 -- Phencyclidine labeled with glucose-6-phosphate dehydrogenase, Tris buffer, bovine serum albumin, preservatives and stabilizers.

Reagent storage location in this laboratory:

Test tubes 12 -16 mm in diameter or sample cups (Cat No. AU1063).

Storage location of test tubes or sample cups in this laboratory:


Emit Calibrator/Control products are packaged individually and sold separately.

Emit Calibrator/Control Level 0 Cat No. 9A509

Emit Calibrator/Control Level 2 (12.5 ng/mL) Cat No. 9A549

Emit Calibrator/Control Level 3 (25 ng/mL) Cat No. 9A569

Emit Calibrator/Control Level 4 (75 ng/mL) Cat No. 9A589

Emit Calibrator/Control Level 5 (100 ng/mL) Cat No. 9A609

Note: The Emit Calibrator/Controls contain stated concentrations of phencyclidine (ng/mL) for calibration of this assay. Refer to package insert for concentration listings.

Storage location of the calibrator in this laboratory:

Preparation


The Emit® II Plus Phencyclidine Assay reagents are packaged in a ready to use liquid form and may be used directly from the refrigerator.

Note: Reagents 1 and 2 are sold as a matched set. They should not be interchanged with components of kits with different lot numbers.

The Emit® Calibrators/Controls are packaged in a ready to use liquid form and may be used directly from the refrigerator. Close the calibrator bottles when not in use. Caps must always be replaced on the original containers.

Precautions:


1. The Emit® II Plus Phencyclidine Assay and Calibrator/Controls are for in vitro diagnostic use.

  1. Reagent 1 contains non-sterile sheep antibodies. Reagent 2 contains non-sterile mouse antibodies. Non-sterile bovine serum albumin is found in both.

3. No known test method can offer complete assurance that products derived from human sources or inactivated microorganisms will not transmit infection. Reagents, calibrators, and human specimens should be handled using prevailing good laboratory practices to avoid skin contact and ingestion.

4. Do not use the reagents or calibrators after the expiration date.

5. This Emit II Plus Phencyclidine Assay is qualified for use only with the Emit Calibrators listed in the Calibrator section

Storage Requirements:


Any reagents not loaded in the reagent refrigerator on the analyzer or any calibrators not in use should be stored at 2-8°, upright, and with caps tightly closed. Do not freeze reagents or calibrators. Avoid exposure to temperatures above 32°C for prolonged periods of time.

Unopened reagents and calibrators are stable until the expiration date printed on the label if stored as directed. Refer to Assay Methodology Sheets for additional on-board stability information.

Improper storage of reagents or calibrators can affect assay performance. Stability depends on handling reagents and calibrators as directed.

Additional storage requirements as designated by this laboratory:

Indications of Deterioration:


Discoloration (especially yellowing) of the reagents or calibrators, visible signs of microbial growth, turbidity, or precipitation in reagents or calibrators may indicate degradation and warrant discontinuance of use.

PERFORMANCE PARAMETERS:


The following performance characteristics represent total system performance and should not be interpreted to refer only to reagents. Studies were performed on the Beckman Coulter AU analyzer series. Results may vary due to analyzer-to-analyzer differences. Positive results were confirmed by GC/MS.

Precision


Within run precision was performed and calculated according to Clinical and Laboratory Standards Institute (CLSI EP5-A) by running 2 replicates of the cutoff Calibrator/Control and positive and negative controls twice a day for 20 days (N=80). Total precision was calculated from this data. Results for these studies are summarized below.




Within-Run Precision

Total Precision




Cutoff Calibrator (25 ng/mL)

Control 75%

Control 125%

Cutoff Calibrator (25 ng/mL)

Control 75%

Control 125%

Mean mAU/min

441

417

471

441

417

471

SD

3.9

2.9

4.0

5.3

4.2

5.7

% CV

0.9

0.7

0.8

1.2

1.0

1.2

Comparison


Clinical urine specimens were tested using Emit II Plus Phencyclidine Assay on an Beckman Coulter AU analyzer and using the corresponding Emit II assay on the SYVA-30R Biochemical System. The results are summarized below showing the number of positive and negative results identified and the percent agreement between analyzers. Specimens positive by either method contained phencyclidine ranging from 14.9 to 78.5 g/mL.

Assay

Positive

Negative

% Agreement

Phencyclidine

49

54

99

ANALYTICAL RECOVERY


Negative human urine specimens were spiked with a concentration of phencyclidine. Specimens spiked with drug concentrations lower than the cutoff concentration were analyzed qualitatively and correctly identified as negative 100% of the time. Specimens spiked with drug concentrations greater than the cutoff were correctly identified as positive 100% of the time. Results from semi-quantitative analysis of the specimens are listed below:

Concentration (ng/mL)

Mean (ng/mL)

12.5

13.0

17.5

16.6

40

45.5

50

58.5

90

86.2

CALIBRATION:

Qualitative Analysis:


Perform a one-point calibration (AB) using a water blank (blue rack) and the EMIT Calibrator/Control for the desired cutoff: Level 3 = 25 ng/mL. Refer to Analyzer Specific Protocol for calibration set-point options and analyzer settings.

Three options are available for Qualitative Calibration:

Option 1: On the “Specific Test Parameters” menu, the “General Tab”, program the Correlation B factor as 0.0. On the same screen, under the “Range Tab” program the Value/Flag Level H as 999999. Under Calibration Specific Parameters menu set the Calibration type to MB. Blank the test using the blue rack. The cutoff calibrator (25) is run in a white rack. Each sample response is compared to the cutoff calibrator response to determine if the sample is positive or negative. Positive samples will not be flagged. Comparison of sample responses is a manual process.

Option 2: On the “Specific Test Parameters” menu, the “General Tab”, program the Correlation B factor as 0.0. On the same screen, under the “Range Tab” program the Value/Flag Level H as 100. Under Calibration Specific Parameters menu set the Calibration type to AB with Formula as Y=ax+b. The Conc. for the calibrator should be entered as 100. Blank the test using the blue rack. Calibrate by placing the designated cutoff calibrator (25) in the assigned position in the calibration rack (yellow rack). Positive samples will be flagged (P) and will printout as greater than or equal to 100.

Option 3: On the “Specific Test Parameters” menu, the “General Tab”, program the Correlation B factor as -100. On the same screen, under the “Range Tab” program the Value/Flag Level H as 0.0. Under Calibration Specific Parameters menu set the Calibration type to AB with Formula as Y=ax+b. The Conc. for the calibrator should be entered as 100. Blank the test using the blue rack. Calibrate by placing the designated cutoff calibrator (25) in the assigned position in the calibration rack (yellow rack). Positive samples will be flagged (P) and will printout as greater than or equal to zero.

Semi-Quantitative Analysis


Perform a multi-point calibration (4AB) using a water blank (blue rack) and the EMIT Calibrator / Controls: Level 2, Level 3, Level 4, and Level 5. Calibration parameters are set to perform calibration and set up the calibration curve. Refer to analyzer User’s Guide or Analyzer Specific Protocol sheets for analyzer settings.

Calibration Stability


Studies have shown the median calibration stability to be at least 14 days. Recalibrate as indicated by control results or with a new lot of reagent. Calibration stability may vary from laboratory to laboratory depending on the following: handling of reagents, maintenance of analyzer, adherence to operating procedures, establishment of control limits, and verification of calibration.

Note: When using a new set of reagents with the same lot number, recalibration may not be required. Validate the system by assaying controls.

QUALITY CONTROL:


During operation of the Beckman Coulter AU analyzer at least two levels of control material should be tested a minimum of once a day. Controls should be performed after calibration, with each new lot of reagent, and after specific maintenance or troubleshooting steps described in the appropriate User’s Guide. Quality control testing should be performed in accordance with regulatory requirements and individual laboratory’s standard procedures. If more frequent verification of test results is required by the operating procedures within your laboratory, those requirements should be met.

Qualitative Analysis


Validate the calibration by assaying controls. Ensure that the result from the negative control is negative (or lower) relative to the Calibrator/ Control set point. Ensure that the result from the positive is positive (or higher) relative to the Calibrator/Control set point. Once the calibration is validated, run urine specimens.

Semi-Quantitative Analysis


Validate the calibration by assaying controls. Ensure that control results fall within acceptable limits as defined by the testing facility. Once the calibration is validated, run urine specimens.

PARAMETERS:


A complete list of test parameters and operating procedures can be found in the appropriate User’s Guide and at www.beckmancoulter.com. The Analyzer Specific Protocol Sheets may also be used.

CALCULATIONS:


None required.

REPORTING RESULTS:

Reference Ranges:


No reference ranges are defined for drugs of abuse testing.

Expected reference ranges in this laboratory:

Procedures for Abnormal Results


The laboratory must define procedures to be used in reporting high concentration (toxic) results to the patient’s physician.

Abnormal results are flagged by the listed analyzers according to the normal values entered by the user into the instrument parameters.

Reporting Format:


Results are automatically printed out for each sample in ng/mL at 37C.

Interpretation of Results


Qualitative Analysis -- When the Emit II Plus Phencyclidine Assay is used as a qualitative assay, the amount of phencyclidine, phencyclidine metabolites, or analogs detected by the assay in any given sample cannot be estimated. The assay results distinguish positive from negative samples only. The Emit® Calibrator/Control Level 3 Cutoff, which contains a concentration of 25 ng/mL phencyclidine, is used as a reference for distinguishing “positive” from “negative” specimens.

Positive Results: A specimen that gives a result equal to or higher than the calibrator set-point value is interpreted as positive: The specimen contains phencyclidine or phencyclidine metabolites or analogs.

Negative Results: A specimen that gives a result lower than the calibrator set-point value is interpreted as negative: Either the specimen does not contain phencyclidine or phencyclidine metabolites or analogs or they are present in concentrations below the cutoff level for this assay.


Semi-Quantitative Analysis -- When used semi-quantitatively, the Emit® II Plus Phencyclidine Assay yields approximate, cumulative concentrations of the drug and metabolites detected by the assay. The semi-quantitation of positive results enables laboratories to determine an appropriate dilution of the specimen for confirmation by GC/MS. Semi-quantitation also permits laboratories to establish quality control procedures and assess control performance.

Immunoassays that produce a single result in the presence of multiple detectable components cannot fully quantitate the concentration of individual components. Interpretation of results must also take into account that urine concentrations can vary extensively with fluid intake and other biological variables. A more specific alternative chemical method must be used to obtain a confirmed analytical result.

Additional reporting information as designated by this laboratory:


LIMITATIONS:


  1. The Emit® II Plus Phencyclidine Assay is designed for use only with human urine.

  2. A positive result from the assay indicates the presence of phencyclidine but does not indicate or measure intoxication.

  3. Boric acid is not recommended as a preservative for urine specimens.

  4. Other substances and/or factors not listed (e.g., technical or procedural errors) may interfere with the test and cause false results.

  5. Interpretation of results must take into account that urine concentrations of phencyclidine can vary extensively with fluid intake and other biological variables.

  6. Immunoassays that produce a single result in the presence of a drug and its metabolites cannot fully quantitate the concentration of individual components.

Sensitivity


The minimum detection limit of the Emit® II Plus Phencyclidine Assay is 1.4 ng/mL. This level represents the lowest concentration of phencyclidine that can be distinguished from 0 ng/mL with a confidence level of 95%.

Specificity


The Emit® II Plus Phencyclidine Assay detects phencyclidine or phencyclidine metabolites or analogs in human urine.

The following tables list the compounds this assay detects and the levels at which the compounds have been found to give a response approximately equivalent to the Emit® Calibrator/Control Level 3, 25 ng/mL. Each concentration represents the reactivity level for the stated compound when it is added to a negative urine specimen. These concentrations are within the range of levels found in urine following use of the compound or, in the case of metabolites, the parent compound. If a specimen contains more than one compound detected by the assay, lower concentrations than those listed may combine to produce a rate approximately equivalent to or greater than that of the cutoff calibrator.

Compound

Concentration Tested

Dextromethorphan

120 g/mL

Dextrorphan

97 g/mL

Meperidine

67 g/mL

Mesoridazine

50 g/mL

N,N-Diethyl-1-phenylcyclohexylamine (PCDE)

234 ng/mL

1-(4-Hydroxypiperdino)phenylcyclohexane

420 ng/mL

1-(1-Phenylcyclohexyl)morpholine (PCM)

41 ng/mL

1-(1-Phenylyclohexyl)pyrrolidine (PCPy)

54 ng/mL

4-Phenyl-4-piperidinocyclohexanol

32 ng/mL

1-[1-(2-Thienyl)-cyclohexyl]morpholine (TCM)

80 ng/mL

1-[1-(2-Thienyl)-cyclohexyl]piperidine (TCP)

37 ng/mL

1-[1-(2-Thienyl)-cyclohexyl]pyrrolidine (TCPy)

83 ng/mL


The next table lists the concentrations of compounds that show a negative response to the Emit® II Plus Phencyclidine Assay at a 25 ng/mL cutoff level.

Compound

Concentration Tested (g/mL)

Acetaminophen

1000

-Acetyl-N,N-dinormethadol (dinor LAAM)

15

L--Acetylmethadol (LAAM)

25

N-Acetylprocainamide (NAPA)

400

Acetylsalicylic Acid

1000

Amitriptyline

125

D-Amphetamine

1000

Benzoylecgonine

1000

Buprenorphine

1000

Caffeine

1000

Chlorpromazine

*

Cimetidine

1000

Clomipramine

2.5

Clonidine

1000

Codeine

500

Cotinine

100

Cyclobenzaprine

62

Desipramine

800

Diphenhydramine

1000

Doxepin

250

2-Ethylidene-1,5-dimethyl-3,3- diphenylpyrrolidine (EDDP)

1000

Fluoxetine

1000

Glutethimide

500

Ibuprofen

1000

Ketamine

100

Ketorolac Tromethamine

1000

Lormetazepam

1.0

LSD

10 ng/mL

Methadone

1000

D-Methamphetamine

35

Methaqualone

1500

Morphine

58

Naproxen

1000

Nortriptyline

1000

Oxazepam

300

Phenytoin

1000

Promethazine

170

Propoxyphene

1000

Ranitidine

1000

Scopolamine

500

Secobarbital

1000

11-nor-9-THC-9-COOH

50

Thioridazine

48

Tramadol

1000

Tyramine

100

Zidovudine (AZT)

2 mg/mL

Zolpidem

100

* While chlorpromazine does not cross-react, patients on this drug may produce positive results with this assay.

REFERENCES:


1. Hawks RL, Chiang CN, eds. Urine Testing for Drugs of Abuse. Rockville, MD: National Institute on Drug Abuse (NIDA), NIDA research monograph 73. Department of Health and Human Services; 1986.

2. Aniline O, Pitts FN. Phencyclidine (PCP): A review and perspectives. CRC Crit Rev Toxicol. 1982; 10:145-177.

3. Finkle HI. Phencyclidine identification by thin-layer chromatography. A rapid screening procedure for emergency toxicology. Am J Clin Pathol. 1978; 70:287–290.

4. Froelich PE, Gross G. Separation and detection of phencyclidine in urine by gas chromatography. J Chromatogr. 1977; 137:135–143.

5. Gupta RC et al. Determination of phencyclidine in urine and illicit street drug samples. Clin Toxicol. 1975; 8:611–621.

6. Oellerich M. Enzyme immunoassays in clinical chemistry: Present status and trends. J Clin Chem Clin Biochem. 1980; 18:197–208.

© Beckman Coulter, Inc. 2010 Page of
All printed copies are considered to be copies of the electronic original. Rev # 1, Dec 31, 10

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