1. SAMPLE VALIDITY AND ADULTERATION: pH- CREATININE – SPECIFIC GRAVITY – OXIDANTS
Why is creatinine measured? What does it mean?
Creatinine is an indicator of sample validity. Creatinine is a natural by-product of protein metabolism in the body, and it is generally produced at a constant amount daily, which differs for each individual. It is always present in the urine if the kidneys are working properly. If a person is eliminating a lot of water, the creatinine will become more dilute temporarily. If a person is dehydrated, the creatinine will become more concentrated. But the amount eliminated will be relatively constant from day to day. For instance, carboxy-THC concentrations are frequently normalized to creatinine concentrations so that fluctuations in urine concentration are minimized. If a patient substituted water for his urine sample, there would be no creatinine, and the sample would be considered not valid. 20 mg/100 ml is the low end of the reference range. Specimens with a lower creatinine may also be valid, but may require closer attention.
Does a low creatinine value indicate dilution or attempted flushing of system?
While most patients have creatinine values greater than 20 mg/100ml, values less than that are not uncommon and occur naturally in some people. Low creatinine may not be a concern until it drops below 10 mg/100 ml. Then it is worthwhile to check specific gravity. If the specific gravity is normal (greater than 1.003) the sample is considered to be valid .
What is Specific Gravity?
Specific gravity indicates the weight of a ml of urine relative to water. (1.000 g per ml) If the urine sample was produced by the kidneys, and the kidneys are working properly, there will always be dissolved salts in the urine, and that increases the weight of the urine. A urine sample is considered valid if the specific gravity is greater than 1.003.
Does drinking a lot of water really affect urine drug tests?
Drinking a lot of water can temporarily dilute urine for a short period of time. If a drug or metabolite is excreted in small concentrations, near the cutoff, it is possible to cause a negative screen. For instance, with a 50 ng/ml cutoff for carboxy-THC, a patient with a metabolite concentration of 52 ng/ml might be able to dilute their urine to 49.2, and have good fortune on that particular screen. However, a person with 5000 ng of oxycodone with a 100 ng/ml cutoff will not significantly change their screening outcome by excessive ingestion of water.
Do commercial ‘cleansing’ products really affect drug screening?
Products ingested orally do not alter the outcome of drug screening. Acidic products can increase the rate of clearance of some drugs, like amphetamine, but they do not affect any of the tests. Products added to the urine sample can drastically change screening outcomes by chemically destroying the drugs and metabolites present in the specimen. Observed collections eliminate this problem. Observed collection is the only way to assure that an outside sample has not been substituted for one provided by the patient.
Does high pH indicate an adulterated urine sample?
Urine samples, on standing at room temperature or at elevated temperatures during shipping, may display a gradual increase in pH to around 9. This is normal. A pH of 10 or more is suggestive of adulteration, but, addition of agents to change the pH of the specimen also change the color and appearance of the specimen, and the specimen may have an unusual odor. The laboratory staff are very careful to watch for these signs.
Does candy or gum affect the outcome of oral fluid screening?
Food items like this which contain sugar can, in the presence of bacteria or yeast, produce sugar by fermentation, and produce high concentrations of alcohol.
2. Tests related:
What is the difference between screening and confirmatory tests?
Screening tests use immunoassay techniques to detect a drug (or drug class). In many cases, screening tests can determine if a sample is positive for a given drug class (e.g. Benzodiazepines) but not which specific drug is present (e.g. Alprazolam). Confirmatory tests are performed using LC/MS/MS, which is highly selective and can determine which specific drug is present, as well as quantify the amount.
My patient tested positive for (drug) in the POC test and laboratory screening. However, the LC/MS/MS did not confirm any drug. Why?
Screening tests (immunoassays) are just that – a screen. In some cases, other related (or even non-related) compounds can “cross react”, i.e. interfere, with the test, resulting in a positive. When the specimen is sent for LC/MS/MS confirmatory testing, there is no interference from the cross-reacting substance. A classic example is over the counter cold medicines resulting in a positive Amphetamine screen due to drugs such as pseudoephedrine or diphenhydramine. However, LC/MS/MS confirmation can distinguish between these cold medications and illicit amphetamine use. Samples that screen positive should be considered presumptive positive and always sent to a laboratory for confirmation.
My patient tested positive for (drug) in the POC test. However, both laboratory screening and LC/MS/MS confirmation did not detect any drug. Why?
Although laboratory screening tests are based upon the same principles as POC test strips, the screening tests in the laboratory can be more sensitive and/or less prone to cross-reactivity from interfering compounds. These interfering compounds can result in a false POC screening result.
My patient tested negative in the POC (or laboratory) screening test(s) but had a positive LC/MS/MS confirmation. Why?
A negative screening result and positive confirmation is typically because the level present is below the screening cutoff level. However, LC/MS/MS is a very sensitive technique and can usually detect and quantify levels below the screening cutoff.
One of the validity tests came back “abnormal.” Was the sample adulterated?
Validity testing is used to detect adulteration. However, an abnormal result does not signify that the sample was definitely adulterated. Diet, hydration levels, and illness/infection are a few conditions that can affect validity results.
What is the difference between confirming negative and confirming the positive? And why use one over the other?
Many clients request to confirm a positive screen (either POC or laboratory EIA). Screening tests are considered presumptive positive and should be confirmed using a more specific and sensitive technique to 1) ensure that it is a true positive and not a false positive due to cross reactivity from another compound; and/or 2) determine which drug in the class is present (e.g. morphine vs. hydrocodone in a positive opiate screen).
When a screening test is negative, a “confirm negative” test may be ordered. There are several reasons this negative confirmation may be requested: 1) Screening tests may have cutoff levels significantly higher than confirmation tests and the practitioner may be interested if any drug is present; 2) When testing for a drug class, some drugs in the class do not react as well as others with the immunoassay screen. For example, the reactivity of Clonazepam (Klonopin) with the benzodiazepine screen is very poor; therefore, a patient may be taking Clonazepam and still show a negative benzodiazepine drug screen. Clonazepam use is easily detected using LC/MS/MS confirmation; 3) Confirmation tests typically detect not only the drug but major metabolites Detection of metabolites are important because they have a longer window of detection versus parent drug. Furthermore, some screening tests do not detect metabolites very well, such as the test for Fentanyl (Duragesic). Fentanyl doses are very low and this drug is rapidly metabolized to Norfentanyl. A person taking Fentanyl may have very low levels of the parent drug, yet higher levels of the Norfentanyl metabolite. However, Norfentanyl has poor reactivity with the Fentanyl screening test so there is a possibility that a Fentanyl drug screen would be negative. Confirmatory tests detect both Fentanyl and Norfentanyl, giving a broader and more accurate depiction of a person’s drug use.
Because of the reasons listed, it is recommended that not only all positive drug screens be confirmed, but also any unexpected negative results.
Why is the POC screen for THC positive but the laboratory results negative?
Some medications (e.g. proton pump inhibitors) cross react with the THC POC test but will not confirm as THC positive. Also, several metabolites of THC are formed and the POC test can react with more than one of these metabolites. The POC screen will therefore measure the cumulative level of many metabolites. The laboratory confirmation targets carboxy-THC (THC-COOH), the metabolite that has been widely accepted as indication of THC use, and the concentration of this level may be below the reporting limits of the confirmatory test.
My patient is prescribed Hydrocodone (or Oxycodone). The test results are positive for the parent drug, but no Hydromorphone (or Oxymorphone) metabolite is present. Do the results indicate that the patient is misusing the prescription?
Many factors affect metabolism, including genetics, renal function, diet, co-administered drugs (both prescription and OTC), etc. Although Hydrocodone is metabolized to Hydromorphone (and Oxycodone to Oxymorphone), the amount of metabolite detected – if any – can vary significantly. About 1-2% of the population are extensive metabolizers and will show higher levels of the metabolite; approximately 10% of the population are poor metabolizers and little metabolite will be present, if any.
3. DRUG SCREENING, CUTOFF CONCENTRATIONS, POC DEVICES
Why are cutoff concentrations used? Are values below the cutoff valid?
Cutoff concentrations are generally accepted in the industry and are chosen to maximize the number of true positive samples, while minimizing the number of false positives. In this way, the maximal number of drug users are detected, while reducing as much as possible false positive screens which require needless confirmation. It is accepted laboratory procedure to report values less than the cutoff as negative. While values less than the cutoff may be real, the convention is to consider them negative. Calibrations, quality control and confidence are established for the cutoff concentrations.
Why do some specimens screen positive but not confirm?
The preliminary screening, whether POC or in the laboratory, is accomplished by using antibodies to the drugs. While the manufacturers take great care to make the antibodies as selective as possible, sometimes other compounds in the specimen will cross-react and cause a false-positive screen. Most of the time the preliminary screening is quite accurate. But high concentrations of other prescribed medications, or other non-prescription agents may cause a positive screen reaction. This is why confirmatory analysis is essential. In that process, specific chemical compounds are uniquely identified and quantitated. The preliminary screen is just an initial snapshot to provide guidance in confirmatory analysis.
Can you distinguish between actual ingestion of a drug versus the addition of some crushed tablet to a urine sample?
It is difficult for a lay person to know how much tablet material to add to a sample. The presence of an extremely high parent drug concentration and the absence of any metabolites in a urine sample might be a indicator of such adulteration, and would require careful examination.
Are POC cups accurate?
Generally, yes. But they are made as guidelines- quick snapshots. Their problem is twofold. First, other compounds may cause a false positive with them occasionally. Second, if drugs are present very near the cutoff, they may read positive or negative, and only LC/MS/MS confirmation can truly establish the presence or absence of the drug.
Why do results sometimes come back as ‘QNS’?
This happens when these is not sufficient sample for analysis in the oral fluid tube or the urine cup. Most of the time this is due to leakage of the specimen container which, in turn, is frequently due to incomplete closure of the top or lid.
Does the presence of bacteria or mold in the sample affect testing?
Bacteria cause a problem only with particular analytes. For instance, bacteria can destroy the alcohol metabolite ethyl glucuronide (EtG). This is why another metabolite (EtS) is also measured, because it is not affected by bacteria. Mold contamination in a sample where sugar is present (urine of a diabetic, patient, or oral fluid obtained after sucking hard candy) can result in the production of alcohol- by way of fermentation of the sample. Alcohol concentrations higher than the fatal range can be observed in these cases.