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NLCP: STATE OF THE SCIENCE - UPDATE # 1
(February 14, 2000)

Subject: Urine Specimen Validity Testing: Evaluation of the Scientific Data Used to
Define a Urine Specimen as Substituted

Background

The Mandatory Guidelines for Federal Workplace Drug Testing Programs published in the Federal Register on June 9, 1994 (59 FR 29908) and the U.S. Department of Transportation (DOT) regulations (49 CFR Part 40) applicable to DOT federally regulated programs permit laboratories to conduct additional tests to determine the validity of a urine specimen. The laboratories certified under the National Laboratory Certification Program (NLCP) have reported that the number of adulterated, diluted, and substituted urine specimens has been increasing. In response, the U.S. Department of Health and Human Services (HHS) and DOT began the process, using the HHS Substance Abuse and Mental Health Services Administrationâ€™s (SAMHSA) Drug Testing Advisory Board (DTAB), to develop standards for the testing and reporting of validity test results for urine specimens tested in federally regulated programs. The scientific consensus reached and guidance provided by the DTAB was issued in the form of National Laboratory Certification Program (NLCP) Program Documents (PDs). These PDs established the standards for performing and reporting tests for adulteration, dilution, and substitution if such testing is conducted on Federal and federally regulated specimens. At this time, validity testing is authorized, utilizing specific NLCP criteria, but is not mandatory for urine specimens collected under Federal employee drug testing programs.

Urine is an aqueous solution. Its major constituents are primarily electrolytes, metabolic excretory products and other substances eliminated through the kidneys. Creatinine is one such metabolic excretory product spontaneously formed from creatine in muscle. Thus, creatinine production is dependent upon muscle mass. Clinically, creatinine is used to assess renal function because it is excreted at a relatively constant rate.

Specific gravity and osmolality assess urine concentration, or the amount of substances dissolved in urine. As increasing amounts of substances are added to urine, the concentration of these dissolved substances and the density, or the weight of substances per unit volume of liquid, increases. Urinary osmolality determines the concentration of the dissolved substances in urine. Specific gravity measures the density of urine relative to the density of pure water. The greater the specific gravity and osmolality, the more concentrated the urine.

Creatinine and specific gravity are two common clinical chemistry parameters which characterize normal human urine specimens for a variety of purposes. Random urine reference ranges are routinely used to evaluate a donorâ€™s urine, especially in drug testing programs. Urine specimens are defined in PD #35 (see attachment 1) as "dilute" if the creatinine concentration is < 20 mg/dL and the specific gravity is < 1.003. A number of urine specimens reported "dilute" by these rules were so extremely low in their creatinine concentration and specific gravity that it raised doubt if, in fact, the specimen was human urine as excreted by the kidney. Some individuals, in an effort to conceal drug use will attempt to suborn any procedure employed to identify the presence of illegal drugs in a collected urine specimen. The need, therefore, to define urine specimens that are "substituted" (i.e., not consistent with normal or dilute human urine) is paramount for an effective drug testing program.

Scientific Review and Evaluation

Before NLCP PDs were issued to provide guidance concerning urine specimen validity testing, an extensive review of the published scientific literature was performed to develop the criteria for defining a "substituted" specimen. The review encompassed relevant studies, the majority of which were done in the 1990s. An analysis of that review resulted in selecting urine creatinine < 5 mg/dL and urine specific gravity <1.001 or >1.020 as the criteria to define a "substituted" specimen. Four different types of studies were evaluated:

1.Normal random urine reference range reports from a leading clinical diagnostic reference text
2.Clinical studies involving the analysis of random urine specimens
3.Medical conditions resulting in overhydration
4.Water loading studies

Normal Random Urine Reference Ranges

The clinically accepted reference ranges for normal random urine specimens are shown in Table 1. "Random" urine specimens refer to those obtained from the general population, including males and females of varying ages, ethnicities, (i.e., Caucasian, African-American, Asian, Hispanic), persons of different body sizes and shapes, dietary habits, and other variables of human lifestyle.

Table 1. Normal random urine reference ranges.¹
Analyte	Random Urine Reference Range
Creatinine	37 – 300 mg/dL (female) ; 44 – 250 mg/dL (male) *
Specific gravity	1.002 - 1.030
Osmolality	50 – 1200 mOsm/kg

*Based on a calculation from 24 hour creatinine and total urine volume reference ranges.

Random Urine Clinical Studies

Eleven different clinical studies enrolling normal subjects for either medical evaluation purposes or substance abuse related conditions that had reported creatinine and specific gravity in random urine specimens were identified in the published literature. A summary of these studies are found in Table 1 in the appendix.

In the urine specimens collected for medical evaluation purposes, there were no reports of urine specimens in which the "paired data" - specimens where both the urine creatinine and urine specific gravity were measured - showed the urine creatinine concentration was < 5 mg/dL and the urine specific gravity was <1.001 or >1.020. In other words, all urine specimens collected for medical evaluation purposes met the biochemical criteria for excreted human urine. Regardless of the medical conditions studied, no donor urine specimen was identified where both the urine creatinine concentration and the urine specific gravity met the criteria for a "substituted" specimen.

Urine specimens collected from donors in the studied substance abuse populations where testing was done on known drug abusers inherently run the risk of being altered by a deliberate attempt aimed at suborning the results of the test and concealing illegal drug use. Nonetheless, no urine specimen identified in these studied substance abuse populations met the substituted urine specimen criteria where both the urine creatinine concentration was < 5 mg/dL and the urine specific gravity was < 1.001 or > 1.020. There were some urine specimens in which one of the measured parameters (i.e., creatinine or specific gravity) met or exceeded the individual criteria, but not both.

Our extensive review of the scientific literature has shown that there were no cases meeting the substituted specimen creatinine/specific gravity criteria in both the medical evaluation population and substance abuse population. Additionally, there were only a small number of either low creatinine or specific gravity test values even in the substance abuse population. These findings, taken together provide the realistic, scientific basis for the selected "substituted" specimen criteria.

Medical Overhydration Studies

Several clinical conditions produce overhydration or polyuria, the production of excessive amounts of urine. The extreme conditions that produce exceedingly dilute urine are described in Table 2, on the next page. Twenty-seven case studies were identified that provided random urine data for creatinine, specific gravity, and/or osmolality. A summary of the medical cases reporting excessive urine production are found in Table 2 in the appendix. In two extreme cases, 19 liters of water were consumed in 6 hours (water intoxication) and 6 liters of water were consumed in "a few" hours (psychogenic polydypsia). The lowest osmolality results from urine specimens collected after consumption of these excessive amounts of water were 142 and 84 mOsm/kg, respectively. Both of these results are comparable to a normal specific gravity (see note below). For those medical case studies where paired creatinine and specific gravity data did exist, no urine specimen was identified in which the urine creatinine concentration was < 5 mg/dL and the urine specific gravity was < 1.001 or > 1.020 (i.e., no specimen was identified as "substituted").

Table 2. Medical conditions that result in polyuria.
Condition	Description
Psychogenic polydipsia	Excessive fluid consumption resulting from a disorder in personality, without a demonstrable organic lesion
Water intoxication	Severe overhydration which may result in convulsions and death due to uncorrected hyponatremia and cerebral edema
Diabetes insipidus	The chronic excretion of very large amounts of pale urine of low specific gravity, accompanied by extreme thirst, resulting from inadequate amounts of pituitary antidiuretic hormone (ADH)
Nephrogenic diabetes	The chronic excretion of very large amounts of pale urine of low specific gravity resulting from the inability of the kidneys to respond to ADH
Iatrogenic diabetes	Diabetes introduced by an unfavorable response to a therapeutic intervention

Note: For those cases shown in Table 2 in the appendix where an osmolality measurement was made in the absence of a specific gravity measurement, it should be noted that an osmolality value of 70 mOsm/kg is equal to a specific gravity of 1.002 or greater.

Water Loading Studies

Water loading studies are those in which excessive amounts of water were ingested in an attempt to dilute the urine. Ten water-loading studies were reported that included random urine creatinine and/or specific gravity data. Table 3 in the appendix shows a summary of the water loading studies. In the most significant study, 3.8 to 4.2 liters (about 1 gallon) of fluid were consumed in 4 hours. Analysis of the creatinine concentration and the specific gravity in the urine specimens provided after consumption of this large amount of fluid did not produce any specimens defined as "substituted" (i.e., urine creatinine concentration < 5 mg/dL and urine specific gravity < 1.001 or > 1.020). In those studies where paired creatinine and specific gravity data were reported, in no case was the "substituted" criteria achieved; that is, no specimen was identified in which the urine creatinine concentration was < 5 mg/dL and the urine specific gravity was < 1.001 or > 1.020. Creatinine or specific gravity values were individually low in a few cases.

Regulatory Actions

The NLCP published PD #35 for reporting urine specimen validity test results.²It addressed adulterated and diluted urine specimens and created a new classification known as "substituted" specimens. By definition, a specimen is called "substituted" (i.e., the specimen does not exhibit the clinical signs or characteristics associated with normal human urine) if the urine creatinine concentration is < 5 mg/dL and the urine specific gravity is < 1.001 or > 1.020.

Subsequently, the NLCP published PD #37 (see attachment 2) to provide analytical criteria for laboratories testing Federal and federally regulated specimens when conducting these biochemical tests.³ To report a specimen as "substituted," at a minimum, creatinine must be measured by at least one quantitative procedure on two different aliquots, both using the specified cutoff of 5 mg/dL. Reported creatinine results must contain one significant decimal place more than that stated in the decision point (i.e., results are reported to the first decimal place). Creatinine values of 5 mg/dL may not be truncated to the nearest whole number.

Also, at a minimum, specific gravity must be determined on one of these aliquots utilizing the specified cutoffs of 1.001 or 1.020. Specific gravity measurements are required to be performed by refractometry and reported to the third decimal place.

Each laboratory must have a written and validated standard operating procedure for performing urine creatinine and specific gravity analyses and is subjected to the same NLCP laboratory inspection protocol as that for evaluating drug testing processes.

Conclusions

In order for a specimen to be reported as substituted, both creatinine and specific gravity must meet defined criteria; that is, urine creatinine < 5 mg/dL and urine specific gravity < 1.001 or > 1.020. This testing requirement provides both an analytical and physiological safeguard. The review of the scientific literature including random clinical studies, medical conditions resulting in severe overhydration or polyuria, and water loading studies confirms that the urine criteria of creatinine < 5 mg/dL and urine specific gravity < 1.001 or > 1.020 represent a specimen condition that is not consistent with normal human urine. In the deductive evaluation of 47 studies, no exception to the criteria defining a "substituted" specimen was reported.

Acknowledgment

The material for this publication was developed under the direction of Division of Workplace Programs, and funded by the Substance Abuse and Mental Health Services Administration (SAMHSA) under contract number 277-99-6033.

Robert L. Stephenson II, M.P.H.
Director (Acting)
Division of Workplace Programs
Appendix tables, references and attachments:

Table 1. Random Clinical Studies
Number of Subjects/Samples	Creatinine Range, (mg/dL)	Specific Gravity Range	Population	Notes	References
14	> 10	1.002 - 1.024	Medical		A1
350	172 + 81		Medical	All creatinines > 10 mg/dL	A1
50	1.1 - 29	1.001 - 1.084	Drug	No paired data	A2
176	1.1 - 361		Drug		A3
10	183 + 85		Medical		A4
9	18 - 532	1.002 - 1.036	Drug	N = 1206	A5
1601		1.000 - 1.055	Drug	0.37% in 1.000 - 1.005 range	A6
423		1.001 - 1.040	Drug	10% in 1.001 - 1.010 range	A7
37	20 - 477		Medical		A8
67	18 - 200		Medical		A9
7	7 - 318	1.001 - 1.029	Drug		A10
6	6 - 360		Drug	N = 955	A11

Table 2. Medical Polyuria Case Studies
Number of Subjects	Condition	Creatinine (mg/dL)	Specific Gravity	Osmolality, (mOsm/kg)	Notes	References
11	Psychogenic Polydipsia		1.002 - 1.024	45 - 530		C1
1	Diabetes Insipidis		1.003	106	12 - 15 L/d	C2
1	Diabetes Insipidis		1.007	296	8.5 L/d	C2
1	Psychogenic Polydipsia			84	> 6 L in "few" h	C3
1	Water Intoxication			237	6 L/d	C4
1	Water Intoxication			142	19 L/6 h	C5
1	Water Intoxication			78	1.44 L ingestion Seizures, coma	C6
1	Diabetes Insipidis	13	1.005	54	4 - 6 L/d	C7
10	Psychogenic Polydipsia	4 - 185	1.000 - 1.017		No paired data	C8
3	Polydipsia			144 +23		C9
1	Iatrogenic Polydipsia			313	15 - 18 L/d	C10
14	Polydipsia			122 +66 112 +57		C11
10	Polydipsia			> 50	20 mL/kg water load in 10 min	C12
1	Psychogenic Polydipsia			154		C13
1	Psychogenic Polydipsia			141		C14
Number of Subjects	Condition	Creatinine (mg/dL)	Specific Gravity	Osmolality, (mOsm/kg)	Notes	References
1	Water Intoxication		1.000	80		C15
1	Water Intoxication			55		C16
3	Water Intoxication		1.000 - 1.002	131 - 590		C17
1	Water Intoxication			87		C18
1	Water Intoxication			203	Ingested 3 L/3 h	C19
1	Water Intoxication		1.000		Ingested 24 L/10 h	C20
4	Psychogenic Polydipsia			143 - 342		C21
2	Water Intoxication		1.001 - 1.006			C22
8	Psychogenic Polydipsia		1.001 - 1.010	56 - 176	Void 5 - 19 L/d	C23
1	Water Intoxication			50 - 60	Void 11 - 17 L/d	C24
11	Diabetes Insipidus			100 - 200		C25
10	Psychogenic Polydipsia			225 - 325		C25
20	Diabetes Insipidus			83 - 303	Void 5 - 12 L/d	C26
4	Nephrogenic Diabetes			64 - 190		C27

Table 3. Water Loading Studies
Number of Subjects	Dose	Creatinine Range, (mg/dL)	Specific Gravity Range	Pairs	Notes	References
6	4 mL/kg over 10 h		1.012 +0.002	No creatinine data	Osmolality: 295 + 69 mOsm/kg	B1
1	1.2 L/1h 0.1L/30 min 1.4 L/75 min	32 - 157	1.004 - 1.014	Lowest pair: 32 creatinine 1.005 SG	Lowest creatinine: 32 mg/dL	B2
6	1.0 - 2.0 L /4 h	> 30	1.003 - 1.010			B3
7	3.8 - 4.2 L/4 h	4 - 266	1.000 - 1.030	Lowest pairs: (4 : 1.003) (5 : 1.003)	SG < 1.001 paired with 8 - 12 mg/dL creatinines	B4
2	3.4 - 3.8 L/4 h		1.000	No creatinine data	Only value claimed	B5
23	0.5 L/15 min	8 - 257			Osmolality: 69 - 1075 mOsm/kg	B6
23	1.0 L/15 min	7 - 257			Osmolality: 45 - 996 mOsm/kg	B6
20	0.8, 1.0 and 1.5 L/BSA (m²)/d		1.025 (0.003) (0.8 L/BSA) 1.022(0.005) (1.0 L/BSA) 1.015(1.010) (1.2 L/BSA)			B7
8	20 mL/kg				Osmolality: 32 - 95 mOsm/kg	B8
9	5.6 +1.8 L/21 h		1.005 - 1.025		Osmolality: 200 - 850 mOsm/kg	B9
8	2.6 L fluids / 2 h post exercise				Osmolality range 1 h post ingestion: 120- 350 mOsm/kg

References

Burtis CA, Ashwood ER, eds. Tietz Textbook of Clinical Chemistry, 3^rd ed. Philadelphia: WB Saunders, 1999.
Program Document (PD) 35 issued on September 28,1998, Notice to the National Laboratory Certification Program (NLCP) Inspectors and HHS Certified Laboratories, "Guidance for Reporting Specimen Validity Test Results".
Program Document (PD) 37 issued on July 28, 1999, Notice to the National Laboratory Certification Program (NLCP) Inspectors and HHS Certified Laboratories, "General Guidance/Criteria for Specimen Validity Testing".

Random Clinical Studies

A1.Needleman SB, Porvaznik M, Ander D. Creatinine analysis in single collection urine specimens. J For Sci 1992; 37:1125-1133.

A2.George S, Braithwaite RA. An investigation into the extent of possible dilution of specimens received for urinary drugs of abuse screening. Addiction 1995; 90:967-970.

A3.Lafolie P, Beck O, Blennow G, BorÃ©us L, Borg S, Elwin CE, Karlsson L, Odelius G, Hjemdahl P. Importance of creatinine analyses of urine when screening for abused drugs. Clin Chem 1991; 37:1927-1931.

A4.Shepard MD, Penberthy LA, Fraser CG. Short- and long-term biological variation in analytes in urine of apparently healthy individuals. Clin Chem 1981; 27:568-573.

A5.Goldberger BA, Loewenthal B, Darwin WD, Cone EJ. Intrasubject variation of creatinine and specific gravity measurements in consecutive urine specimens of heroin users. Clin Chem 1995; 41:116-117.

A6.Park J, Park S, Lho D, Choo HP, Chung B, Yoon C, Min H, Choi MJ. Drug testing at the 10^th Asian games and the 24^th Seoul Olympic games. J Anal Tox 1990; 14:66-72.

A7.Chan SC, Torok-Both GA, Billay DM, Przybyski PS, Gradeen CY, Pap KM, Petruzelka J. Drug analysis at the 1988 Olympic games in Calgary. Clin Chem 1991; 37:1289-1296.

A8.Hasday J, Grum C. Nocturnal increase of urinary uric acid: creatinine ratio. Am Rev Respir Dis 1987; 135:534-538.

A9.Gokce C, Gokce O, Baydic C, Ilhan N, Alasehirli E, Ozkucuk F, Tasci, Atikeler K, Celebi H, Arslan N. Use of random urine samples to estimate total urinary calcium and phosphate excretion. Arch Int Med 1991; 151:1587-1588.

A10.Cone EJ, Lange R, Darwin WD. In vivo adulteration: excess fluid ingestion causes false-negative marijuana and cocaine urine test results. J Anal Tox 1998; 22:460-473.

A11.Huestis MA, Cone EJ. Differentiating new marijuana use from residual drug excretion in occasional marijuana users. J Anal Tox 1998; 22:445-454.

Medical Polyuria Case Studies

C1.Cronin RE. Psychogenic polydipsia with hyponatremia: report of eleven cases. Am J Kid Dis 1987; 9:410-416.

C2. Kern KB, Meislin HW. Diabetes insipidus: occurrence after minor head trauma. J Trauma 1984; 24:69-72.

C3.Ligtenberg JJM, Wymenga ANM, Tulleken JE, van der Werf TS, Zijlstra JG. A lethal complication of psychogenic polydipsia: cerebral edema and herniation. Intensive Care Med 1998; 24:644-655.

C4.Santonastase P, Sala A, Favaro A. Water intoxication in anorexia nervosa: a case report. It J Eat Disord 1998; 24:439-442.

C5.Garigan TP, Ristedt DE. Death from hyponatremia as a result of acute water intoxication in an Army basic trainee. Mil Med 1999; 164:234-238.

C6.Robson WL, Shashi V, Nagaraj S, Norgaard JP. Water intoxication in a patient with the Prader-Willi syndrome treated with desmopression for nocturnal enuresis. J Urol 1997; 157:646-647.

C7.Buridi A, Corman L, Redinger R. Hypokalemic nephropathy and nephrogenic diabetes insipidus due to excessive consumption of a soft drink. S Med J 1998; 91:1079-1082.

C8.Vieweg WVR, David JJ, Rowe WT, Peach MJ, Veldhuis JD, Kaiser DL, Spradlin WW. Psychogenic polydipsia and water intoxication - concepts that have failed. Biol Psychiatry 1985; 20:1308-1320.

C9.Musch w, Thimpoint J, Vandervelde D, Verhaeverbeke L, Berghmans T, Decaux G. Combined fractional excretion of sodium and urea better predicts response to saline in hyponatremia than do usual clinical and biochemical parameters. Am J Med 1995; 99:348-355.

C10.Olapade-Olaopa EO, Morley RN, Ahiaku EKN, Bramble FJ, Iatrogenic polydipsia: a rare cause of water intoxication in urology. Brit J Urol 1997; 79:488.

C11.Goldman MB, Robertson GL, Luchins DJ, Hedeker D, Pandey GN. Psychotic exacerbations and enhanced vasopression secretion in schizophrenic patients with hyponatremia and polydipsia. Arch Gen Psychiatry 1997; 54:443-449.

C12.Goldman MB, Robertson GL, Luchins DJ, Hedeker DJ. The influence of polydipsia on water excretion in hyponatremic, polydipsic, schizophrenic patients. Clin Endocrinol Metab 1996; 81:1465-1470.

C13.Singh S, Padi MH, Bullard H, Freeman H. Water intoxication in psychiatric patients. Br J Psychiatry 1985; 146:127-131.

C14.Bourgeois P, Leys D, Petit H. Water intoxication in psychiatric patients. Br J Psychiatry 1985; 147:323-324.

C15.Staunton SB, Van Allen MW. Water intoxication. J Iowa Med Soc 1967; 57:665-667.

C16.Manley MRS. Further thoughts on self-induced water intoxication. Am J Psychiatry 1981; 138:392-393.

C17.Rinard G. Water intoxication. Am J Nurs 1989; 89:1635-1638.

C18.Estzioni A, Benderley A, Levi Y. Water intoxication by the oral route in an infant. Arch Dis Child 1979; 54:551-553.

C19.Klonoff DC, Jurow AH. Acute water intoxication as a complication of urine drug testing in the Workplace. JAMA 1991; 265:84-85.

C20.Frizzell RT, Lang GH, Lownce DC, Lathan R. Hyponatremia and ultramarathon running. JAMA 1986; 255:772-774.

C21.Tallis GA. Hyponatremia in psychiatric patients. Med J Aust 1989;150:151-153.

C22.Nickman SL, Buckler BM, Weiner LB. Further experiences with water intoxication. Pediatrics 1968; 41:149-151.

C23.Okura M, Morii S. Polydipsia, polyuria and water intoxication observed in psychiatric inpatients. Tokushima J Exp Med 1986; 33:1-5.

C24.Yonemura K, Hishida A, Miyajima K, Tawarahara K, Mizoguchi K, Nishimura Y, Ohishi K. Water intoxication due to excessive water intake: observation of initiation stage. Jpn J Med 1987; 26:249-252.

C25.Saito T, Ishikawwa S, Ita T, Oda H, Ando F, Higashiyama M, Nagasaka S, Hieda M, Saito T. Urinary excretion of aquaporin-2 water channel differentiates psychogenic polydipsia from central diabetes insipidus. J Clin Endocrinol Metab 1999; 84:2235-2237.

C26.Tetiker T, Sert M, Kocak M. Efficacy of indapamide in central diabetes insipidus. Arch Intern Med 1999; 159:2085-2087.

C27.Kirchlechner V, Koller DY, Seidl R, Waldhauser F. Treatment of nephrogenic diabetes insipidus with hydrochorothiazide and amiloride. Arch Dis Child 1999; 80:548-552.

Water Loading Studies

B1.Zambraski Ej, Rofrano TA, Ciccone CD. Effects of aspirin treatment on kidney function in excercising man. Med Sci Sports Exerc 1982; 14:419-423.

B2.Coleman DE, Baselt RC. Efficacy of two commercial products for altering urine drug test results. Clin Tox 1997; 35:637-642.

B3.Needleman SB, Porvaznik M, Ander D. Creatinine analysis in single collection urine specimens. J For Sci 1992; 37:1125-1133.

B4.Cone EJ, Lange R, Darwin WD. In vivo adulteration: excess fluid ingestion causes false-negative marijuana and cocaine urine test results. J Anal Tox 1998; 22:460-473.

B5.Homer GM, Born B. A discussion of creatinine analysis in single collection urine specimens. J For Sci 1993; 38:501-502.

B6.Lafolie P. Beck O, Blennow G, BorÃ©us L, Borg S, Elwin CE, Karlsson L, Odelius G, Hjemdahl P. Importance of creatinine analyses of urine when screening for abused drugs. Clin Chem 1991; 37:1927-1931.

B7.Abbott K, Barr J, Fasciano A, Gouge S. Evaluation of gender differences in urine specific gravity and serum electrolytes in response to varied fluid intake and ibuprofen use. Mil Med 1993; 158:131-135.

B8.Allon M. Renal Abnormalities in sickle cell disease. Arch Int Med 1990; 150:501-504.

B9.Armstrong LE, Herrera Soto JA, Hacker FT, Casa DJ, Kavouras SA, Maresh CM. Urinary indices during dehydration, exercise, and rehydration. Int J Sport Nutr 1998; 8:345-355.

B10.Kovacs EMR, Senden JMG and Brouns F. Urine color, osmolality and specific electrical conductance are not accurate measures of hydration status during post exercise rehydration. J Sports Med Phys Fitness 1999; 39:47-53.

ATTACHMENT #1
NLCP PROGRAM DOCUMENT #35
September 28, 1998
NOTICE TO HHS CERTIFIED AND APPLICANT LABORATORIES
Subject: Guidance for Reporting Specimen Validity Test Results

The Mandatory Guidelines for Federal Workplace Drug Testing Programs published in the Federal Register on June 9, 1994 (59 FR 29908) and the Department of Transportation (DOT) regulations (49 CFR Part 40) applicable to DOT federally regulated programs permit laboratories to conduct additional tests to determine the validity of a specimen. To ensure maximum consistency, the following guidance is provided for all laboratories in determining the validity of a specimen. We have consulted with the Department of Transportation. It agrees with these procedures and definitions and recommends that such procedures be followed for its federally regulated programs.

A.Single and/or Primary (Bottle A) Specimens

1.Guidance

a.A laboratory may determine for each specimen (i.e., from either a single specimen collection or the primary specimen (Bottle A) from a split specimen collection) the nitrite concentration, creatinine concentration, specific gravity, and pH. These tests shall follow scientifically suitable methods and produce results which are accurately quantified.

b.When a laboratory suspects the presence of an interfering substance/adulterant that could make a specimen unsuitable for testing and the laboratory is unable to identify the interfering substance/ adulterant (e.g., glutaraldehyde, surfactant, bleach), the laboratory may send the specimen to another HHS certified laboratory that has the capability of conducting scientifically suitable validity tests to identify the interfering substance/adulterant.

c.A laboratory shall make every effort to conserve the specimen volume for possible future testing.

2.Definitions

Based on information gathered from a review of current clinical and forensic toxicology literature and recommendations made by the Substance Abuse and Mental Health Services Administrationâ€™s Drug Testing Advisory Board, a specimen is defined to be:

a.Dilute if the creatinine is < 20 mg/dL and the specific gravity is < 1.003, unless the criteria for a substituted specimen are met.

b.Substituted (i.e., the specimen does not exhibit the clinical signs or characteristics associated with normal human urine) if the creatinine concentration is < 5 mg/dL and the specific gravity is < 1.001 or > 1.020.

c.Adulterated if the nitrite concentration is > 500 Fg/mL.

d.Adulterated if the pH is < 3 or > 11.

e.Adulterated if an exogenous substance (i.e., a substance which is not a normal constituent of urine) or an endogenous substance at a higher concentration than normal physiological concentration is present in the specimen.

3.Reporting Results

The Federal custody and control form (CCF) requires laboratories to report drug test results as either Negative, Positive (for a specific drug), or Test Not Performed. Additionally, the laboratory must include an appropriate comment on the "Remarks" line in Step 7 on the CCF when the specimen is dilute, adulterated, substituted, or not tested for drugs (e.g., presence of a fatal flaw or uncorrected flaw). If the additional comments cannot be fully described on the "Remarks" line, the laboratory may attach a separate sheet describing the problem, and reference the attachment on the "Remarks" line.

Note: NLCP Program Document #009 (dated October 10, 1991) and DOT memorandum (dated June 1, 1992) titled "Operating Guidance for DOT Mandated Drug Testing Programs" provide recommendations for rejecting specimens for testing if procedural errors occur.

The following guidance is provided to report a specimen as Negative, Positive, or Test Not Performed:

Negative. The "Negative" box in Step 7 on the CCF is checked when a negative drug test result is obtained on the initial test or on the confirmatory test. If the specimen is also dilute, the laboratory includes the following statement on the "Remarks" line: "Dilute Specimen."

Note: A negative drug test result is not reported when the specimen has been determined to be adulterated or substituted.

Positive. The "Positive" and the specific drug/drug metabolite(s) boxes in Step 7 on the CCF are checked when a positive drug test result is obtained on an initial test and a confirmatory test. If the specimen is also dilute, the laboratory includes the following statement on the "Remarks" line: "Dilute Specimen."

Note: A positive drug test result is not reported when the specimen has been determined to be adulterated or substituted; however, the laboratory may conduct and complete the confirmatory test.

Test Not Performed. The "Test Not Performed" box is checked in Step 7 on the CCF if the specimen is (1) not tested because of a fatal flaw (e.g., broken seal; specimen ID numbers do not match), (2) not tested because of an uncorrected flaw (e.g., a collectorâ€™s signature was omitted and a signed statement is not received to correct the error), (3) unsuitable for testing or contains an unidentified interferant because a valid drug test result cannot be obtained, (4) adulterated, or (5) substituted.

Note: The "Test Not Performed" box is checked regardless of whether there is a negative or positive drug test result if a specimen has been determined to be adulterated or substituted.

If the "Test Not Performed" box in Step 7 on the CCF is checked, one of the following statements is to be included on the "Remarks" line:

1. "Fatal Flaw, " (with the flaw stated)

2. "Uncorrected Flaw, " (with the flaw stated)

3. "Specimen Unsuitable: Cannot obtain valid drug test result"

4a. "Specimen Adulterated: Nitrite is too high"

4b. "Specimen Adulterated: pH is too high (or too low)"

4c. "Specimen Adulterated: Presence of (specify) detected"

5. "Specimen Substituted: Not consistent with normal human urine"

Note: The quantitative results for validity tests (e.g., nitrite concentration, creatinine concentration, actual specific gravity, or actual pH) may not be routinely reported to the MRO, but may be provided to the MRO upon request on a case by case basis.

B.Split (Bottle B) Specimens

1.Guidance

a.When a donor requests, through the MRO, to have the split (Bottle B) specimen tested, a second laboratory (Laboratory B) tests the split specimen for the drug/drug metabolite detected in the primary specimen.

b.If Laboratory B is unable to reconfirm the presence of the drug/drug metabolite that was reported positive in the primary specimen by Laboratory A, Laboratory B must conduct validity tests in an attempt to determine the reason for being unable to reconfirm the presence of the drug/drug metabolite. Laboratory B should conduct the same validity tests as it would conduct on a primary (Bottle A) specimen.

Note: Occasionally, Laboratory B is unable to reconfirm the presence of a drug/drug metabolite (i.e., the confirmatory test results fail to satisfy the criteria established by Laboratory B to report a positive test result) but the laboratory believes that the drug/drug metabolite is present. In this case, Laboratory B may decide to continue testing the split specimen in an attempt to get a valid confirmatory test result. If it appears that Laboratory B may possibly use the entire split specimen in an attempt to get a valid confirmatory test result, Laboratory B must contact the MRO and explain the problem. Laboratory B and the MRO must decide if the remaining amount of the split specimen should be sent to a Laboratory C for the confirmatory test. If the decision is made to use a Laboratory C, Laboratory B sends the split specimen using chain of custody procedures to Laboratory C without reporting a result to the MRO.

c.If Laboratory B is unable to conduct the validity tests, Laboratory B must send the split (Bottle B) specimen and Copy 3 of the Federal custody and control form using chain of custody procedures to a third laboratory (Laboratory C) that has the capability to conduct the validity tests. If the validity tests conducted by Laboratory C do not determine the reason for being unable to reconfirm the presence of the drug/drug metabolite in the split specimen, Laboratory C must test the split (Bottle B) specimen for the drug/metabolite found in Bottle A by Laboratory A.

2.Definitions

Same definitions as in section A.2 of this Program Document.

3.Reporting Split Specimen Results

The CCF requires laboratories to report split (Bottle B) specimen test results as either Reconfirmed (notating the specific drug), Failed to Reconfirm, or Test Not Performed. Additionally, the laboratory must include an appropriate comment on the "Remarks" line in Step 7 on Copy 3 of the CCF if it finds that the specimen is adulterated or substituted, or when a drug test was not performed.

The following guidance is provided to report a specimen as Reconfirmed, Failed to Reconfirm, or Test Not Performed:

Reconfirmed. The "Reconfirmed" and the specific drug/drug metabolite boxes are checked in Step 7 on Copy 3 when the laboratory confirms the presence of the drug/drug metabolite that was reported positive in the primary specimen.

Failed to Reconfirm. The "Failed to Reconfirm" box in Step 7 on Copy 3 of the CCF is checked if (1) the drug/drug metabolite is not detected, (2) the specimen is adulterated, or (3) the specimen is substituted.

If the "Failed to Reconfirm" box is checked, one of the following statements must be included on the "Remarks" line:

1. "Drug/Drug metabolite not detected"

2a. "Specimen Adulterated: Nitrite is too high"

2b. "Specimen Adulterated: pH is too high (or too low)"

2c. "Specimen Adulterated: Presence of (specify) detected"

3. "Specimen Substituted: Not consistent with normal human urine"

Test Not Performed. The "Test Not Performed" box in Step 7 on Copy 3 of the CCF is checked if (1) the specimen is not tested for drugs or (2) the testing could not be completed successfully.

If the "Test Not Performed" box is checked, one of the following statements must be included on the "Remarks" line:

1a. "Fatal Flaw, ________ (with the flaw stated)"

1b. "Uncorrected flaw, _________ (with the flaw stated)"

2a. "Specimen Unsuitable: Cannot obtain valid confirmatory test result"

2b. "Insufficient specimen volume to complete testing"

This Program Document supersedes and replaces PD #033, and should be used in conjunction with DOT memorandum ("MRO Guidance for Interpreting Specimen Validity Test Results") dated September 28, 1998.

If you have any questions regarding this guidance, please contact my staff at (301) 443-6014.

Robert L. Stephenson II, M.P.H.
Director (Acting)
Division of Workplace Programs

ATTACHMENT # 2
NLCP PROGRAM DOCUMENT #37
July 28, 1999

NOTICE TO HHS CERTIFIED LABORATORIES AND INSPECTORS

Subject: Specimen Validity Testing

The Department of Health and Human Servicesâ€™ (HHS) Mandatory Guidelines for Federal Workplace Drug Testing Programs require laboratories to test urine specimens for only those drugs included in agency Drug-Free Workplace plans. Additionally, the Guidelines permit laboratories to conduct other tests to determine the validity of the specimen.

Certified laboratories reported that the number of adulterated, substituted, and diluted specimens have been increasing. HHS and the Department of Transportation (DOT) began a process utilizing the HHS Substance Abuse and Mental Health Services Administrationâ€™s Drug Testing Advisory Board (DTAB) to establish a policy for testing, reporting, and interpreting validity test results for specimens tested under federally regulated programs. A team of program staff and consultants determined the normal ranges for the routine clinical measurements that could be conducted on urine specimens and selected levels that were outside the normal range for each clinical measurement. As a result of this effort, National Laboratory Certification Program (NLCP) Program Document #35 was issued on September 28, 1998, to provide guidance to laboratories in determining the validity of urine specimens.

General Guidance/Criteria

Specimen validity testing is the evaluation of the specimen to determine if it is consistent with normal human urine. Validity testing is used to determine if adulterants or foreign substances were added to the urine specimen or if the specimen was substituted. Specimen validity can be determined by establishing parameters that are consistent with normal human urine and/or by testing for the presence of an abnormal or foreign substance in the urine. Specimen validity testing may be conducted on Bottle A and must be conducted on Bottle B if Bottle B fails to reconfirm for the requested drug/analyte. Specimen validity tests may include, but are not limited to, tests for creatinine concentration, specific gravity, pH, nitrite concentration, pyridine, glutaraldehyde, bleach, and soap. These tests must be performed using methods that are validated by the laboratory.

Specimen validity testing shall be conducted utilizing the following criteria:

1.For dilute specimens, at a minimum, creatinine and specific gravity must be measured by quantitative procedures at a cutoff of 20 mg/dL and 1.003, respectively.

2.For substituted specimens, at a minimum, creatinine must be measured by at least one quantitative procedure on two different aliquots both utilizing the specified cutoff of 5 mg/dL. At a minimum, specific gravity must be performed on one of these aliquots utilizing the specified cutoffs of 1.001 or 1.020.

3.For adulterated specimens, concerning pH and nitrites, at a minimum, two procedures must be performed for pH and nitrites. One procedure must be quantitative and utilize the specified cutoff. The second procedure may be qualitative, must be at least as sensitive as the quantitative procedure, and must be performed on a separate aliquot.

4.For adulterant analytes without a specified cutoff (e.g., glutaraldehyde, bleach, surfactant), at least one procedure must be performed on two separate aliquots.

5.All specimen validity testing methods must be characterized by demonstrating precision and accuracy. Where cutoffs are specified, the limit of quantitation (LOQ) and linearity must be determined. The limit of detection (LOD) must be experimentally determined for qualitative methods.

6.All methods used to characterize and validate these tests must be documented in the laboratoryâ€™s SOP.

Specific Issues/Comments

Issue 1: Is a certified laboratory required to implement validity testing?

Comment: Currently, validity testing is optional. If a laboratory chooses to conduct validity tests, the laboratory must use the definitions provided in PD35 to report results for specimens that are dilute/adulterated/substituted.

Issue 2: A laboratory may send a specimen to another HHS certified laboratory that has the capability of identifying the presence of an interfering substance/adulterant. Does the laboratory send an aliquot or the entire specimen to the other certified laboratory?

Comment: If a certified laboratory suspects the presence of an interfering substance/adulterant that it is unable to identify and decides to send the specimen to another laboratory, it must send the entire specimen to the other certified laboratory. The selection of the other laboratory must be made in coordination with the MRO. When transferring a single specimen bottle/split specimen bottles to another certified laboratory, the single specimen/primary (Bottle A) specimen must be resealed. All specimen bottles and chain of custody forms received from the collection site must accompany the specimen bottle(s) to the other laboratory (i.e., copies 1, 2, and 3 of the CCF and all internal chain of custody documents). The primary laboratory should retain copies of all original documents sent to the second laboratory. When the transfer occurs, the primary laboratory must not report any result to the Medical Review Officer (MRO).

Note: The process of transferring specimens to another laboratory may add several days to the reporting time. It is strongly recommended that specimens be kept refrigerated during the transfer to the other laboratory to minimize degradation or changes caused by any adulterants or interfering substances.

Issue 3: When a specimen is sent to a second laboratory, what results does the second laboratory report to the MRO?

Comment: The second laboratory reports results of its drug testing and/or validity testing to the MRO in accordance with PD35. The original laboratory must not report any results to the MRO.

Issue 4: Is a certified laboratory required to accept and test specimens sent to it by another laboratory without prior notification?

Comment: No. Although the NLCP requires every certified laboratory to have the capability to perform reconfirmation testing, a certified laboratory is not required to accept specimens for reconfirmation testing or to accept specimens for validity testing unless this has been agreed upon before the specimens are sent by the first laboratory. Each laboratory should establish prior agreements with a few selected laboratories to ensure that transfers of specimens are handled expeditiously. The transfer of specimens must be made in coordination with the MRO. If a laboratory chooses not to accept a specimen for retesting, it must contact the sender and make arrangements to forward the specimen to an alternate laboratory.

Issue 5: How does a laboratory interpret quantitative specimen validity results?

Comment: Truncating a quantitative value has been acceptable with ">", ">", and "<" decision points or cutoffs. However, truncating a quantitative value is not acceptable with "<" decision points or cutoffs. In "<" scenarios, truncating would change the result from acceptable to unacceptable (e.g., truncating a pH reading of 3.2 to 3 or a creatinine of 5.4 mg/dL to 5 mg/dL). Values from tests for creatinine (<5 mg/dL) or pH (<3) should contain one significant decimal place more than that specified in the stated decision point. For specific gravity (< 1.001), the method must measure to the third (3^rd) decimal place. This will require refractometry because spectrophotometric and "paper/stick" procedures are not sensitive enough to accurately discriminate in that range.

Issue 6: What are the minimum quality control requirements for conducting a specimen validity test?

Comment: There should be at least one control in the "acceptable" range and one control in the "unacceptable" range analyzed with each batch of validity test specimens. Assays that have more than one decision point (i.e., creatinine, specific gravity, and pH) require more than one control in the unacceptable range: creatinine <20 mg/dL and <5 mg/dL; specific gravity >1.020, < 1.003, and <1.001; and pH <3 and >11. Controls should be prepared in an appropriate urine matrix and validated according to the laboratoryâ€™s standard operating procedure (SOP) manual. In the case of pH controls, an appropriate buffer matrix may be used and the controls validated according to the laboratoryâ€™s SOP manual. All controls must be validated prior to use.

Issue 7: Many laboratories have observed a significant increase in specimens which have a creatinine of <5 mg/dL, but have a specific gravity that is acceptable between 1.003 and 1.019. The specimens appear to be saline. These do not fit the definitions of "dilute" or "substituted" as stated in PD35. How are these specimens to be reported?

Comment: For specimens of this type, the laboratory should provide a "Specimen Unsuitable: Unable to Obtain Valid Drug Test Results" comment in block 7 of the CCF without reporting a "negative" drug test result.

Issue 8: Is more than one comment allowed when multiple adulterants are identified and the specimen is reported "Test Not Performed" or "Failed to Reconfirm"?

Comment: Although it is sufficient to provide only one comment listed in PD35 under "Test Not Performed" or "Failed to Reconfirm" to support an "adulterated" or "substituted" result, the laboratory may provide multiple comments if it has validity test results that require multiple comments.

Issue 9: Some manufacturers of immunoassay test kits have established an acceptable range for the pH of a specimen (e.g., 4.5 - 9). Can a laboratory reject a specimen as "unsuitable for testing" based on pH without determining whether it is adulterated (i.e., < 3 or > 11)?

Comment: Yes, a laboratory can reject specimens that do not meet its specimen acceptance criteria. Specimen rejection criteria are separate from specimen validity testing. Rejected specimens are reported as "Test Not Performed" with the comment "Specimen Unsuitable: Unable to Obtain Valid Drug Test Results." This comment is appropriate with a number of specimen rejection criteria, such as, the observation of foreign objects, unacceptable coloration, unacceptable viscosity, unacceptable odor, or when the pH indicates that the specimen is outside the recommended pH range established by the immunoassay test kit manufacturer. These criteria are separate from specimen validity testing and are not associated with the definitions of PD35.

Issue 10: Can a laboratory reject a specimen as "unsuitable for testing" based on pH instead of reporting it as "adulterated" when pH is <3 or >11?

Comment: No, if a laboratory measures pH as a component of its specimen validity testing, it must adhere to the reporting criteria specified in PD 35. Moreover, the procedures utilized must be validated by the laboratory and follow the criteria outlined in paragraph 3. A. of this document.

Issue 11: Some laboratories indicate that assay performance is adversely affected when nitrite is present but <500 Âµg/mL. Can a laboratory report "unsuitable for testing" when nitrite is <500 Âµg/mL?

Comment: If the nitrite concentration is <500 Âµg/mL and the laboratory is unable to obtain a valid confirmatory test result, the laboratory may report "Test Not Performed." In addition, the comment "Specimen Unsuitable: Cannot obtain valid drug test result" must be entered on the comment line indicating that it is the laboratoryâ€™s belief that the failure to obtain a valid confirmatory test result is caused solely by the presence of nitrite. However, if the laboratory is uncertain that nitrites are the cause for the failure to confirm because there may be an unidentified interfering substance/adulterant in the specimen, the laboratory may send the entire specimen to another certified laboratory (see issue 2 above).

Issue 12: Can "Adulterated" take precedence over "Reconfirmed the presence of ..." for a split specimen? For example, if a split specimen is positive for nitrite, is the split specimen reported "adulterated" even if the laboratory has reconfirmed an analyte that may or may not be affected by nitrite?

Comment: No. Since the primary (Bottle A) specimen was reported "positive" for a specific drug, laboratory B is always required to conduct the confirmatory test for that drug and to report it as "reconfirmed" if it reconfirms the analyte. If laboratory B is unable to reconfirm the presence of the drug, it must perform specimen validity testing to attempt to determine the reason for being unable to reconfirm the presence of the drug (PD35, Paragraph B.1.b).

Issue 13: The primary specimen (Bottle A) is reported substituted or adulterated. What must the laboratory do with Bottle B?

Comment: When a primary specimen (Bottle A) is reported adulterated or substituted, the laboratory must retain both Bottle A and Bottle B for a minimum of 12 months.

If you have any questions regarding these issues or comments, please contact my staff at (301) 443-6014.

Robert L. Stephenson II, M.P.H.
Director (Acting)
Division of Workplace Programs

Drug Testing

NLCP: STATE OF THE SCIENCE - UPDATE # 1 (February 14, 2000)

Subject: Urine Specimen Validity Testing: Evaluation of the Scientific Data Used to Define a Urine Specimen as Substituted

NLCP: STATE OF THE SCIENCE - UPDATE # 1
(February 14, 2000)

Subject: Urine Specimen Validity Testing: Evaluation of the Scientific Data Used to
Define a Urine Specimen as Substituted