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Vitamin D Deficiency and Toxicity: Laboratory Support of Diagnosis and Management

Vitamin D Deficiency and Toxicity Laboratory Support of Diagnosis and Management

Clinical Focus

Vitamin D Deficiency and Toxicity

Laboratory Support of Diagnosis and Management

  

Contents:

Clinical Background  - Table 1 - Table 2

Individuals Suitable for Testing  - Table 3

Test Availability  - Table 4

Test Selection

Test Interpretation

References
 

Clinical Background [return to contents]

Vitamin D-Associated Disorders

Vitamin D deficiency, as defined by the Institute of Medicine and professional organizations such as the Endocrine Society, is common in the general population. The most severe deficiencies are associated with rickets in children and osteomalacia and osteoporosis in adults; vitamin D deficiency is also associated with muscle weakness and secondary hyperparathyroidism, both of which can exacerbate these bone disorders and/or their clinical impact.1,2  In many cases, however, significant vitamin D deficiency can exist without immediate clinical symptoms, presenting a future risk to bone health if the condition is not diagnosed for many years.

Evidence, mostly from observational studies, suggests a link between vitamin D deficiency and non-bone diseases as well. These diseases include cancer (especially colorectal and breast cancer), cardiovascular disease, autoimmune diseases, diabetes, depression, and schizophrenia.1,3,4

In rare cases, ingestion of large doses of vitamin D results in vitamin D toxicity, which can manifest as hypercalcemia, hypercalciuria, or kidney stones.1,5  The risk of vitamin D toxicity may be increased in individuals who have hyperparathyroidism, sarcoidosis, tuberculosis, or lymphoma and are taking vitamin D supplements.5

Physiology

Vitamin D is a fat-soluble vitamin that promotes bone health by enhancing intestinal absorption of calcium and phosphorus1,6; without sufficient absorption of calcium and phosphorous, parathyroid hormone (PTH) levels can increase and result in bone resorption.6  More recent discoveries suggest that vitamin D may have additional biological functions. For example, vitamin D receptors have been found in a number of tissues including colon, breast, prostate, and brain. The active form of vitamin D (1,25-dihydroxyvitamin D) is involved in the control of more than 200 genes, including those involved in immune function7 and regulating cell growth and apoptosis.1

Vitamin D occurs in 2 forms: vitamin D2 and vitamin D3. Both forms enter the body in small amounts through diet, but vitamin D3, the main form in humans, is produced in the skin in response to sunlight.6  Vitamins D2 and D3 are rapidly metabolized in the liver to their respective 25-hydroxyvitamin D (25[OH]D) forms, 25(OH)D2 and 25(OH)D3, which are converted in the kidneys to their corresponding active forms (Table 1).4 Experts disagree about the relative potencies of vitamins D2 and D3; some experts claim that the 2 forms are equally effective in raising 25(OH)D levels, whereas other experts assert that vitamin D2 is less effective than vitamin D3 in elevating 25(OH)D.8,9

Table 1. Important Vitamin D Forms

Name

Description

Abbreviation

Description

Ergocalciferol Vitamin D2 NA Plant-based form
Cholecalciferol Vitamin D3 NA Animal-based form; form produced in human skin
Ercalcidiol 25-hydroxyvitamin D2 25(OH)D2 Main circulating form of vitamin D2
Calcidiol 25-hydroxyvitamin D3 25(OH)D3 Main circulating form of vitamin D3
Ercalcitriol 1,25-dihydroxyvitamin D2 1,25(OH)2D2 Active form of vitamin D2; has a short half-life in the blood
Calcitriol 1,25-dihydroxyvitamin D3 1,25(OH)2D3 Active form of vitamin D3; has a short half-life in the blood
3-epimer 3-epi-25-hydroxyvitamin D 3-epi-25(OH)D Low-activity form; significant levels present in infants

Recommended Vitamin D Intake

The American Association of Clinical Endocrinologists (AACE) recommends vitamin D and calcium supplementation to prevent osteoporosis in postmenopausal women.2 In conjunction with calcium, vitamin D supplementation increases bone mineral density and reduces the risk of fractures and falls in postmenopausal women.2,10  The American Academy of Pediatrics also recommends supplementation with vitamin D to prevent rickets in children, especially for breastfed infants.11  Indeed, vitamin D supplementation/therapy is now the standard of care for preventing and treating rickets.6,11

Optimal intake levels, however, remain a subject of debate. In 2011, the Institute of Medicine (IOM) increased the recommended daily vitamin D intake from 200-600 IU/d to 400-800 IU/d, depending on age.12  Other experts believe that these increases were not sufficient; they recommend as much as 1,000-2,000 IU/d (Table 2).6  These same experts believe that an intake up to 10,000 IU/d is safe.

Table 2. Institute of Medicine (IOM) and Endocrine Society Recommendations for Vitamin D Intake6,12
Life-stage Groupa

IOM Recommendations (IU/d)

 

Endocrine Society Recommendations (IU/d)b

Intake

Upper Limitc

Intake

Upper Limitc

0-6 months 400d 1,000 400-1,000 2,000
6-12 months 400d 1,500 400-1,000 2,000
1-3 years 600 2,500 600-1,000 4,000
4-8 years 600 3,000 600-1,000 4,000
9-18 years 600 4,000 600-1,000 4,000
19-30 years 600 4,000 1,500-2,000 10,000
31-50 years 600 4,000 1,500-2,000 10,000
51-70 years 600 4,000 1,500-2,000 10,000
71+ years 800 4,000 1,500-2,000 10,000
Pregnant or lactating women (14-18 years) 600 4,000 600-1,000 4,000
Pregnant or lactating women (19-50 years) 600 4,000 1,500-2,000 10,000

a  Includes normal healthy individuals of both genders unless otherwise specified.

b  Estimated intake needed to maintain blood 25(OH)D levels above 30 ng/mL.

c  Maximum level that is expected to have no risk of adverse effects to healthy individuals. 1 μg of vitamin D is equivalent to 400 IU.
d  Refers to adequate intake (intake estimated to maintain protective 25(OH)D levels in a group of healthy individuals with limited sun exposure and vitamin D stores) instead of recommended intake, which could not be established because of insufficient evidence.

Obtaining Sufficient Amounts of Vitamin D

An estimated 68% to 77% of patients have suboptimal (<30 ng/mL) levels of vitamin D.13-15  Sufficient amounts can be obtained through adequate sunlight exposure and/or a diet containing enough vitamin D-rich foods such as oily fish. Other animal-based foods provide small amounts of vitamin D3, and some plant-based foods provide small amounts of vitamin D2.6 For individuals who do not get enough vitamin D by these means, supplementation is available. Vitamins D2 and D3 are both available in fortified foods and in over-the-counter supplements that range in dose from 400 to 50,000 IU. Vitamin D2 is also available by prescription in liquid (8,000 IU) or high-potency (50,000 IU) capsule formulations. For vitamin D deficiency, the recommended treatment dosage, length of treatment, and maintenance therapy dosage differ based on whether the patient is an adult, child, or infant.9 Higher vitamin D doses may be required for people who are obese, have a malabsorption syndrome (eg, cystic fibrosis, celiac disease, Crohn’s disease),1 have undergone bariatric surgery, or are taking medications that affect vitamin D metabolism.6

Determining Vitamin D Status

Laboratory measurement of 25(OH)D in blood is the accepted means for determining vitamin D status.6 25(OH)D levels can be used in: 1) diagnosing vitamin D insufficiency or deficiency, thus identifying individuals who may benefit from supplementation; 2) monitoring response to vitamin D supplements; and 3) diagnosing vitamin D toxicity. There is no consensus about the optimal 25(OH)D level, but many experts accept a range 30 to 60 ng/mL as optimal.1,2,6  Suboptimal levels are broken into 2 groups: insufficiency (21 to 29 ng/mL) and deficiency (≤20 ng/mL).

Individuals Suitable for Testing [return to contents]

  • Individuals with suspected vitamin D deficiency (eg, those with persistent, nonspecific musculoskeletal pain)

  • Individuals at increased risk for vitamin D deficiency (eg, pregnant, lactating, dark-skinned, elderly, obese, or housebound individuals; those dwelling in latitudes higher than 33 degrees north or 33 degrees south; and infants who are breastfeeding without vitamin D supplementation or from vitamin D-deficient mothers)

  • Individuals being treated with vitamin D2 or vitamin D3 supplementation

  • Individuals with disorders associated with reduced 25(OH)D levels (Table 3)

  • Individuals taking certain medications associated with reduced 25(OH)D levels (Table 3)

  • Individuals with suspected toxicity (eg, those with hypercalcemia of obscure origin)

Table 3. Effect of Various Disorders and Medications on 25-Hydroxyvitamin D and 1,25-Dihydroxyvitamin D Concentrations1,6,7,16-22

Disorder

25(OH)D
Concentration

1,25(OH)2D
Concentration

Chronic kidney disease ↓ or N
Fat malabsorption disorders, short bowel syndrome
Hypercalcemia of cancer N
Hyperparathyroidism or N ↑ or N
Intestinal diseases causing excessive loss of vitamins
D2 and D3
Lymphoma, granulomatous disorders ↓ or N
Medications that increase vitamin D metabolism (eg, anticonvulsants, antiretrovirals, and glucocorticoids)
Nephrotic syndrome
Nutritional rickets ↓ or N
Obesity
Osteomalacia ↓or N
Secondary hyperparathyroidism
Severe parenchymal liver disease (impaired
25-hydroxylation of vitamin D)
Vitamin D-dependent rickets, type Ia N
Vitamin D-dependent rickets, type IIb N
Vitamin D (25[OH]D) toxicity (uncommon) or N

25(OH)D, 25-hydroxyvitamin D; 1,25(OH)2D, 1,25-dihydroxyvitamin D; N, normal; ↑, elevated; ↓, reduced.
a  Also called pseudo-vitamin D-deficiency rickets.
b  Also called hereditary vitamin D-resistant rickets.

Test Availability [return to contents]

Tests offered by Quest Diagnostics to assist in the diagnosis of vitamin D deficiency or toxicity are presented in Table 4. This table is provided for informational purposes only and is not intended as medical advice. A physician’s test selection and interpretation, diagnosis, and patient management decisions should be based on his/her education, clinical expertise, and assessment of the patient.

Table 4. Tests Available to Determine Vitamin D Levels
Test Code Test Name Method Description
17306 QuestAssureD™ 25-Hydroxyvitamin D (D2, D3), LC/MS/MS LC/MS/MS Reference method; accurately quantifies and reports both forms of vitamin D: 25(OH)D2 and 25(OH)D3; total 25(OH)D reported as the sum of the 2 forms; useful for diagnosing vitamin D insufficiency or deficiency and for monitoring vitamin D2 or D3 therapya
91935 QuestAssureD™ for Infants, 25-Hydroxyvitamin D, LC/MS/MS LC/MS/MS Accurately quantifies 25(OH)D2, 25(OH)D3, and total 25(OH)D in infants/toddlers <36 months of age; 3-epimer, which is present at relatively high levels in infants, is separated and excluded from measurements in this assay; useful for diagnosing vitamin D insufficiency or deficiency and for monitoring vitamin D2 or D3 therapya
16558 Vitamin D, 1,25-Dihydroxy, LC/MS/MS LC/MS/MS Accurately quantifies 1,25(OH)2D; useful in differential diagnosis of vitamin D-related diseases and for monitoring vitamin D therapy in patients with chronic kidney disease
16761 QuestAssureD™ 25-Hydroxy and 1,25-Dihydroxyvitamin D, LC/MS/MS LC/MS/MS See individual analytes

LC/MS/MS, liquid chromatography tandem mass spectrometry.
a May also be used to confirm vitamin D toxicity, which occurs rarely.

Test Selection [return to contents]

25(OH)D

Measurement of 25(OH)D, the main form of circulating vitamin D, is used to determine vitamin D status and to monitor most patients receiving vitamin D therapy.6 Several methods are available, each with its own advantages and disadvantages. Immunoassays are widely used and include RIAs, electrochemiluminometric assays, and immunochemiluminometric assays. Disadvantages of immunoassays include the use of radioactive isotopes (RIAs), method-to-method variation, and laboratory-to-laboratory variation.23 Sample matrix effects and cross-reactivity with interfering substances, such as other vitamin D metabolites, account for some of the variability. Moreover, some immunoassays overestimate total 25(OH)D levels, and some underestimate total levels due to low detection of 25(OH)D2.24-26

LC/MS/MS, in contrast, accurately and precisely measures 25(OH)D2, 25(OH)D3, and total 25(OH)D concentrations27 and is the reference method endorsed by experts at the National Institute of Standards and Technology, the Centers for Disease Control and Prevention, and the National Institutes of Health.28-30 It has an analytical sensitivity greater than that of many immunoassays and comparable to that of an RIA, without the need for radioisotopes.25,26

Quest Diagnostics uses an LC/MS/MS method to measure 25(OH)D blood levels. Concentrations of both 25(OH)D forms are independently reported, and the 2 forms are summed to provide the total 25(OH)D concentration. Thus, the QuestAssureDTM 25-Hydroxyvitamin D (D2, D3), LC/MS/MS assay differentiates the separate contributions of vitamins D2 and D3 to vitamin D status. It detects 25(OH)D2 and 25(OH)D3 at an analytical sensitivity of 4 ng/mL, and does not cross-react with the D2 or D3 forms of vitamin D, 25-hydroxyvitamin D, or 1,25-dihydroxyvitamin D. This test can be used to diagnose vitamin D insufficiency or deficiency and to monitor patients receiving vitamin D2 or D3 supplementation.

Unlike adults, infants may have significant levels of the 25(OH)D3 3-epimer (also known as C3-epimer), a form with reduced activity.31 In one study, about three quarters of infants under 4 months of age and 25% to 40% of infants 4 to 12 months of age had measurable levels (≥3 ng/mL) of the 3-epimer. Although these data indicate that accounting for 3-epimer is most appropriate for infants younger than 12 months of age, a notable proportion (18%) of toddlers between 12 and 36 months old also had measurable levels.32 LC/MS/MS tests that do not remove the 3-epimer component can overestimate total vitamin D levels, which could lead to undertreatment. One group found that including the 3-epimer in total vitamin D quantitation led to misclassification of 9% of infants as vitamin D sufficient.31 The QuestAssureD for Infants 25-Hydroxyvitamin D, LC/MS/MS assay differs from the standard 25(OH)D assay in that it chromatographically separates the 3-epimer, so that the epimer can be excluded from the reported 25(OH)D3 and total 25(OH)D concentrations.

1,25(OH)2D

Measurement of 1,25(OH)2D is not used to diagnose vitamin D deficiency because most individuals have normal levels due to tight regulation.6,10 Furthermore, 1,25(OH)2D may even be elevated in people with secondary hyperparathyroidism because PTH enhances the conversion of 25(OH)D to 1,25(OH)2D.1 1,25(OH)2D measurement is reserved for distinguishing some cases of primary hyperparathyroidism from hypercalcemia of cancer and for the differential diagnosis of vitamin D-dependent rickets (type I vs type II) (Table 3). 1,25(OH)2D quantitation is also useful for monitoring vitamin D therapy in patients with chronic kidney disease, who may have normal 25(OH)D levels. As kidney disease progresses, the ability of the kidney to produce 1,25(OH)2D decreases; supplementation with vitamin D is thus no longer effective, and patients require administration of 1,25(OH)2D. Although 1,25(OH)2D may be increased or decreased in a number of other disorders, levels are typically used for confirmation rather than diagnosis of these conditions (Table 3).

Test Interpretation [return to contents]

Optimal 25(OH)D levels are 30 to 60 ng/mL, whereas levels between 21 ng/mL and 29 ng/mL indicate vitamin D insufficiency and levels ≤20 ng/mL indicate deficiency.6 It is important to note that the effect of 25(OH)D levels between 21 and 29 ng/mL on long-term bone health in children is not yet well understood.33 Vitamin D insufficiency and deficiency may both lead to elevated PTH levels (secondary hyperparathyroidism),1 and the most severe forms of deficiency may be associated with hypocalcemia, hypophosphatemia, and elevated alkaline phosphatase.

In children and adults, low 25(OH)D levels are most commonly associated with lack of dietary intake and/or lack of sun exposure. They are also associated with disorders that are characterized by decreased absorption or excessive loss in the gastrointestinal tract, increased vitamin D metabolism, or impaired conversion of vitamin D to 25(OH)D.1,2 Table 3 lists expected 25(OH)D and 1,25(OH)2D levels associated with various disorders and medications. In infants, low 25(OH)D levels are associated with dark skin pigmentation, maternal vitamin D deficiency, and breastfeeding without vitamin D supplementation.6,11

High 25(OH)D levels are suggestive of vitamin D toxicity, but expert opinions vary regarding an appropriate toxicity threshold. The Institute of Medicine cites reports of adverse events at 25(OH)D levels ≥50 ng/mL and recommends relatively low vitamin D intake levels.12 However, many experts such as the authors of the Endocrine Society clinical practice guideline consider levels up to 100 ng/mL safe and assert that vitamin D toxicity only occurs at 25(OH)D levels ≥150 ng/mL.6,33

References [return to contents]

  1. Holick MF. Vitamin D deficiency. N Engl J Med. 2007;357:266-281.

  2. Watts NB, Bilezikian JP, Camacho PM, et al. American Association of Clinical Endocrinologists medical guidelines for clinical practice for the diagnosis and treatment of postmenopausal osteoporosis. Endocr Pract. 2010;16(Suppl 3):1-37.

  3. Peterlik M, Cross HS. Vitamin D and calcium insufficiency-related chronic diseases: an emerging world-wide public health problem. Int J Environ Res Public Health. 2009;6:2585-2607.

  4. Thatcher TD, Clarke BL. Vitamin D insufficiency. Mayo Clin Proc. 2011;86:50-60.

  5. Vieth R. Vitamin D supplementation, 25-hydroxyvitamin D concentrations, and safety. Am J Clin Nutr. 1999;69:842-856.

  6. Holick MF, Binkley NC, Bischoff-Ferrari HA, et al. Evaluation, treatment, and prevention of vitamin D deficiency: an Endocrine Society clinical practice guideline. J Clin Endocrin Metab. 2011;96:1911-1930.

  7. Bouillon R. Vitamin D: from photosynthesis, metabolism, and action to clinical applications. In: Jameson JL, De Groot LJ, et al, eds. Endocrinology. 6th ed. Philadelphia, PA: Saunders; 2010:1089-1110.

  8. Holick MF, Biancuzzo RM, Chen TC, et al. Vitamin D2 is as effective as vitamin D3 in maintaining circulating concentrations of 25-hydroxyvitamin D. J Clin Endocrinol Metab. 2008;93:677-681.

  9. Armas LAG, Hollis BW, Heaney RP. Vitamin D2 is much less effective than vitamin D3 in humans. J Clin Endocrinol Metab. 2004;89:5387-5391.

  10. Cranney A, Horsley T, O’Donnell S, et al. Effectiveness and Safety of Vitamin D in Relation to Bone Health. Rockville, MD: Agency for Healthcare Research and Quality (US); 2007. Available at: http://www.ncbi.nlm.nih.gov/books/NBK38416. Accessed January 27, 2012.

  11. Wagner CL, Greer FR; American Academy of Pediatrics Section on Breastfeeding; American Academy of Pediatrics Committee on Nutrition. Prevention of rickets and vitamin D deficiency in infants, children, and adolescents. Pediatrics. 2008;122:1142-1152.

  12. Ross AC, Manson JE, Abrams SA, et al. The 2011 Report on Dietary Reference Intakes for Calcium and Vitamin D from the Institute of Medicine: What Clinicians Need to Know. J Clin Endocrinol Metab. 2011;96:53-58.

  13. Ginde AA, Liu MC, Camargo CA, et al. Demographic differences and trends of vitamin D insufficiency in the US population, 1988-2004. Arch Intern Med. 2009;169:626-632.

  14. Kumar J, Muntner P, Kaskel FJ, et al. Prevalence and associations of 25-hydroxyvitamin D deficiency in US children: NHANES 2001-2004. Pediatrics. 2009;124:e362-e370.

  15. Lai JK, Lucas RM, Clements MS, et al. Assessing vitamin D status: pitfalls for the unwary. Mol Nutr Food Res. 2010;54:1062-1071.

  16. Bringhurst FR, Demay MB, Kronenberg HM. Hormones and disorders of mineral metabolism. In: Melmed S, Polonsky KS, Larsen PR, Kronenberg HM, eds. Williams Textbook of Endocrinology. 12th ed. Philadelphia, PA: Elsevier Saunders; 2011:1237-1304.

  17. Lorenzo JA, Canalis E, Raisz LG. Metabolic bone disease. In: Melmed S, Polonsky KS, Larsen PR, Kronenberg HM, eds. Williams Textbook of Endocrinology. 12th ed. Philadelphia, PA: Elsevier Saunders; 2011:1305-1349.

  18. Silverberg SJ, Bilezikian JP. Primary hyperparathyroidism. In: Jameson JL, De Groot LJ, et al, eds. Endocrinology. 6th ed. Philadelphia, PA: Saunders; 2010:1176-1197.

  19. Horwitz MJ, Stewart AF. Malignancy-associated hypercalcemia and medical management. In: Jameson JL, De Groot LJ, et al, eds. Endocrinology. 6th ed. Philadelphia, PA: Saunders; 2010:1198-1211.

  20. National Kidney Foundation. KDOQI clinical practice guidelines for bone metabolism and disease in chronic kidney disease. New York, NY: National Kidney Foundation; 2004.

  21. St-Arnaud R, Glorieux FH. Genetic defects in vitamin D metabolism and action. In: Jameson JL, De Groot LJ, et al, eds. Endocrinology. 6th ed. Philadelphia, PA: Saunders; 2010:1236-1249.

  22. Demay MB, Krane SM. Disorders of calcification: osteomalacia and rickets. In: Jameson JL, De Groot LJ, et al, eds. Endocrinology. 6th ed. Philadelphia, PA: Saunders; 2010:1311-1329.

  23. Binkley N, Krueger D, Cowgill CS, et al. Assay variation confounds the diagnosis of hypovitaminosis D: a call for standardization. J Clin Endocrinol Metab. 2004;89:3152-3157.

  24. Hollis BW. Comparison of commercially available 125I-based RIA methods for the determination of circulating
    25-hydroxyvitamin D. Clin Chem. 2000;46:1657-1661.

  25. Glendenning P, Taranto M, Noble JM, et al. Current assays overestimate 25-hydroxyvitamin D3 and underestimate 25-hydroxyvitamin D2 compared with HPLC: Need for assay-specific decision limits and metabolite-specific assays. Ann Clin Biochem. 2006;43:23-30.

  26. Herrmann M, Harwood T, Gaston-Parry O, et al. A new quantitative LC tandem mass spectrometry assay for serum 25-hydroxy vitamin D. Steroids. 2010;75:1106-1112.

  27. Maunsell Z, Wright DJ, Rainbow SJ. Routine isotope-dilution liquid chromatography-tandem mass spectrometry assay for simultaneous measurement of the 25-hydroxy metabolites of vitamins D2 and D3. Clin Chem. 2005;51:1683-1690.

  28. Tai SS, Bedner M, Phinney KW. Development of a candidate reference measurement procedure for the determination of 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 in human serum using isotope-dilution liquid chromatography/tandem mass spectrometry. Anal Chem. 2010;82:1942-1948.

  29. Chen H, McCoy LF, Schleicher RL, et al. Measurement of 25-hydroxyvitamin D3 (25OHD3) and
    25-hydroxyvitamin D2 (25OHD2) in human serum using liquid chromatography-tandem mass spectrometry and its comparison to a radioimmunoassay method. Clin Chem Acta. 2008;391:6-12.

  30. Yetley EA, Pfeiffer CM, Schleicher RL, et al. NHANES monitoring of serum 25-hydroxyvitamin D: a roundtable summary. J Nutr. 2010:140:2030S-2045S.

  31. Bailey D, Veljkovic K, Yazdanpanah M, et al. Analytical measurement and clinical relevance of vitamin D(3) C3-epimer. Clin Biochem. 2013;46:190-196.

  32. Goldman MM, Viec KV, Caulfield MP, et al. The measurement of 3-epimer 25-hydroxyvitamin D by mass spectrometry in clinical specimens detects inconsequential levels in adult subjects. J Invest Med. 2014;62:690-695.

  33. Vogiatzi MG, Jacobson-Dickman E, Deboer MD, et al. Vitamin D supplementation and risk of toxicity in pediatrics: a review of current literature. J Clin Endocrinol Metab. 2014;99:1132-1141.
     

Content reviewed 03/2014

 

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* The tests listed by specialist are a select group of tests offered. For a complete list of Quest Diagnostics tests, please refer to our Directory of Services.