A growing challenge for nephrologists is to quickly identify patients with chronic kidney disease (CKD). Experts believe there is now “coherent, undisputable evidence” that timely treatment of CKD can delay the onset of end-stage renal failure (ESRF).
The challenge of timely diagnosis
Several studies suggest that early identification and management of CKD can reduce the risk of kidney failure progression and halve co-morbidities such as cardiovascular disease. The latter is a significant achievement on its own, since cardiovascular complications are estimated to increase mortality in CKD patients by 2-4 times over that of the general population.
Nevertheless, early diagnosis of CKD has not proven to be easy. Chronic kidney disease tends to develop slowly, with few evident symptoms. As a result, most patients seek clinical expertise only after the disease has advanced to a stage where dialysis becomes unavoidable.
Epidemiological data limited
Confounding the problem is a lack of epidemiological data. In the US, the Centers for Disease Control (CDC) can only estimate that “more than 20 million people may have CKD.”
The picture is even less encouraging in Europe. In a first-of-its-kind effort in 2010 to explore what they labelled a “silent epidemic,” Dutch and Italian researchers were “struck by the fact that epidemiological data for CKD existed only in a tiny minority of European countries.” More puzzling was official data from the European Commission or the bulk of EU Member States, which “did not even mention CKD as an issue of public health concern.”
Attempts to establish aggregated CKD data in Europe have led to mixed results. The ambitious EUGLOREH (Status of Health in the European Union) survey established prevalence rates of (higher risk) Stages 3-5 CKD ranging from 3.6% in Norway to 7.2% in Germany, but little about earlier Stages (1 and 2) where treatment best delays progression.
Prevalence of Stages 3-5 CKD was similar for males and females in Germany and Italy, but higher for females in other countries – Belgium, England, Iceland and Norway. In countries for which data was available, prevalence also rose with age.
However, EUGLOREH underlined the total lack of time-trend epidemiological data in Europe, unlike the US where the so-called NHANES III and IV surveys showed prevalence of Stages 1-5 CKD rising from 14.5% in 1988-94 to 16.8% in 1999-2004, while that of Stages 3-5 remained near unchanged, at about 6%.
Delaying dialysis
Recent clinical practices, which advise delaying the onset of dialysis as far as possible, are driving the need for early identification of CKD. In 2003, ‘Kidney International’, the Official Journal of the International Society of Nephrology, stated that “dialysis delayed is death prevented.”
The authors noted that hundreds of thousands of renal failure patients lived in developing countries with inadequate resources or infrastructure; however, many Western patients too were unsuitable for dialysis, “because of age, frailty, or a co-morbid illness with poor prognostic outcome.”
Costs of CKD
The financial impact of CKD is high. In the US, the cost of CKD and its co-morbidities in the pre-ESRF phase are estimated to be as much as $26,000 per patient each year.
Delaying the onset of ESRF provides significant, additional benefits. For example, Italy spends 1.8% of its total health care budget on ESRF patients, who represent only 0.083% of the general population. In the UK, ESRF accounts for 2% of health spending.
The situation in the US is dramatic, with kidney treatment accounting for 24% of Medicare spending, higher than congestive heart failure.
Testing for CKD
Typically, detection and monitoring of CKD requires both urine and blood tests in the laboratory. The former typically focus on kidney function and the latter on damage, according to the US National Institutes of Health (NIH). Key markers for CKD are abnormal urinary levels of albumin (albuminuria) and other proteins (proteinuria), along with a persistent reduction in the glomerular filtration rate (GFR). Variations in urine concentration are corrected by urine albumin-to-creatinine ratio (ACR) or protein-to-creatinine ratio (PCR) on a spot specimen. However, there is considerable debate about the clinical and cost-effectiveness evidence of ACR versus PCR.
So far, CKD testing has largely been driven by diabetes patients, with best practices based on care standards from professional bodies such as the US National Kidney Foundation, the American Diabetes Association and the National Institute for Health and Care Excellence (NICE) in the UK. At-risk groups considered to be typical targets for screening consist of patients with hypertension, cardiovascular disease or a family history of kidney failure. In the US, the CDC estimates that about “1 of 3 adults with diabetes and 1 of 5 adults with high blood pressure has CKD.”
Limitations to screening
With some exceptions, testing urine is not recommended in the general population. In the US, the NIH explains that the “benefit of CKD screening in the general population is unclear.”
The reasons for this restraint are also practical. Albumin levels fluctuate over the day due to metabolism and diet. As a result, urine samples are collected on a random, 4-hourly, or overnight basis. If abnormalities are indicated, re-confirmation is required via a timed, 24-hour collection cycle, accompanied by the labelling and refrigerated storage of the samples. All this clearly poses logistical challenges for large scale CKD screening.
Secondly, any mass screening would clearly use colour-changing dipsticks, the first point of call for urine tests. Though the National Kidney Foundation considers dipsticks satisfactory for a first screening, it warns clinicians to be especially cognizant of false negative results, and specifies the need for lab analyses to quantify urine PCR and ACR ratios. Indeed, while ACR of over 30 mg/g is considered to indicate CKD, the sensitivity level of dipsticks begins at 300 mg/g.
The only alternative to urine is a blood sample, but this too has evident limitations as a mass screening tool for CKD.
Expanding screening: recent developments
Nevertheless, there is growing evidence that larger-scale screening for CKD is likely in the future.
In 2005, Dutch researchers sought to address the challenge of a 24-hour urine collection cycle. They reported that a level of about 11 mg/L urinary albumin concentration (UAC) in a single spot morning urine sample could identify microalbuminuria, and do this as effectively as ACR. They suggested the above as a cut-off point for requiring subsequent urine sample collections, to avoid “huge numbers of individuals” having to undergo “the cumbersome procedure of a 24-hour urine collection” in any mass screening program.
In 2008, the UK’s NICE formally enlarged the scope of testing. It recommended that apart from diabetes, screening for CKD be undertaken in patients with hypertension, cardiovascular diseases, structural renal tract disease, renal calculi or prostatic hypertrophy and multisystem diseases such as systemic lupus erythematosus. The list extended to people with a family history of Stage 5 CKD or hereditary kidney disease.
In its guidelines, NICE also attempted to come to grips with vexing questions on CKD markers such as albuminuria and proteinuria, and the selection and sequencing of tests. The Institute however left open room for further interpretation. Its consensus recommendation was that “ACR should be the test of choice” , but also said there would “often be good clinical reasons for subsequently using PCR to quantify and monitor” proteinuria.
In 2012, the Australasian Proteinuria Consensus Working Group took what may be one of the biggest steps so far to scale up screening. Based on a retrospective longitudinal cohort study of 5,586 CKD patients, it extended the UK NICE screening envelope to include obesity and smoking, and “strongly” advocated targeted opportunistic testing for CKD risk in “all adults attending an appointment with their health care practitioner”. The Working Group also concluded that ACR from a morning (or random) spot urine sample was as good as PCR and 24-hour urinary albumin and protein measurements, and recommended testing for albuminuria rather than proteinuria in individuals at risk of CKD.
The group advised “all pathology laboratories in Australia” to implement its recommendations as part of an “integrated national approach to CKD detection.”
The UK: A ‘World Leader’ in early detection of CKD
Although the Australian recommendations appear to be much more explicit than the NICE guidelines, the UK has ensured that CKD is incorporated into its wider Quality and Outcomes Framework (QOF). As a result, GPs are now rewarded based on how well they identify and manage patients with CKD. CKD is given 27 points in QOF, alongside 9 points for diabetes related to kidney disease. This is considered to provide significant support for the Renal National Service Framework (NSF), which aims to minimize the impact of kidney disease in its early stages.
Indeed, the EUGLOREH report mentioned previously considers the CKD-directed steps in the Quality and Outcomes Framework to have made the UK “a world leader in this field.”
Telediagnosis may offer breakthrough in mass CKD screening
Meanwhile, a breakthrough in the US could remove the final barriers to a true mass screening program for CKD.
In August 2013, researchers from the University of California at Los Angeles (UCLA) announced they had developed a compact, field-portable device to conduct albumin tests and transmit data via smartphone, thus reducing the need for “frequent office visits by people with diabetes and others with chronic kidney ailments” or the use of “bulky and costly benchtop urine analysers” which limit testing and diagnosis to laboratory settings. The new system projects beams of visible light through two attached fluorescent tubes, one of which contains a control liquid and the other a urine sample mixed with fluorescent dyes. The smartphone camera captures the fluorescent light after it passes through an additional lens.
The device measures albumin concentration to less than 10 micrograms per millilitre, which its inventor states is “more than 3 times lower than the clinically accepted normal range.” It weighs less than 1 kg and is estimated to cost $50 to $100, with tests taking about 5 minutes.
Whipple´s disease, a systemic and ultimately fatal infection with Tropheryma whipplei, is usually easily treated if diagnosed early enough. A novel real-time PCR protocol and fluorescence in situ hybridization provide an improved diagnosis. All results, however, need careful interpretation. We recommend involving specialized centres in the initial diagnosis and patient follow-up.
by Alexandra Wießner, Dr Annette Moter and Dr Judith Kikhney
Whipple’s disease: a fatal infectious disease
Tropheryma whipplei causes a rare, but fatal, bacterial infection: Whipple’s disease. This systemic disease can usually be cured by antibiotic therapy if detected early enough. The key challenge for physicians and microbiologists is to recognize the bacterial origin of various clinical symptoms in time. Diagnosis is still not trivial owing to the rarity of the disease, diverse and unspecific clinical symptoms, the fastidious nature of T. whipplei and the absence of non-invasive serological tests [1]. Improved diagnostic assays for the detection of T. whipplei are very valuable in combination with expertise to interpret the results for fast initiation of treatment.
T. whipplei belongs to the Gram-positive class of Actinobacteria and can be detected intracellularly in vacuoles or as extracellular bacteria in the tissue [2]. It is a slender rod shape, readily visible with Periodic Acid–Schiff (PAS) staining. T. whipplei strains can be cultured in an axenic culture medium supplemented with amino acids, but the slow growth rate means that culture is not an option for routine diagnosis of T. whipplei infection.
Symptoms of Whipple’s disease
In classical Whipple’s disease patients suffer from chronic diarrhoea, weight loss and fever. Molecular methods have detected isolated or systemic T. whipplei infection in almost every organ [joints, central nervous system (CNS), heart valves, skin, eye, lymph node, bone and lung], even in the absence of intestinal involvement. Depending on the location of infection, the symptoms may vary substantially. Often, the diagnosis of Whipple’s disease is delayed as the result of misdiagnosis as sero-negative rheumatoid arthritis, culture-negative endocarditis or neurological disorders. The involvement of the CNS is especially dramatic, as damage caused by the bacteria is often irreversible and antibiotic treatment may no longer be effective enough to cure the infection [3].
Transmission and asymptomatic carriage
To complicate the picture even more T. whipplei has been found in healthy carriers at an estimated prevalence in the population of <1–4% [4, 5]. This means that the detection of T. whipplei in stool or saliva may not be indicative of Whipple’s disease and in this case does not necessarily require antibiotic treatment. A higher prevalence has been found in high risk populations for direct or indirect faecal–oral transmission, such as sewage workers [5], homeless people and family members of Whipple´s disease patients. As T. whipplei is common in the environment, it is assumed that Whipple’s disease patients must have an immunological predisposition for developing a chronic infection instead of being only transiently colonized [6].
The current transmission model assumes that T. whipplei is taken up orally, probably in early childhood, leading to temporary asymptomatic carriage, self-limiting gastroenteritis, fever, or cough [1, 7, 8]. In most cases a protective humoral and cellular immune response prevents T. whipplei infection. However, in predisposed persons T. whipplei may spread systemically over the years resulting in Whipple´s disease.
Diagnosis of Whipple’s disease
Currently, Whipple’s disease is most often detected through PAS staining of biopsies from the lower duodenum or jejunum showing PAS-positive macrophages in the lamina propria. However, PAS staining can give false-positive results because of other infections, for example with nontuberculous mycobacteria, and also false-negative results because of low bacterial load [9]. Therefore, every positive PAS result should be confirmed by an independent method. Here, molecular techniques such as PCR are, so far, irreplaceable for providing a direct, valid species diagnosis. Several in-house PCR protocols are now successfully used to detect T. whipplei DNA [10, 11]. In patients without gastrointestinal manifestation of classical Whipple’s disease, sample specimens from the clinically affected organs, e.g. heart valves, lymph nodes, synovial tissue, cerebrospinal fluid (CSF) or brain biopsies, may be PAS-positive, whereas duodenal biopsies remain negative [1]. PCR was suggested for screening stool and saliva samples as the prevalence and load of T. whipplei is far higher in Whipple´s disease patients than in healthy controls [4]. Here, however, positive PCR results are no proof of infection compared to the direct detection of T. whipplei DNA in affected organs. Analysis of peripheral blood is also possible, but a negative PCR result will not rule out infection [12]. As with all PCR assays, results need to be carefully interpreted as the assay is prone to laboratory contamination (especially nested PCR protocols) or false-positive results because of nonspecific reaction conditions or primer design. Importantly, some positive PCR results in the past have been shown to be due to cross-reactivity, e.g. with Actinomyces odontolyticus [13].
Improved diagnostics of T. whipplei
A break-through for the diagnosis of Whipple’s disease that is specific and less prone to contamination is modern real-time PCR [5, 14]. We evaluated a real-time PCR assay targeting T. whipplei-specific segments within the rpoB gene on test strains and over 1000 clinical specimens in a national reference laboratory [14]. This assay proved to be specific, sensitive and substantially faster than a conventional in-house assay. The protocol includes two specific hybridization probes and, to our knowledge for the first time in T. whipplei diagnostics, a melting curve analysis. Both are crucial for the robustness and reliability of the assay. This applies especially to polymicrobial samples, such as saliva or stool, which contain numerous uncultured bacterial species with unknown DNA sequences. Here, the problem of unexpected probe binding with false-positive results remains and, therefore, PCR results should always be interpreted in the context of clinical and histopathological findings. An initial diagnosis of Whipple´s disease should not rely on only one isolated PCR result, and a confirmatory PCR (using a different target sequence, sequence analysis of ribosomal RNA sequence or genotyping PCR) is mandatory. In inconclusive cases a second PCR with an independent sample specimen is recommended.
Emerging techniques for the detection of T. whipplei
Besides PCR and PAS staining, additional methods such as immunohistochemistry or fluorescence in situ hybridization (FISH) are offered by specialized laboratories. These techniques are, as yet, not part of the routine work-up but provide promising insights. FISH uses fluorescently labelled probes that hybridize specifically with their target sequence in the intact bacterial cells (usually the 16S rRNA). Thus, FISH not only provides direct identification of T. whipplei but also visualizes the pathogen directly in the tissue context. Surprisingly, we found T. whipplei to be by far the most abundant cause of culture-negative endocarditis among the rare pathogens [15]. FISH revealed impressive infected areas in heart valves densely scattered with T. whipplei. In gut biopsies FISH reveals the amount and localization of single microorganisms in the tissue (Fig. 1). As with all microscopic techniques, however, FISH is less sensitive than PCR and will only give information on post-operatively obtained tissue and exclusively on the section investigated. Thus, a low bacterial load in the tissue might be missed. However, FISH is so far the only method bridging the gap between specific molecular biology and histopathology and, thus, might find broader application in the future.
Sampling for T. whipplei
Tissue specimens, such as small bowel biopsies in classical Whipple´s disease or samples of the affected organ in isolated T. whipplei infection, should be examined by PAS staining and PCR (Fig. 2). In the event of positive results, CSF should be tested by PCR to check for CNS involvement. For isolated T. whipplei infections gastrointestinal involvement should be controlled as well. Fluid samples, such as CSF, etc., should be examined by PCR.
For histological examination, PAS staining and FISH the samples should be fixed in 10% formalin and transported at room temperature. For PCR the samples need to be native (no formalin pre-treatment!) and can be transported at room temperature within one day. Samples can be stored for a few days at 4°C and should be kept at –80°C for long-term storage.
Conclusions
The recent development of real-time PCR protocols with hybridization probes for the specific detection of T. whipplei provides accurate and fast results in the challenging clinical situation of Whipple´s disease. However, due to the variety of clinical symptoms, asymptomatic carriage, isolated and systemic infection, as well as false positive and negative results all examinations need careful interpretation in specialized centres. Clinical and histopathological facts always have to be taken into account. Emerging techniques such as FISH might in the future close the gap between molecular biology and histopathology. Together clinical and microbiological expertise are the key to the fast and successful treatment of Whipple´s disease. Similarly, after initial diagnosis and initiation of treatment, it is highly recommended to follow each patient in specialized centres during and after antibiosis to keep relapses at bay.
Acknowledgements
We thank the Robert Koch Institute for its continuous support.
Funding Sources
This study was supported by the Robert Koch Institute (RKI). The epifluorescence microscope was a gift from the Sonnenfeld-Stiftung.
References
1. Moos V, Schneider T. Changing paradigms in Whipple’s disease and infection with Tropheryma whipplei. Eur J Clin Microbiol Infect Dis. 2011; 30: 1151–1158.
2. Raoult D, Birg ML, La Scola B, Fournier PE, Enea M, Lepidi H, et al. Cultivation of the bacillus of Whipple’s disease. N Engl J Med. 2000; 342: 620–625.
3. Lagier JC, Lepidi H, Raoult D, Fenollar F. Systemic Tropheryma whipplei: clinical presentation of 142 patients with infections diagnosed or confirmed in a reference center. Medicine 2010; 89: 337–345.
4. Fenollar F, Laouira S, Lepidi H, Rolain JM, Raoult D. Value of Tropheryma whipplei quantitative polymerase chain reaction assay for the diagnosis of Whipple disease: usefulness of saliva and stool specimens for first-line screening. Clin Infect Dis. 2008; 47: 659–667.
5. Fenollar F, Trani M, Davoust B, Salle B, Birg ML, Rolain JM et al. Prevalence of asymptomatic Tropheryma whipplei carriage among humans and nonhuman primates. J Infect Dis. 2008; 197: 880–887.
6. Martinetti M, Biagi F, Badulli C, Feurle GE, Müller C, Moos V et al. The HLA Alleles DRB1*13 and DQB1*06 Are Associated to Whipple’s Disease. Gastroenterology 2009; 136: 2289–2294.
7. Moos V, Schneider T. The role of T cells in the pathogenesis of classical Whipple’s disease. Expert Rev Anti Infect Ther. 2012; 10: 253–255.
8. Schneider T, Moos V, Loddenkemper C, Marth T, Fenollar F, Raoult D. Whipple’s disease: new aspects of pathogenesis and treatment. Lancet Infect Dis. 2008; 8: 179–190.
9. Müller SA, Vogt P, Altwegg M, Seebach JD. Deadly carousel or difficult interpretation of new diagnostic tools for Whipple’s disease: case report and review of the literature. Infection 2005; 33: 39–42.
10. Hinrikson HP, Dutly F, Nair S, Altwegg M. Detection of three different types of ‘Tropheryma whippelii’ directly from clinical specimens by sequencing, single-strand conformation polymorphism (SSCP) analysis and type-specific PCR of their 16S-23S ribosomal intergenic spacer region. Int J Syst Bacteriol. 1999; 49: 1701–1706.
11. Relman DA, Lepp PW, Sadler KN, Schmidt TM. Phylogenetic relationships among the agent of bacillary angiomatosis, Bartonella bacilliformis, and other alpha-proteobacteria. Mol Microbiol. 1992; 6: 1801–1807.
12. Marth T, Fredericks D, Strober W, Relman DA. Limited role for PCR-based diagnosis of Whipple’s disease from peripheral blood mononuclear cells. Lancet 1996; 348: 66–67.
13. Rolain JM, Fenollar F, Raoult D. False positive PCR detection of Tropheryma whipplei in the saliva of healthy people. BMC Microbiol. 2007; 7: 48.
14. Moter A, Schmiedel D, Petrich A, Wiessner A, Kikhney J, Schneider T et al. Validation of an rpoB gene PCR assay for detection of Tropheryma whipplei: 10 years’ experience in a National Reference Laboratory. J Clin Microbiol. 2013; 51: 3858–3861.
15. Geißdörfer W, Moos V, Moter A, Loddenkemper C, Jansen A, Tandler R et al. High frequency of Tropheryma whipplei in culture-negative endocarditis. J Clin Microbiol. 2012; 50: 216–222.
16. Mallmann C, Siemoneit S, Schmiedel D, Petrich A, Gescher DM, Halle E et al. Fluorescence in situ hybridization to improve the diagnosis of endocarditis: a pilot study. Clin Microbiol Infect. 2010; 16: 767–773.
The authors
Alexandra Wießner1, Annette Moter1* MD, Judith Kikhney1,2 PhD
1 Center for Biofilms and Infection, German Heart Institute Berlin, Berlin, Germany
2 Institut für Mikrobiologie und Hygiene, Charité University medicine Berlin, Berlin, Germany
*Corresponding author
E-mail: moter@dhzb.de
The measurement of 25-hydroxyvitamin D [25(OH)D] levels is affected by assay interference and variability. This article describes the steps that are being taken to improve assay standardization.
by Prof. Etienne Cavalier and Dr Jean-Claude Souberielle
Background
The number of 25-hydroxyvitamin D [25(OH)D] determinations has dramatically increased over the last ten years. This increase can be explained by the growing awareness of the medical community (as well as the general population) of the high prevalence of vitamin D deficiency and its potential impact on numerous diseases beyond bone pathologies, such as cardiovascular diseases, autoimmune diseases, diabetes, malignancies, etc. [1]. This increasing number of requests has led most of the clinical laboratories to move from the DiaSorin radioimmunoassay (RIA), the most widely used method in the late 20th and early 21st centuries, to methods presenting a larger throughput, i.e. automated immunoassays or liquid chromatography tandem mass spectrometry (LC-MS/MS). Also, one has to remember that analytical 25(OH)D determination is far from an easy task – several important problems have to be overcome to correctly assess this parameter.
Pratical difficulties
Among them, the very high lipophilic nature of the molecule and its strong association with vitamin D binding protein (VDBP) and, to a lesser extent, albumin necessitates a thorough separation step and, for the one-phase immunoassays, a good equilibrium between the analyte and the antibodies used in the kits [2]. VDBP can be present at different concentrations according to some physiological or pathological conditions, such as race [3] , pregnancy or chronic kidney disease, which could influence the kinetics of 25(OH)D release [4, 5]. Vitamin D can be found as 25-hydroxyvitamin D2 [25(OH)D2] or 25-hydroxyvitamin D3 [25(OH)D3] and an assay should measure both forms [6]. Other different metabolites of vitamin D, i.e. C3-epimer or 24,25-dihydroxyvitamin D3 can be present in the serum of the patients at different levels, possibly interfering with either immunoassays or LC-MS/MS methods [7]. As with any other immunoassays, vitamin D assays are prone to heterophilic antibody interference, leading to potentially spurious results [8]. Last but not least, standardization of the different assays remains a major problem. Indeed, previously, when the DiaSorin RIA was the only test kit available, all the results obtained could be transposable throughout the world – even though analytical skill and inter-laboratory variation can be a problem with such a manual method – and all the clinicians could compare the results obtained in their patients with the cut-offs obtained in the observational or interventional studies that had used this assay. The increased use of chromatographic methods has shown that the results obtained with the DiaSorin RIA were often lower than those obtained with chromatographic methods. These chromatographic methods were most often ‘home-brewed’ methods and were calibrated against a curve obtained by gravimetric addition of known concentrations of 25(OH)D. Unfortunately, the results generated by these methods presented a high variability, mainly due to extraction and calibration problems, and the results obtained by LC-MS/MS could not be compared from one lab to the other.
Assay standardization
Different standards were then issued by the National Institute of Standards and Technology (NIST 972) and LC-MS/MS methods could thus be calibrated against this material. Unfortunately, due to different matrix problems, it was impossible for immunoassays manufacturers to use these standards to calibrate their assays. They thus chose to indirectly calibrate their tests on serum standards whose concentration had been established with a LC-MS/MS method traceable to the NIST 972 or to remain indirectly calibrated against the DiaSorin RIA. To overcome this problem, a worldwide Vitamin D Standardization Program (VDSP), coordinated by the Centers for Disease Control and Prevention (CDC), the NIST and the University of Ghent, is ongoing: 40 serum samples obtained in apparently healthy single donors and containing 25(OH)D amounts determined with a LC-MS/MS method accepted now as the only reference method for 25(OH)D determination are distributed to laboratories and manufacturers and are expected to allow an unambiguous calibration of the commercially available or the ‘home-brewed’ methods, as well as all the immunoassays [9].
Limitations of assay standardization
This ambitious analytical programme will certainly reduce the variation observed between the methods and the laboratories in healthy individuals. Nevertheless, other problems listed below will remain unsolved:
1. ‘Special’ populations
In patients presenting special patterns of the serum, such as pregnant women or hemodialysed patients, the standardization seems to be less efficient [4, 5].
2. 25(OH)D2 standardization and recovery
As none of the VDSP calibration samples contain significant amount of 25(OH)D2, matrix effects have been described with the NIST samples that have been spiked with 25(OH)D2 and cross-reactivity of the immunoassays with 25(OH)D2 is difficult to establish with precision.
3. C3-epimer
The 3-epi-25-hydroxyvitamin D [3-epi-25(OH)D] presents an identical mass/charge ratio as 25(OH)D. This epimer has an unknown physiological role but may be found in some particular conditions, i.e. in neonates, in patients suffering from liver deficiency or in those presenting high levels of 25(OH)D. Even if not extensively studied, the immunoassays do not seem to cross react with the C3-epimer. However, the separation of 25(OH)D3 and 25(OH)D2 from their epimeric forms 3-epi-25(OH)D with LC-MS/MS methods can only be achieved by derivatization, use of chiral or cyano chromatographic columns and longer runs. Unfortunately, many LC-MS/MS methods (commercial or ‘home-brewed’) have not paid attention to the C3-epimer, resulting in an overestimation of 25(OH)D levels. Thus, even if ‘standardized’, those methods will provide inaccurate results.
4. 24,25-dihydroxyvitamin D
The production of 24,25(OH)2D is linked to the activation of the CYP24A1 in the kidney to inactivate vitamin D. This enzyme is tightly regulated, mainly by PTH and FGF23, showing that this pathway is of physiological importance. 24,25(OH)2D can be present at variable concentrations in the serum, but may be up to 10% of 25(OH)D concentrations. Of note is that 24,25(OH)2D concentrations are proportionally higher in the higher range of 25(OH)D. If there is no interference of 24,25(OH)2D with LC-MS/MS methods, a cross-reactivity, of differing extent is observed with immunoassays, particularly at higher concentrations of 25(OH)D. This can thus result in an overestimation of the ‘true’ 25(OH)D value being observed with immunoassays.
5. Impact of re-standardization on clinical cut-offs
Traditionally, values of 20 or 30 ng/mL are used as clinical cut-offs to define vitamin D sufficiency. These cut-offs derive from studies that generally used the DiaSorin RIA for 25(OH)D measurements. Using these cut-offs with immunoassays or LC-MS/MS methods that are differently calibrated is thus hazardous. Re-standardization will improve the method-to-method variability, but will also consequently impact the value of the cut-offs, that will also need to be updated according to the new standardization.
Conclusion
In conclusion, vitamin D assays need to be standardized, and also improved in terms of accuracy. The VDSP is an ongoing programme that will improve the lab-to-lab consistency, even if different problems will, however, persist.
References
1. Holick MF. Vitamin D deficiency. N Engl J Med. 2007; 357:.266–281.
2. Wallace AM, et al. Measurement of 25-hydroxyvitamin D in the clinical laboratory: current procedures, performance characteristics and limitations. Steroids 2010; 75: 477–488.
3. Powe CE, et al. Vitamin D-binding protein and vitamin D status of black Americans and white Americans. N Engl J Med. 2013; 369: 1991–2000.
4. Heijboer AC, et al. Accuracy of 6 routine 25-hydroxyvitamin D assays: influence of vitamin D binding protein concentration. Clin Chem. 2012; 58: 543–548.
5. Depreter B, et al. Accuracy of three automated 25-hydroxyvitamin D assays in hemodialysis patients. Clin Chim Acta 2013; 415: 255–260.
6. Cavalier E, et al. Cross-reactivity of 25-hydroxy vitamin D2 from different commercial immunoassays for 25-hydroxy vitamin D: an evaluation without spiked samples. Clin Chem Lab Med. 2011; 49: 555–558.
7. Kobold U. Approaches to measurement of vitamin D concentrations – mass spectrometry. Scand J Clin Lab Invest Suppl. 2012; 243: 54–59.
8. Cavalier E, et al. Human anti-animal interference in DiaSorin Liaison total 25(OH)-vitamin D assay: towards the end of a strange story? Clin Chim Acta 2012; 413: 527–528.
9. Sempos CT, et al. Vitamin D status as an international issue: national surveys and the problem of standardization. Scand J Clin Lab Invest Suppl. 2012; 243: 32–40.
The authors
Etienne Cavalier*1 PhD, PharmD, European Specialist in Laboratory Medicine (EuSpLM) and Dr Jean-Claude Souberielle2 PhD, EuSpLM
1 Department of Clinical Chemistry, University of Liège, Liège, Belgium
2 Hôpital Necker-Enfants malades, Paris, France
*Corresponding author
E-mail: etienne.cavalier@chu.ulg.ac.be
March 2026
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