July 2018ISSN 1650-3414

Communications and Publications Division (CPD) of the IFCC
Editor-in-chief: Prof. János Kappelmayer, MD, PhD
Faculty of Medicine, University of Debrecen, Hungary
e-mail: ejifcc@ifcc.org

The
Journal of theInternational
Federation ofClinicalChemistry andLaboratory
Medicine

Volume 29 Number 2

In this issue

Laboratory investigation of vitamin D metabolites and bone metabolism markers
Harjit Pal Bhattoa

103

Vitamin D and its metabolites: from now and beyond
Etienne Cavalier, Jean-Claude Souberbielle

105

Vitamin D and endocrine disorders: routine laboratory diagnostic implications
Giovanna Muscogiuri, Harjit Pal Bhattoa

111

Laboratory aspects and clinical utility of bone turnover markers
Harjit Pal Bhattoa

117

Practical guide for identifying unmet clinical needs for biomarkers
Phillip J. Monaghan, Sarah Robinson, Daniel Rajdl, Patrick M.M. Bossuyt,
Sverre Sandberg, Andrew St John, Maurice O’Kane, Lieselotte Lennartz,
Ralf Röddiger, Sarah J. Lord, Christa M. Cobbaert, Andrea R. Horvath

129

Prevalence of anemia and its associated factors among children in Ethiopia:
a protocol for systematic review and meta-analysis
Zegeye Abebe, Wubet Worku Takele, Degefaye Zelalem Anlay,
Daniel Tekelia Ekubagewargies, Zegeye Getaneh, Molla Abebe, Mulugeta Melku

138

Acute myeloid leukemia with severe coagulation disorder and concomitantcentral nervous system bleeding – a clinical diagnostic case report
S. Hatzl, B. Uhl, M. Hinterramskogler, S. Leber, F. Eisner, M. Haring, P. Jud146

Vitamin B12 immunoassay interference in a patient with multiple myeloma –troubleshooting in a two step reagent kit based on enhanced chemiluminescence testing
V. Pant, A. Tumbapo, B. Kumar Yadav
152

In this issue: Laboratory investigation of vitamin D and bone metabolism markers

Laboratory investigation of vitamin D metabolites
and bone metabolism markers
Guest Editor: Harjit Pal Bhattoa

Department of Laboratory Medicine, Faculty of Medicine, University of Debrecen, Hungary

ARTICLE INFOEDITORIAL

Corresponding author:
Harjit Pal Bhattoa
Department of Laboratory Medicine
Faculty of Medicine
University of Debrecen
Hungary
E-mail: harjit@med.unideb.hu

It may well be stated that routine laboratories havebeen overwhelmed with requests for determinationof 25-hydroxyvitamin D, the analyte reflecting vitamin D status. The number of test requests is increasing pretty much from year to year in essentially all
known laboratory settings.
Given the pandemic of vitamin D insufficiency experienced on a global scale it is important to understandthe laboratory implications pertaining to the determination of this not so easy analyte.
Similarly to quite a few other hormone determinations, harmonization and standardization is a valid issue and professional bodies have set up task forces toresolve the discrepancies experienced between the
different currently available methodologies.
In the first article of three from this thematic issue,
Cavalier et al. have comprehensively reviewed alldata implicated in vitamin D testing that is relevant to
the practicing laboratory professional.
Professor Etienne Cavalier is Head of the Departmentof Clinical Chemistry, University of Liege, Belgium. He
is currently the president of the Belgian Royal Societyof Laboratory Medicine. He is member of the Editorial
Board of leading Journals in the field of Laboratory
Medicine and has over 230 publications with an hindex of 28. He is member of numerous National andInternational Working Groups. Being a professor ofPage 103eJIFCC2018Vol29No2pp103-104

Harjit Pal Bhattoa
Laboratory investigation of vitamin D metabolites and bone metabolism markers

Clinical Chemistry, he has published extensively
on ground breaking research in routine laboratory practice and has dealt not only with researchissues but also the practical implication of novel
biomarkers particularly in the field of musculoskeletal endocrinology and metabolism. His current research interests include Bones markers,
vitamin D, PTH, vascular calcification markers,
markers of acute kidney diseases, glomerular filtration rate (estimation, biomarkers), markers offrailty and sarcopenia, LC-MS/MS methods forsteroids and peptides quantification. Given theirprofessional achievements, Professor Cavalierand colleague are one of the most authentic toaddress this very important and relevant topic
of vitamin D determinations.
Vitamin D is known to have various non-skeletal effects including its role in endocrinologicalconditions. The second article summarizes theimportance of vitamin D in common endocrinological diseases penned by Muscogiuri et al.
Dr. Giovanna Muscogiuri graduated in Medicine
from the Catholic University, Rome in 2006 andthen carried out postgraduate residency inEndocrinology and Metabolic Disease at thesame university, obtaining her degree of specialization in 2012. She attended the post-doctoral
fellowship at University of Texas Health science
Center at San Antonio, TX – USA. She is currently
focused on the study of vitamin D and relateddiseases. She has received several awards forher scientific achievements in the metabolic
research field. She has written or co-authoredover 100 papers in peer-reviewed scientific journals. Muscogiuri et al place in perspective what
our correct knowledge on vitamin D is with relevance to endocrinology. Furthermore, given theargument supporting achievement of optimal
vitamin D status in the common endocrinological diseases dedicatedly discussed, the laboratory issues that may arise are briefly enumerated.
In the third article, Bhattoa summarizes the importance of determination of biochemical markers of bone turnover in metabolic bone diseases.
Given the sensitive nature of their determination, pre-analytical, analytical and post-analyticalissues are extensively discussed and their clinicalutility is placed in perspective.

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In this issue: Laboratory investigation of vitamin D and bone metabolism markers

Vitamin D and its metabolites:from now and beyond
Etienne Cavalier1, Jean-Claude Souberbielle2
12

Department of Clinical Chemistry, University of Liege, CHU de Liège, Belgium
Laboratoire d’Explorations fonctionnelles, Hôpital Necker-Enfants malades, Paris, France

ARTICLE INFOABSTRACT

Corresponding author:
Etienne CavalierDepartment of Clinical Chemistry
University of Liège
4000 Liège
Belgium
E-mail: Etienne.cavalier@chuliege.be

Total 25-hydroxyvitamin D is currently considered as
the most representative metabolite of vitamin D status. There are a multitude of challenges that still deserve to be addressed and despite recent technological advances its determination remains complicated.
This current review gives an abbreviated overview ofthe phases of development that vitamin D metabolite
determination has gone through and discusses thedifficulties that still require resolving. Furthermore,
given the different platforms and methodologies
available, the critical issue of standardization and allefforts made as far towards its realization have been
discussed. And last but not least, the concepts of ‘free’and ‘bioavailable’ vitamin D along with the ‘Vitamin D
Metabolism Ratio’ have been discussed.

Key words:
25 hydroxyvitamin D,
1,25 dihydroxyvitamin D,
standardization,
Vitamin D Metabolism Ratio (VMR)

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Etienne Cavalier, Jean-Claude Souberbielle
Vitamin D and its metabolites: from now and beyond

INTRODUCTION
Until recently, 25(OH)-vitamin D (25-OHD) was
merely the only vitamin D (VTD) metabolite ofinterest to explore vitamin D status and metabolism. Unfortunately, the determination of thisVTD metabolite, as well as the levels that need
to be achieved in healthy or diseased individuals are quite problematic and remain an important matter of debate [1.2].
Recently, other VTD metabolites, like 24,25OH2D, “bioavailable” or “free” vitamin D, cholecalciferol itself and 1,25-OH2D, have emerged
as potential new players to better understandthe important vitamin D pathway. In this paper,
we provide a brief overview on the issues regarding 25-OHD assays and standardization andwe will evoke 24,25(OH)2D and Vitamin D ratio(VMR) as potential metabolites of choice to explore vitamin D deficiency.
25-HYDROXY VITAMIN D DETERMINATION
25-OHD is still currently considered as the mostrepresentative metabolite of vitamin D status.
Unfortunately, its determination remains complicated despite recent technological advances[3]. The reasons why this metabolite is so complicated to be correctly assessed are multiple.
First, 25-OHD assays need to recognize 25-OHD2
and 25-OHD3. Second, 25-OHD is a very hydrophobic molecule that circulates bound to vitamin D binding protein (DBP), albumin (ALB) andlipoproteins and a thorough dissociation of theanalyte from its ligands is mandatory prior tomeasurement. This step is particularly complicated for automated immunoassays where, incontrast to radio-immunoassays, binding-protein or chromatographic assays, organic solvents
cannot be used for extraction. Hence, automated immunoassays need alternative releasing
agents, which do not always achieve total dissociation of 25-OHD. In particular physiologicalor pathological conditions such as pregnancy,

estrogen therapy or renal failure, automated immunoassays often fail to correctly quantify 25OHD [4-7]. Third, 25-OHD2 and 25-OHD3 havedifferent affinity constants for the carriers andthe dissociation step must be highly efficient toobtain an accurate quantification of both forms.
Forth, in-vitro recovery experiments with themolecule give spurious results with immunoassays since it is not clear whether exogenous metabolites bind to all the different carriers in thesame proportions as endogenous metabolites.
Under-recovery of exogenous 25-OHD has beenreported in automated immunoassays [8-9] andeven liquid chromatography tandem mass spectrometry (LC-MS/MS) methods [10].
The different methods available for the quantification of 25-OHD use chromatographic separation (HPLC with UV or LC-MS/MS detectors),
antibodies or binding-proteins. Binding protein
assays have been used in the early eighties andpresented clinically acceptable analytical sensitivity and imprecision. They were based on thedisplacement of H3-labelled 25-OHD and necessitated a chromatographic purification after organic extraction. These home-made methods
were very time-consuming and performed insome reference laboratories only. Hence, they
have been superseded by radio-immunoassays(RIA) methods. The first commercially available
RIA was based on a method described by Hollis
et al. in 1993 [11] and the DiaSorin RIA method
has been the most widely used method for bothroutine diagnostic testing as well as for clinicalstudies until recently. Traditional 25-OHD cutoffs in use today for vitamin D deficiency (either
20 or 30 ng/ml) have been defined on the basisof studies (and meta-analyses of studies) that
predominantly used this assay. However, due
to the logarithmic increase in 25-OHD requests
observed during the last decade, laboratories
have opted for more automated immunoassays
and less than 1% of laboratories participating in
the DEQAS still use this RIA assays nowadays.

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Etienne Cavalier, Jean-Claude Souberbielle
Vitamin D and its metabolites: from now and beyond

The first automated immunoassay for 25-OHD
determination was launched in 2001 by Nichols
Diagnostics on the Advantage platform. This
assay used a competitive ligand binding technique with acridinium-ester labelled anti-DBP.
Nowadays, most of the major in-vitro diagnostic companies have launched their own methods for 25-OHD determination. These methods use a competition design, except the one
from Fujirebio on the Lumipulse, which is a
non-competitive (sandwich) method based on
antimetatype monoclonal antibodies against
a hapten–antibody immunocomplex using an
ex vivo antibody development system, namely
the Autonomously Diversifying Library system,a process which has recently been validated[12]. A large number of studies have evaluatedthe different automated assays by comparison
with RIA, HPLC or, more recently, with LC-MS/
MS methods. Conclusions regarding the accuracy of the assays have also been based on the
results of large external proficiency testing programs, such as DEQAS or CAP which now use areference method to measure the samples sent
to the participants, allowing a true calculationof the bias. In a recent study coordinated by the
Vitamin D Standardization Program (VDSP) group[13], a set of 50 healthy individuals donor samples were analyzed by 15 different laboratories
to provide results for total 25-OHD using bothimmunoassays and LC-MS/MS methods. The results were compared with those obtained by two
reference methods, namely the Ghent University
and the U.S. National Institute of Standards andTechnology (NIST) methods. Results showed thatall but 2 LC-MS/MS achieved VDSP criteria ofperformance (namely CV ≤ 10% and mean bias ≤5%), whereas only 50% of immunoassays met thecriteria. These results can be regarded as optimistic for immunoassays. First, it is obvious fromthese results that standard deviation around thebias is much more important for immunoassays
than LC-MS/MS. As an example laboratory 2athat used an immunoassay and laboratory 10
used a LC-MS/MS method which both presented
an excellent mean bias of -1%. But the standard
deviation around this bias was 14% for the immunoassay vs. 5% for the LC-MS/MS method. As
a consequence, the LC-MS/MS will have 75% of
its value within the 5% boundaries whereas theimmunoassay will only have 29%. Second, thisstudy has been performed on serum obtainedin healthy donors and not in patients. Indeed,patients with chronic kidney disease, dialysis patients, pregnant women, different ethnic groups,
patients in intensive care with fluid shifts present
differences in their serum matrix compared tohealthy individuals and this can impact the performance of automated 25-OHD immunoassays.
Recently, we have shown good clinical concordance between 4 different immunoassays anda VDSP-traceable LC-MS/MS method in healthy
subjects. However, significantly poorer agreement with the same LC-MS/MS method has
been found in other clinical populations [4, 14].
In the past years, the IFCC has made great efforts to promote standardization of laboratory
assays. Indeed, standardization is importantto achieve comparable results across different
methods and manufacturers. For 25-OHD assays, clinical cut-offs are generally used as target
values, and applying common cut-offs on results
generated with poorly standardized assays will
inevitably lead to inconsistent patient classification and inappropriate therapeutic decisions.
Hence, in 2010, the Vitamin D Standardization
Program (VDSP) was established to improve thestandardisation of 25-OHD assays. The aim of
VDSP is that 25-OHD measurements are accurate and comparable over time, location, andlaboratory procedure to the values obtained using reference measurement procedures (RMPs)
developed at the NIST [15] and Ghent University[16]. As mentioned earlier, a method is considered as standardized if the CV is <10% and thebias <5% [17]. Each candidate receives a set ofPage 107eJIFCC2018Vol29No2pp105-110

Etienne Cavalier, Jean-Claude Souberbielle
Vitamin D and its metabolites: from now and beyond

10 samples 4 times a year and has to run thesesamples in duplicate on 2 consecutive days. In
January 2018, 27 methods, coming either from
IVD companies or clinical laboratories wereconsidered as standardized against the RMPs.
However, the proportion of the 40 samples that
met the bias criterion (<5%) in 2017 was quite
different from one method to the other andranged from 23 to 85%, with LC-MS/MS methods presenting better results than immunoassays. The list of these standardized methods
can be found on the CDC website (http://www.
cdc.gov/labstandards/pdf/hs/CDC_Certified_
Vitamin_D_Procedures.pdf).
Although substantial progress has been made, arange of important issues like standardization of
25-OHD2 and 24,25-(OH)2D as well as improvement of (immuno)assays performance on samples from diseased patients or subjects from different ethnic groups still needs to be achieved.
It may thus be tempting to think that immunoassays are outdated and that LC-MS/MS should
replace these methods. There are clear limitations to this simplistic view. Indeed, performinga LC-MS/MS is complex and needs experienced
and very well trained people. Notably, extensive
validation of the LC-MS/MS and sample preparation are of extreme importance. To run a LCMS/MS is much more complicated than “crash
the proteins, inject and obtain the results in 2
minutes”. A detailed review on how complex
running a LC-MS is out of the scope of this present paper, but can be found in a previous report [18]. Finally, laboratories that run LC-MS/
MS do not run “the” reference method, even
if their method is certified by the VDSP. As an
illustration of this assertion, one can see thatsome VDSP-certified LC-MS/MS methods present a percentage of samples out of the bias
criterion that is lower than immunoassays andmuch lower than other LC-MS/MS. Also DEQAS
results show that LC-MS/MS methods present
CVs that are as high as immunoassays.

24,25-(OH)2D DETERMINATION
AND THE VITAMIN D METABOLITE RATIO
One advantage of LC-MS/MS methods over immunoassays is the possibility to simultaneously
quantify 25-OHD and 24,25-(OH)2D allowing
to calculate the 25-OHD/24,25-(OH)2D ratio,
also known as the Vitamin D Metabolism Ratio(VMR). Indeed, some light has recently been
shed on the potential interest of this vitamin D
metabolite to better reflect vitamin D deficiency[19]. In summary, CYP24A1, the enzyme allowing the degradation of 25-OHD and 1,25-(OH)2D
into 24,25-(OH)2D and 1,24,25-(OH)3D sees its
expression increased when there is an increased
binding and activation of the VDR in response to
1,25-(OH)2D [20]. Hence 24,25-(OH)2D concentration may thus reflect VDR activity which is notreally the case with 25-OHD. It has recently been
demonstrated that lower 24,25-(OH)2D concentration and lower VMR were associated withincreased hip fracture risk in community-living
older men and women, whereas 25-OHD was
not associated with hip fracture risk. Anotherpoint of interest with 24,25-(OH)2D and VMR
is that, although concentrations of 25-OHD and24,25-(OH)2D strongly correlate with each otherand are both lower in black Americans than inwhites, blacks and whites have equivalent median VMR values [21]. In CKD patients, Bosworth etal have shown that 24,25-(OH)2D was better associated with PTH than 25-OHD or 1,25-(OH)2D[22]. These findings are of great interest but
still need to be confirmed by other studies.
On the other hand, it is clearly demonstrated
that biallelic mutations in CYP24A1 led to idiopathic infantile hypercalcemia [23], a phenotype
characterized by profound hypercalcemia, suppressed intact parathyroid hormone, hypercalciuria and nephrocalcinosis. Many heterozygous
mutations of CYPA24A1 have recently been described [24]. If they are associated with a lessdramatic phenotype than homozygous mutations, patients suffering from these mutations

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Etienne Cavalier, Jean-Claude Souberbielle
Vitamin D and its metabolites: from now and beyond

often present with hypercalcemia, suppressed
PTH and renal stones [25]. Hence, in patients
presenting with a non-parathyroid hypercalcemia (without evident clinical cause), CYP24A1
mutations should be investigated and simultaneous 24,25-(OH)2D and VMR ratio should bemeasured. A ratio higher than 50, or even 80
should lead to a genetic research of a CYP24A1
mutation. Again, this measurement should bestandardized. Fortunately, one candidate reference measurement procedure (RMP) has been
published [26] and NIST standard reference material (SRM) 2972a includes 4 standards withcertified values (unfortunately, these 4 values are very close to each other) [27]. DEQAS
data report that about 10 laboratories provide
24,25-(OH)2D results. These data show quite alarge variability, which can partially be attributed to the low concentration of the analyte, butalso to the lack of ongoing standardization program. This latter will be (probably) even more
important than the 25-OHD itself since small
variations in 24,25-(OH)2D have a dramatic impact on the VMR.
CONCLUSION
The assessment of vitamin D status is a changing landscape [19]. Although 25-OHD is still recommended as the marker of choice by virtually
all scientific bodies growing evidence indicatessignificant limitations that hamper the utility ofthis analyte in clinical practice. Issues related
to the use of 25-OHD include analytical aspects
and the interpretation of results. While in normal individuals the agreement of results generated with automated assays is improving, comparability of results in distinct populations, suchas children, pregnant women, hemodialysis patients or intensive care patients, remains problematic. The relationships between 25-OHD
and various clinical indices are also rather weakand not consistent across races. Recent studies
have provided new insights in physiological andanalytical aspects of vitamin D that may change
the way how we will assess vitamin D status in
the future. The VMR (25-OHD/24,25-OH2D ratio), but also ‘free’ and ‘bioavailable’ vitamin D
are all interesting markers that have expanded
our knowledge about vitamin D metabolism andsome of these analytes may now be considered
for routine use (at least in specialized centers).
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1. Ross AC, Manson JE, Abrams SA, et al. (2011) The 2011
report on dietary reference intakes for calcium and vitamin D from the Institute of Medicine: what cliniciansneed to know. J Clin Endocrinol Metab 96:53–58.
2. Holick MF, Binkley NC, Bischoff-Ferrari HA, et al. (2012)
Guidelines for preventing and treating vitamin D deficiency and insufficiency revisited. J Clin Endocrinol Metab
97:1153–1158.
3. Hollis BW (2004) The determination of circulating
25-hydroxyvitamin D: No easy task. J Clin Endocrinol
Metab 89:3149–3151.
4. Moreau E, Bächer S, Mery S, et al. (2015) Performance
characteristics of the VIDAS® 25-OH Vitamin D Total assay – comparison with four immunoassays and two liquid
chromatography-tandem mass spectrometry methods ina multicentric study. Clin Chem Lab Med.
5. Depreter B, Heijboer AC, Langlois MR (2013) Accuracyof three automated 25-hydroxyvitamin D assays in hemodialysis patients. Clin Chim Acta 415:255–260.
6. Heijboer AC, Blankenstein M a., Kema IP, Buijs MM(2012) Accuracy of 6 routine 25-hydroxyvitamin D assays:
Influence of vitamin D binding protein concentration. Clin
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7. Cavalier E, Lukas P, Crine Y, et al. (2014) Evaluation of
automated immunoassays for 25(OH)-vitamin D determination in different critical populations before and after
standardization of the assays. Clin Chim Acta 431:60–65.
8. Carter GD, Jones JC, Berry JL (2007) The anomalous behaviour of exogenous 25-hydroxyvitamin D in competitive
binding assays. J Steroid Biochem Mol Biol 103:480–482.
9. Horst RL (2010) Exogenous versus endogenous recovery of 25-hydroxyvitamins D2 and D3 in human samples
using high-performance liquid chromatography and the
DiaSorin LIAISON Total-D Assay. J Steroid Biochem Mol
Biol 121:180–182.
10. Lankes U, Elder P a., Lewis JG, George P (2015) Differential extraction of endogenous and exogenous 25-OHvitamin D from serum makes the accurate quantification

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Med 52:151–160.
11. Hollis BW, Kamerud JQ, Selvaag SR, et al. (1993) Determination of vitamin D status by radioimmunoassay
with an 125I-labeled tracer. Clin Chem 39:529–533.
12. Omi K, Ando T, Sakyu T, et al. (2015) Noncompetitive
Immunoassay Detection System for Haptens on the Basisof Antimetatype Antibodies. Clin Chem 61:627–635.
13. Wise SA, Phinney KW, Tai SSC, et al. (2017) Baseline
Assessment of 25-Hydroxyvitamin D Assay Performance:
A Vitamin D Standardization Program (VDSP) Interlaboratory Comparison Study. J AOAC Int 100:1244–1252.
14. Cavalier E, Lukas P, Bekaert A-C, et al. (2016) Analyticaland clinical evaluation of the new Fujirebio Lumipulse®G
non-competitive assay for 25(OH)-vitamin D and threeimmunoassays for 25(OH)D in healthy subjects, osteoporotic patients, third trimester pregnant women, healthy
African subjects, hemodiadialyzed and intensive care patients. Clin Chem Lab Med 54(8):1347-55.
15. Tai SS-C, Bedner M, Phinney KW (2010) Developmentof a candidate reference measurement procedure forthe determination of 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 in human serum using isotope-dilution
liquid chromatography-tandem mass spectrometry. Anal
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16. Stepman HCM, Vanderroost A, Van Uytfanghe K,
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Chromatography-Tandem Mass Spectrometry. Clin Chem
57:441–448.
17. Stöckl D, Sluss PM, Thienpont LM (2009) Specifications for trueness and precision of a reference measurement system for serum/plasma 25-hydroxyvitamin D
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18. Herrmann M, Farrell C-JL, Pusceddu I, et al. (2017) Assessment of vitamin D status – a changing landscape. Clin
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19. Ginsberg C, Katz R, de Boer IH, et al. (2018) The
24,25 to 25-hydroxyvitamin D ratio and fracture riskin older adults: The cardiovascular health study. Bone
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20. Tryfonidou M a, Oosterlaken-Dijksterhuis M a, Mol J
a, et al. (2003) 24-Hydroxylase: potential key regulator in
hypervitaminosis D3 in growing dogs. Am J Physiol Endocrinol Metab 284:E505-13.
21. Berg AH, Powe CE, Evans MK, et al. (2015) 24,25-Dihydroxyvitamin D3 and Vitamin D Status of Community-Dwelling Black and White Americans. Clin Chem
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22. Bosworth CR, Levin G, Robinson-cohen C, et al. (2012)
The serum 24,25-dihydroxyvitamin D concentration, amarker of vitamin D catabolism, is reduced in chronic kidney disease. Kidney Int 82:693–700.
23. Schlingmann KP, Kaufmann M, Weber S, et al. (2011)
Mutations in CYP24A1 and Idiopathic Infantile Hypercalcemia. N Engl J Med 365:410–421.
24. Molin A, Baudoin R, Kaufmann M, et al. (2015) CYP24A1 mutations in a cohort of hypercalcemic patients:
Evidence for a recessive trait. J Clin Endocrinol Metab
100:E1343–E1352.
25. Ferraro PM, Minucci A, Primiano A, et al. (2016) A
novel CYP24A1 genotype associated to a clinical pictureof hypercalcemia, nephrolithiasis and low bone mass.
Urolithiasis 25:1–4.
26. Tai SS-C, Nelson M a. (2015) Candidate Reference
Measurement Procedure for the Determination of (24R ),25-Dihydroxyvitamin D3 in Human Serum Using Isotope-Dilution Liquid Chromatography–Tandem Mass
Spectrometry. Anal Chem 87:7964–7970.
27. Wise SA, Tai SSC, Burdette CQ, et al. (2017) Role ofthe national institute of standards and technology (NIST)in support of the Vitamin D initiative of the national institutes of health, office of dietary supplements. J AOAC Int
100:1260–1276.

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In this issue: Laboratory investigation of vitamin D and bone metabolism markers

Vitamin D and endocrine disorders:routine laboratory diagnostic implications
Giovanna Muscogiuri1, Harjit Pal Bhattoa2

Department of Clinical Medicine and Surgery, Endocrinology Unit, Federico II University,
Medical School of Naples, Italy2
Department of Laboratory Medicine, Faculty of Medicine, University of Debrecen, Hungary
1

ARTICLE INFOABSTRACT

Corresponding author:
Giovanna Muscogiuri
Department of Clinical Medicine and Surgery
Endocrinology Unit
Federico II University
Medical School of Naples
Italy
E-mail: Giovanna.muscogiuri@gmail.com

The role of vitamin D in calcium-phosphorus metabolism regulation is the most highlighted, nonethelessthere is enormous literature on the extra-skeletal effects of vitamin D, and lately new insight into the roleof vitamin D in endocrine disease mechanisms has
seen light of day. The present narrative review gives
an overview of the proposed roles of vitamin D in the
etiology of Hashimoto’s thyroiditis, Grave’s disease,
Addison’s disease and primary hyperthyroidism. The
implications as pertaining to the routine laboratory
practice are readily applicable to this patient groupas well, and do not pose any additional challenge.

Key words:
Vitamin D, Hashimoto’s thyroiditis,
Grave’s disease, Addison’s disease,primary hyperthyroidism

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Giovanna Muscogiuri, Harjit Pal Bhattoa
Vitamin D and endocrine disorders: routine laboratory diagnostic implications

INTRODUCTION
The role of vitamin D in calcium-phosphorus metabolism regulation is the most highlighted, nonetheless there is enormous literature on the extraskeletal effects of vitamin D [1]. It is known that
non-skeletal cells, including those of the endocrine system, too express vitamin D receptor andas such interact with the active form of vitamin D,
i.e., 1,25-dihydroxyvitamin D, furthermore, mostof these cells express extra-renal forms of 25-hydroxyvitamin D-1α-hydroxylase enzyme and,eventually the active form of vitamin D is known
to regulate quite a number of genes, including
those implicated in proliferation, differentiation
and apoptosis [2-5]. Recently, it has been described that the pathophysiology of autoimmune
thyroid disease, adrenal disease and hyperparathyroidism has a Vitamin D component [6-12].
The present review discusses the association between vitamin D status and the aforementioned
endocrine conditions and delineates the routine
laboratory assessment of vitamin D metabolites.
VITAMIN D AND HASHIMOTO’STHYROIDITIS
Hashimoto’s thyroiditis is an autoimmune disease with defective suppressor T-cell function.
Thyrocytes in this disease condition express major histocompatibility complex class II (MHC II)
surface HLA-DR antigens, as a result of cytokines
produced by T helper 1 (Th1) cells, making themprone to immunologic attack as such triggering
the autoimmune process. Subsequently the Tcell activated B lymphocytes produce autoantibodies reacting with thyroid antigens [9,13,14].
The active form of vitamin D suppresses the
autoimmunity in Hashimoto’s thyroiditis at anumber of stages. Initially it may suppress dendrocyte mediated T lymphocyte activation, maycause blunting of Th1 cell proliferation and its interferone gamma secretion. By the inhibition ofcytokine production, it may block MHCII surface

HLA-DR expression on thyrocytes. Additionally,
B cell proliferation, as a result of activation by T
cells, may be seduced and B cell apoptosis may
be accelerated by 1,25-hydroxyvitamin D. Hence,
active vitamin D may decrease the autoantibody
load [8,13,14].
Lately, studies have reported increased risk ofHashimoto’s thyroiditis where vitamin D levels
may be low, and in conditions where vitamin D
functionality may be compromised, as in certain
gene polymorphisms of the vitamin D receptor(VDR) and abnormality in vitamin D-binding
protein [15,16]. Nonetheless, solid data arenecessary to clarify the link between vitamin D
status and Hashimoto’s thyroiditis.
VITAMIN D AND GRAVE’S DISEASEIn Grave’s disease TSH receptor autoantibodies
cause hyperthyroidism. Reflected in the increasing interest in the role of vitamin D in susceptibility to autoimmune conditions, potentiallythis autoimmune disease may also be linked tovitamin D. This may be candidly assumed since
it is known that vitamin D suppresses activatedT cell proliferation and encourages macrophage
phagocytic ability [17,18].
Grave’s diseases’ etiology has been reported
to be associated with VDR and vitamin D binding protein gene polymorphism, presumably byvitamin D function reduction, that may consequently have inhibitory effects on immune regulation [19,20].
These effects appear to be ethnicity specific,
Asians with ApaI, BsmI and FokI polymorphisms
seem to have a higher susceptibility as comparedto their Caucasian counterparts [21]. Additionally,it has also been reported that autoimmune thyroiditis is associated with BsmI and TaqI polymorphism, whereas ApaI and FokI are not [22].
Those with increased ultrasonography measured thyroid volume and early-onset disease

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are reported to have low vitamin D levels [23].
Additionally, those in remission were reported
to have higher vitamin D levels as comparedto their non-remittent counterparts [24].
Although data at present is meager, further
studies on the role of vitamin in Grave’s disease
are worthwhile.
VITAMIN D AND ADDISON’S DISEASE
Addison’s disease results from destruction of theadrenal cortex due to an autoimmune process,and may present solitarily or as a polyendocrine
syndrome. Although elusive, the etiology of thedisease currently suggests that environmental
factors along with a genetic component may be
responsible for the destruction of the adrenal
cortex mediated by CD8 lymphocytic infiltration
and autoantibodies against the 21 hydroxylase
enzyme [25]. Genetic susceptibility is identified
primarily at the HLA locus, but VDR and CYP27B1
genes have also been implicated [26,27].
Given the genetic background it may be assumed that vitamin D is involved in major pathophysiological pathways, as active vitamin D may
downregulate CYP21A2 and upregulate CYP17A1
and CYP11A1, as such disrupt steroidogenesis.
Additionally, vitamin D also acts on the adrenal
tissue along with the immune system in adrenal
cell models [28].
Although vitamin D levels comparing controls andAddison’s disease patients are not available, theassociation between vitamin D status and susceptible gene loci may allow one to presume thevitamin’s disease modifying role. Nonetheless,there is a need for studies investigating the function and relevance of vitamin D.
VITAMIN D AND PRIMARYHYPERPARATHYROIDISM
The tight regulation of serum ionized calcium
level is a result of orchestrated regulation by theparathyroid hormone (PTH), calcitonin and active

vitamin D. The biosynthesis of active vitamin D is
primarily achieved by the renal CYP27B1, whichis stimulated by PTH. Conversely, active vitamin
D and ionized calcium downregulate PTH.
The inactive, 25-hydroxyvitamin D is a potent regulator of parathyroid tissue. In contrast to othertissue, parathyroid cells readily take up vitamin D
binding protein along with its 25-hydroxyvitaminD, as such guaranteeing better hold of the circulating 25-hydroxyvitamin D.
Additionally, the gland possesses CYP27B1 ensuring 1,25 dihydroxyvitamin D production forparacrine action. This proficient access to both
circulating 25-hydroxyvitamin D and 1,25 dihydroxyvitamin D, in addition to its inherent production of 1,25 hydroxyvitamin D induces suppression of PTH secretion and proliferation ofparathyroid cells [29].
It is known that sizable parathyroid adenomas
have poor feedback by calcium and active vitamin D, as a result in primary hyperthyroidism,
1,25 dihydroxyvitamin D correlate positively
with 25 hydroxyvitamin D [30]. Primary hyperthyroidism remains latent, i.e., normocalcemic
primary hyperparathyroidism, when circulating
vitamin levels are low [31]. Increased 25 hydroxyvitamin D levels and elevated PTH activate
renal CYP27B1 accelerating 1,25 dihydroxyvitamin D production in approximation to the
25 hydroxyvitamin D and consequently causing hypercalcemia. This relationship highlights
a fundamental aspect of the vitamin D system:
its operation under first-order reaction kinetics, namely, the yield of the product (1,25 dihydroxyvitamin D) is proportional to the supply of
the substrate (25 hydroxyvitamin D).
As such, the yield of 1,25 dihydroxyvitamin D
is proportional to the supply of 25 hydroxyvitamin D and hence qualifying the kinetics ofthe vitamin D system as a first-order reaction.
Consequently, the vitamin D system enzymes
modify their function depending on the supply

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Giovanna Muscogiuri, Harjit Pal Bhattoa
Vitamin D and endocrine disorders: routine laboratory diagnostic implicationsof 25 hydroxyvitamin D. Depending on severity,
primary hyperparathyroidism can disrupt the adaptation, resulting in elevated 1,25 dihydroxyvitamin D and increased intestinal calcium absorption. In primary hyperthyroidism this functional
adaptation may be disrupted causing increased
1,25 dihydroxyvitamin D production and as suchincreased intestinal absorption of calcium. Given
the relatively high prevalence of parathyroid adenomas, the hypercalcemia is not a manifestation of overt vitamin D toxicity rather a hypersensitivity to the high dose of vitamin D [32].
In the healthy, increasing 25 hydroxyvitamin Dlevels are known to decrease serum PTH concentrations. As such, PTH levels may serve as
determinants for vitamin D level adequacy. This
association does not hold in primary hyperparathyroidism, where the high PTH levels cause unregulated overproduction of 1,25 dihydroxyvitamin D, a potent hypercalcemic hormone.
Nonetheless, the role of vitamin D in primary
hyperthyroidism needs clarification.
ROUTINE LABORATORYDIAGNOSTIC IMPLICATIONSGiven the implication of vitamin D, as summarized in the above sections, the number of samples sent for measurement of total 25-hydroxyvitamin D to the routine laboratory may increase
from patients suspected or diagnosed with theabove mentioned common endocrine disorders.
Apart from the laboratory tests routinely requested from patients suffering from endocrine
conditions, 25-hydroxyvitamin D measurements
may well become part of the routine diagnostic
and follow-up panel for this patient group.
An additional sampling tube need not be drawn
for the procedure, since the primary tube sent
for the routine endocrine laboratory tests would
suffice for total 25-hydroxyvitamin D measurement as well. Furthermore, no extraordinary
preanalytical requirements need to be fulfilledapart from those already expected when drawing samples for routine endocrine testing. Total
25-hydroxyvitamin D measurement methodology has long enjoyed the boons of automatization and test results would ideally be delivered
within a short turnaround time.
Lately, the technological advancements in themeasurement of 1,25 dihydroxyvitamin D havemade the methodology commercially available
allowing application to platforms in use at theroutine albeit specialized laboratory setting,although the gold-standard 1,25-dihydroxyvitamin D liquid chromatography-tandem mass
spectrometry (LC-MS/MS) assay may not bereadily available at the basic laboratory level, a
recently introduced automated chemiluminescence immunoassay may increase the popularity of this testing [33-36].
Nonetheless, data on 1,25 dihydroxyvitamin
D and its implications in various disease contexts, including endocrine disorders, is limited
and there will probably be a lapse before massrequests for 1,25 dihydroxyvitamin D measurements overwhelm the routine diagnostic laboratory. Technically, the short half-life of about 4hours and its minute concentrations in the pmol/L
range have limited its utility in a routine setting.
Given the data presented in literature, the 75
nmol/L rather than the 50 nmol/L would plausibly suffice as the desired target value to reap
all benefits of vitamin D as implied in its various
non-skeletal effects [37].
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S, Todd JA, Franklyn JA, Gough SC. Lack of association of
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22. Feng M, Li H, Chen SF, Li WF, Zhang FB. Polymorphisms in the vitamin D receptor gene and risk of autoimmune thyroid diseases: a meta-analysis. Endocrine
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10. Lopez ER, Zwermann O, Segni M, Meyer G, Reincke
M, Seissler J, Herwig J, Usadel KH, Badenhoop K. A promoter polymorphism of the CYP27B1 gene is associatedwith Addison’s disease, Hashimoto’s thyroiditis, Graves’disease and type 1 diabetes mellitus in Germans. Eur J of
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23. Yasuda T, Okamoto Y, Hamada N, Miyashita K, Takahara M, Sakamoto F, Miyatsuka T, Kitamura T, Katakami
N, Kawamori D et al. Serum vitamin D levels are decreased and associated with thyroid volume in female
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11. Muscogiuri G, Mari D, Prolo S, Fatti LM, Cantone
MC, Garagnani P, Arosio B, Di Somma C, Vitale G. 25 Hydroxyvitamin D deficiency and its relationship to autoimmune thyroid disease in the elderly. Int J Environ Res Public Health 2016;13. pii: E850.

24. Yasuda T, Okamoto Y, Hamada N, Miyashita K, Takahara M, Sakamoto F, Miyatsuka T, Kitamura T, Katakami N,
Kawamori D et al. Serum vitamin D levels are decreasedin patients without remission of Graves’ disease. Endocrine 2013;43:230–232.

12. Muscogiuri G, Altieri B, Penna-Martinez M, Badenhoop K. Focus on vitamin D and the adrenal gland. Horm
Metab Res 2015;47:239-246.

25. Bratland E, Husebye ES. Cellular immunity and immunopathology in autoimmune Addison’s disease. Mol Cell
Endocrinol 2011;336:180–190.

13. Szyper-Kravitz M, Marai I, Shoenfeld Y. Coexistence of
thyroid autoimmunity with other autoimmune diseases:
friend or foe? Additional aspects on the mosaic of autoimmunity Autoimmunity 2005;38:247–255.

26. Skinningsrud B, Lie BA, Lavant E, Carlson JA, Erlich
H, Akselsen HE, Gervin K, Wolff AB, Erichsen MM, Lovas K et al. Multiple loci in the HLA complex are associated with Addison’s disease. J Clin Endocrinol Metab
2011;96:E1703–E1708.

14. Bozkurt NC, Karbek B, Ucan B, SahinM, Cakal E, Ozbek
M, Delibasi T. The association between severity of vitamin D deficiency and Hashimoto’s thyroiditis. Endoc Pract
2013;19:479–484.
15. Tamer G, Mesci B. Role of vitamin D in the immune
system. Turk J Endocrin Metab 2013;17:5–7.
16. Kivity S, Agmon Levin N, Zisappl M, Shapira Y, Nagy EV,
Dankó K, Szekanecz Z, Langevitz P, Shoenfeld Y. Vitamin
D and autoimmune thyroid diseases. Cell Mol Immunol
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17. Christakos S, Raval-Pandya M, Wernyj RP, Yang W. Genomic mechanisms involved in the pleiotropic actions of
1,25-dihydroxyvitamin D3. Biochem J 1996;316:361–371.

27. Pani MA, Seissler J, Usadel KH, Badenhoop K. Vitamin
D receptor genotype is associated with Addison’s disease.
Eur J Endocrinol 2002;147:635–640.
28. Lundqvist J, Norlin M, Wikvall K. 1a,25-Dihydroxyvitamin D3 affects hormone production and expression of steroidogenic enzymes in human adrenocortical NCI-H295R
cells. Biochim Biophys Acta 2010;1801:1056–1062.
29. Segersten U, Correa P, Hewison M, Hellman P, Dralle
H, Carling T, Akerström G, Westin G. 25-Hydroxyvitamin
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30. Souberbielle JC, Bienaime´ F, Cavalier E & Cormier C.
Vitamin D and primary hyperparathyroidism (PHPT). Annales d’Endocrinologie 2012;73:165–169.
31. Cusano NE, Silverberg SJ, Bilezikian JP. Normocalcemic primary hyperparathyroidism. J of Clin Densitom
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32. Amir E, Simmons CE, Freedman OC, Dranitsaris G,
Cole DE, Vieth R, Ooi WS, Clemons M. A phase 2 trial exploring the effects of high-dose (10,000 IU/day) vitamin
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33. Casetta B, Jans I, Billen J, Vanderschueren D, Bouillon R. Development of a method for the quantificationof 1alpha,25(OH)2-vitamin D3 in serum by liquid
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34. Strathmann FG, Laha TJ, Hoofnagle AN. Quantification of 1α,25-dihydroxy vitamin D by immunoextraction and liquid chromatography-tandem mass spectrometry. Clin Chem 2011;57(9):1279-1285.
35. van Helden J, Weiskirchen R. Experience with thefirst fully automated chemiluminescence immunoassay
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In this issue: Laboratory investigation of vitamin D and bone metabolism markers

Laboratory aspects and clinical utilityof bone turnover markers
Harjit Pal Bhattoa

Department of Laboratory Medicine, Faculty of Medicine, University of Debrecen, Hungary

ARTICLE INFOABSTRACT

Corresponding author:
Harjit Pal Bhattoa
Department of Laboratory Medicine
Faculty of Medicine
University of Debrecen
Hungary
E-mail: harjit@med.unideb.hu

With an aging population, there is a marked increasein prevalence of metabolic bone diseases, especiallyosteoporosis. Perhaps the most dreaded complication of metabolic bone disease, fractures typically
impose a huge burden on the ailing body and areassociated with high co-morbidity and mortality.
The consequent public health and socioeconomic
burden warrant timely diagnosis, treatment and follow-up of these disorders. Knowing the limitationsof radiological techniques, biochemical markers ofbone turnover measurements come handy since
the changes in their levels readily reflect bone physiology. Bone biomarkers typically analyzed in highthroughput automated routine laboratories are collagen degradation products, reflecting osteoclast
activity, and the collagenous or non-collagenous
proteins produced by the osteoblasts. Since bone
biomarker levels vary considerably due to quite a few
endogenous and exogenous pre-analytical factors,
knowledge of these limitations is mandatory prior
to clinical utilization since these variabilities complicate test result interpretation. Standardization toharmonize different assay methodologies is desired,
and the primary aims of the IFCC/IOF bone marker standards working group are also presented.
Current literature data advocate bone markers as
best used in monitoring anti-osteoporosis therapy

Key words:
bone turnover, bone formation,bone resorption,bone biomarkers,
biochemical markers of bone turnover

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Laboratory aspects and clinical utility of bone turnover markers

efficacy and compliance, nonetheless, there isabundant data supporting their role in predicting bone loss and fracture risk. Furthermore,they have widespread clinical utility in osteoporosis, renal osteodystrophy, and certain oncological conditions and rheumatic diseases.


INTRODUCTION
The interplay of the bone cells, namely, the osteoclasts and the osteoblasts is generally termed
as bone turnover. Knowingly, the macrophagelineage derived osteoclasts are destined to carry out bone resorption and the mesenchymelineage derived osteoblasts are responsible forbone formation ideally in a coupled fashion (1,2).
The imbalance in their functioning ultimately disrupts bone turnover and is characteristically noticed in metabolic bone disease (3). Per se diagnosis, monitoring, disease severity and treatment
efficacy is generally challenging due to the silent,
symptomless nature of these disorders, usuallyat onset, and primarily because the radiologicalfeatures do not promptly reflect changes in bonemetabolism. The high morbidity and mortality
associated with these conditions mandate diagnostic procedures that would ideally reflect theactual state of the bone. The present narrativemini-review presents the biomarkers characteristic of osteoblast and osteoclast functioning,
namely the markers of bone formation and thoseof bone resorption, respectively. Ideally changes
in levels of the biochemical markers of bone turnover coupled with radiological findings and fracture risk assessment in individual patients ideally
identify the patient most susceptible to suffera non-traumatic fracture. Although dual energy
x-ray absorptiometry (DEXA) till date is the gold
standard methodology to measure bone mineraldensity (BMD), it is known that decrease in bonemass does not solely account for fracture risk
(4,5). Realizing this limitation, the University of

Sheffield, UK launched the fracture risk assessment tool (FRAX) in 2008 under the guidance ofProfessor John A. Kanis (6), where apart fromthe femur neck BMD 11 other easily assessablerisk factors are included. The FRAX algorithms
give the 10-year probability of hip fracture andthe 10-year probability of a major osteoporotic
fracture (clinical spine, forearm, hip or shoulder
fracture) (7). Lately, the addition of bone turnover marker levels to the FRAX algorithms has
also been advocated (8). Nonetheless, it is realized that bone mass changes measurable byradiographical techniques, including DEXA, aredetectable almost a year following change at
the cellular level (9).
Apart from mineralization, which is rather aphysicochemical affair, bone turnover reflectsbone cellular activity and is a dynamic biological process (10). Given the difficulties of assessing dynamic processes at a static interval, bone
histomorphometry using tetracycline doublelabeling is the gold standard in determining thisfeature of bone biology (11,12). Although considered the gold standard, bone histomorphometry has its own limitations, including sampling
error, invasiveness, costs, and lack of availability
at the primary level. As such, due to limitationsof both radiographic techniques and bone histomorphometry, measurement of biomarkers
readily assessable from blood samples is an attractive alternative to evaluate bone turnover.
Since all metabolic bone diseases usually present with alterations in osteoblast and osteoclast
activity, biochemical markers of bone turnoverreflecting these activities mirror real time boneturnover. Furthermore, bone biomarker levels
also provide an index of disease activity in certain tumorous and rheumatological diseases affecting bone. The present review summarizes
the pre-analytic, analytic and post-analytic implications of bone turnover biomarkers, sinceknowledge of these limitations is mandatory incorrect test result interpretation. Furthermore,

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the clinical utility of these biomarkers has beensummarized in renal osteodystrophy, certain
oncological conditions, rheumatic diseases andPaget’s Disease of the bone.
MARKERS OF BONE TURNOVER
Bone biomarkers typically analyzed in highthroughput automated routine laboratories
are collagen degradation products, reflecting
osteoclast activity, and collagenous or non-collagenous proteins produced by the osteoblasts(table 1). All these markers can be quantitatedwell from blood samples, serum being the preferred sample of choice. Although assays forurine examination were developed for quite afew markers, blood sampling generally detours
the pre-analytic issues usually involving urine
sampling (13). The most commonly used bone
resorption and bone formation markers are
discussed below.
BIOMARKERS OF BONE RESORPTION
C- and N-terminal telopeptideof type I collagen
During bone degradation, osteoclast derived
tartrate-resistant acid phosphatase (TRAP) andTable 1

cathepsin K breakdown the bone matrix, including the triple helices of the mature type I collagen,to release carboxy- and nitrogen telopeptide containing fragments (CTx and NTx). The assay designed determines specific amino acid sequenceof the telopeptide of Type I collagen termed as
crosslaps, and those with β-aspartic acid as βCTx
(14). Although its counterpart the N-terminal
telopeptide (NTx) can also be measured fromurine samples, CTX has gained increased popularity as it can be measured from blood samples
on automated platforms, and given the increasing body of literature dealing with this biomarker
it may perhaps be stated that it has turned out tobe the biomarker of choice to examine osteoclastic bone resorption activity (15). CTx and NTx areboth cleared by the kidneys, as such its clinicalusefulness in CKD is significantly limited.
At the dawn of bone turnover biomarker development tartrate-resistant acid phosphatase,collagen cross-link molecules pyridinoline anddeoxypyridinoline, hydroxyproline and bone sialoprotein saw light of day and were examined tomeasure osteoclast activity (16). All the aforementioned markers have since been supersededby the more sensitive and specific telopeptidesof type I collagen, namely the C-terminal telopeptide (CTx).

Biochemical markers of bone turnover
Bone formation markers

Bone resorption markers

Osteocalcin

C-Telopeptide of Collagen Cross-links (CTx)

Bone Specific Alkaline Phosphatase (BSAP)

N-Telopeptide of Collagen Cross-links (NTx)Carboxyterminal propeptide of Type I Collagen(P1CP)
Aminoterminal propeptide of Type I Collagen(P1NP)

Pyridinolines
Deoxypyridinoline
Tartrate-Resistant Acid Phosphatase (TRAP)

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BIOMARKERS OF BONE FORMATION
N- and C-terminal propeptidesof type I collagen
Osteoblasts secrete type I collagen as a procollagen which forms a triple helix (containing
two α- and β-chain), and contains the N- andC-terminal propeptides (P1NP and P1CP), thesepropeptides are immediately cleaved in its extracellular vicinity eventually entering the blood
circulation (17). As such, the N- and C-terminal
propeptides qualify themselves as being biochemical markers of bone formation (18). The
cleaved products are initially in the trimeric
form that are eventually broken down to themonomeric form in the circulation the trimeric
P1NP is cleared by hepatic uptake, while themonomeric form is cleared via the kidneys.
Assays measure the monomeric and trimericforms (total P1NP) or only the trimeric form
(intact P1NP) (19). Being dependent on renal
clearence, the monomeric form of P1NP accumulates in chronic kidney disease (20,21).
Osteocalcin
Osteocalcin is the most abundant non-collagenous protein in the bone composed of 49 aminoacids and is secreted by the mature bone formation cells the osteoblasts. It is also known asbone gamma-carboxyglutamic acid-containing
protein since it contains 3 glutamic acids at
positions 13, 17 and 20 that undergo gammacarboxylation in a vitamin-K dependent fashion (22). It is noteworthy here that patients on
vitamin-K antagonists (e.g., warfarin) show decreased osteocalcin concentrations. Osteocalcin
has appeared again recently in the reflector
light, following the identification of its role as abone derived hormone influencing male fertility, glucose metabolism, and its actions on thecentral nervous system and muscle in animal
experiments (23-25). Although primarily identified as a marker of bone formation, due to its

tight correlation with bone formation measurements by bone histomorphometry, it may well
be considered a marker reflecting both formation and resorption, i.e, a marker of bone turnover since it is also liberated during osteoclastic
bone resorption (19, 26).
Due to its labile 6-amino acid C-terminal sequence samples for osteocalcin determination
have traditionally required special collection
and transportation requirements, this has been
overcome by development of assays that determine the more stable N-MID fragment (27,28).
Nonetheless, osteocalcin measurement has limited value in patients with reduced renal function since its mainly cleared by the kidneys (29).
Bone-specific alkaline phosphatase
In the healthy adult, almost half of the circulation total alkaline phosphatase is derived
from the bone, i.e., produced by the osteoblasts and the remainder is constituted by thefraction produced by the hepatocytes (30,31).
Bone-specific alkaline phosphatase (BSAP) primarily inactivates the mineralization inhibitor
pyrophosphate (32). Although commercial assays are available to measure BSAP, they show
cross-reactivity with liver alkaline phosphate, assuch in patients with liver disease BSAP measurements have limited applicability (19). BSAP
show good correlation with fracture risk in CKD
populations (33). Although a disease of the osteoclasts, it has been reported that BSAP proved
to be sensitive in monitoring disease progress inpatients suffering from Paget’s disease (34).
In summary, as compared to the bone resorption biomarkers, there is a larger repertoire ofbiomarkers of bone formation, reflecting osteoblast activity, that can be used in automatedhigh throughput laboratories, namely serumbone-specific alkaline phosphatase, osteocalcin
and procollagen type I N-terminal propeptide(PINP). Although produced by the osteoblasts,

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osteocalcin may be defined as a bone turnovermarker reflecting both bone formation andbone resorption, since it is also released fromthe bone matrix during bone resorption. P1NP
is more extensively described in literature arecompared to the other bone formation biomarkers (35).
PRE-ANALYTICAL AND ANALYTICALCONSIDERATIONS IN ROUTINELABORATORY DETERMINATION
OF BIOMARKERS OF BONE TURNOVER
Biomarkers of bone turnover are quite sensitive to a number of pre-analytical and analyticalissues.
Technical pre-analytical issues pertaining tosample collection are implicated mainly in urinary sample collection (13). As mentioned earlier, due to the cumbersomeness of spot or 24 hr
urine sample collection and the need for correction for creatinine, urine sampling has started
to go out of fashion and blood sampling is thepreferred mode of sample collection.
Perhaps the major challenge is the over-comingof biological factors that cause variability in test
results. Although trivial for the professional athome with the markers of bone turnover, one
needs to be reminded of a number of endogenous and exogenous factors that should be
mandatorily considered before interpreting test
results.
Normal blood levels of bone turnover markers
is usually higher in children, depending on thebiomarker these elevations may well be a multifold of those expected in the adult population(16,36). The levels in the elderly usually show
a decline, but there usually is an elevation in
women following menopause (16,37,38). Levels
are albeit usually higher in men as compared
to women (16,38). The ethnic background of
the patient is also to be considered since datasuggest that the Caucasians usually have lower

levels as compared to their age and sex matched
adult counterparts (16,39).
Marker levels are elevated during pregnancy and lactation and tend to normalize after a few months following weaning (40,41).
Marked elevations have been reported inmarker levels in those immobilized or bedridden for any reason (42). Marker levels may
be significantly elevated even at 6 months following a bone fracture (43-45). Patient withconcomitant comorbidities such as primary
hyperparathyroidism, Paget’s disease, multiple myeloma and metastatic prostate andbreast cancer usual present with higher levels (46-52). Abnormal kidney function resultsin elevated marker levels, particularly monomeric P1NP, CTx and osteocalcin, these mainly undergo renal clearance (53).
Biomarker levels vary considerable due to quite
a few endogenous factors, as such one needs
to take into consideration the circadian rhythm,
the phase of the menstrual period, seasonalvariation, physical exercise and diet.
Bone markers, particularly the resorption markers, follow a circadian rhythm where the peaklevels are typically observed in the early morning hours and the levels tapper off during theday (16,37).
Biomarkers of bone formation are characteristically elevated following ovulation, i.e., during
the luteal phase (54). On the other hand, resorption markers are elevated during the follicularphase of the menstrual period (55). Biomarker
levels of both resorption and formation reflect
vitamin D status in the winter months; this usually translates into these levels being higher during this time of the year (56). Given the literature till date, there is no clear consensus on theeffect of exercise on bone turnover biomarker
levels (57,58). A meat or gelatin rich diet usual
results in elevated marker levels (59). Patients
may be advised to have an overnight fast before

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the examination. High basal bone biomarkers
levels are usually observed in current smokers
and those with low body mass index (60). Since
most bone turnover markers are also present inother non-skeletal organs with type I collagen,cardiac conditions and systemic sclerosis, e.g.,
have also shown to present with elevated bone
biomarker concentrations (61-63).
There is marked analytical variability of bone
biomarkers. The methodology is not standardized and using different assays from various
manufactures generally present a huge difference in test results from the same sample. Task
forces or working groups like the International
Federation of Clinical Chemistry and Laboratory
Medicine (IFCC) bone marker standards in affiliation with the International Osteoporosis
Foundation (IOF) are working arduously to harmonize various assay methodologies (64-66).
Additionally, participation in external quality
control schemes could help minimize inter-laboratory variations (15).
IFCC WORKING GROUPON STANDARDISATION OF BONE
MARKER ASSAYS (WG-SBMA)
IN COLLABORATION WITH IOF
The WG-SBMA is currently headed by Professor
Etienne Cavalier, and the following are theterms of reference and projects for the period
2018-2020, as presented in the IFCC Handbook
2018-2020 (67).
Terms of reference•

To standardise or harmonise clinical assays
available for routine and research use, forthe following two bone turnover markers;
the serum assay for CTx and P1NP.

Current projects•

Review literature and current status ofavailable assays in order to develop andundertake a project to establish a reference
measurement system for serum β-CTx or harmonisation of the assays for serum β-CTx as
appropriate.
•

Review literature and current status of available assays in order to develop and undertake a project to establish a reference
measurement system for serum P1NP or harmonisation of the assays for serum P1NP as
appropriate.

•

Review and identify data required for the
regulatory authorisation of these modified
assays.

•

Review literature and consider the criticaldecision limits and potential target levels of
serum β-CTx and serum P1NP for treatmentof postmenopausal osteoporosis and othercauses of osteoporosis as appropriate•

IOF-IFCC study to summarize fracture prediction strength of reference bone turnover markers.

The above is in line with their position paper
published in 2001, where they advocate useof P1NP and CTx (i.e., one formation and one
resorption marker) in clinical trials and otherstudies to improve our understanding of bone
biomarkers and their application in every day
clinical practice (65).
CLINICAL UTILITY OF BONE
BIOCHEMICAL MARKERS
Monitoring anti-osteoporosis
therapeutic efficacy and compliance
Perhaps the greatest part of our knowledge on
the clinical utility of bone markers is based on
the results achieved in various pharmaceuticalstudies on novel anti-osteoporotic drugs (68-78).
The nature of their change in response to treatment is well characterized, and can be utilized toforecast increases in bone mineral density andtherapeutic efficacy in reducing fracture risk.

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Anti-resorptive drugs sabotage bone resorption
by driving osteoclasts into apoptosis and hence
resulting in rapid decrease in bone resorption
marker levels. The dose and mechanism of action
dictate the degree of inhibition of bone resorption, as such the resorption marker levels. Sincebone resorption and formation are coupled processes, inhibition of bone resorption results indecrease of bone formation, as such decrease inbone formation marker levels.
It is now evident that following patients every
3-6 months with their bone marker levels canmonitor drug adherence and efficacy. This is anadvantage over bone mineral density examinations where a follow-up of within 1-year period is
not productive, since changes in BMD take longer
to happen than bone markers, follow-up DEXA
scans are limited by their estimation of least significant change (LSC) and BMD changes explain
only a part of fracture reduction (66,68). Table 2
summarizes the changes in bone marker levels
during different anti-osteoporosis therapeutic regimes. Treatment with anti-resorptive drugs, e.g.,
estrogen, selective estrogen receptor modulators, like raloxifene, bisphosphonates, like alendronate and risedronate, and denosumab areassociated with decrease in markers of bone
resorption and formation. Human recombinant
PTH treatment is associated with increase in
Table 2bone formation markers followed by increase inbone resorption marker levels (74).
Both baseline bone marker levels and changes
following initiation of therapy predict BMD
changes. Early decrease in bone marker levels with bisphosphonates and denosumab are
known to correlate with 2-3-year increases in
BMD (70, 71).
Fracture risk reduction at the spine and hip has
been reported where early marker level reduction(P1NP, BSAP, CTx) was demonstrated upon commencing alendronate therapy (70). Raloxifene
induced changes in osteocalcin predicted spine
fracture risk reduction better that changes in
BMD (79, 80). Similar association was observed
also between P1NP and zolendronate (81).
Predicting bone loss and fracture risk
It has been recognized, based on populationbased studies, that elevated marker levels
predict accelerated bone loss and increased
non-traumatic fracture risk independent ofunderlying co-morbidities, age or sex (82,83).
Nonetheless, the implementation of these population-based observations has been difficult on
the individual patient level. Prospective randomized clinical trials designed to assess the efficacy
and cost-effectiveness of screening programs are

Changes in bone biomarker levels
during different anti-osteoporosis therapeutic regimes

Bone marker

Type

Therapy

TargetlevelsFollow-up period

ß Crosslaps

Resorption
markerAnti-resorptivemin. 35% Baseline and every 6 months

Total P1NP

FormationmarkerAnti-resorptivemin. 40% Baseline and every 6 months

Anabolicmin. 40% ↑Baseline and every 6 months

Osteocalcin

TurnovermarkerAnti-resorptivemin. 20% Baseline and every 6 months

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missing, and currently use of bone marker measurements are not recommended to identify patients at increased risk of bone loss as a publichealth measure (84).
Use in Nephrology
End-stage renal failure is usually associated withrenal osteodystrophy. The hallmarks of renal osteodystrophy include low serum calcium levels
and elevated PTH (secondary hyperparathyroidism). The high bone turnover characteristicof the condition results in high bone turnovermarker levels. TRAP and BSAP are the only biomarkers not cleared by the kidney and as such
reflect the state of bone turnover. Monomeric
P1NP, osteocalcin and CTx typically are elevated, and these elevations do not reflect the true
nature of bone turnover. PTH levels are known
to show good association with bone turnover(85). Practically speaking serum BSAP ad PTH
are the only reliable marker in patients suffering from kidney disease.

constellation in marker levels has been reported
in rheumatoid arthritis, polymyalgia rheumatic,
psoriatic arthritis, ankylosing spondylitis, and reactive arthritis (89,90). Furthermore, the resorption marker levels usually show good correlation
with disease activity indices of different rheumatological conditions (91-93).
Use in Paget’s disease of the bone
Paget’s disease of the bone is bone metabolic
disorder characterized by extremely high boneturnover causing expansion and deformationof affected bones. Patients usually present withmarked increase in all bone turnover marker levels (94). P1NP concentrations have been demonstrated to correlate with disease activity andto anti-resorptive therapeutic response, as suchbone turnover markers present traits of both
diagnostic and disease monitoring in Paget’s
disease (95).

Use in Oncology

CONCLUSION

Solid tumors like prostate, lung and breast cancer
typically metastasize to the bone. Furthermore,primary involvement of bone is characteristicof multiple myeloma. Depending on the tumor, the bone involvement may be osteolytic
or osteoblastic. This potentially suggests that
resorption marker elevations dominate duringosteolytic presentation and formation markers
are elevated when osteoblastic bone lesions are
manifested (86). Furthermore, bone biomarkers
function as tumor markers when secreted directly by primary bone tumor. Osteoid osteoma
secretes osteocalcin (87). BSAP can be secretedby osteosarcoma (88).

Biochemical markers of bone turnover reflectbone homeostasis, i.e., the activity of osteoblasts and osteoclasts in both physiological andpathophysiological conditions. Although quitesensitive to a multitude of exogenous and endogenous pre-analytical factors, bone markers
are best used in monitoring anti-osteoporosis
therapy efficacy and compliance. Combinationof BMD measurement by DEXA with biochemical markers of bone turnover levels, at least
one bone resorption and one bone formation
marker, may potentially improve early detection of individuals at increased risk for boneloss and eventually non-traumatic bone fracture. Furthermore, they have widespread clinical utility in osteoporosis, renal osteodystrophy,
certain oncological conditions and rheumatic
diseases.

Use in Rheumatology
The inflammatory pathogenesis of most rheumatological disorders promotes bone resorption
and suppresses bone formation. This uncoupled

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Practical guide for identifying
unmet clinical needs for biomarkers

Phillip J. Monaghan1, Sarah Robinson2, Daniel Rajdl3,
Patrick M.M. Bossuyt4, Sverre Sandberg5, Andrew St John6,
Maurice O’Kane7, Lieselotte Lennartz8, Ralf Röddiger9, Sarah J. Lord10,11,
Christa M. Cobbaert12,15, Andrea R. Horvath13,14,15
The Christie Pathology Partnership, The Christie NHS Foundation Trust, Manchester, United Kingdom
Thermo Fisher Scientific. Clinical Mass Spectrometry Clinical Diagnostics Systems, United Kingdom
3
Department of Clinical Biochemistry and Hematology, Medical Faculty in Pilsen,
Charles University, Czech Republic
4
Department of Clinical Epidemiology, Biostatistics & Bioinformatics, Academic Medical Center,
University of Amsterdam, The Netherlands
5
Laboratory of Clinical Biochemistry, Haukeland University Hospital, Helse Bergen HF, Bergen, Norway,
Norwegian Quality Improvement of Laboratory examinations (Noklus), Haraldsplass Deaconess Hospital,
Bergen, Norway and Department of Global Health and Primary Health Care, University of Bergen, Norway
6
ARC Consulting, Perth, Australia
7
Clinical Chemistry Department, Altnagelvin Hospital, Western Health and Social Care Trust, Londonderry,
N. Ireland, United Kingdom
8
Abbott Diagnostics, Wiesbaden, Germany
9
Medical and Scientific Affairs, CPS, Roche Diagnostics GmbH, Mannheim, Germany
10
School of Medicine, University of Notre Dame, Sydney, Australia
11
National Health and Medical Research Council Clinical Trials Centre, University of Sydney, Australia
12
Department of Clinical Chemistry and Laboratory Medicine, Leiden University Medical Centre,
The Netherlands
13
New South Wales Health Pathology, Department of Clinical Chemistry and Endocrinology,
Prince of Wales Hospital and School of Medical Sciences, University of New South Wales, Australia
14
School of Public Health, University of Sydney, Australia
15
For the Test Evaluation Working Group of the European Federation of Clinical Chemistry
and Laboratory Medicine
1
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Phillip J. Monaghan et al.
Practical guide for identifying unmet clinical needs for biomarkers

ARTICLE INFOABSTRACT

Corresponding author:
Phillip J. Monaghan
The Christie Pathology Partnership
The Christie NHS Foundation Trust
Manchester
United Kingdom
Phone: (+44) 161 918 2466
E-mail: phillip.monaghan@nhs.net

The development and evaluation of novel biomarkers
and testing strategies requires a close examination ofexisting clinical pathways, including mapping of current pathways and identifying areas of unmet need.
This approach enables early recognition of analyticaland clinical performance criteria to guide evaluation
studies, in a cyclical and iterative manner, all the time
keeping the clinical pathway and patient health outcomes as the key drivers in the process.

Key words:
unmet clinical need, clinicalpathway, test evaluation
Author contributions:
All the authors have accepted responsibility
for the entire content of this submitted
manuscript and approved submission.
Financial support:
The European Federation of Clinical Chemistry
and Laboratory Medicine (EFLM) provided
financial support to the Test Evaluation
Working Group for the preparation of thispaper through payment of travel expenses
to attend Working Group meetings. Abbott,
Roche Diagnostics, and Thermo Fisher Scientifichave provided independent educational grant
to EFLM (EFLM grant reference numbers
2017/01, 2017/02 and 2017/04 respectively).
Role of sponsor:
Neither the funding organization (EFLM)
nor the independent educational support
by Roche Diagnostics, Abbott and Thermo
Fisher Scientific influenced the content
of this paper. This publication reflects thecollective view of the Working Group. The
authors include representatives from Roche
Diagnostics and Abbott Diagnostics as they
provided important insights from the IVD
industry, a key stakeholder in the researchand development of new biomarker assays.
Employment or leadership:
None declared.

The Test Evaluation Working Group of the European
Federation of Clinical Chemistry and Laboratory
Medicine (EFLM TE-WG) https://www.eflm.eu/site/
page/a/1158 has published a conceptual framework of the test evaluation cycle which is driven by
the clinical pathway, inherent to which is the test
purpose and role within the pathway that are defined by clinical need.
To supplement this framework, the EFLM TE-WG has
also published an interactive checklist for identifying
unmet clinical needs for new biomarkers; a practical
tool that laboratories, clinicians, researchers and industry can equally use in a consistent manner when
new tests are developed and before they are released
to the market. It is hoped that these practical tools
will provide consistent and appropriate terminology
in this diverse field and offer a platform that facilitates
greater consultation and collaboration between all
stakeholders. The checklist should assist the work of
all colleagues involved in the discovery of novel biomarkers and implementation of new medical tests.
The tool is aligned with the IOM recommendations
and the FDA and CE regulating body’s requirements.

Honorarium:
None declared.
Competing interests:
The funding organization(s) played norole in the study design; in the collection,
analysis, and interpretation of data; in thewriting of the report; or in the decision
to submit the report for publication.

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INTRODUCTION
Clinical laboratory scientists and pathologists,
responsible for the provision of in vitro medical
tests, are regularly approached by industry colleagues about the availability of new tests. Thislate notification sometimes poses problems;e.g. the new test does not seem to fulfil an unmet clinical need, the evidence on the clinicaleffectiveness of the biomarker is not yet available or controversial and therefore the new biomarker may not be commissioned or get on thereimbursement schedule.
Unmet clinical needs for new biomarkers areoften discussed at clinical meetings within thehealth care setting, yet these perceived needs
are rarely communicated to R&D and industry
colleagues as key stakeholders in the biomarker
development process.
Furthermore, laboratory professionals are more
likely to experience pressure from the hospitalboard to reduce the costs of pathology testing
and to rationalize test requesting rather thanadding new tests to the laboratory’s repertoire;the new test should be shown to improve patient care and outcomes or the cost-effectiveness of care.
To support laboratories in evidencing the valueof tests, the Test Evaluation Working Group of
the European Federation of Clinical Chemistry
and Laboratory Medicine (EFLM TE-WG) has published a conceptual framework of the test evaluation cycle which is driven by clinical need andthe clinical pathway of managing patients [1].
The aim of the working group is to supplement
this framework with practical tools that laboratories, clinicians, researchers and industry
can equally use in a consistent manner when
new tests are developed and before they are
released to the market. The first such tool is achecklist for identifying unmet clinical needs for
new biomarkers [2].

The goal of the test evaluation framework andthe unmet clinical needs checklist is to provide
consistent and appropriate terminology in thisdiverse field and to offer a platform that facilitates greater consultation and collaboration between all stakeholders.
THE CYCLICAL FRAMEWORKOF TEST EVALUATION
The test evaluation framework of the EFLM TEWG [1] is intended to be applied after a potential biomarker has been discovered in basic research (so-called ‘proof of concept’) studies andis ready for further development and evaluationin clinical settings. The framework describes
biomarker evaluation as a cycle, where key elements of the process, i.e. assessment of theanalytical performance, clinical performance,clinical effectiveness, cost-effectiveness, andoverall impact of the new test, are primarily
driven by clinical needs and the clinical pathwaythat should lead to improved health outcomesor greater health care efficiency.
This dynamic framework reflects well-known
steps in test evaluation but unlike most otherlinearly staged test evaluation models, it places
the clinical pathway and thus testing-related
patient outcomes into the centre (Figure 1).
This is a critical component of the cyclical test
evaluation framework, since the relationshipbetween laboratory testing and subsequent patient outcomes is, more often than not, indirect.
In most cases, only if test results are utilised to
inform and guide effective downstream clinicaldecisions can patient outcomes be improved.
Thus, clear identification of the test purpose (i.e.
intended clinical application and how the test information will be used to improve clinical management; e.g. diagnosis, prognosis, monitoring,screening, treatment selection, etc.) and testrole within the clinical pathway (i.e. how the test
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Practical guide for identifying unmet clinical needs for biomarkers

Figure 1

Framework for the evaluation of in vitro medical tests

The cyclical framework illustrates the interplay between the key elements of the test evaluation process and that all
are dynamically linked to one another in a cycle driven by the intended use of a test in the clinical pathway.
Adapted from Horvath, et al [1].

pathway; e.g. replacement, triage, add-on test)
are essential.
NOVEL APPROACH TO TESTEVALUATION IN PRACTICE
The problem with many frameworks publishedso far is that they describe what needs to bedone, but they do not offer clear explanation of
how each of the key steps should be undertaken. The EFLM TE-WG therefore aims to provide
practical tools for each step of this framework to
help operationalize the theory and the key principles described. The working group found thatclinical pathway mapping is a useful method for

identifying clinical needs and management decisions and to link information from testing tohealth outcomes.
Unmet need for medical tests is relatively vaguely defined and its assessment, in general, is acomplex process that could be very subjective
depending on the background, practice, experience and interest of stakeholders. For example,
a representative from a reimbursement organization with strict funding and under government pressure for cost-effectiveness of healthcare services could see the need for a new biomarker from a very different perspective, compared to a researcher who has just discovered

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a promising new biomarker of potential clinical effectiveness; or indeed a clinician who isstruggling to manage patient treatment in theabsence of a reliable biomarker of treatment
effectiveness.
To ensure that the differing perspectives ofvarious stakeholders involved in the biomarker
translation pipeline are captured, we concluded that a checklist of specific questions, withcheckpoints, would be a practical and informative tool, rather than a set of generic recommendations. The checkpoint could also act asa catalyst for open dialogue between various
Figure 2

stakeholders to identify and assess unmet needsin view of the clinical pathway.
The TE-WG used a 4-step process to develop
the unmet clinical needs checklist:
1. scoping literature review;
2. face-to-face meetings to discuss scope,strategy and checklist items;
3. iterative process of feedback and consensus to develop the checklist;
4. testing and refinement of checklist items
using case scenarios.

The EFLM TE-WG 14-item checklist organized into 4 domains

The checklist is intended to achieve more efficient biomarker development and translation into practice [2].

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Clinical pathway mapping was utilised to identify clinical management decisions linking testing
to health outcomes and the 14-item checklist
was set around 4 key domains:
1. Identifying the unmet needin the current clinical pathway;

e-Learning platform: https://elearning.eflm.eu/
course/view.php?id=11. The platform also contains a short video showing how to use the interactive checklist, including worked examples.
SYNERGY WITH THE NATIONAL ACADEMYOF MEDICINE BIOMARKER REPORT

2. Verifying the unmet need;
3. Validating the intended use; and4. Assessing the feasibility of the new
biomarker to influence clinical practiceand health outcomes.
The checklist presents an outcome-focused
approach that can be used by multiple stakeholders for any medical test, irrespective of the
purpose and role of testing (Figure 2). In eachmain domain there are more specific questions
that need to be discussed and answered bystakeholders in order to facilitate a structured,considered judgment process. The checklist isbuilt with checkpoints in such a way that if theanswers to certain key questions are unfavorable, then the whole process should stop andthe medical need for the biomarker and further
evaluation of the test are not justified.
Based on the working group’s experience, achecklist such as the AGREE checklist to assess
the methodological quality of guidelines [3], or
the STARD checklist that guides researchers on
how to design and report diagnostic accuracy
studies [4], is a very effective tool in providing
clear guidance and a standardized way of handling complex evidence-based clinical decisions.
The central strength of the checklist is thatwhilst it takes into consideration the perspectives of all stakeholders, it prioritizes the clinicalpathway and health outcomes of the patient at
the centre of the needs evaluation process.
In collaboration with the EFLM Working Groupfor Distance Education and e-Learning, we havedeveloped an interactive version of this checklist, now openly available through the EFLM

The National Academy of Medicine (formerly theInstitute of Medicine [IOM]) has issued several
very useful documents over the years which influenced the development of the unmet clinicalneeds checklist of the EFLM TE-WG. These include the 2011 IOM report on the “Evaluation of
Biomarkers and Surrogate Endpoints in Chronic
Disease” [5] and recommendations for:•effective biomarker evaluation and improving evidence-based regulation;•

development of biomarker-based tools forcancer (2006) [6];•improving diagnosis in health care (2015)[7]; and most recently,•

biomarker tests for molecularly targeted
therapies (2016) [8], a document that wasissued after the EFLM checklist had alreadybeen completed.

Equally the TE-WG was very strongly influenced
by the new in vitro diagnostic (IVD) regulatorychanges in Europe [9] and the US [10], which
demand more clinical evidence before newtests enter the market. Significant consideration was also given to the work of the Global
Harmonization Task Force (GHTF) [11], which issued a number of valuable documents that have
set the definitions, principles and key elementsof and proposed processes for more effective
biomarker evaluation before market approval.
The EFLM checklist provides a practical ‘how to’
tool that addresses the goals and principles set
out by the above groups and regulatory bodies of
the United States Food and Drug Administration(FDA) and Conformité Européenne (CE) in the

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European economic area. It is anticipated, thatthe checklist will facilitate inter-disciplinary,multi-stakeholder collaboration for efficient biomarker development and pre- and post-market
evaluation. Critically, the checklist will help toverify and validate the purpose and role of abiomarker in the context of the clinical pathway,
thus providing the necessary evidence for the
proposed intended use of the test. These important clinical considerations should then guide theanalytical and clinical performance requirements
and the generation of evidence of clinical effectiveness and value, as promulgated in IOM recommendation 3 [8].
PROFESSIONAL UPTAKE OF THE TOOLS
Initially, it is expected that the checklist will
be used by a number of stakeholders such as
clinical laboratory professionals, and that it will
be pilot-tested with other colleagues, including those staff providing direct clinical care andindustry representatives when reviewing theneed for new biomarkers or new intended usesof existing medical tests.
Good communication with stakeholders in research and development is needed so that thebiomarker development pipeline is aligned withevolving unmet clinical needs. The checklist
will ideally drive multidisciplinary collaboration
to break down the conventional working silos,
and contribute to making biomarker evaluationa more efficient and targeted process, thus becoming an enabler for the adoption of innovative tests. Such collaboration will promote robust implementation planning proportionate to
the clinical pathway, so test results are available
and acted upon in an appropriate and timely
manner, with a strong link to clinical intervention and outcomes.
There are numerous publications reporting biomarker failures and even harm caused by poorly
performing biomarkers [12-18].

It is known that only a very tiny fraction of themany newly discovered ‘omics’ markers find theirway ‘from bench to the bedside’. Laboratory professionals are under increasing pressure from clinicians and health care administration to provethe value of existing tests in terms of impact
on various health, organisational and financial
outcomes.
It is also envisaged that IVD companies involved
in research and biomarker development will use
this checklist before investing in setting up major
work for releasing new tests for novel biomarkers to the market.
Due to stricter regulations both by the FDA andCE marking authorities in Europe, IVD companies
are under increasing pressure to provide data on
the clinical performance of biomarkers before
regulatory approval. Such studies are complex,
costly and time-consuming. Notwithstanding
the complexities of translational research, it has
been stated to take on average 17 years for research evidence to reach clinical practice [19].
Therefore, it is in the interest of the IVD industry that the unmet clinical need and the purpose and role of new biomarkers in a clinicalpathway and the potential impact of testing on
various outcomes are thoroughly considered.
This would reduce research waste and preventthe release of useless or even harmful tests tothe market.
REGIONAL IMPLEMENTATION,
DIFFERENCES AND EXAMPLES
OF THOSE USING THE CHECKLISTThe strength of the working group’s checklist
is that, instead of providing recommendations
which may match the healthcare setting of one
country or region but not that of another, it
asks open questions that can be answered withfull consideration given to the local health caresetting.

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For example, the checklist asks the user of a new
test to consider their current local practice, thelimitations of current practice and to map out
the current clinical pathway to see where the
new test would fit in and what value it would
add to current practice.
Obviously this clinical pathway can be very different even for the same medical condition in
Europe, the US, or Africa or Australia.
Even within one country the care pathway andthe utility of or the need for a test may depend
on whether the relevant health care is provided
in a metropolitan or in a rural care setting.
For example a point-of-care (POC) Troponin test
may not fit well into the clinical pathway of a
metropolitan hospital, which has 24/7 access toa higher sensitivity and more reliable Troponinassay with a <1hr turn-around-time in its central laboratory. Nevertheless, it may do so in arural setting where there is limited access tolaboratory testing and where a POC Troponintest may save lives by selecting patients whoneed urgent transport to a hospital, where appropriate care for an acute myocardial injury
can be provided.
The checklist also asks whether the new biomarker is feasible in practice technically, commercially, economically, and organizationally,
and what other local, cultural, social, etc. barriers may exist to its implementation. These again
can be locally determined issues and the answers tailored to each setting may define medical need for the same test completely differently
in various countries.
Indeed, unmet clinical need is a crucial primary component of the wider value proposition
framework of laboratory medicine [20], taking
into account the impact on clinical, operationaland economic outcomes to assure feasibility of
implementation.

SUMMARY
The unmet clinical need checklist produced by
the EFLM TE-WG is a practical tool that should
assist the work of all stakeholders involved in
the discovery or implementation of new biomarkers and testing strategies.
We encourage pilot testing and regular use ofthis new interactive tool. The checklist can beused before new biomarkers are developed or
fully validated for clinical use as well as whenassessing the clinical need for and the clinicalutility of existing tests. The TE-WG would appreciate feedback to inform future refinements ofthe checklist based on user experience.
REFERENCES
1. Horvath AR, Lord SJ, StJohn A, Sandberg S, Cobbaert
CM, Lorenz S, Monaghan PJ, Verhagen-Kamerbeek WD,
Ebert C, Bossuyt PM; Test Evaluation Working Group of
the European Federation of Clinical Chemistry Laboratory Medicine. From biomarkers to medical tests: thechanging landscape of test evaluation. Clin Chim Acta.
2014;427:49–57
2. Monaghan PJ, Lord SJ, St John A, Sandberg S, Cobbaert CM, Lennartz L, Verhagen-Kamerbeek WD, Ebert C,
Bossuyt PM, Horvath AR; Test Evaluation Working Groupof the European Federation of Clinical Chemistry andLaboratory Medicine. Biomarker development targeting
unmet clinical needs. Clin Chim Acta. 2016;460:211–19.
3. Brouwers MC, Kerkvliet K, Spithoff K; AGREE Next
Steps Consortium. The AGREE Reporting Checklist: a tool
to improve reporting of clinical practice guidelines. BMJ.
2016;352:i1152.
4. Bossuyt PM, Reitsma JB, Bruns DE, Gatsonis CA, Glasziou PP, Irwig L, Lijmer JG, Moher D, Rennie D, de Vet HC,
Kressel HY, Rifai N, Golub RM, Altman DG, Hooft L, Korevaar DA, Cohen JF; STARD Group. An Updated List of Essential Items for Reporting Diagnostic Accuracy Studies.
BMJ. 2015;351:h5527.
5. Christine M. Micheel and John R. Ball, Editors; Evaluation of Biomarkers and Surrogate Endpoints in Chronic
Disease. 2010 Washington, DC: The National Academic
Press. http://doi.org/10.17226/12869
6. Institute of Medicine. Developing Biomarker-BasedTools for Cancer Screening, Diagnosis, and Treatment:
The State of the Science, Evaluation, Implementation, and Economics: Workshop Summary. 2006.

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Washington, DC: The National Academies Press. http://
doi.org/10.17226/11768

12. Benowitz S. Biomarker boom slowed by validation
concerns. J Natl Cancer Inst. 2004;96:1356-7

7. National Academies of Sciences, Engineering, andMedicine. Improving Diagnosis in Health Care. 2015.
Washington, DC: The National Academies Press. http://
doi.org/10.17226/21794

13. Diamandis EP. Cancer Biomarkers: Can we turn recent
failures into success? J Natl Cancer Inst. 2010;102:1462-7.

8. National Academies of Sciences, Engineering, andMedicine, Biomarker Tests for Molecularly Targeted
Therapies: Key to Unlocking Precision Medicine. 2016.
Washington, DC: The National Academies Press. http://
doi.org/10.17226/21860

14. Diamandis, EP. The failure of protein cancer biomarkers to reach the clinic: why, and what can be done to address the problem? BMC Med. 2012;10:87.
15. Kern SE. Why your new cancer biomarker may neverwork: Recurrent patterns and remarkable diversity in biomarker failures. Cancer Res. 2012;72:6097-101

9. Regulation (EU) 2017/746 of the European Parliamentand of the Council of 5 April 2017 on in vitro diagnostic medical devices and repealing Directive 98/79/EC and Commission Decision 2010/227/EU. https://eur-lex.europa.
eu/legal-content/EN/TXT/?uri=CELEX%3A32017R0746

16. Ioannidis, JPA. Biomarker failures, Clin Chem.
2013;59:202–4.

10. Discussion Paper on Laboratory Developed Tests(LDTs) January 13, 2017. https://www.fda.gov/MedicalDevices/ProductsandMedicalProcedures/InVitroDiagnostics/LaboratoryDevelopedTests/default.htm

18. Ioannidis JPA, Bossuyt PMM. Waste, Leaks, and Failuresin the Biomarker Pipeline. Clin Chem. 2017;63(5):963-72.

11. Study Group 5 of the Global Harmonization Task Force.
Clinical evidence for IVD medical devices — key definitions
and concepts. GHTF/SG5/N6;2012:1–11. http://www.imdrf.org/docs/ghtf/final/sg5/technical-docs/ghtf-sg5-n62012-clinical-evidence-ivd-medical-devices-121102.pdf

17. Ioannidis, JPA. Why Most Clinical Research Is Not Useful. PLoS Med. 2016;13:e1002049

19. Morris ZS, Wooding S, Grant J. The answer is 17 years,
what is the question: understanding time lags in translational research. J R Soc Med. 2011;104:510-20.
20. St John, A, Cullen L, Julicher P, Price CP. Developing
a value proposition for high sensitivity troponin testing.
Clin Chim Acta. 2018;477:154-159.

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Prevalence of anemia and its associatedfactors among children in Ethiopia:
a protocol for systematic review and meta-analysis
Zegeye Abebe1,*, Wubet Worku Takele2, Degefaye Zelalem Anlay2,
Daniale Tekelia Ekubagewargies2, Zegeye Getaneh3, Molla Abebe4,
Mulugeta Melku3,*

Department of Human Nutrition, Institute of Public Health, College of Medicine and Health Sciences,
University of Gondar, Ethiopia
2
School of Nursing, College of Medicine and Health Sciences, University of Gondar, Ethiopia
3
Department of Hematology and Immunohematology, School of Biomedical and Laboratory Sciences,
College of Medicine and Health Sciences, University of Gondar, Ethiopia
4
Department of Clinical Chemistry, School of Biomedical and Laboratory Sciences, College of Medicine
and Health Sciences, University of Gondar, Ethiopia*Contributed equally to this protocol for sytematic review and meta-analysis
1

ARTICLE INFOABSTRACT

Corresponding author:
Mulugeta Melku
Department of Hematology
and Immunohematology
School of Biomedical and Laboratory Sciences
College of Medicine and Health Sciences
University of Gondar
P.O. Box 196, Gondar
Ethiopia
E-mail: mulugeta.melku@gmail.com

BackgroundKey words:
anemia, children, Ethiopia, meta-analysis,
protocol, systematic review

Anemia is one of the global public health problems
that affect more than one third of the world population. It has been strongly associated with poor growthand development, limited psychomotor development, and poor long-term performance in cognitive,social, and emotional functioning in children. There is
not a single national figure about childhood anemia
in Ethiopia. There is also inconclusive evidence about
factors associated with anemia. Moreover, the present meta-analysis will generate concrete evidence
in which the result may urge policy makers and program managers to design appropriate intervention
to control childhood anemia. Thus, the aim of thisreview is to estimate the pooled prevalence and to
identify associated factors of anemia among children
in Ethiopia.
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Z. Abebe, Wubet W. Takele, Degefaye Z. Anlay, Daniale T. Ekubagewargies, Z. Getaneh, M. Abebe, M. Melku
Prevalence of anemia and its associated factors among children in Ethiopia

MethodBACKGROUND

Published relevant cross-sectional studies will
be searched using comprehensive search strings
through PubMed/Medline, SCOPUS, HINARI,EMBASE, Web of Science databases. In addition,
Google Scholar and Google will be searched forgrey literature.

Anemia is recognized as a major public healthproblem globally, mostly affecting children,
women of childbearing age and pregnant women [1]. In developing countries, anemia can bea result of a number of causes; but nutritional
anemia, particularly iron deficiency, is the mostcommon cause. It is primarily caused by insufficient dietary intake and/or insufficient absorption, and suboptimal utilization of micronutrients like vitamin or mineral and/or due tofrequent infections [1, 2].

Reference lists and communication with content experts will be used to get additional
relevant studies. Two groups of review authors will independently appraise the studies for scientific quality and extract the datausing the Joanna Briggs Institute (JBI) tools.
The pooled estimate will be determined using random effect model. Heterogeneity between the studies will be assessed using theI2 statistics. Sensitivity and subgroup analysis
will be employed in the case of heterogeneity.
Publication bias will be assessed by visual inspection of the funnel plot, and using Egger’sand Begg’s statistical tests.
Discussion
Childhood anemia is known to have negative
consequences on mental, physical and social
development of children. The burden and its
associated factors are greatly varied as to thesocial, economic, and geographical differencesof the target population.
Therefore, the proposed systematic review will
generate evidence about the pooled prevalenceof anemia and its associated factors among children in Ethiopia.
Protocol registration
The protocol was registered at PROSPERO
International Prospective Register of Systematic
Reviews.
(Registration number: CRD42018088223).
The health implication of anemia is numerous.
It is associated with impaired mental, physical,
motor and language development; and poor
coordination, scholastic achievement and immune function [3-5]. In addition, the outcomesof anemia can be considered from a variety ofperspectives, including detrimental impacts on
economic and social development [6].
According to World Health organization (WHO)2015 report, about 43% of under five children
were anemic, with regional variations of 62.3%
in African, 53.8% in South-East Asia and 21.9%
in Western Pacific Region [7].
In Ethiopia, though the prevalence of severe tomoderate anemia in the last fifteen years has
significantly declined, children and pregnant
women are still suffering from the consequences of anemia due to high iron requirements,
low intake of iron from foods, and frequentepisodes of infection [8, 9]. As many as six in
ten under five children in Ethiopia are anemic.
Moreover, according to the local conditions,the proportion varies across the regions of thecountry due to feeding, wealth and cultural differences. As an illustration, the highest level ofchildhood anemia was found in Somali Region(83%), followed by Affar (75%) and Dire Dawa
(72%), but the lowest was found in the Amhara
Region (42%) [10].

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Prevalence of anemia and its associated factors among children in Ethiopia

The government of Ethiopia has been working
to reduce childhood anemia. Accordingly, it endorsed the national nutrition program, bimanual school deworming, developed micronutrient deficiency prevention and control guideline
and implemented micronutrient fortification.
But studies from different corners of the country have shown that childhood anemia is still amajor public health problem. In addition, there
is not a single national figure about childhood
anemia and also inconclusive evidence about
factors associated with it. Systematic review
and meta-analysis generates concrete evidence
in which the evidence may urge policy makers
and program managers to design appropriate
intervention to control and minimize the negative consequences of childhood anemia. As far
as the authors are aware, there is no publishedsystematic review and meta-analysis that generated the pooled estimated prevalence ofanemia and the pooled effect size of factors
associated with childhood anemia in Ethiopia.
Therefore, the aim of this systematic review andmeta-analysis is to estimate the pooled prevalence of childhood anemia and its associatedfactors among children in Ethiopia.
OBJECTIVES
The objectives of this systematic review and meta-analysis are:•To estimate the pooled prevalence of anemia
among children in Ethiopia;•

To identify factors associated with childhood
anemia in Ethiopia.

The protocol of this systematic Review andMeta-analysis has been registered in the
PROSPERO, international prospective register ofsystematic reviews, with registration number of
CRD42018088223.
Search strategy
Relevant published articles will be searched in
PubMed/Medline, HINARI, SCOPUS, EMBASE and
Web of Sciences electronic databases. Google
scholar, and Google will be searched for grey literature. In addition, conference paper and thesisor research final report will be accessed, professional annual research conference log book, andresearch and publication directorate from each
University in Ethiopia upon request.
The search terms will be developed in accordancewith the Medical Subject Headings (MeSH) thesaurus using a combination of key terms. Hand
searching of articles published in Ethiopian journal of health sciences, Ethiopian Medical journal,
Ethiopian journal of Health and Development,
and Ethiopian Journal of Health and Biomedical
Sciences will be conducted.
Reference lists of retrieved articles will be probed
to identify any studies that are not retrieved fromelectronic databases. Content experts will becontacted to get additional studies that are notretrieved by electronic database searching andreference list scrutinizing. Two author groups:
group one (MM, ZA) and group two (DTE, WWT),
will independently search the articles. In the caseof lack of necessary data from studies, we willcontact the corresponding author through email.
Search Strategy:

METHODOLOGY•Design and protocol registration
This protocol is designed according to thePreferred Reporting Items for Systematic Reviews
and Meta-Analysis Protocols (PRISMA-P 2015
Guidelines) [11].

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anemia OR iron deficiency anemia OR nutritional anemia OR hemoglobin OR nutritional
status OR hematological parameters OR determinant factors of anemia OR associatedfactors of anemia AND children OR adolescent OR preschool AND Ethiopia

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Prevalence of anemia and its associated factors among children in Ethiopia

Study selection and quality appraisal
All articles retrieved through search strategy will
be imported to EndNote X7 (Thomson Reuters,
New York, USA). After excluding the duplicate articles, titles and abstracts will be independently
screened for inclusion in full text appraisal by
two groups of review authors: group one (MM,ZA) and group two (DTE, WWT). Differences
between two groups will be resolved throughthorough discussion. In case of disagreement
between the two groups of review authors’ reports, the decision will be determined by thirdgroup of review authors (ZG, MA, DZA).
For articles deemed to appear relevant, the fulltext will be appraised for inclusion in systematic
review and meta-analysis.
The quality of articles will be assessed using
Joana Brigg’s Institute (JBI) critical appraisal
checklist for simple prevalence [12] and analytical cross-sectional studies [13] having nine andeight checklist items, respectively. Studies withan overall quality assessment score greater than
50% will be included. The discrepancies during
critical appraisal will be solved as similar manner for title/abstract screening phase.
Participants
Studies reporting the prevalence and/or factors
associated of anemia among children in Ethiopia.
Eligibly criteria
Inclusion criteria
Studies will be included in this systematic review if they met all the below inclusion criteria:•

Studies reporting the outcome of interest among children (up to 18 years old) in
Ethiopia•

Observational studies like cross-sectional,
prospective cohort studies and repeatedcross-sectional studies. For prospective cohort and repeated cross-sectional studies

that reported the outcomes of interest, thebaseline data will be used for our systematic
review and meta-analysis.
Exclusion Criteria
Studies will be excluded if they have met at least
one of the exclusion criteria below:•

Studies conducted in healthcare facilities•

Studies that used Visual comparative method (Sahli-hellige method, and MBS hemoglobinometer color scale) and Copper
Sulphate densitometer to ascertain the outcome (anemia)•

Studies conducted in children living with
HIV/AIDS

Outcomes of the study
The primary outcome of this systematic review
is to determine the prevalence of anemia amongchildren in Ethiopia. It will also identify factors
associated with the burden of anemia amongchildren in Ethiopia. Anemia is defined as hemoglobin value below the established reference
interval for the sex and age of the population.
Data extraction
The JBI tool will be adopted for data extraction.
Two groups of review authors, group one (MM,ZA) and group two (DTE, WWT), will extract thedata independently. In case where there are differences between the two authors’ groups withregard to the extracted data, the difference will
be solved via discussion.
Unless resolved via discussion, the decision will
rely on the third review authors’ group (ZG, MA,
DZA) report. Information such as name of firstauthor, year of publication, age group of children, study year, study area/region, study design, total number of children, number of anemic children, and number of anemic cases andnon-anemic cases for the reported associatedfactors will be extracted.

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Z. Abebe, Wubet W. Takele, Degefaye Z. Anlay, Daniale T. Ekubagewargies, Z. Getaneh, M. Abebe, M. Melku
Prevalence of anemia and its associated factors among children in Ethiopia

The data will be recorded in a Microsoft excel
spreadsheet. When authors find multiple publications from the same dataset, the article reported the prevalence and factors associated ofanemia in extractable form will be used.
Moreover, for prospective cohort and repeatedcross-sectional studies that reported the outcomes of interest, the baseline data will be used
for our systematic review and meta-analysis tofacilitate comparability of results across studies
and to reduce loss to follow-up bias.
Data analysis
The data extracted from primary eligible studies
will be entered into Microsoft excel, and thenwill be exported to STATA version 14 (StataCorp
LLC, Texas, USA) for analysis.
The magnitude of heterogeneity between included studies will be quantitatively measured
by index of heterogeneity (I2 statistics) [14].
I2 values of 25%, 50% and 75% are assumed torepresent low, medium and high heterogeneity,respectively.
The significance of heterogeneity will be determined by p-value of I2 statistics; and a p-valueof <0.05 will be an evidence of heterogeneity.
If I 2 value is greater than 50%, we will use
Dersimonian and Liard random effect model todetermine the pooled estimates [15].
Sensitivity analysis will be carried out if there isheterogeneity between studies. Subgroup analysis will be done considering age group, regionand study design as grouping variables.
Small-study effects and publication bias will be
evaluated using the visual funnel plot test, andEgger’s and Begg’s statistics in the random effect model. In the evidence of small-study effect, trim and fill methods (Duval and Tweedie’s)
will be used to estimate the pooled prevalence
anemia and to estimate effect size of factors associated with childhood anemia, as the method

is robustness for estimation due to publication
bias [16]. Odds ratio with its 95% confidence
will be used to estimate the measure of association between anemia and associated factors.
The results will be presented both in text andForest plot.
Ethical statement and dissemination
As this systematic review will based on published data, ethical approval is not required.
The final report will be disseminated throughpublication in a peer-reviewed scientific journal
and will also be presented at Local, regional, national and international conferences.
DISCUSSION
Anemia has been shown to be a public healthproblem affecting low, middle and high-income
countries. It has also been significantly associated with negative consequences on health, socialand economic development [17, 18]. Globally, it
is a moderate to severe public health problem
in children.
As to the WHO 2015 estimate, the highest prevalence of anemia is found in children, 42.6% (95%
CI: 37- 47%) of children were suffering from anemia [19]. The consequence of anemia is worse,
as it limits the physical growth [20], mental [21],social [22] as well as the behavioral development of children [23].
Anemia prevalence data remains an important
indicator of public health as anemia is related
to morbidity and mortality, particularly in vulnerable segments of the population such as inchildren [10].
In Ethiopia, the magnitude of anemia is expected to be higher among children who are malnourished and economically disadvantaged; andamong children who are living in areas where
infectious diseases are highly prevalent anddrought is common.

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Z. Abebe, Wubet W. Takele, Degefaye Z. Anlay, Daniale T. Ekubagewargies, Z. Getaneh, M. Abebe, M. Melku
Prevalence of anemia and its associated factors among children in Ethiopia

Previous studies highlighted that the prevalenceof anemia among children in Ethiopia ranged
from 5.83% [24] to 83% [10].
At a global level, anemia prevalence data is a
useful indicator to assess the impact of widespread or highly effective interventions and to
track the progress made towards the goal of reducing anemia in children [25].
In order to design more adaptable intervention and control strategies to the local context,
national estimate of anemia and its associatedfactors needs to be generated. Given the importance of such data, there is no publishedmeta-analysis estimate on the prevalence ofanemia and its associated factors among children in Ethiopia.
Therefore, the authors believe that, this systematic review and meta-analysis will generate concrete evidence on the national burden of anemia and its associated factors among children.
The review will provide substantial evidence forthe government and other stakeholders working on child health; and it will help them in designing intervention and control strategies toreduce anemia in a better adaptable and more
effective manner.

Strengths••

Limitations•

A potential limitation of this systematic willbe the heterogeneity between publishedstudies that can be raised from differencesin study design, study period and geographic areas. Besides, studies reported in otherthan English language may not be included.

Amendments of the protocol•

In case there is a need to amend this protocol, the reasons for the amendment and thedate when it has been amended will be explicitly described.
Conflicts of Interest
The authors declared that they have no competing interests.
Availability of data and materials
Not applicable.
Authors’ contributions
MM, ZA, ZG and WWT: have been developing thesearch strategy;

STRENGTH AND LIMITATION
OF THIS SYSTEMATIC REVIEW•

for Systematic reviews and Meta-Analyses
protocol (PRISMA-P 2015 Statement).

This will be the first published systematic review and meta-analysis that comprehensively estimate the burden of anemia, and its associated factors among children in Ethiopia.
The involvement of experts from publichealth, biomedical and clinical fields during
searching, screening, appraisal, and data extraction processes will help to make the evidence generated more comprehensive.
The data reporting will adhere as to the guideline outlined in the Preferred Reporting Items

MM, ZA, ZG, DTE, WWT, DZA and MA: will search,
screen and appraise the studies, and will extract
the data;
MM, ZA, DZA, MA and WWT: will analyze the data;
ZA, MM, ZG, WWT and MA: involved during thedrafting of the protocol.
All authors read and approved the final protocol
for publication.
Funding Statement
No funding was secured for this review.

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Z. Abebe, Wubet W. Takele, Degefaye Z. Anlay, Daniale T. Ekubagewargies, Z. Getaneh, M. Abebe, M. Melku
Prevalence of anemia and its associated factors among children in Ethiopia

REFERENCES
1. Harika R, Faber M, Samuel F, Mulugeta A, Kimiywe
J, Eilander A. Are Low Intakes and Deficiencies in Iron,
Vitamin A, Zinc, and Iodine of Public Health Concern
in Ethiopian, Kenyan, Nigerian, and South African Children and Adolescents? Food and Nutrition Bulletin
2017; 38:405-427.
2. Herrador Z, Sordo L, Gadisa E, Buño A, Gómez-Rioja R, Iturzaeta JM, de Armas LF, Benito A, Aseffa A,
Moreno J, Cañavate C. Micronutrient Deficiencies andRelated Factors in School-Aged Children in Ethiopia:
A Cross-Sectional Study in Libo Kemkem and Fogera
Districts, Amhara Regional State. PLoS ONE 2014;
9:e112858.
3. Walter T. Effect of iron-deficiency anemia on cognitive skills and neuromaturation in infancy and childhood. Food and Nutrition Bulletin 2003; 24:S104-110.
4. WHO. Worldwide prevalence of anaemia 1993–2005.
Geneva: World Health Organization; 2008. Accessed at:
http://apps.who.int/iris/bitstream/10665/43894/1/
9789241596657_eng.pdf; accessed date: January 20,
2018.
5. Santos JN, Rates SPM, Lemos SMA, Lamounier JA.
Consequences of anemia on language development ofchildren from a public day care center. Rev Paul Pediatr
2009; 27:67-73.
6. Plessow R, Arora NK, Brunner B, Tzogiou C, Eichler
K, Brügger U, Wieser S. Social costs of iron deficiency
anemia in 6–59-month-old children in India. PLoS One
2015; 10:e0136581.
7. WHO. The global prevalence of anaemia in 2011. Geneva: World Health Organization; 2015. Accessed at:
http://www.who.int/nutrition/publications/micronutrients/global_prevalence_anaemia_2011/en/;
Accessed date: January 20, 2018.
8. Getaneh Z, Enawgaw B, Engidaye G, Seyoum M, Berhane M, Abebe Z, Asrie F, Melku M. Prevalence of anemia and associated factors among school children in
Gondar town public primary schools, northwest Ethiopia: A school based cross-sectiona lstudy. PLoS ONE
2017; 12:e0190151.
9. Deribew A, Alemseged F, Tessema F, Sena L, Birhanu
Z, Zeynudin A, Sudhakar M, Abdo N, Deribe K, Biadgilign S. Malaria and Under-Nutrition: A Community
Based Study Among Under-Five Children at Risk of Malaria, SouthWest Ethiopia. PLoS ONE 2010; 5:e10775.
10. Central Statistical Agency [Ethiopia] and ICF International. Ethiopia Demographic and Health Survey
2011. Addis Ababa, Ethiopia and Calverton,Maryland,
USA: Central Statistical Agency and ICF International.
2016.

11. Shamseer L, Moher D, Clarke M, Ghersi D, Liberati A, Petticrew M, Shekelle P, Stewart LA, PRISMA-P
Group. Preferred reporting items for systematic review
and meta-analysis protocols (PRISMA-P) 2015: elaboration and explanation. BMJ 2015; 350:g7647.
12. Munn Z, Moola S, Lisy K, Riitano D, Tufanaru C.
Methodological guidance for systematic reviews ofobservational epidemiological studies reporting prevalence and incidence data. International journal of evidence-based healthcare 2015; 13:147–153.
13. JBI. The Joanna Briggs Institute Critical Appraisal
tools for use in JBI Systematic Reviews: Checklist for
Analytical Cross Sectional Studies. 2016. Accessed at:
http://joannabriggs.org/research/critical-appraisaltools.html; accessed date: January 21, 2018.
14. Higgins J, Thompson SG. Quantifying heterogeneity in a meta‐analysis. Statistics in Medicine 2002;
21:1539-1558.
15. DerSimonian R, Laird N. Meta-analysis in clinical
trials. Controlled clinical trials 1986; 7:177-188.
16. Tweedie R, Duval S. A Nonparametric “Trim andFill” Method of Accounting for Publication Bias in Meta-Analysis. Journal of the American Statistical Association 2000; 95:89-98.
17. Smith RE Jr. The Clinical and Economic Burden of
Anemia. Am J Manag Care 2010; 16:S59-S66.
18. Alcázar L. The economic impact of anaemia in Peru.
Lima: Group for the Analysis of Development and Action Against Hunger; 2013. Accessed at: http://www.
grade.org.pe/upload/publicaciones/archivo/download/pubs/LIBROGRADE_ANEMIAENG.pdf; accessed
on: February 11, 2018.
19. WHO. The global prevalence of anaemia in 2011.
WHO, Geneva, Switzerland, 2015. Accessed at: http://
www.who.int/nutrition/publications/micronutrients/
global_prevalence_anaemia_2011/en/; Accessed date:February 11, 2018.
20. Soliman AT, De Sanctis V, Kalra S. Anemia andgrowth. Indian J Endocrinol Metab 2014; 18:S1-S5.
21. Hurtado EK, Claussen AH, Scott KG. Early childhood
anemia and mild or moderate mental retardation. Am
J Clin Nutr 1999; 69:115-119.
22. Chang S, Wang L, Wang Y, Brouwer ID, Kok FJ, Lozoff
B, Chen C. Iron-Deficiency Anemia in Infancy and Social
Emotional Development in Preschool-Aged ChineseChildren. Pediatrics 2011; 127:e927–e933.
23. Lozoff B, Corapci F, Burden MJ, Kaciroti N, AnguloBarroso R, Sazawal S, Black M. Preschool-Aged Children
with Iron Deficiency Anemia Show Altered Affect andBehavior The Journal of Nutrition 2007; 137:683–689.

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Z. Abebe, Wubet W. Takele, Degefaye Z. Anlay, Daniale T. Ekubagewargies, Z. Getaneh, M. Abebe, M. Melku
Prevalence of anemia and its associated factors among children in Ethiopia

24. Gashu D, Stoecker BJ, Adish A, Haki GD, Bougma K,
Marquis GS. Ethiopian pre-school children consuming a
predominantly unrefined plant-based diet have low prevalence of iron-deficiency anaemia. Public Health Nutrition
2016; 19:1834–1841.

25. Kraemer K, Zimmermann MB. Nutritional Anaemia. Basel,
Switzerland: Sight and Life Press; 2007. Available at: http://
ernaehrungsdenkwerkstatt.de/fileadmin/user_upload/EDWText/TextElemente/Ernaehrungswissenschaft/Naehrstoffe/nutritional_anemia_book.pdf; accessed on: Feb. 12, 2018.

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Acute myeloid leukemia with severe coagulation
disorder and concomitant central nervous system
bleeding – a clinical diagnostic case report
S. Hatzl1, B. Uhl1, M. Hinterramskogler2, S. Leber3, F. Eisner4,
M. Haring1, P. Jud5

Division of Hematology, Department of Internal Medicine, Medical University of Graz, Austria
Department of Anesthesiology and Intensive Care Medicine, Medical University of Graz, Austria
3
Department of Radiology, Medical University of Graz, Austria
4
Intensive Care Unit, Department of Internal Medicine, Medical University of Graz, Austria
5
Division of Angiology, Department of Internal Medicine, Medical University of Graz, Austria
1
2

INFO

ABSTRACT

Corresponding author:
Stefan Hatzl
Division of Hematology
Department of Internal Medicine
Medical University of Graz
AustriaE-mail: stefan.hatzl@medunigraz.at

We report a case of severe central nervous system
bleeding in a patient with acute monocytic leukemia.
The patient was admitted to our emergency department because of massive back pain and positivemeningeal signs. MR imaging yielded a spontaneous
epidural hematoma of the thoracic vertebral column.
Coagulation studies revealed fibrinogen levels below
the linear measuring range and blood smears showed
myeloid blast cells in the peripheral blood. The diagnosis of acute monocytic leukemia was confirmed byflow cytometric analysis. Despite of substitution withmore than 12 g fibrinogen per day over 3 days plasma
fibrinogen levels couldn’t be stabilized. After starting
the induction chemotherapy with cytarabine, laboratory coagulation test results were improved. Despite
all intensive medical efforts, the patient died due tocerebral epidural hematoma.

Key words:
acute myeloid leukemia, disseminatedintravascular coagulopathy, bleeding
Conflict of interest:
All authors declare that noconflict of interest exists.
Funding:
This research received no specific grantfrom any funding agency in the public,commercial, or not-for-profit sectors.

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S. Hatzl, B. Uhl, M. Hinterramskogler, S. Leber, F. Eisner, M. Haring, P. Jud
Acute myeloid leukemia with severe coagulation disorder and central nervous system bleeding

INTRODUCTION

Figure 1A

Acute myeloid leukemia (AML) is a result of malignant transformation of hematopoietic stem orprogenitor cells and is the most frequent acuteleukemia in adults. (1, 2) DIC is one of the mostfeared and fatal complication in acute promyelocytic leukemia (APL), however it is rare in allother subtypes of acute myelogenous leukemia(AML). (3) The incidence of disseminated intravascular coagulation (DIC) induced by hematologic malignancies was 12.7% in a large cohortof patients, studied by Okajima et al., with an
overall mortality of more than 50% (4) A typicalfeature of DIC in acute leukemia is hyperfibrinolysis and reduction of natural anticoagulants
like protein C and antithrombin III. (5, 6)

Magnetic resonanceimaging of the thoracic,
lumbar and sacral spine*

In our case report we show that early eradication of the leukemia by using high dose chemotherapy is the foremost option to control DIC andimprove the coagulation test results as well as
overall survival.
CLINICAL-DIAGNOSTIC CASE
A 36-year-old woman presented to the emergency department with acute back pain, fatigue
and fever. Physical examination revealed positive meningeal signs (Lhermitte, Laseque). For
further evaluation of the back pain, a magnetic
resonance imaging was performed, which yielded massive epidural bleeding from the first thoracic vertebral body to the fourth lumbar vertebral body. (Figure 1 A)
Laboratory testing revealed leukocytosis [18,520/µL] and mild thrombocytopenia [100.000 G/l],
additionally a severely decreased fibrinogen level[< 80 mg/dL] using the Clauss method as well asa massively elevated D-Dimer value of >33 mg
FEU/L could be detected. A hematological causeof hyperfibrinolysis was suspected and bloodsmear as well as flow-cytometric analysis (FACS)
was performed. Because of the severe clinical andlaboratory findings pointing towards disseminated*Revealing epidural hematoma reaching fromthoracic vertebral level 1 to sacral vertebral
level 1 and compressing thoracic spinal cord
as well as conus medullaris (arrows)

intravascular coagulation, the most probable diagnosis was APL and a therapy with all-trans-retinoic
acid 45mg/m2/day was administered. The patient
was transferred to the intensive care unit and substitution of fibrinogen was initiated.

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S. Hatzl, B. Uhl, M. Hinterramskogler, S. Leber, F. Eisner, M. Haring, P. Jud
Acute myeloid leukemia with severe coagulation disorder and central nervous system bleeding

During the first day of hospitalization, a total
amount of 16 g fibrinogen was given, despite
substitution fibrinogen levels in plasma remained undetectable, no signs of bleeding could
be detected. The patients’ condition improved
and was hemodynamically stable with pain controlled by continuous morphine infusion.
During the second day, results of the blood smears
including cytochemistry revealed 10% myeloid
blasts with Auer rods, 15% atypical monocytes,
40% lymphocytes as well as 35% band stage andmature neutrophil granulocytes. (Figure 1B) FACS
confirmed these results resulting in the diagnosis of an acute monocytic leukemia (FAB M4 according to French American British classification
Figure 1Bof acute leukemia). On the second day, again 12 g
fibrinogen and 1 g tranexamic acid were administered without any effect on plasma fibrinogenlevel (Figures 1C; 2A).
After confirmation of the diagnosis FAB M4, abone marrow biopsy was performed and chemotherapy with cytarabine 200mg/m²/day wasadministered. Due to DIC and possible tumor
lysis syndrome, daunorubicine 60mg/m² was
postponed to day 5.
Directly after administration of cytarabine and fibrinogen, plasma fibrinogen levels increased into
normal ranges for the first time after initial diagnosis (Figure 1C). Due to chemotherapy, platelet
count started to decrease and one irradiated

Peripheral blood smear showing myeloid differentiated blast cells

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S. Hatzl, B. Uhl, M. Hinterramskogler, S. Leber, F. Eisner, M. Haring, P. Jud
Acute myeloid leukemia with severe coagulation disorder and central nervous system bleeding

Figure 1C

Time course oflevel and thrombocyte countig fibrinogenr 1A
during AML induction chemotherapy

thrombocyte concentrate wastoF administered
1C
keep thrombocyte count above 50.000 G/L.
On the third day of hospitalization, the patientdeveloped pronounced macrohematuria andmultiple disseminated skin hematomas. Four
irradiated erythrocyte concentrates were needed to maintain a hemoglobin level of 7.0 g/L.
Subsequently, the patients’ neurological deficits
were relieved, reaching a Glasgow Coma Scalescore of 15 points.
However, on the morning of the fourth day thepatient was comatose and did not respond to any
stimulation reaching a Glasgow coma scale of 3
points. Due to neurological impairment, immediate endotracheal intubation was performed

and total intravenous anesthesia with propofol
and remifentanil as continuous infusion was
initiated.
Cranial computer tomography (CCT) revealed atemporally right sided newly developed epidural
bleeding causing a midline-shift of the brain towards left. Emergency craniotomy was successfully performed for intracranial decompression.
A Thrombelastography (TEG) was performed to
re-evaluate the coagulation status again revealing low fibrinogen levels and additionally a newly
developed thrombocytopathia/thrombocytopenia was detected. This triggered a subsequent
continuous infusion of fibrinogen (3 g/h) together with administration of tranexamic acid3g daily plus Factor XIII concentrate with the aim

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S. Hatzl, B. Uhl, M. Hinterramskogler, S. Leber, F. Eisner, M. Haring, P. Jud
Acute myeloid leukemia with severe coagulation disorder and central nervous system bleeding

Figure 2A

Initial TEG (citrated kaolin
and citrated functional
fibrinogen)*

Figure 2B*Before administration of fibrinogenand factor XIII concentrate

Follow Up TEG day 4(citrated kaolin and citratedfunctional fibrinogen)**After administration of fibrinogenand factor XIII concentrate

to enhance the clot stability which was achieved
as demonstrated in following TEGs. (Figure 2B)
On the fifth day after the diagnosis of AML, intravenous anesthesia was stopped and the patient was extubated. On this day, the patient reached again a Glasgow Coma Scale score of 15
points, the coagulation status remained stableand fibrinogen substitution was stopped. Seven
hours later, however, the patient’s neurologicalstatus deteriorated and mechanical ventilation
was necessary again. CCT scan showed a massive haemorrhagic infarction of the occipital lobe
with massive brain edema.
In the evening of the sixth day after the diagnosis, electroencephalography revealed zero-line
consistent with the brain death of the patient.

DISCUSSION
The standard of care therapy for acute myeloid
leukemia is a 7 + 3 days chemotherapy regimen including continuous infusion of cytarabine (200 mg/m²) for seven days combined withbolus infusion of an anthracycline for threedays. (1)All other forms of acute leukemia like acute lymphoblastic leukemia and acute promyelocytic
leukemia also respond to administration of cytarabine. (7, 8)
The paradigm for treating acute promyelocytic
leukemia with impaired coagulation test results
is that even if acute promyelocytic leukemia isjust suspected, treatment with all-trans retinoic
acid should be immediately started.

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S. Hatzl, B. Uhl, M. Hinterramskogler, S. Leber, F. Eisner, M. Haring, P. Jud
Acute myeloid leukemia with severe coagulation disorder and central nervous system bleeding

However, the main issue in treatment of patients with acute leukemia associated DIC and without correct subtype diagnosis is that all-transretinoic acid is only effective in acute promyelocytic leukemia.
In our case, the patient did not receive any effective treatment for more than one day with active central nervous bleeding. Additionally, plasma fibrinogen level did not respond to massive
replacement. We could observe that coagulation parameters ameliorated and leukemic blast
cells decreased after starting chemotherapy. In
summary, the main goal to stop DIC and bleeding complications must be the early eradication of the leukemia. Coagulation managementin these patients is very difficult to handle, thus
by using frequent TEG testing a better control of
bleeding complications could be achieved.
We conclude that immediate continuous infusion with cytarabine would have been adequate
to start with because of expectable response inany form of acute leukemia. This might have allowed an early control of DIC even before receiving results of acute leukemia subtype diagnostics. An early detection of DIC and early
improvement of the coagulation status can potentially improve the outcome of such patients.
TAKE HOME MESSAGES/LEARNING POINTS•

In case of unknown differentiation of acuteleukaemia, Cytarabine can be used as thecyto­reductive agent of choice because of thehigh effectiveness in all forms of acute leukaemia whether lymphoblastic or myelogenous.
•

TEG result-based consequent substitution of
pro-coagulative agents like fibrinogen, factor
XIII and tranexamic acid independently may
help to improve coagulation in such patientswith DIC and underlying haematologicalmalignancy.

REFERENCES
1. Dohner H, Weisdorf DJ, Bloomfield CD. Acute Myeloid
Leukemia. N Engl J Med. 2015 Sep 17;373(12):1136-52.
2. Hatzl S, Geiger O, Kuepper MK, Caraffini V, Seime T,
Furlan T, et al. Increased Expression of miR-23a Mediates a Loss of Expression in the RAF Kinase Inhibitor
Protein RKIP. Cancer Res. 2016 Jun 15;76(12):3644-54.
3. Lo-Coco F, Ammatuna E, Montesinos P, Sanz MA. Acutepromyelocytic leukemia: recent advances in diagnosis
and management. Semin Oncol. 2008 Aug;35(4):401-9.
4. Okajima K, Sakamoto Y, Uchiba M. Heterogeneity
in the incidence and clinical manifestations of disseminated intravascular coagulation: a study of 204
cases. Am J Hematol. 2000 Nov;65(3):215-22.
5. Franchini M, Di Minno MN, Coppola A. Disseminated intravascular coagulation in hematologic malignancies. Semin Thromb Hemost. 2010 Jun;36(4):388-403.
6. Hunt BJ. Bleeding and coagulopathies in critical care. N
Engl J Med. 2014 Feb 27;370(9):847-59.
7. Gokbuget N, Hoelzer D. Treatment of adult acutelymphoblastic leukemia. Semin Hematol. 2009
Jan;46(1):64-75.
8. Lo-Coco F, Avvisati G, Vignetti M, Breccia M, Gallo
E, Rambaldi A, et al. Front-line treatment of acutepromyelocytic leukemia with AIDA induction followedby risk-adapted consolidation for adults younger than
61 years: results of the AIDA-2000 trial of the GIMEMA Group. Blood. 2010 Oct 28;116(17):3171-9.

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Vitamin B12 immunoassay interference
in a patient with multiple myeloma –troubleshooting in a two step reagent kit
based on enhanced chemiluminescence testing
V. Pant, A. Tumbapo, B. Kumar Yadav

Department of Biochemistry, Institute of Medicine (IOM), Tribhuvan University Teaching Hospital (TUTH),
Kathmandu, Nepal

INFO

ABSTRACT

Corresponding author:
Dr. Vivek Pant
Department of Biochemistry
Institute of Medicine (IOM)
Tribhuvan University Teaching Hospital (TUTH)
Kathmandu, Nepal
E-mail: drvpant@gmail.com

Immunoassays are widely used for quantification ofserum analytes however they are subjected to interference by endogenous antibodies. The laboratory
procedures used to identify these endogenous antibodies is the demonstration of response to dilution or
use of nonimmunoglobulin protein to block the interfering antibodies or the use of an alternate immunoassay. We report a clinical-diagnostic situation where
serum vitamin B12 determination was interfered in
an immunoassay due to excess of endogenous antibodies from monoclonal gammopathy that resulted
in excess of analyte concentration. Reporting of suchcases may be beneficial when assaying sera of multiple myeloma to avoid false results and in addition
to avoid costs due to unnecessary repeat testing andfurther delay reporting of results.

Key words:
endogenous antibody, monoclonal
gammopathy, immunoassay
Informed consent:
Informed consent was given by the patient
for the publication of this case report.

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V. Pant, A. Tumbapo, B. Kumar Yadav
Vitamin B12 immunoassay interference in a patient with multiple myeloma

CLINICAL-DIAGNOSTIC CASE
Quantification of Serum B12 was ordered by theon-duty doctor from a forty-five years old female inpatient admitted at medical ward with
provisional diagnosis of multiple myeloma.
The patient serum sample was processed fortesting B12 using Vitros “ECiQ Immunodiagnostic
system” Enhanced Chemiluminescence Immuno
assay (ECI) after performing daily maintenanceand running internal quality control samples from
Bio-Rad, which were found to be within the acceptable range [Level 1 range = 268-608 pg/mL].
The analyser failed to give a result for the sample
and the error message displayed “No Result.”
Repeated testing of the sample failed again. A
new sample from the patient was requested andFigure 1

Immunofixation showing
monoclonal gammopathy in
gamma globulin region andkappa light chains (arrows)

processed, yielding again no result. The test was
repeated with a dilution series of this sample,
after 25 folds dilution of the sample a result of3512.0 pg/mL was obtained (Normal range: 239931 pg/mL) [1].
After reporting results to the clinician, the patient’s medical records were reviewed, and it
was observed that the patient was diagnosed
with grade III Multiple Myeloma. Serum protein
electrophoresis on cellulose acetate paper revealed a significant M-gradient. On immunofixation, the immunological nature of M-gradientwas found to be a monoclonal gammopathy in
gamma globulin region with corresponding lightchain correlates in kappa light chains [Figure 1].
Beta 2 microglobulin in serum was increased up
to 11494 ng/mL (Reference: 609.0-2366.0 ng/mL) [1], based on which the grading of multiple
myeloma was done. Beta 2 microglobulin wasquantified using Siemens Nephelometer. There
was absence of myeloproliferative disorder,
eosinophilia, and iatrogenic intake of cyanocobalamin in this patient. Absence of heterophileantibody interference was confirmed using heterophile antibody blocking tube.
To eliminate the interference generated by endogenous antibodies from multiple myeloma,
the serum of patient was then subjected to precipitation with Polyethylene Glycol (PEG) 6000
to remove interfering antibodies after performing control studies with another patient’s serum

Table 1

Testing procedure and obtained values of serum vitamin B12
Testing of Serum B12

Value (pg/mL)

Undiluted serum sample (repeat testing)

Not determinable

Diluted serum sample (1:25)

3521

PEG precipitation (1:2)

887(1:25) = 25 times dilution; (1:2) = 2 times dilution

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V. Pant, A. Tumbapo, B. Kumar Yadav
Vitamin B12 immunoassay interference in a patient with multiple myeloma

sample. This resulted in the value of 887.0 pg/mL
which was within normal range. (Normal range:
239-931 pg/mL) [1]. Values of serum vitamin B12
obtained after various methods applied in ECI isshown in Table 1.
DISCUSSION
Immunoassays are based on antigen-antibody
reaction. Despite advances in immunoassay,
they may be subjected to interference, depending on the assay used. According to International
Federation of Clinical Chemistry (IFCC), interference in immunoassay is described as a systematic error of analytic measurement caused by asample tested, which does not allow for a signal in the measuring system. [2] Interferences
can rise from the presence of endogenous antibodies like heterophilic antibodies, anti-animal
antibodies or autoantibodies. [3] In most cases,
these interfering antibodies tend to produce
false high values of the analyte tested. [4] Vitros
Enhanced Chemiluminescence Immunoassay(ECI) is based on competitive binding immunoassay technique.
First, there is a competition between vitamin
B12 present in the patient’s serum sample witha Horseradish peroxidase (HRP)-labeled vitamin
B12 conjugate for binding sites on a biotinylated
Intrinsic Factor. When this competitive binding
occurs, the vitamin B12 Intrinsic Factor complexes are formed which are captured by streptavidin on the wells. The complex bound with HRP
thus formed is then measured by a luminescent
reaction. This amount of complex bound with
HRP is indirectly proportional to the concentration of vitamin B12 present. [5] We report a clinical-diagnostic situation where serum vitamin
B12 testing was interfered in this immunoassay
due to excess endogenous antibodies produced
from monoclonal gammopathy which resulted
in excess analyte concentration.

On average, 15-20 samples are analysed daily for
Vitamin B12 in our Central Biochemistry Laboratoryof the Institute of Medicine by Vitros Diagnostic
immunoassay which is based on the principle of
ECI. High serum vitamin B12 concentrations may
be seen in myeloproliferative disorders, hyper
eosinophilic syndromes, hepatic diseases, renal
disease, and presence of heterophile antibodies
or iatrogenic intake of cyanocobalamin for long
duration. [6]In this patient, myeloproliferative disorder
was absent as bone marrow biopsy was normal. There was no raised eosinophil count inblood and patient had normal liver and renal
function test. The patient was not taking cyanocobalamin during and six months before thesampling. Absence of heterophilic antibodies
interference for the cause of the increased vitamin B12 in our patient was confirmed using
heterophilic antibody blocking tubes. Possible
cause for the excess vitamin B12 in this patientserum was due to high endogenous antibodies
saturating all available sites on the IF in the first
incubation step.
Subsequently, in the second incubation step
there were no vacant sites on the IF for Vitamin
B12 to bind. Thus, no signal was generated. In thiscase the patient has high endogenous antibodies from monoclonal gammopathy. Presence ofexcess IgG immunoglobulin also forms cobalamin complex and results in high value of vitamin
B12 in serum and interferes with its assay. [7]High vitamin B12 concentration in this patient
was due to immune complexes composed of
IgG as seen in immunofixation and to prove thiswe treated the serum with PEG and measured
B12 in the supernatant. PEG precipitation test
can be used as an alternative to size exclusion
chromatography and is an easy method to confirm presence of immunocomplex. [8] An equal
quantity of 25% PEG was added to 200 μl of theB12-elevated serum sample.

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V. Pant, A. Tumbapo, B. Kumar Yadav
Vitamin B12 immunoassay interference in a patient with multiple myeloma

After full mixing, the mixture was centrifuged
at 1500×g for 30 minutes, and then the supernatant was isolated for B12 analysis. This gave
a value of 887.0 pg/mL which was within thenormal range. This decrease in serum B12 level
after PEG treatment can be due to effect of
PEG on assay system and not precipitation ofinterfering antibody.
So, control studies with other samples weredone to investigate this observation and no effect of PEG was seen in the assay system. Thus,
we present a case of positive interference in the
Ortho B12 assay because of paraprotein interference in a patient with monoclonal gammopathy.
To our knowledge, this particular paraprotein
interference has not been previously reported.
Thus, for troubleshooting for assay interference
when other samples can be quantitatively measured by two step reagent kit based on ECI, various steps can be taken.
Obtaining history of patient regarding the clinical status and dilution of sample should be done
if there is a prior history of intravenous administration of Vitamin B12. Likewise, the single
step incubation method may be used. Others
have also used heterophilic antibody blocking
tubes, protein G–sepharose, size-exclusion High
Performance Liquid Chromatography (HPLC),
sodium dodecyl sulphate–polyacrylamide gelelectrophoresis (SDS-PAGE) and Matrix-Assisted
Laser Desorption/Ionization Time-of-Flight Mass
Spectrometry (MALDI-TOF MS). [7]But these methods are expensive and time consuming. The proper algorithmic knowledge isessential. Communication and collaborative approach between clinicians and laboratory staff
is crucial in such cases yielding benefit for thepatient. Furthermore, reporting these types ofcases help clinicians and laboratory staffs to beaware of unnecessary repeated investigations
or even false interpretation of results.

TAKE HOME MESSAGES/LEARNING POINTS•This case presents a positive interference in
the Ortho B12 assay due to paraprotein interference in a patient with monoclonal gammopathy. Laboratory staff should be awareof such a phenomenon to avoid false results,
unnecessary delay in reporting and unnecessary reagent wastage due to repeat tests.

•

In such situations, the laboratory staff should
consider dilution of sample and PEG precipitation before analysis.

REFERENCES
1. Price CP, Bossuyt PM, Bruns DE. Introduction to laboratory medicine and evidence-based laboratory medicine.
Tietz textbook of clinical chemistry and molecular diagnostics, 4th ed. St. Louis: Elsevier Saunders. 2006:323-51.
2. Buttner J, Borth CR, Boutwell JH, Broughton PM,
Bowyer RC. International Federation of Clinical Chemistry. Clinica Chimica Acta. 1979; 98:135F-62F.
3. Bolstad N, Warren DJ, Nustad K. Heterophilic antibody
interference in immunometric assays. Best Pract Res Clin
Endocrinol Metab. 2013 ;27(5):647-61.
4. Gulbahar O, Konca Degertekin C, Akturk M, Yalcin MM,
Kalan I, Atikeler GF et al. A case with immunoassay interferences in the measurement of multiple hormones. Clin
Endocrinol Metab. 2015 Apr 21;100(6):2147-53.
5. Wild D, editor. The immunoassay handbook: theory
and applications of ligand binding, ELISA and related
techniques. Newnes; 2013 Jan 21.
6. Ermens AA, Vlasveld LT, Lindemans J. Significance ofelevated cobalamin (vitamin B12) levels in blood. Clin
Biochem. 2003 Nov 30;36(8):585-90.
7. Bowen RA, Drake SK, Vanjani R, Huey ED, Grafman J,
Horne MK, 3rd. Markedly increased vitamin B12 concentrations attributable to IgG-IgM-vitamin B12 immune
complexes. Clin Chem. 2006;52(11):2107-14.
8. Remacha AF, Zapico E, Sarda MP, Rojas E, Simó M,
Remacha J et al. Immune complexes and persistenthigh levels of serum vitamin B12. Int j Lab Hematol.
2014 Feb 1;36(1):92-98

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Editor-in-chief
János Kappelmayer
Department of Laboratory Medicine, University of Debrecen, Hungary

Assistant Editor 				Case Editor			Harjit Pal Bhattoa				Department of Laboratory Medicine		
University of Debrecen, Hungary		Reinhard B. Raggam
Department of Internal Medicine
Division of Angiology, University of Graz, Austria

Editorial Board

Khosrow Adeli, The Hospital for Sick Children, University of Toronto, Canada
Borut Božič, University Medical Center, Lubljana, Slovenia
Edgard Delvin, CHU Sainte-Justine Research Center, Montréal, Québec, Canada
Nilda E. Fink, Universidad Nacional de La Plata, Argentina
Ronda Greaves, School of Health and Biomedical Sciences, RMIT University, Victoria, Australia
Mike Hallworth, Shrewsbury, United Kingdom
Andrea R. Horvath, Prince of Wales Hospital and School of Medical Sciences, University of New
South Wales, Sydney, Australia
Ellis Jacobs, Abbott, Orlando, FL, USA
Allan S. Jaffe, Mayo Clinic, Rochester, USA
Bruce Jordan, Roche Diagnostics, Rotkreuz, Switzerland
Gábor L. Kovács, University of Pécs, Hungary
Evelyn Koay, National University, Singapore
Tamas Kőszegi, University of Pécs, Hungary
Janja Marc, University of Ljubljana, Slovenia
Gary Myers, Joint Committee for Traceability in Laboratory Medicine, USA
Tomris Ozben, Akdeniz University, Antalya, Turkey
Maria D. Pasic, Laboratory Medicine and Pathobiology, University of Toronto, Canada
Maria del C. Pasquel Carrera, College of Chemists, Biochemists and Pharmacists, Pichincha, Ecuador
Oliver Racz, University of Kosice, Slovakia
Rosa Sierra Amor, Laboratorio Laquims, Veracruz, Mexico
Sanja Stankovic, Institute of Medical Biochemistry, Clinical Center of Serbia, Belgrade, Serbia
Danyal Syed, Ryancenter, New York, USA
Grazyna Sypniewska, Collegium Medicum, NC University, Bydgoszcz, Poland
Jillian R. Tate, Queensland Royal Brisbane and Women’s Hospital, Herston, Australia
Peter Vervaart, LabMed Consulting, Australia
Stacy E. Walz, Arkansas State University, USA

				

			

				
		
		

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