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\begin{document}

\title{Differences in analytical standardization may lead to misinterpretation
and medical flaws}



\author[1]{Jacquelien Hillebrand}%
\author[1]{Marleen de Lange}%
\author[1]{Norah van Mello}%
\author[2]{Edwin Ten Boekel}%
\author[1]{Annemieke Heijboer}%
\affil[1]{Amsterdam UMC Location AMC}%
\affil[2]{Northwest Clinics Alkmaar}%


\vspace{-1em}



  
  \date{June 10, 2024}


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\textbf{Differences in analytical standardization may lead to
misinterpretation and medical flaws}

Jacquelien J. Hillebrand\textsubscript{a}\textsuperscript{*}, Marleen.
E. de Lange\textsubscript{b}, Norah M. van Mello\textsubscript{b} ,
Edwin ten Boekel\textsubscript{c}, Annemieke C.
Heijboer\textsubscript{a,d}

Affiliations:

a Amsterdam University Medical Centers, University of Amsterdam,
Endocrine Laboratory, Department of Clinical Chemistry, Amsterdam
Gastroenterology Endocrinology Metabolism Meibergdreef 9, 1105 AZ
Amsterdam, the Netherlands. j.j.hillebrand@amsterdamumc.nl

b Amsterdam University Medical Centers, University of Amsterdam,
Department of Obstetrics and Gynecology, Meibergdreef 9, 1105 AZ
Amsterdam, the Netherlands. m.e.delange@amsterdamumc.nl,
n.vanmello@amsterdamumc.nl.

c Northwest Clinics Alkmaar, Department Laboratory for Clinical
Chemistry, Hematology and Immunology, Wilhelminalaan 12, 1815 JD
Alkmaar, The Netherlands. E.ten.boekel@nwz.nl

d Amsterdam University Medical Centers, Vrije Universiteit Amsterdam,
Endocrine Laboratory, Department of Clinical Chemistry, Amsterdam
Gastroenterology Endocrinology Metabolism, Meibergdreef 9, 1105 AZ
Amsterdam, the Netherlands. a.heijboer@amsterdamumc.nl

\textbf{*Corresponding author: Jacquelien J Hillebrand, PhD}

Amsterdam University Medical Centers, University of Amsterdam, Endocrine
Laboratory, Department of Clinical Chemistry, Amsterdam Gastroenterology
Endocrinology Metabolism, Meibergdreef 9, 1105 AZ Amsterdam, the
Netherlands

Telephone +31 20 56 63127, Fax +31 20 69 17682,
j.j.hillebrand@amsterdamumc.nl

Short title: Differences in analytical standardization

Methods to measure hormones in serum are notorious for their differences
in analytical standardization, which may lead to different laboratory
results for the same blood sample. Although not a new finding, many
healthcare professionals may not be aware of it. Hormones such as
glycoprotein hCG circulate in many isoforms or variants in the serum,
fluctuating with physiological state or illness.\textsuperscript{1} For
hCG, not only the intact heterodimer hCG, but also the free \selectlanguage{greek}α \selectlanguage{english}and \selectlanguage{greek}β
\selectlanguage{english}chain, (nicked) degradation products and (hyper)glycosylated variants
occur in the serum. This great variety of immunoreactive hCG complicates
analytical standardization of the laboratory assay, in absence of a
reference ``gold standard'' analytical method and higher-order reference
materials. Indeed, (commercially available) hCG immunoassays do not
detect hCG variants to the same extent.\textsuperscript{2} In 1994
international initiatives started to improve hCG standardization. This
led i.a. to clarified nomenclature, reference reagents, and a highly
purified fifth international reference standard (IS 07/364) to replace
the impure fourth standard (WHO 75/189).\textsuperscript{3} Some IVD
manufacturers, like Beckman Coulter responded by re-standardizing their
immunoassay, but unfortunately others, like Roche Diagnostics did not.

Many medical decisions depend on laboratory results. Clinical guidelines
on e.g. diagnosis and management of ectopic pregnancy describe hCG
ranges for treatment ranges and targets during follow-up, but often
without mentioning assay differences.\textsuperscript{4,5} Given the
standardization differences, specific assay information and assay
differences should be mentioned in both original studies and clinical
guidelines. As diagnostic work-up and post-treatment follow-up of
patients is not always performed in a single hospital, knowledge of
assay differences is important. Also the percentage decrease in hCG
during follow-up is hard to interpret and may cause incorrect medical
decisions when different assays are used.

This can be illustrated by the following case of a 39-year-old woman
presenting with abdominal pain in our hospital. She reported being five
weeks pregnant, serum hCG was 870 U/L. Transvaginal ultrasound
examination confirmed ectopic pregnancy. Because of a history of complex
abdominal surgeries, MTX treatment (50 mg/m\textsuperscript{2} im) was
preferred over laparoscopic surgery. Seven days after MTX treatment
serum hCG was 1824 U/L (measured in another laboratory). This apparent
rise in hCG led to a second treatment with MTX (50
mg/m\textsuperscript{2} im) in our hospital the day after. After the
second MTX treatment was administered, the hCG level measured in our
laboratory, came back as 406 U/L, showing actually a good response
(\textgreater{}15\% reduction) to initial MTX treatment. Only then it
was realized that both laboratories used different immunoassays for
total hCG. Retrospective analysis indeed showed significant reduction in
hCG over time for both assays (Figure 1). Fortunately, the patient
experienced no complications of the unnecessary MTX treatment.

In conclusion, erroneous interpretation of hCG results and incorrect
medical decisions can be prevented by state-of-the-art standardization
of (commercial) assays. We urge IVD manufacturers to act and take
responsibility. Meanwhile one should be aware of the existence of assay
differences for hCG as well as for other hormones to prevent incorrect
interpretation of laboratory results in clinical practice, research and
during guideline development.



\textbf{Acknowledgements}

Dr J Fischer, Amsterdam UMC, University of Amsterdam, Department of
Clinical Chemistry, Amsterdam, The Netherlands for laboratory analyses.

\textbf{Disclosure of interest}

none

\textbf{Contribution to authorship}

\textbf{J.J. Hillebrand:} conceptualization, investigation,
writing-original draft preparation, \textbf{M.E. de Lange, N.M. van
Mello, E. ten Boekel:} investigation, writing-reviewing and
editing,\textbf{A.C. Heijboer:} conceptualization, writing-reviewing and
editing.

\textbf{Ethical approval}

This letter includes a case description. The patient involved gave
informed consent for publishing.

\textbf{Funding}

none

References:

Stenman UH, Tiitinen A, Alfthan H, Valmu L. The classification,
functions and clinical use of different isoforms of HCG. \emph{Hum
Reprod Update} 2006; \textbf{12} : 769-84. DOI:~10.1093/humupd/dml029

Whittington\textsuperscript{~}J,~ Fantz CR,~ Gronowski AM,~McCudden
C,~Mullins R,~Sokoll L,~et al. The analytical specificity of human
chorionic gonadotropin assays determined using WHO International
Reference Reagents. \emph{Clin Chim Acta} 2010; \textbf{411} : 81-5.
DOI:~10.1016/j.cca.2009.10.009

Sturgeon CM, Berger P, Bidart JM, Birken S, Burns C, Norman RJ, et al.;
IFCC Working Group on hCG. Differences in recognition of the 1st WHO
international reference reagents for hCG-related isoforms by diagnostic
immunoassays for human chorionic gonadotropin. \emph{Clin Chem} 2009;
\textbf{55} : 1484-91. DOI:~10.1373/clinchem.2009.124578

Elson CJ, Salim R, Potdar N, Chetty, M, Ross JA, Kirk EJ on behalf of
the Royal College of Obstetricians and Gynaecologists. Diagnosis and
management of ectopic pregnancy. \emph{BJOG} 2016; \textbf{123} :
e15-e55. DOI:~10.1111/1471-0528.14189

American College of Obstetricians and Gynecologists.: Tubal Ectopic
Pregnancy, Obstetrics \& Gynecology: \emph{Obstet Gynecol}
2018;\textbf{131} : e91-e103. DOI:~10.1097/AOG.0000000000002560

Figure 1. hCG concentrations following MTX treatment in a patient with
ectopic pregnancy.

HCG was measured (partly in retrospect) in laboratory 1 with the Roche
Diagnostics hCG+\selectlanguage{greek}β \selectlanguage{english}assay (WHO 75/189, triangles, dashed line) assay and
in laboratory 2 with the Beckman Coulter total \selectlanguage{greek}β\selectlanguage{english}hCG assay (IS 07/364,
squares, dashed line). The solid line represent the hCG concentrations
as interpreted during treatment follow-up. MTX was given following blood
sampling at day 1 and day 8.\selectlanguage{english}
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