An Immunoassay for Human Chorionic Gonadotropin in Cerebrospinal Fluid: Validation of a Modified-Approved Method for Accreditation by the College of American Pathologists

Introduction

Intracranial germ cell tumors are primary malignant central nervous system (CNS) tumors, which account for 0.5 to 3.0% of pediatric brain tumors in North America and Europe.^[1] In Asian countries, the incidence of these tumors is even higher, representing up to 11% of all pediatric CNS tumors.^[2] These tumors can be divided into two types, germinomas and nongerminomatous germ cells, both of which have a male preponderance of between 2:1 to 3:1 and tend to arise along the anatomical midline.^[1,3] Measurement of serum human chorionic gonadotropin (hCG) is an important adjunct method in the differential diagnosis of these tumors when the lesions contain syncytiotrophoblastic cells, which secrete hCG.^[4] Due to the low concentration in serum, the detection of hCG concentrations in cerebrospinal fluid (CSF) is a more robust and sensitive indicator of the presence of tumors and can be a reliable source for guiding treatment and monitoring response to treatment.^[5-7]

Currently, all quantitative hCG assays in China are approved by the China Food and Drug Administration (FDA) for application in serum only. Furthermore, it is important to note that matrix effects can influence test results when alternative sample types are used. Therefore, standard methods used outside their intended scope, and validated methods that are subsequently modified, should be validated. The accreditation program of the College of American Pathologists (CAP) states, in the common checklist, that modified FDA-cleared/approved tests or laboratory-developed tests (LDTs) should be validated for the assay before clinical use.

Here, we validated an immunoassay for the detection of hCG in CSF by using a Roche Diagnostics Cobas e 602 analyzer and illustrated a model to validate modified-approved methods in agreement with the CAP accreditation requirement, according to LDTs.

Methods

Results

The LoQs for hCG were determined to be 0.25 IU/L. The intra-assay precision was 0.64 and 0.57% and the intermediate imprecision was 4.26 and 3.54% at the concentration 10 and 250 IU/L, respectively. Recovery ranged from 109 to 123%. The dynamic range for hCG assay was set at 0.2 to 1,200 IU/L. The CSF hCG level in 120 samples ranged from < 0.1 U/L to 0.55 U/L. The 95th percentile value was 0.4 IU/L. All results of the validation procedure are reported in ►Table 1. The details of both the imprecision and recovery studies are detailed in ►Table 2, while linearity is reported in ►Fig. 1. Finally, demographic characteristics and hCG concentrations in CSF are presented in ►Table 3. Differences in concentrations among the four groups were not statistically significant (p > 0.05).

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Fig. 1

Linearity of hCG. hCG, human chorionic gonadotropin.

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Ten patients were ranging from 10 to 32 years at diagnosis. Eight patients were diagnosed with neurohypophyseal germinoma or intracranial ectopic germinoma on the basis of a satisfactory response to treatment with radiotherapy. Diagnosis of two cases of nongerminomatous germ cell tumors was confirmed by the histopathological findings. Six patients were positive for hCG in both serum and CSF before treatment, but cases 1, 2, 4, and 5 showed elevated hCG only in the CSF. After therapy, there was a marked drop of the CSF hCG level in three patients. The clinical data of all 10 patients are summarized in ►Table 4.

Discussion

Indeed, the validation of an immunoassay for hCG in CSF has been previously described,^[9,10] but that validation was not performed as per the CAP or any other international accreditation. The CAP checklist provides the criteria needed to validate a laboratory method in terms of performance characteristics and clinical claims. Clinical claims include statements regarding the diagnostic sensitivity and specificity of a test.

To adequately support a claim about the ability to predict the risk of germinoma, we defined the RIs and processed the clinical validation, which included 10 positive clinically diagnosed cases. An established reference value of CSF hCG is useful for detecting the early slight elevation of CSF hCG. When examining the CSF hCG level using the “2 IU/L” upper limit of serum male normal, patient 1 would has been misdiagnosed. With no CSF measurements, patients 1, 2, 4, and 5 would also escape diagnosis because of the low serum hCG concentrations. Repetitive CSF hCG levels in three patients after therapy achieved the results below 0.5 IU/L, which reconfirmed the relevance of the established reference value. Therefore, the CSF hCG assay (intact hormone, free β-fraction, or both) for detecting intracranial germinoma is more sensitive than any other serum parameters. It is a minimally invasive diagnostic method, yet capable enough to ensure crucial information concerning not only the mere presence of intracranial hCG secreting neoplasms but also their closer identification which is fundamental for consecutive therapy and for the patient's prognosis.

The laboratory improvement programs initiated 70 years ago by the CAP, are currently implemented in more than 100 countries, with approximately 8,000 accredited laboratories providing proficiency testing to 20,000 laboratories worldwide.^[11] As of 2019, 59 medical laboratories in China have received accreditation from the CAP. The CAP accreditation is a competitive process, which describes specific requirements for the quality and technical aspects of a medical laboratory. The requirements related to the analytical methods used in the candidate laboratories are subject to stringent evaluation, with the main objective to validate analytical methods and to demonstrate their fit-for-purpose.^[12] Validation is defined as “where the specified requirements are adequate for the intended use.”^[13] Nevertheless, the majority of methods are validated by manufacturers; all the procedures, namely laboratory development methods, standard methods used outside their intended scope, and subsequent modified validated methods—usually termed “home-made methods”—need to be validated.

In conclusions, the CAP accreditation requirements concerning the validation of LDTs and modified-cleared/approved methods require a medical laboratory to perform a series of procedures to guarantee that the performance specifications achieved are relevant for the purpose of the test. The CAP accreditation program is a valuable resource that allows researchers to demonstrate the impact of a medical laboratory in a clinical context.