COMPARISON BETWEEN IMMUNOMETRIC METHODS FOR THE DETERMINATION OF FSH, LH, TSH AND PRL HORMONES IN CSF

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1 General Endocrinology COMPARISON BETWEEN IMMUNOMETRIC METHODS FOR THE DETERMINATION OF FSH, LH, TSH AND PRL HORMONES IN CSF Mariana Purice 1 *, Monica Gheorghiu 1,2, Andra Caragheorgheopol 1, Ruxandra Tanasescu 2 1 C.I.Parhon, Institute of Endocrinology Bucharest, Romania, 2 Carol Davila University of Medicine and Pharmacy, Bucharest, Romania Introduction: The necessity to determine the levels of pituitary hormones in the cerebrospinal fluid is motivated both by difficulties in the diagnosis of different neuroendocrine disorders, as well as by research purposes such as understanding the transport mechanisms of hormones through the blood brain barrier. Objective: The aim of this study was to compare the results obtained with different immunometric methods for some pituitary hormones in the serum and CSF in patients with neuroendocrine tumors. Materials and methods: The levels of LH, FSH, PRL and TSH were determined simultaneously in the serum and CSF with 3 different immunometric methods using commercial kits: IRMA, FIA and Chemiluminescence for 36 patients. The validation of IRMA method on CSF samples was performed using the dilution test. Results: The dilution test - as one of validation criteria of an immunoassay - proved that the results obtained in the CSF using the commercially available kits were correct. This enables the use of this sensitive method to accurately demonstrate abnormal levels of pituitary hormones beyond the blood-brain barrier. The correlation between IRMA and FIA: Hormonal concentrations values obtained by IRMA correlate well with those measured by FIA for serum with no significant differences of the mean concentration value for FSH (r=0.93, p: NS) and LH (r=0.99, p: NS), but with a significant difference for PRL (r=0.88, p=0.002). In CSF, mean concentrations values correlate well and we found no differences for TSH (r =0.97, p: NS) and LH (r = 0.94, p: NS), but significant differences for PRL (r= 0.98, p=0.001) and FSH (r=0.98, p=0.001). Statistically significant differences were also found for the CSF/serum ratio for PRL (p=0.08), FSH (p=0.001) and LH (p=0.001). The ratios CSF/serum are significantly higher with IRMA method than with FIA for all the hormones. Except for one patient for all the others we found the ratio CSF/serum less than 1 showing that the pathologic significance of this parameter is not modified due to the type of immunometric assay. A statistically significant difference (p<0.001) was found for mean FSH concentration in CSF with FIA and Chemiluminescent assays. Conclusions: Any immunometric method currently used for the determination of *Correspondence to: Mariana Purice, Institute of Endocrinology, 36 Bd.Aviatorilor, , Bucharest, Romania, mpurice_endo@yahoo.co.uk Acta Endocrinologica (Buc), vol. II no. 2, p ,

2 Mariana Purice et al. hormones in the serum or plasma has to be validated accordingly in order to be used on CSF. For obtaining results that can be interpreted and compared it is preferred that the same immunometric method is used on both serum and CSF, inside one group of patients. The presence of a control group for the results determined with that method is strongly recommended. Key words: immunometric methods, IRMA, FIA, Chemiluminescence, cerebrospinal fluid, pituitary hormones. INTRODUCTION Immunoassays are the most widely used analytical techniques and they have been successfully applied to an extensive range of substances, including both large and small molecules, cells, cellular components and viruses. They depend on the use of selected specific antibodies such as reagents. Because antibodies can display high specificity and react with high affinity, immunoassays are capable of measuring substances in complex matrices without pretreatment, extraction, purification or concentration. The measurement of the concentration of a molecule in any biological environment has to take into account the influence of its structure on the immunologic reaction. Minimizing the interaction that might occur, specific or not, is one of the first objectives when elaborating an immunoassay. Regardless of the method used the reaction medium has to use the antigen-antibody reaction, which is influenced by the ph and the ionic strength (1, 2).This is why in order to be applied for another matrix (saliva, urine, or CSF) than those for which they were designed (usually serum or plasma), the commercial immunoassay kits have to be carefully used and validated accordingly. A matrix (biological environment) is required for standards (calibrators) as well as for control samples. A suitable matrix is one without any analyte and which mimics the behavior of the sample matrix in the assay (2). The determination of pituitary hormones in CSF is motivated both by the diagnosis of various neuro endocrine disorders and by the follow-up after treatment of pituitary adenomas. For research purposes, measuring the exact values of polypeptidic hormones in the serum and cerebrospinal fluid enables investigating the blood-brain barrier and the transport mechanisms for polypeptidic hormones, both in normal conditions and in different disorders (3, 4, 5, 6, 7). The aims of this study were to validate for CSF samples a IRMA method, commonly used for the measurement of hormonal concentrations on serum or plasma and to evaluate the correlation between three different immunometric assay methods: IRMA (a radioimmunoassay), FIA and CHEMI (non radioimmunoassays) that we used for hormones determination in CSF and serum. 152

3 Comparison between immunometric methods for peptidic hormones in CSF MATERIALS AND METHODS Samples All blood (by i.v puncture) and cerebrospinal fluid (by lumbar puncture) from 36 patients with anterior pituitary adenomas were simultaneously sampled. Both serum and CSF samples were distributed in several aliquots and stored at -40 o C until assayed. Informed consent was obtained from each investigated case. Methods and protocol For validating the IRMA method by using the dilution test and for the IRMA FIA comparison we used 18 samples (both serum and CSF). The FIA- CHEMI comparison was performed on 36 samples (both serum and CSF). Internal control serum was considered for the calibration curves for each assay. The immunoradiometric assay (IRMA) was performed using MAIAclone kits (ADALTIS Italy) that have been designed for the quantitative determination of a polypeptidic hormone in human serum or plasma. Two high affinity monoclonal antibodies (MAb) are used: one, labelled with 125 I, that attaches quickly to a unique site on the molecule, the second one linked to fluorescein binds at a discrete site on the hormone molecule forming a molecular sandwich which is then separated in a magnetic field. This gives a convenient separation of the antibody-bound and free antigen (8,9). The time-resolution fluoroimmunoassay (FIA) was done with DELFIA kits (Pharmacia Wallac, Finland) designed for the quantitative determination of polypeptidic hormones in human serum or plasma in a solid phase using a monoclonal antibody labeled with Europium (Eu) that binds to the investigated hormone in a sandwich reaction. A second antibody, directed against mouse IgG, is coated into the solid phase and binds the Ab-Ag-Ab complex (10). The chemiluminometric immunoassay (CHEMI) was performed with ACS kits (Bayer, UK) designed for the polypeptidic hormone determination in serum or plasma is a two site sandwich, a direct assay using an antibody linked to acridine ester and the second one in a solid phase ( a polyclonal sheep antibody against the polypeptide human hormone). The performances of the immunoassay kits presented above are shown in Table 1. The immunometric assays were performed as follows: IRMA: the standard curve was determined in duplicate, the hormonal concentrations in the patient samples were simultaneously measured: in serum, in CSF and in diluted CSF (1:2) directly in the test tube. The quality control (QC) for the reaction was internal Serotest M. For the dilution of the samples we used the standard zero of the kit (bovine serum with sodium azide < 0.02%) for TSH, LH, FSH and the assay diluent (bovine serum with sodium azide < 0.02%) for PRL. In this way we increased the total protein concentration of the CSF samples to a level closer to the protein concentration of the serum sample. 153

4 Mariana Purice et al. FIA: the standard curve was determined in duplicate and the patient samples (serum and CSF) were simultaneously investigated, using for the assay an internal quality control serum. Table 1. Immunassay kits performances Method Producer Sensitivity Measurement Range IRMA (125 I) MAIAclone FIA (Eu) DELFIA Chemiluminescence (Acridin ester) ACS ADALTIS (Italy) LKB-Wallac (Finland) Bayer (UK) (Detection limit) TSH = 0.02 µui/ml PRL = 0.3 ng/ml LH = 0.15 mui/ml FSH = 0.25mUi/ml TSH = 0.03 µui/ml PRL = 0.04 ng/ml LH = 0.12 mui/ml FSH = 0.05 mui/ml PRL = 0.3 ng/ml FSH = 0.3 mui/ml TSH: µui/ml PRL: ng/ml LH: mui/ml FSH: mui/ml TSH: µui/ml PRL: ng/ml LH: mui/ml FSH: mui/ml PRL: ng/ml FSH: ng/ml CHEMI: after the initial calibration, hormone concentrations were simultaneously determined in patient samples (serum and CSF). The reaction was performed with external QC using Tri level serum as internal QC. Due to economic reasons for validation the IRMA assay on CSF we performed only the dilution test. The dilution test consists in measuring of specimen concentration at various dilutions in the matrix and assesses linearity. Poor recovery or nonlinearity indicates inaccurate calibration or an inappropriate matrix or both. We measured the hormonal levels in undiluted CSF and at 1:2 dilution and calculated the recovery (concentration multiplied by dilution factor) for the following hormones: TSH, PRL, FSH and LH. Statistical analysis Correlation coefficients for the dilution test were calculated from the regression equation. For describing the relation between the results of the three immunometric methods we used paired means compared by two-tailed Student s test and r (correlation coefficient) calculated with Pearson s test. RESULTS A developed assay protocol requires validation to establish suitability to practical application. In broad terms validation should test the assay in the recovery and the dilution tests. Due to economic reasons we performed only the dilution test. We measured the hormonal levels in undiluted CSF and at dilutions 1:2 and calculated the recovery (concentration multiplied by dilution factor) for the following hormones: TSH, PRL, FSH and LH. The results are shown in Fig. 1. Further we compared the concentration results obtained in the serum, in CSF and the fraction of CSF/ serum concentration (R CSF/serum ) obtained with different immunoassay methods: 154

5 Comparison between immunometric methods for peptidic hormones in CSF Figure 1. Dilution tests on CSF by IRMA for TSH, PRL, FSH and LH. For IRMA-FIA correlation the results are shown in Figs. 2, 3 and 4. Figure 2. Correlation between IRMA and FIA on CSF for TSH and PRL. 155

6 Mariana Purice et al. For FIA- CHEMI comparison we preset data for 36 samples (serum and CSF) for which we determined the concentration of FSH. The correlation coefficient was r =0.97 on serum (n=23) and r=0.99 on CSF (n=25). For PRL only three values with CHEMI method could be obtained, consequently the statistical analysis could not be performed. Correlation IRMA-FIA for LH in serum (r=0.99) and CSF (r= 0.93) IRMA-FIA correlation for serum FSH (n=12). Figure 3. Correlation between IRMA and FIA on CSF for FSH and LH. Correlation IRMA-FIA in CSF for FSH (n=11) Figure 4. CSF concentration mean values for TSH,PRL,FSH and LH with IRMA and FIA methods. 156

7 Comparison between immunometric methods for peptidic hormones in CSF The results for FSH concentrations in serum and CSF with the 3 methods are shown in Fig. 5. Figure 5. Comparison between IRMA, FIA and CHEMI methods for FSH values in serum and CSF. We also calculated the fraction R CSF/serum values both for IRMA and FIA for PRL, FSH and LH ( Fig. 6) and compared the mean values (Fig. 7). Statistical correlation and significance of the mean concentrations for serum, CSF and R CSF/serum fraction obtained with the 3 methods are synthesized in Table 2. PRL: CSF/serum ratio FSH: CSF/serum ratio LH: CSF/serum ratio Figure 6. IRMA- FIA correlation of CSF/serum ratio for PRL, FSH and LH. 157

8 Mariana Purice et al. DISCUSSION CSF levels of endogenous pituitary hormones are normally under those found in the blood, in healthy, non menopausal subjects: TSH is about 44%, PRL is about 20%, LH is about 8.6% and FSH is about 14%. This proportion remains constant under conditions of persistent steady-state and the CSF/serum ratio is less than one (3). In pathological conditions (as local tumor secretion or a modification of the BBB permeability) the situation is modified and CSF/serum ratio could be over 1. In both cases for low and respectively high CSF concentration, a reliable, sensitive and high specific method for quantification of this ratio is forced necessary. The dilution test method (2) as one of the validation criteria performed for IRMA on CSF showed that the results obtained by us using the commercially available kits (designed for human serum or plasma) are correct. The values determined in CSF for each hormone (PRL, TSH, FSH and LH) correlate statistically with those calculated after the dilution of the initial samples. The use of IRMA MAIAclone on CSF samples enables to demonstrate abnormal levels of pituitary hormones beyond the blood-brain barrier, inside the central nervous system. Even though the concentration of total proteins and albumin are different in CSF than in plasma and the biological matrix is not the same, by using immunometric kits with high sensibility and specificity (due to the monoclonal antibodies) and efficient systems of separation of the antigen-antibody complexes, the results obtained on CSF are correct. They are also in good correlation with the serum levels and the clinical status. Figure 7. Mean values of CSF/serum ratio obtained with IRMA and FIA methods for RL,FSH and LH. Comparison between methods on serum and CSF The hormonal concentrations on serum and CSF using IRMA method correlate statistically with those obtained by FIA, for all assayed hormones. In the study group (n=18) the mean values concentrations of TSH and LH (on serum and on CSF) are not significantly different between IRMA and FIA. These results are well correlated with our previous one (11). For serum FSH the mean values are 158

9 Comparison between immunometric methods for peptidic hormones in CSF not significantly different between the two methods, but the results on CSF show values that are statistically different (p =0.001). Regarding PRL concentrations in serum and in CSF, the mean values are statistically different (p=0.002, respectively p=0.001) between the two methods. This can be due to the presence in samples of macroprl. Macroprolactine can be due either to anti-prl auto antibodies IgG (IgG-macroPRL) or other PRL containing molecules without IgG-PRL immuncomplexes (nigg-macroprl), the composition of which is not fully clarified (12, 13).These epitopes can interfere within antigen-antibody sandwich reaction in the assay and they cannot be recognized separately by anti-prl antibody. Table 2. Correlation coefficients (r) and statistical significance (p) between immunoassay methods for hormones concentrations (serum and CSF) and CSF/serum ratio Methods TSH PRL FSH LH IRMA - FIA Serum IRMA - FIA CSF IRMA - FIA CSF/serum ratio FIA - Chemi Serum FIA - Chemi CSF - r=0.88 n=10 p=0.002 r=0.97 n=11 p:ns r=0.98 n=17 p= r=0.1 p=0.08 r=0.97 n=12 p: NS r=0.98 n=11 p=0.001 r=0.76 p= n=3 r=0.85 n=23 p: NS - n=3 r=0.81 n=25 p<0.001 r=0.99 n=10 p: NS r=0.94 n=12 p=ns r=0.94 p= The correlation FIA CHEMI was performed only for FSH (n=25) obtaining a weak correlation (r = 0.4) for serum with a medium concentration value for the group not statistically different (p=ns). For FSH concentration in CSF the mean values were significantly different (p<0.001) between the methods. Comparison of CSF/serum ratio with IRMA and FIA The values of CSF/serum ratios with IRMA are constantly higher than those obtained using FIA for PRL, LH and FSH. The Student s t test shows that the values of CSF/serum ratio are significantly different between IRMA and FIA for FSH (p= 0.001) and LH ( p= 0.001) but p=0.08 for CSF/serum ratio for PRL. With both methods we obtained CSF/serum ratios <1 for all the hormones, with one exception: for LH with IRMA the CSF/serum ratio was 1.17 ( >1)and with FIA the CSF/serum ratio was 0.71 (<1). Consequently, the pathological significance of this ratio is not modified if we use a different immunometric assay, either it is IRMA or FIA

10 Mariana Purice et al. CONCLUSIONS We recommend the validation on CSF of any commercial kit (designed for serum/plasma) at least by the dilution test and, if possible, by the recovery test (measure of the concentration in specimens before and after adding a known amount of pure analyte). In order to obtain results that can be compared and interpreted inside one group of patients, it is preferred to use the same immunometric method both for measurements in serum and in CSF (14,15). This ensures the uniformity of the results and conveys a correct image both of the local secretion of the investigated hormone and of the integrity of the blood-brain barrier by interpreting the CSF/serum ratio (3). The normal ranges proposed by the kit producers are for serum samples, because the kits are designed for serum or plasma. When using a commercial kit for CSF samples it is strongly recommended to first obtain an appropriate range from a reference (control) group. References 1. Barbier Y. (coordonateur). Les Immunodosages. Lyon : Les editions de l Acomen, 1992; Edwards R. (editor) Immunoassays.Essential Data.Chichester: JohnWiley& Sons, 1996; Coculescu M. Clinical Endocrinology (in Romanian), Bucharest: Ed Stiintifica si Enciclopedica, 1986, Coculescu M, Temeli E, Oprescu M, Dimitriu V, Simionescu N. Blood cerebrospinal fluid barrier for prolactine in empty sella syndrome. Rev Roum Endocrinol 1981; 19: Coculescu M, Poiana C, Pop A, Oprescu M, Constantinovici A, Simionescu N. Altered specificity of blood-cerebrospinal fluid barrier for pituitary hormones in patients with tumoral hypothalamohypophyseal diseases. Rev. Roum. Physiology 1985; 22: Coculescu M. Blood - Brain Barrier for Human Growth Hormone and Insulin like Growth Factor - I. J Ped Endocr Metab 1999;12: Purice M, Suta A, Badiu C, Danaila L, Coculescu M, The evidence of the modification of the blood brain barrier for Zn, Cu and some hormones in some cerebral and anterior pituitary tumors.1999; The IX National Symposium of Psychoneuro endocrinology, 1999, Bucharest 8. Zuber E, Mathis G, Flandrois JP. Homogeneous two-site immunometric assay kinetics as a theoretical tool for data analysis. Anal Biochem 1997; 15;251(1): Rattle SJ, Purnell DR, Williams P, Siddle K and Forrest GC. New separation mothod for monoclonal immunoradiometric assays and its application to assay for thyrotropin and human choriogonadotropin. Clin Chem 1984; 30: Soini E, Kojola H.Time-resolved fluorometer for lanthanides chelates - a new generation of non isotopic immunoassay. Clin Chem 1983; 29:65-68 (1-2): Purice M, Alexiu F, Caragheorgheopol A, Cirnu R. Non conventional immunometric assays. Comparative studies. 1998; Revista de medicina si farmacie, UMF Tg Mures, Vol 44 supl

11 Comparison between immunometric methods for peptidic hormones in CSF 12. Amadori P, Dilberis C, Marcolla A, Pinamonti M, Menapace P, Valentini A. Identification of IgGimmunocomplex macroprolactin with an immunometric sandwich system: technical and clinical considerations. J Endocrinol Invest 2004; 27(11): Amadori P, Dilberis C, Marcolla A, Pinamonti M, Menapace P, Dal Bosco F. Macroprolactinemia: predictability on clinical basis and detection by PEG precipitation with two different immunometric methods. J Endocrinol Invest 2003; 26(2): Rasmussen AK, Hilsted L, Perrild H, Christiansen E, Siersbaek-Nielsen K, Feldt-Rasmussen U. Discrepancies between thyrotropin (TSH) measurement by four sensitive immunometric assays. Clin Chim Acta 1997; 18: Taylor AE, Khoury RH, Crowley WF Jr. A comparison of 13 different immunometric assay kits for gonadotropins: implications for clinical investigation. J Clin Endocrinol Metab 1994; 79(1):

12 162 Mariana Purice et al.