Hormone measurements: diagnostic errors related to preanalytical factors. 1. Introduction: Ph.D. Thesis

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1 Hormone measurements: diagnostic errors related to preanalytical factors Ph.D. Thesis Doctoral School of Medical Sciences Semmelweis University, Budapest Clinical endocrinology and its experimental aspects Program leaders: Prof. Rudolf de Chatel MD, DSc. Prof. Géza Szilágyi MD, DSc. Written by: Dr. Erzsébet Toldy (Mrs. Salamon) Central Laboratory, Markusovszky Hospital of County Vas and of Szombathely, University Teaching Hospital Adviser: Prof. Dr. Gábor L. Kovács corresponding member: Hungarian Academy of Sciences Markusovszky Hospital of County Vas and of Szombathely, and University of Pécs Szombathely, Introduction: The special preanalytical and analytical factors have their impacts on the clinical evaluation of hormonal findings. Due to the specific features of the immunoassay technique (cross-reactivity of the antibodies, specificity, technology-dependent sensitivity limits, the matrix effect, etc.), bare numbers alone on the laboratory report might be misleading in these laboratory tests. In many instances, the numeric result is mutually influenced by the serum concentration of the analyte, as well by the chemical environment in the patient serum. The main aim of this thesis was to investigate and quantify the power of preanalytical influences on hormone (immunoassay) measurements in the two most common endocrine diseases, such as thyroid dysfunctions and hyperprolactinemia. 2. Specific aims of the study: 2.1. The analysis of thyroid hor mones: Diagnostic strategies following TSH measurements: The specific aims of the studies were to analyze:? the routine clinical practice of thyroid requests in County Vas and to compare this practice between the periods before versus after the introduction of the sensitive TSH assay by our laboratory,? whether the introduction of the 3 rd generation TSH assays gives an added diagnostic value and whether it changes the algorithm of thyroid requests,? whether the introduction of the direct measurement of free thyroid hormones - gives an added diagnostic value in comparison to the calculation of indirect hormone indices,? how often T3 toxicosis is diagnosed and whether free T3 measurement is necessary at all The clinical validity of thyroid hormone measurements in unusual protein environments. The specific aims of the studies were to analyze:? in what direction abnormal circulating TBG levels would affect thyroid results,? whether serum albumin and IgG levels (changing in vivo or in vitro) affect free thyroid hormone and TSH levels The analysis of prolactin levels. The specific aims of the studies were:? to analyze serum prolactin levels before and after the elimination of the high molecular weight macroprolactin,? to introduce to the Hungarian practice a new method of biologically active prolactin, 1

2 ? to investigate the analytical validity of this method (effects of dilution and nonspecific precipitation) to investigate the clinical validity of this method, 3. Materials and methods: 3.1. Laboratory (and some other diagnostic) methods All laboratory parameters were investigated with routine methods in our laboratory. thyreotropin stimulating hormone (TSH): IFMA: 2 nd and 3 rd generation assays, ELISA, MEIA: 2 nd generation assay, ECLMA: 3. generation assay; total thyroxine (TT4 ), total triiodothyronine (TT3): FIA; T3 uptake (T3U): RIA; free thyroxine (ft4); free triiodothyronine (ft3): ELISA (one-step tracer analogue assay), FIA, MEIA (two-step back-titration methods), RIA and ECLIA (one-step antibody-labeled assay); free T4 index (ft4i = TT4xT3U or TT4/TBG or TT4/TBK), free T3 index (ft3i = TT3xT3U): calculated; albumin: colorimetric end-point method; IgG: laser nephelometry; thyroxin binding globulin (TBG), thyroxin binding capacity (TBC): ELISA; prolactin (PRL): ECLMA. The other diagnostic tools Complete case history, physical examinations, radiological reports (ultrasound, scintigraphy, MRI-scan), aspiration cytology investigation and the medication were evaluated together with the laboratory reports Patients sera investigations The analysis of thyroid hormones: Hormone analysis, in vitro algorithms:? Altogether 2637 patient samples referred to the laboratory for thyroid assays were analyzed.? TSH was reanalyzed with a 3 rd generation TSH assay in 446 patient sera, where the hormone appeared to be undetectable (suppressed < 0.05 mu/l) or subnormal (< 0.42 mu/l) with the 2 nd generation assay. It has also been analyzed, whether very low TSH levels have any clinical significance. The 2 nd and 3 rd generation TSH assays were compared for their sensitivities and specificities in screening for thyroid dysfunctions.? In order to find out its significance in the diagnosis of hyperthyroidism, free thyroid hormones were measured directly as well as calculated indirectly (hormone indices) in 150 patient sera.? In order to establish T3 hyperthyroidism, ft3 direct measurement or indirect calculation of ft3i has also been investigated in 171 patient samples, all of them with subnormal or suppressed TSH levels (measured with a 2 nd generation assay) accompanied by normal ft4 levels The effect of transport proteins on hormone levels. Thyroid hormone parameters have been studied:? In 199 clinicaly euthyreoid patients with high (> 325 nmol/l) levels of TBG (155 pregnant patients in the 2 nd and 3 rd trimester of gravidity and 44 women taking oral contraceptives). There patients were compared to 35 euthyroid patients with normal ( nmol/l) TBG concentrations,? In 44 patients with low albumin (<34 g/l) levels (all patients suffering from different clinical stages of liver cirrhosis; stages A, B, C, according to the Child classification), compared to 33 sera of healthy control individuals.? In 33 patients with low serum albumin (<34 g/l) or low serum IgG (< 10 g/l) levels, or the combination of these symptoms. Either albumin, or IgG was then stepwise added in vitro to the samples and thyroid parameters were intra-assay analyzed in the manipulated sera The analysis of prolactin:? 1051 patient sera with high (> 700 mu/l) serum prolactin levels were selected and monomeric biologically active prolactin also measured following PEG precipitation in the supernatant.? The frequency of macroprolactinemia and the clinical validity of the PEGprecipitation method have been evaluated.? The analytical characteristics of the PEG-precipitation method have been studied, by measuring the effect of sample dilution (245 sera), estimating the value of nonspecific precipitation (NSP) in samples containing exogenously added human recombinant prolactin standard to the samples. Free prolactin (f-prl) values were calculated (N = 745) and the correlations were evaluated in 256 patients with known MRI findings. 4. Results: 4.1. The analysis of thyroid hormones: In routine requests for thyroid dysfunction? In 70 % of the cases, where the screening algorithm was based on the measurement of sensitive TSH, no further thyroid test was needed. Thus the number of thyroid tests/patient decreased by 43 % as a result of the algorithm.? In 61 % of the samples with undetectable TSH values (2 nd generation assay), TSH became detectable with the sensitive (3 rd generation) assay. 2

3 ? There was a significant (P<0.001) difference between TSH levels of manifest (0.020 ± mu/l) and subclinical (0.100 ± mu/l) hyperthyroidism. The 3 rd generation TSH assay exhibited a sensitivity of 93 % and a specificity of 67 % in differentiating between the two stages of hyperthyroidism.? Using the 2 nd generation TSH assay 187 patient samples appeared to contain detectable, however subnormal TSH values (< 0.42 mu/l). Repeating the measurement with a 3 rd generation TSH assay, 104 of the 187 samples could be reclassified as euthyroid into the reference range. Furthermore, euthyroidism was confirmed in 97 samples by ft4 and ft3 measurements. Thus, the more sensitive 3 rd generation TSH assay decreased the frequency of subclinical hyperthyroidism (TSH < 0.27 mu/l, accompanied by normal ft4 and ft3 levels).? A 100 % sensitivity value was accompanied by 80 % specificity with the 2 nd generation assay, while a specificity of 98 % was reached with the 3 rd generation TSH test.? The correlation between measured free thyroid hormones and calculated free thyroid indices was statistically significant, albeit far from being optimal (r = 0.60).? In a certain proportion of the cases (20 % of the untreated and 40 % of the treated thyroid patients), calculated thyroid indices are misleading the diagnosis. In 0.4 % of the untreated and 3-times more frequently in the treated thyroid patients, laboratory reports do not seem to follow the well-known negative feed-back regulation.? The correlation between ft3 and TT3 values was weak (r = 0.36), whereas between ft3i and ft3 was medium strong (r = 0.58).? The discrepancy between ft3 and ft3i was more frequent in patients treated with thyreostatic medication than in untreated patients (44 and 19 %, respectively).? The ratio of ft4/ft3 was significantly (P < 0.01) lower in patients on thyreostatic medication, than in untreated thyroid patients or in healthy individuals.? There was no significant correlation between logtsh and TT3 or logtsh and ft3i values. Between logtsh and ft3, on the other hand, the negative correlation was significant.? Summarizing, 1.5 % and 3.9 % of the 466 patients with subnormal TSH values turned out to have T3 toxicosis, using calculated free hormone indices or measuring free hormones, respectively. Based on ft3 measurement, T3 toxicosis can be observed in 2.2 % of the untreated patients, while in 7.3 % of the patients receiving methimazole treatment. T3 toxicosis was present in 0.9 % of the total laboratory requests for thyroid screening (N = 2100) was The effect of transport proteins on hormone levels: The effect of increased circulating TBG levels on thyroid test results:? In patients with high TBG levels, the ft4 values irrespective of the three various assay technologies were significantly (P < 0.001) lower than those in control patients with normal TBG. The biggest difference was described in pregnant patients (32 nd week of pregnancy), especially if ft4 was measured with the one-step tracer analogue method (control: 15.6 ± 2.8; group taking contraceptives: 12.5 ± 2.3; pregnancy, week 16 th : 10.4 ± 1.4; week 32 nd : 6.5 ± 1.1 pmol/l). The difference resulting from high TBG was the smallest using two-step assay designs.? Investigating the pregnant patients (week 16 th ), the two step assays for ft4 gave the expected results i.e. euthyroidism. Using the one-step tracer analogue method or the antibody labeled technology, on the other hand, 82 % and 13 % of the patients (respectively), would have been falsely diagnosed as hypothyroidism. In later stages of pregnancy (32 nd week), thyroid values indicating hypothyroidism were present in 100 % with the tracer analogue method, in 86 % with the antibody labeled method and only in 3 % with the two-step assay technology. In all these cases, however, TSH remained in the reference range.? With the one-step methods, there was a strong negative correlation between TBG and ft4 values (r = -0.81). On the other hand, the negative correlation between ft4 and TSH (r =-0.34) was the strongest using the two-step method The effect of low serum albumin levels:? Using the two-step assay for free thyroid hormone measurements, there was a significantly higher T3U and TT4/TT3 ratio, as well as significantly lower TT3, TBK, ft3i and ft3 values in patients with low serum albumin (liver cirrhosis), as compared to the control patients.? In 13 % of the cirrhotic patients TSH was pathologically elevated. Existence of the low T3 syndrome depends on the principle of the used method.? There was a good correlation between clinical stages of cirrhosis (Child classification) and the values of TT3, T3U and T3i The effect of in vitro manipulations of the albumin and/or IgG serum levels:? There was a significant positive correlation between in vitro elevation of either serum IgG, or serum albumin levels and the bias of ft4 measurement (true for both the one-step ECLIA and the two-step MEIA method). Whereas the bias values of ft4 did not exceed 10 % following manipulation of the IgG levels, they were as high as 60 % following manipulation of albumin. The two-step MEIA assay was more sensitively affected than the one-step ECLIA method. 3

4 ? Similarly, there was a positive correlation between the bias of ft3 and serum IgG (8-30 %) as well as serum albumin ( %) levels. Unlike with ft4, the ECLIA method was more sensitively affected by both protein level.? The effect of albumin on the bias of both free thyroid hormones was much more pronounced, when serum IgG levels were low, if using MEIA method.? If using the one-step ECLIA method, the bias of TSH correlated with serum albumin levels. This was not observed with the two-step MEIA method The analysis of prolactin levels:? In 23 % of the investigated serum samples with high prolactin levels, the presence of macroprolactinemia (MPRL, < 40 % of total circulating prolactin is biologically active) can be diagnosed.? The ratio of total prolactin to the biologically active monomeric prolactin fraction follows a Gaussian distribution in patients with moderately high prolactin levels ( mu/l). If total prolactin exceeds 700 mu/l, the distribution of this ratio becomes bimodal.? Most frequently (in 70 % of the cases), MPRL occurs if total plasma prolactin is between mu/l. In patients with prolactin levels over 2000 mu/l, MPRL is less typical.? In patients with true hyperprolactinemia (thprl, serum prolactin remains pathologically elevated following PEG-precipitation of macroprolactin), pathological MRI-scan of pituitary adenoma is twice as frequent as MPRL patients (serum prolactin in the reference range following PEG-precipitation of macroprolactin).? In 15 % of the sera with macroprolactin, thprl and MPRL occurred simultaneously. Following the correction for NSP, the simultaneous occurrence of thprl and MPRL increased to 28 %.? In women, the incidence of MPRL was significantly (P < 0.01) higher in advanced than in younger ages. 5. Conclusions: 5.1. The analysis of thyroid hormones:? The more reliable less sensitive for preanalytical factors measurement of TSH may compensate for the uncertainty in the measurement of peripheral thyroid hormones. Thus, the use of thyroid algorithm is both financially and analytically meaningful.? The 3 rd generation TSH assay makes 2/3 of the undetectably low TSH values (measured with the 2 nd generation assay) detectable. This indicates the reevaluation of the TRH test. Since both generations are being used in Hungary, this finding points to the importance of the functional sensitivity of the TSH assay.? The more sensitive TSH assay also better proves the well-known clinical experience that subnormal TSH may sometimes indicate euthyroidism in patients treated with thyreostatic medication.? Using the 3 rd generation TSH assay, subnormal TSH accompanied by normal peripheral hormone levels occur less frequently. As a consequence, less ft4 and ft3 test is required.? Direct measurement of free hormone levels has a much higher clinical relevance than indirect calculation of free hormone indices. Indirect indices may be especially misleading in patients on methimazole therapy.? If the symptom of T3 toxicosis is diagnosed upon the laboratory measurements, the incidence of this disease is highly dependent on the assay (the incidence is 2- fold higher when ft3 is measured directly, as compared to the indices method). In moderately iodine-deficient geographical areas (like County Vas in Hungary) 0.9 % of total thyroid dysfunctions may be regarded as T3 toxicosis.? In patients treated for thyroid disease with methimazole, T3 hyperthyroidism occurs 3-fold more frequently than in untreated thyroid patients. This finding may indicate an effect of methimazole on the conversion. Alternatively, it may also suggest that the blocking effect of this drug on the organification of iodine may be more pronounced than in areas with an under optimal iodine supply.? In the sera with high TBG levels ft4i might be a more reliable measure than ft4, while measurement of ft3 or ft3i is inevitable, since TT3 is even more misleading than the TT4 value.? The exact reference range of free thyroid hormones should be established in 2 nd and 3 rd trimester of pregnancy, as well in patients taking oral contraceptives.? Since both pregnancy and contraceptive therapy decreased ft4 values (with all available assay techniques), one might suppose that part of these changes may not be related to compensatory mechanisms of the increased metabolic demand, but can be considered as a laboratory artifact.? In patients suffering from alcoholic liver cirrhosis, the occurrence of low T3 syndrome correlates well with the severity of the clinical symptoms, but it isn t observed by other T3 methods. The frequency of low T3 syndrome depends on the principle of assays.? Hypothyroidism occurs more frequently in patients with alcoholic liver cirrhosis, than in healthy individuals. The level of TBG tends to decrease only in more advanced stages of liver cirrhosis. T3U is a more sensitive parameter, which indicates quantitative and qualitative changes of the binding proteins. 4

5 ? The in vitro and in vivo results of severely ill (albeit clinically euthyroid) patients raise the possibility that altered thyroid parameters may not only derive from the natural defense mechanisms, but also from laboratory artifacts.? Free thyroid hormone levels depend on albumin and IgG concentration of the serum. One should, therefore, establish reference ranges valid for special clinical situations, e.g. NTI. As an alternative, a correction formula for the albumin and IgG levels may also be useful The analysis of prolactin:? If the prolactin level exceeds 700 mu/l, PEG-precipitation test should be carried out, because macroprolactin may be present in 23 % of these cases. By excluding MPRL with laboratory methods, other much more expensive diagnostic investigations (e.g. MRI-scan), or unnecessary bromocriptine therapy may be avoided.? In women, ageing results in a higher incidence of MPRL. This observation may be linked to the age-related increase in autoimmun susceptibility.? The dilution, an unavoidable consequence of the PEG-precipitation test, does not decrease the accuracy of prolactin estimation. One should, however, correct the results with the NSP values.? The joint occurrence of MRPL with thprl in 15 % (or in 28 % with free prolactin) of the patients with hyperprolactinemia is a very important finding which should be considered in the clinical practice.? The 5 % of the cases with 60 % PRL recovery has free PRL levels in the reference range. *The bold type indicated the new recognition 6. Publications of E. Toldy: 6.1. Original in extenso publications related to the thesis Az értekezéssel kapcsolatos in extenso közlemények 1. Toldy E., Locsei Z., Héber S., Gundy K., Varga L., Kovács G.L.: Új stratégia a pajzsmirigyfunkció diagnosztikájában. Orvosi Hetilap, 1993; 134: Locsei Z., Toldy E., Varga L., Kovács G.L.: A TSH ultrasensitiv-assay alkalmazásának klinikai jelentosége. Orvosi Hetilap, 1994; 135: Toldy E., Locsei Z., Kalmár I., Varga L., Kovács G.L.: A pajzsmirigy elleni autoantitestek diagnosztikus értéke. Orvosi Hetilap, 1996; 38: Toldy E., Riba M., Locsei Z., Varga L., Kovács G.L.: Szükséges-e a szabad trijódthyronin meghatározása? Klinikai és Kísérletes Laboratóriumi Medicina, 1998; 25: Locsei Z., Toldy E., Varga L., Kovács G.L.: A pajzsmirigyfunkció vizsgálata májcirrhosisos betegekben. Magyar Belorvosi Archivum, 1996; 4: Toldy E., Locsei Z., Petky Á., Móricz A., Kneffel P., Varga L., Kovács G.L.: Pajzsmirigyfunkció vizsgálata egészséges nok különbözo reprodukciós állapotaiban. Magyar Belorvosi Archívum, 1999; 52: Kovács, G.L., Toldy E., Locsei Z.: Az endokrin lelet értékelése. Lege Artis Medicinae. 2000; 10: Kovács G.L., Toldy E., Locsei Z., Soroncz M., Kovács F.: Az immunoassay csapdái. Klinikai és Kísérletes Laboratóriumi Medicina, 2001; 28: Toldy E., Góth M.: Háziorvoslás: hypophysis (A tévesen hyperprolactinaemiának ítélt macroprolactinaemiáról ). Magyar Orvos, 2003; 11: Toldy E., Locsei Z., Szabolcs I., Kneffel P., Góth M., Szoke D., Kovács G.L.: A macroprolactinaemia és a hyperprolactinaemia differenciáldiagnosztikája, Orvosi Hetilap, 2003; 43: Toldy E., Locsei Z., Szabolcs I., Góth M. I., Kneffel P., Szoke D., Kovács G.L.: Macroprolactinemia: the consequences of a laboratory pitfall. Endocrine, 2003; 22: Toldy E., Locsei Z., Rigó E., Kneffel P, Szabolcs I., Kovács G.L.: Comparative analytical evaluation of thyroid hormone levels in pregnancy and in women taking oral contraceptives: a study from an iodine deficient area. Gynecological Endocrinology, 2004; 8: Toldy E., Locsei Z., Szabolcs I., Bezzegh A., Kovács G.L.: Protein interference in thyroid assays: an in vitro study with in vivo consequences. Clinical Chemistry and Laboratory Medicine, (Submitted) Book chapters related to the thesis: 1. Kovács G.L., Toldy E., Locsei Z.: Általános módszertan. Az endokrin betegségek diagnosztikájában használatos laboratóriumi módszerek. A klinikai endokrinológia és anyagcsere-betegségek kézikönyve (Dr. Leövey András szerk.) Medicina Budapest Toldy E., Kovács G.L., Locsei Z.: A leggyakrabban alkalmazott laboratóriumi tesztek ismertetése és értékelése. Az endokrin betegségek diagnosztikájában használatos laboratóriumi módszerek. A klinikai endokrinológia és anyagcsere- 5

6 betegségek kézikönyve. (Dr. Leövey András szerk.) Medicina, Budapest Toldy E. A macroprolactinaemia diagnosztizálása. Anyagcsere-Endokrinológiai Útmutató Sanofi Synthélabo, Budapest, Published abstracts related to the thesis 1. Toldy E., Locsei Z., Varga L., Kovács G.L.: Assessment of serum free thyroxin and triiodothyronin with hormone-indices and direct methods, Klinikai és Kisérletes Laboratóriumi Medicina, 1995; 22: Locsei Z., Toldy E., Varga L., Kovács G.L.: Evaluation of thyroid status in patients with liver cirrhosis. Klinikai és Kisérletes Laboratóriumi Medicina, 1995; 22: Riba M., Toldy E., Locsei Z., Varga L., Kovács G.L.: Szükséges-e a free T3 meghatározása? Magyar Belorvosi Archívum, 1996; 49.Suppl. 1: Toldy E., Riba M., Locsei Z., Kovács G.L.: Szükséges-e a szabad T3 meghatározása? Klinikai és Kísérletes Laboratóriumi Medicina, 1996; 23: Gábor S., Toldy E., Bárdos É., Babos Sz., Kovács G.L.: Szabad thyroxin és totál triiodthyronin mérése ES -300 és Axsym automatán. Klinikai és Kísérletes Laboratóriumi Medicina, 1996; 23: Locsei Z., Toldy E., Petky Á., Móritz A., Kneffel P., Varga L., Kovács G.L.: Pajzsmirigyfunkció vizsgálata egészséges nok különbözo reprodukciós állapotaiban. Magyar Radiológia, 1997; Suppl. 1: Toldy E., Locsei Z., Kovács G.L.: Immuno-assay módszerek összehasonlítása, gazdaságosság és betegcentrikusság szempontjából.: Magyar Radiológia, 1997; Suppl.1: Toldy E.: Funkcionális szenzitivitás. Magyar Belorvosi Archivum, 1997; I: Bárdos É., Farkas Cs., Dobos Iné, Szabadffy Kné, Toldy E., Kovács G.L.: Tapasztalataink az Elecsys 2010-es immuno-assay automatával. Magyar Radiológia, 1997; Suppl. 1.: Toldy E., Locsei Z, Varga L., Kovács G.L.: A 3. generációs TSH assay klinikai értéke. Magyar Belorvosi Archivum, 1998; VI.Suppl.3: Kovács G.L., Toldy E., Petky A., Locsei Z., Varga L.: Laboratory evaluation of thyroid functions in hyperestrogenism. Clinical Chemistry and Laboratory Medicine, 1999; 37 S Toldy E., Locsei Z., Varga L., Kovács G.L.: Significance of third-generation TSH assay in the differential diagnosis of subclinical hyperthyreosis. Clinical Chemistry and Laboratory Medicine, 1999; 37 S Toldy E., Locsei Z., Kovács G.L.: Az endokrin lelet értékelése. Klinika i és Kísérletes Laboratóriumi Medicina, 1999; 26: Toldy E., Kovács F., Bárdos É., Dobos Iné, Locsei Z., Kovács G.L.: Összehasonlító mérések Architect és Elecsys 2010-es automatával. Klinikai Kísérletes Laboratóriumi Medicina, 2000; 26: Toldy E., Locsei Z., Kovács F., Kovács G.L.: Az endokrin lelet értékelése. Orvosi Hetilap, 2000; 141: 22: Toldy E., Locsei Z., Kovács F., Kovács G.L.: Clinical value of hormonal laboratory findings. Biochemia Medica God., 2000; 10: Toldy, E., Locsei, Z., Rigó, E., Bezzegh A., Kovács F., Kovács G.L.: Protein interference in immuno-assay methods. Clinical Chemistry and Laboratory Medicine 2001; 39: S149; 18. Toldy, E., Kovács, F., Locsei, Z., Rigó, E., Kovács G.L.: Immuno-assay methods and protein-interference: in vivo and in vitro experiences. Clinical Chemistry, 2001; 47: S6.: Toldy E., Locsei Z., Kovács G.L.: Hormon meghatározások protein interferenciája: in vitro kísérlet, in vivo konzekvencia. Orvosi Hetilap 2002; 143: Suppl.1.: Kovács G.L., Toldy E.: Hormonlaboratóriumi vizsgálatok az ezredforduló után. Orvosi Hetilap 2002; Suppl. 1.: Toldy E., Locsei Z., Rimanóczy É., Kneffel P., Jencsics Zs., Kovács G.L.: A macroprolactinaemia analitikája és klinikai jelentosége. Orvosi Hetilap 2002; 143: Suppl.1.: Kneffel P., Toldy E., Locsei Z., Rigó E., Kovács G.L.: Macroprolactinaemia analitikája és klinikai jelentosége. Nogyógyászati és Szülészeti Továbbképzo Szemle 2002; Suppl Toldy E., Locsei Z., Rimanóczy É., Kneffel P., Jelencsics Zs., Kovács G.L.: A macroprolactinaemia analitikája és klinikai jelentosége. Magyar Belorvosi Archivum, 2002; Suppl.3. : Toldy E., Locsei Z., Kneffel P., Szabolcs I., Szoke D., Skrapits J., Kovács G.L.: Macroprolactinemia: the consequences of a laboratory pitfall. Clinical Chemistry and Laboratory Medicine, 2003; 41: S Szoke D., Toldy E., Hartmann J., Locsei Z., Kovács G.L.: Protein interference in thyroid hormone assays: in vitro experiments with in vivo consequences? Clinical Chemistry and Laboratory Medicine, 2003; 41: S Kovács G.L., Toldy E., Locsei Z., Kneffel P., Szabolcs I., Szoke D., Macroprolactinemia: a laboratory pitfall with clinical consequences. Clinical Chemistry, 2003; 49: A Toldy E., Sárkány E., Kovács G.L.: Hormonmérések: tanulságok az elmúlt évi hazai minoségi kontroll adatokból. Orvosi Hetilap 2004; 145: 20 Suppl.3.: Locsei Z., Toldy E., Szabolcs István és Kovács G.L.: Szabad prolactin szintek értékelése. Orvosi Hetilap 2004; 145: 20 Suppl.3.:

7 29. Rigó E., Locsei Z., Toldy E. és Kovács G.L.: Endokrin paraméterek mérésének megbízhatósága szubnormális tartományban. Orvosi Hetilap 2004; 145: 20 Suppl.3.: Original in extenso publications unrelated to the thesis 1. Toldy E.,Horváth B.: Kombinált hatású hüvelytabletta formulálása. Gyógyszerészet 1987; 31: Toldy E., Küttel D., Horváth B., Réffy A.: Kísérlet a hüvely normál kémhatásának helyreállítására Lactobact-lactose tartalmú hüvelytablettával. Gyógyszerészet 1988; 32: Nagy J., Toldy E., Molnár I, Kovács G.L.: A prostata specifikus antigén mérésével szerzett tapasztalataink. Magyar Urológia. 1992; 4: Varga L., Baranyai M., Locsei Z., Toldy E., Brittig F.: Hepatitis B és C vírus marker vizsgálatok alkohol okozta májbetegségekben. Orvosi Hetilap. 1994; 135: Náfrádi L., Toldy E., Varga L.: Csontvesztés mértéke rheumatoid arthritisben Orvosi Hetilap 140., Kovács G.L., Nagy L., Toldy E.: Analitycal evaluation of the Abbott AxSYM Myoglobin Assay and Monitoring of Patients with Chest Pain at the University Teaching Hospital of County Vas in Hungary. Abbott Diagnostics Educational Services Lakner L., Jáger R., Toldy E., Sarang K., Varga L., Kovács G. L., Döbrönte Z. : A Helicobacter pylori infekcio szeroepidemiologiai vizsgálata Vas megyében. Belorvosi Archívum-.LII. évf / Salamon A., Józsa L., Toldy E., Réffy A., Renner A.: The role of different factors in pathogenesis of Dupuytren s disease. (8 th Congress of the International Federation of Societies for Surgery of the Hand, Istanbul, Turkey, Edited by Ridvan Ege: p: Printed by THK, Ankara, Kovács I., Császár A., Vereczkey G., Farkas A., Toldy E., Tarján J.: Quinapril és lozartan hatása posztinfarktusos betegek endothel funkciójára és inflammatorikus faktoraira. Cardiologia Hungarica 2002; 32: Kovács G.L., Toldy E.: Basal and isoproterenol-stimulated camp levels in mouse hippocampus and lymphocytes during alcohol tolerance and withdrawal. Alcohol and Alcoholism, 38: 11-17, Iszlai É.,Kiss E., Toldy E., Ágoston S., Sipos B., Vén L., Rácz F., Szerafin L: Helicobacter pylori szeroprevalencia és anti-caga pozitivitás Szabolcs-Szatmár- Bereg megyében. Orvosi Hetilap 2003; 144: