In-house Immunofluorometric Assay Research Articles

SUN-LB61 Differential and Opposite Regulation of Acid Labile Subunit (ALS) Versus Insulin-Like Growth Factor I (IGF-I) by Oral Estrogens in Premenopausal Women

Background: The impact of estrogens (E2) on the growth hormone (GH)/IGF-I axis is known to depend on route of administration: While oral E2 increases GH and decreases IGF-I, transdermal E2 has only limited or no effect. However, data concerning the impact of E2 on IGF binding protein 3 (IGFBP 3) and ALS are less clear. One study in girls demonstrated higher ALS with oral E2, while the opposite was suggested for postmenopausal women. No data are available for healthy premenopausal women.Methods: We measured IGF-I, IGFBP 3 and ALS in fasted healthy adults (93 males (M), 35 premenopausal women without E2-containing oral contraception (FPRE), 37 premenopausal women with E2-containing oral contraception (FPREOC) and 34 postmenopausal women (FPOST)). IGF-I and IGFBP 3 were measured using the IDS-iSYS chemiluminescence immunoassay, and ALS by an in-house immunofluorometric assay (limit of quantification (LoQ) < 50 mU/ml, range 50 - 4000 mU/mL).Results: Median age (range) was 33 (20 - 76), 28 (20 - 44), 24 (21 - 36) and 56 (49 - 70) years for M, FPRE, FPREOC and FPOST, respectively. As expected, IGF-I was lower in FPREOC compared to FPRE (median IGF-I xULN (IQR) 0.56 (0.45 - 0.73) and 0.72 (0.63 – 0.80), P = 0.0017, Kruskal-Wallis). ALS was significantly higher in FPREOC compared to all other groups (mean ALS in M, FPRE, FPREOC and FPOST: 636, 708, 861 and 648 mU/mL, respectively, ANOVA P < 0.0001, Dunnett’s post-hoc test: M vs FPREOC: P < 0.0001, FPRE vs FPREOC: P = 0.0007, FPOST vs FPREOC: P < 0.0001). IGFBP 3 was not different in females with and without oral E2 (median IGFBP 3 xULN (IQR) FPREOC vs FPRE: 0.62 (0.54 - 0.67) vs 0.60 (0.49 – 0.76), Kruskal-Wallis P = 0.295, Dunn’s post-hoc test: P > 0.9999). This was also true between all other groups (Dunn’s post-hoc test: P ≥ 0.4). In our adult cohort, ALS exhibited negative correlation with age (Pearson r = -0.282, P = 0.0003), similar to IGF-I and IGFBP 3. While IGF-I exhibited a moderate negative correlation to BMI (Pearson r = -0.25, P = 0.0013), IGFBP 3 and ALS were not significantly related to BMI.Conclusion: While IGF-I, IGFBP 3 and ALS all are known to be secreted in response to GH, and IGF-I and ALS are assumed to be produced by the same cells in the liver (hepatocytes), the three GH dependent biomarkers appear to be differently regulated by metabolic factors and oral E2. Only IGF-I has some modest association with BMI. Oral E2 is associated with reduced IGF-I, unchanged IGFBP 3 but increased ALS. While the mechanism behind the differential regulation remains to be uncovered, E2 therapy must be taken into account when interpreting IGF-I and ALS concentrations.

SUN-LB62 Differential Regulation of 22 kDa and 20 kDa Growth Hormone Isoforms in Response to Acute Glucose Load and Moderate Intensity Exercise

Background: Among growth hormone (GH) isoforms, the 22kDa (22kD) is the most abundant (~90%), followed by 20 kDa GH ((20kD) ~10%). 22kD has known diabetogenic potential by increasing insulin resistance. Although of equal somatotropic activity, 20kD has been suggested to be less diabetogenic. Under physiological conditions, both isoforms are largely secreted in parallel, but data on regulation of both isoforms in the context of glucose load and utilization are scarce. Methods: We measured 20- and 22kD GH in fasted healthy adult females during two scenarios: (A) Premenopausal females (n = 25; age (mean (range)) 27.8 (22-52), body mass index (BMI) (kg/m2) 21.8 (18.2-27.3), insulin sensitivity index (ISI) 7.9 (3.2-19.7) underwent an oral glucose tolerance test (OGTT, 75g, sampling at baseline, 30, 60, 90, 120 and 180 minutes (min); (B) Premenopausal females within 1 year postpartum (n=28, age 36 (29-44), BMI 23.7 (19.4-41.3), ISI (7.2- (2.4-13.2)) performed moderate intensity exercise (fixed workload 60% VO2max, sampling at -15, -10, -5, 0, 3, 6, 10, 15, 20, 30 and 45 min and after 5, 10 and 15 min of resting). 20- and 22kD were measured by specific immunoassays (22kD: IDS-iSYS GH CLIA, limit of quantification (LoQ) 0.05 ng/mL; 20kD: in-house immunofluorometric assay (IFMA), LoQ 0.025 ng/mL), and the 20/22kD ratio was calculated. Results: As expected, both, 22kD and 20kD concentrations decreased after glucose load. However, the 20/22kD ratio increased significantly (Friedman test, P = 0.04, Kendall’s W 0.69, Conover’s post test: 0 vs. 60, 90 and 120 min; P = 0.018, 0.032 and 0.018, respectively. After 120 min, 20/22kD returned to baseline.With exercise, both, 20- and 22kD increased. In contrast to glucose load, the 20/22kD ratio significantly decreased (Friedman test, P = 0.025, Kendall’s W 0.59, Conover’s post test: -10 vs. 10 and 20 min; P = 0.015 and 0.022, respectively), reaching a nadir at 10 min and returning to the baseline after cessation of exercise (20 vs. 50, 55 and 60 min; P = 0.02, 0.005 and 0.016, respectively. Conclusion: Glucose load and exercise lead to opposite changes in the 20/22kD ratio, suggesting a tight regulation by glucose demands. We speculate that the putative less diabetogenic isoform (20kD) is preferentially secreted during situations of glucose excess, while the opposite happens during exercise, when increased energy demands are associated with predominant upregulation of the more diabetogenic 22kD GH.

MR spectroscopy of hepatic fat and adiponectin and leptin levels during testosterone therapy in type 2 diabetes: a randomized, double-blinded, placebo-controlled trial

Men with type 2 diabetes mellitus (T2D) often have lowered testosterone levels and an increased risk of cardiovascular disease (CVD). Ectopic fat increases the risk of CVD, whereas subcutaneous gluteofemoral fat protects against CVD and has a beneficial adipokine-secreting profile. Testosterone replacement therapy (TRT) may reduce the content of ectopic fat and improve the adipokine profile in men with T2D. A randomized, double-blinded, placebo-controlled study in 39 men aged 50-70 years with T2D and bioavailable testosterone levels <7.3 nmol/L. Patients were randomized to TRT (n = 20) or placebo gel (n = 19) for 24 weeks. Thigh subcutaneous fat area (TFA, %fat of total thigh volume), subcutaneous abdominal adipose tissue (SAT, % fat of total abdominal volume) and visceral adipose tissue (VAT, % fat of total abdominal volume) were measured by magnetic resonance (MR) imaging. Hepatic fat content was estimated by single-voxel MR spectroscopy. Adiponectin and leptin levels were measured by in-house immunofluorometric assay. Coefficients (b) represent the placebo-controlled mean effect of intervention. TFA (b = -3.3 percentage points (pp), P = 0.009), SAT (b = -3.0 pp, P = 0.006), levels of adiponectin (b = -0.4 mg/L, P = 0.045), leptin (b = -4.3 µg/mL, P < 0.001), leptin:adiponectin ratio (b = -0.53, P = 0.001) and HDL cholesterol (b = -0.11 mmol/L, P = 0.009) decreased during TRT compared with placebo. Hepatic fat content and VAT were unchanged. The effects of TRT on cardiovascular risk markers were ambiguous. We observed potentially harmful changes in cardiovascular risk parameters, markedly reduced subcutaneous fat and unchanged ectopic fat during TRT and a reduction in adiponectin levels. On the other hand, the decrease in leptin and leptin:adiponectin ratio assessments could reflect an amelioration of the cardiovascular risk profile linked to hyperleptinaemia in ageing men with T2D.

O21. First trimester serum placental growth factor and hyperglycosylated human chorionic gonadotropin are associated with later pre-eclampsia

Introduction First trimester serum placental growth factor (PlGF) predicts early-onset pre-eclampsia (diagnosis Objectives To study the accuracy of using combined maternal serum %hCG-h, PlGF, PAPP-A and maternal risk factors in prediction of pre-eclampsia. Methods We determined gestational-age-adjusted concentrations of PlGF, PAPP-A, hCG (Perkin Elmer Wallac Oy, Turku, Finland) and hCG-h (in-house immunofluorometric assay) in maternal serum at 8–13 weeks of gestation. The study consisted of 98 women who later developed pre-eclampsia and 177 pregnant controls. Of the cases, 24 developed preterm pre-eclampsia (diagnosis 160/110mmHg, proteinuria>5g/24h and/or hemolysis, elevated liver enzymes and low platelet count –syndrome). Results Serum PlGF, %hCG-h and PAPP-A were lower in women with later preterm or severe pre-eclampsia than in controls. In receiver-operating characteristics (ROC) curve analysis the area under the curve (AUC) was 0.680 for PlGF, 0.699 for %hCG-h and 0.714 for PAPP-A to predict preterm pre-eclampsia. AUC values for prediction of severe pre-eclampsia were 0.677, 0.702 and 0.677, respectively. When PlGF, %hCG-h and PAPP-A were combined by logistic regression analysis the AUC value was 0.830 for preterm pre-eclampsia and 0.824 for severe pre-eclampsia. Combination of PlGF, %hCG-h, PAPP-A and maternal risk factors including nulliparity and first-trimester mean arterial pressure (MAP) gave an AUC value of 0.805 for preterm pre-eclampsia and 0.882 for severe pre-eclampsia. When PlGF was removed from the analysis the AUC value was 0.803 for preterm pre-eclampsia and 0.878 for severe pre-eclampsia. When %hCG-h was removed the AUC values were 0.756 and 0.840, respectively. Conclusions Combining %hCG-h with PlGF, PAPP-A and maternal risk factors in first trimester improves prediction of later preterm or severe pre-eclampsia. %hCG-h tended to give better AUC values than PlGF when combined with PAPP-A and maternal risk factors. Thus, in first trimester %hCG-h might be superior to PlGF for prediction of preterm or severe pre-eclampsia and it should be further investigated in risk models of pre-eclampsia.

Testosterone therapy decreases subcutaneous fat and adiponectin in aging men

Testosterone therapy increases lean body mass and decreases total fat mass in aging men with low normal testosterone levels. The major challenge is, however, to determine whether the metabolic consequences of testosterone therapy are overall positive. We have previously reported that 6-month testosterone therapy did not improve insulin sensitivity. We investigated the effect of testosterone therapy on regional body fat distribution and on the levels of the insulin-sensitizing adipokine, adiponectin, in aging men with low normal bioavailable testosterone levels. A randomized, double-blinded, placebo-controlled study on 6-month testosterone treatment (gel) in 38 men, aged 60-78 years, with bioavailable testosterone <7.3 nmol/l, and a waist circumference >94 cm. Central fat mass (CFM) and lower extremity fat mass (LEFM) were measured by dual X-ray absorptiometry. Subcutaneous abdominal adipose tissue (SAT), visceral adipose tissue (VAT), and thigh subcutaneous fat area (TFA) were measured by magnetic resonance imaging. Adiponectin levels were measured using an in-house immunofluorometric assay. Coefficients (b) represent the placebo-controlled mean effect of intervention. LEFM was decreased (b = -0.47 kg, P = 0.07) while CFM did not change significantly (b = -0.66 kg, P = 0.10) during testosterone therapy. SAT (b = -3.0%, P = 0.018) and TFA (b = -3.0%, P < 0.001) decreased, while VAT (b = 1.0%, P = 0.54) remained unchanged. Adiponectin levels decreased during testosterone therapy (b = -1.3 mg/l, P = 0.001). Testosterone therapy decreased subcutaneous fat on the abdomen and lower extremities, but visceral fat was unchanged. Moreover, adiponectin levels were significantly decreased during testosterone therapy.

Serum Leptin and Ghrelin Levels in Active and Inactive Acromegalic Patients During an Oral Glucose Tolerance Test

Elevated BMI, fat mass and food intake increases leptin whereas ghrelin is reduced. Ghrelin stimulates growth hormone (GH) secretion. This prospective cross-sectional study was performed to investigate the influence of GH excess on leptin and ghrelin levels at baseline and after glucose load in a large group of acromegalic patients. Leptin, ghrelin, GH, glucose and insulin concentrations were measured during a 3 h OGTT in 17 active, 25 inactive patients without medication (inact) and 8 inactive patients treated successfully with somatostatin analogues (inact-SA). Leptin concentration was measured using an in-house immunofluorometric assay, total serum ghrelin by a commercially available radioimmunoassay. Leptin was corrected for BMI. During OGTT, no significant difference of baseline, nadir or AUC of leptin/BMI was seen between the groups (baseline leptin/BMI: act: 0.26 microgm(2)/lkg (0.06-0.87); inact: 0.34 microgm(2)/lkg (0.03-1.79); inact-SA: 0.29 microgm(2)/lkg (0.03-1.47). A slight but significant reduction of leptin/BMI occurred in all groups during OGTT (active p<0.001, inact p<0.001, inact-SA p<0.05). Inact had higher ghrelin levels (130 ng/L (69-292) compared to active (102 ng/L (27-232); p=n.s.). Inact-SA had significantly lower ghrelin levels (88 ng/L (72-113)) than inact (p<0.001). There was a significant decline of ghrelin during OGTT in all groups (active p<0.001, inact p<0.001, inact-SA p<0.05). GH only has a slight influence on leptin secretion. Ghrelin levels are lowered by GH and SA. Nevertheless, the physiological decreasing effect of glucose load on ghrelin secretion is preserved.

Urinary Epidermal Growth Factor and Interleukin-6 Levels in Patients with Painful Bladder Syndrome/Interstitial Cystitis Treated with Cyclosporine or Pentosan Polysulfate Sodium

To measure urinary epidermal growth factor (EGF) and interleukin-6 (IL-6) levels in painful bladder syndrome/interstitial cystitis (PBS/IC) patients randomized to either cyclosporine A (CyA) or pentosan polysulfate sodium (PPS) treatment for 6 months and compare the results with treatment response. Urine samples from PBS/IC patients were collected before (n = 37) and after treatment (n = 34). The concentration of EGF was determined by an in-house immunofluorometric assay and that of IL-6 by the Immulite assay. The clinical response to treatment was determined by the subjective global response analysis (GRA, scale 1 to 6). Participants who reported categories 5 (much better) or 6 (cured) were considered responders. According to GRA, 72% of CyA patients and 16% of PPS patient responded to their treatment (P <0.001). In the CyA group, post-treatment urinary EGF levels were significantly reduced (from 35 +/- 15.8 to 28.3 +/- 17.9 ng/mg creatinine; P <0.034), whereas the urinary IL-6 levels were not affected by CyA or PPS treatment in the whole group. In older patients (aged more than 52 years) who had higher IL-6 levels and longer duration of disease, these were reduced by successful CyA therapy. Treatment of PBS/IC patients with CyA resulted in significantly decreased urinary levels of EGF. Interleukin-6 levels did not change significantly in all treated patients after either CyA or PPS treatment, but in older patients the levels were reduced after CyA treatment.

Gender Differences of Oligomers and Total Adiponectin during Puberty: A Cross-Sectional Study of 859 Danish School Children

Pubertal stages have been shown to influence total adiponectin (ADPN) levels. Furthermore, testosterone has been shown to alter the isomer distribution of ADPN. The goal of this study was to investigate whether pubertal stages and testosterone levels influenced total serum ADPN levels and the distribution of ADPN isomers. This is a cross-sectional study. The study included 859 children and adolescents (396 males) aged 6-20 yr. Total ADPN and ADPN isomers were measured using a validated in-house immunofluorometric assay. Fractioning of the ADPN into the three major molecular fractions was performed in representative subgroups of pre- and postpubertal males and females (n = 40, 10 in each group) using a validated fast protein liquid chromatography method. Total ADPN levels before puberty were 13.4 (11.1-15.9) mg/liter (median and interquartile range) and 14.7 (12.3-18.1) mg/liter (P = not significant), in males and females, respectively. After puberty, ADPN levels were significantly reduced in males, 9.7 (8.2-12.0) mg/liter but remained unchanged in females, 12.1 (9.7-15.3) mg/liter (P < 0.0001). Concomitantly, a reduction was seen in the ratio of high-molecular-weight (HMW) isomers to total ADPN (HMW ratio) when comparing prepubertal and postpubertal males. Also, postpubertal males had lower HMW ratios than corresponding females (P = 0.038). Finally, a negative correlation was seen between HMW ratio and testosterone (r = -0.430, P = 0.007). Serum total ADPN levels decrease through puberty in males. Also, a reduced HMW ratio is seen in males at the onset of puberty. We speculate that the suppression of HMW ADPN may be caused by testosterone.

Valores de paratormônio obtidos com ensaios imunométricos dependem da especificidade do anticorpo amino terminal empregado

Introduction of 2nd generation immunometric assays for the measurement of serum parathyroid hormone (PTH), turned them more available, simple and rapid. These methods, based on double identification of the PTH molecule, supposedly measure the intact, bioactive molecule, with the sequence 1-84. Recent works showed that they also measure forms with amino-terminal deletions, like the 7-84 form, which are not able to activate the traditional PTH receptor (PTH1R). Thus, an important practical aspect is the definition of the PTH forms measured by the immunometric assays, a fact that depends on the specificity of the antibodies employed. In this report we compare the results obtained with an in-house immunofluorometric assay that presents a cross-reactivity of 50% with the 7-84 PTH sequence, and two commercial 2nd generation assays, that react 100%. In a first study, 135 samples were measured using our assay and an electrochemiluminescent assay, resulting in a correlation coefficient of 0.961 (P<0.0001) and medians of 35.0 and 51.0 ng/L (P<0.0001). In a second study, 252 samples were analyzed using our assay and an immunochemiluminometric assay, resulting in a correlation of 0.883 (P<0.0001) and medians of 36.0 and 45.5 ng/L (P<0.0001). In both studies results obtained with the in-house assay were significantly lower, as expected by the specificity of the anti-amino-terminal antibody employed. Our data support the need of a precise description of the specificity of the amino-terminal antibodies employed in 2nd generation PTH assays in order to better compare results and define normal ranges.

Preoperative hCGbeta and CA 72-4 are prognostic factors in gastric cancer.

In gastric cancer, the role of tumour markers in assessment of prognosis is unconfirmed. In our study, we evaluated the prognostic significance of serum tumour markers carcinoembryonic antigen (CEA), CA 19-9, CA 72-4, CA 242 and free beta subunit of human chorionic gonadotropin (hCGbeta) in gastric cancer. Preoperative serum samples were obtained from 146 patients with gastric cancer, including 29 with stage I, 11 with stage II, 42 with stage III and 64 patients with stage IV cancer. Quantitation of CEA, CA 19-9, CA 72-4 and CA 242 in serum was performed with commercial assays. HCGbeta was measured with an in-house immunofluorometric assay based on monoclonal antibodies specific for the free beta-subunit of hCG. Survival analysis was performed with Kaplan-Meier life-tables and log-rank test, and with multivariate Cox regression analysis. Disease-specific cumulative 2-year survival rate was 40%. Serum levels of CEA, CA 72-4, CA 242 and hCGbeta showed significant correlation with stage (p<0.027); for CA 19-9 the association was of borderline significance (p=0.056). Of the studied markers, CA 19-9, CA 72-4, CA 242 and hCGbeta were found to be prognostic factors in univariate analysis (p< 0.022). In multivariate analysis, stage had the statistically most significant association with prognosis followed by hCGbeta, tumour histology according to the Laurén classification and by CA 72-4. In gastric cancer, tumour markers hCGbeta and CA 72-4 are independent prognostic factors in addition to stage and histological type of the tumour.

Serum HCGβ and CA 72-4 Are Stronger Prognostic Factors than CEA, CA 19-9 and CA 242 in Pancreatic Cancer

Objective: In pancreatic cancer, the extent of the spread of the disease is considered to be the strongest prognostic factor. In addition, tumor markers, particularly CA 19-9, may also provide prognostic information. In this study, we evaluated the prognostic value of serum tumor markers CEA, CA 19-9, CA 242, CA 72-4 and hCGβ in pancreatic cancer. Methods: Preoperative serum samples were obtained from 160 patients with pancreatic cancer, including 10 with stage I, 25 with stage II, 24 with stage III and 101 patients with stage IV cancer. Quantitation of CEA, CA 19-9, CA 242, and CA 72-4 in serum was performed with commercial assays. HCGβ was measured with an in-house immunofluorometric assay based on monoclonal antibodies specific for the free β-subunit of hCG. Survival analysis was performed with univariate Kaplan-Meier life-tables and log-rank test, and with multivariate Cox regression analysis. Results: Of the tumor markers studied, CA 19-9 was most frequently elevated. Overall 2-year survival was 10%. Stage, tumor location and size, curative resection, and CEA, CA 72-4 and hCGβ were all found to be prognostic factors (p < 0.026) in univariate analysis. In multivariate analysis, each marker had independent prognostic value (p < 0.011) when analyzed individually but adjusting for stage. When all the covariates were included in the same model, the strongest prognostic factor was hCGβ followed by CA 72-4 and stage. The other clinical characteristics and serum tumor markers contributed insignificant prognostic information. Conclusions: All the tumor markers studied (CEA, CA 19-9, CA 242, CA 72-4, and hCGβ) had prognostic value in pancreatic cancer, and hCGβ, CA 72-4, and stage were the strongest independent prognostic factors in this study.

Testing and validating a homogeneous immunometric assay for interference.

We analyzed 95 sera, demonstrating interference in a previous study, with the Kryptor homogeneous time-resolved fluorescence resonance energy transfer carcinoembryonic antigen (CEA) immunoassay (Brahms AG, Berlin, Germany). Only one serum differed, i.e., 6.0 microg/l for Kryptor vs. 13.3 microg/l for a microtiter plate in-house immunofluorometric assay (IFMA), using both aggregated mouse immunoglobulins as blocker and capture monoclonal antibody (Mab) F(ab')2-fragments to reduce interference. Sera were further analyzed with experimental IFMAs with intact capture Mabs and without blocker. The Kryptor-CEA assay Mab GFR44 (capture) had elevated results in 71% of sera with the Kryptor Mab G15 (tracer) or in 81% with our tracer Mab. G15 (capture) had 65% elevated results with GFR44 (tracer) or 99% with our tracer Mab. The latter was reduced from 99% to 62% by adding Kryptor blocker to the buffer or to 30% by adding our blocker. Finally, combining both assay Mabs and assay buffer from Kryptor still gave interference in 16% of sera. Kryptor-CEA assay results thus agreed with our in-house CEA assay results, showing no interference. As the Kryptor-CEA assay antibodies were sensitive to interference and the Kryptor-CEA assay buffer did not reduce interference as efficiently as our in-house assay buffer, the Kryptor-CEA assay format was crucial for the absence of interference.

Effects of growth hormone replacement therapy on levels of cortisol and cortisol-binding globulin in hypopituitary adults.

To determine if human growth hormone (hGH) replacement therapy alters pharmacokinetics of hydrocortisone (CS) substitution in hypopituitary adults. To this aim, we analysed serum and salivary CS profiles 270 min after oral CS administration at baseline and 6 and 12 months after initiation of hGH replacement therapy. Serum IGF-I, cortisol-binding globulin (CBG), thyroxine-binding globulin (TBG) and sex hormone-binding hormone (SHBG) were measured using commercially available radioimmunoassays. In-house immunofluorometric assays were employed for measurements of CS and hGH. hGH replacement did not change total serum CS bioavailability (area under the serum cortisol profile curve). Interference of orally administered CS with salivary measurement of free CS (fCS) caused significant bias. Therefore, fCS levels were calculated from their total CS and cortisol-binding globulin (CBG) levels. CBG decreased by approximately 30% after both 6 and 12 months of hGH replacement therapy (n=20, P<0.01). A significant negative correlation between deltaCBG (CBG6months-CBGbaseline) and deltaIGF-I (IGF-I6months-IGF-Ibaseline) was observed (P=0.04). The calculated values of free CS tended to increase with physiological hGH replacement, but this effect was marginal and did not reach statistical significance. In contrast to the CBG concentrations, plasma levels of sex hormone-binding globulin and thyroxine-binding globulin were essentially stable. Given that no clinically relevant alterations in pharmacokinetics of CS were evoked by initiation of hGH replacement in hypopituitary adults, we conclude that CS substitution does not require dose adjustment after initiation of hGH replacement.

STABILITY OF FREE AND TOTAL PROSTATE SPECIFIC ANTIGEN IN SERUM FROM PATIENTS WITH PROSTATE CARCINOMA AND BENIGN HYPERPLASIA

Purpose Instability of prostate specific antigen (PSA) in serum might complicate the interpretation of the free-to-total PSA ratio. We studied the in vitro stability of free PSA and total PSA in serum of patients with prostate cancer or benign prostate hyperplasia (BPH), and of elderly man without known prostate disease. Furthermore, we investigated conditions to stabilize the in vitro values in serum. Materials and Methods The effects of storage at 4C on free and total PSA were investigated in serum of 32 men with prostate cancer, 25 with BPH and 29 older than 70 years. All had total PSA less than 25 micro g./l. The influence of total PSA levels on in vitro changes in free-total PSA was studied in serum of 39 other prostate cancer patients (total PSA 1.7 to 298 micro g./l.). Stabilization studies were performed in yet another series of samples from 54 prostate cancer patients (total PSA 1.3 to 238 micro g./l.) by adjustment of serum pH to 5.5 before storage. Free and total PSA was measured by a commercial immunofluorometric assay, as well as by in-house immunofluorometric assays. Statistical analyses of the results were performed by analysis of variance with repeated measures. Results We found no difference between the results obtained by the 2 assay systems. After 7 days at 4C there was a slight decrease in total PSA in sera of prostate cancer patients, BPH patients and men older than 70 years. A decrease in mean free PSA values occurred in all groups (21.3, 15.7 and 14.6%, respectively). The decrease of free PSA with time was significant (p <0.0001) in all groups but there was no significant difference among the groups (p = 0.16). The concomitant decrease in free-to-total PSA ratio was significant in all groups (p <0.0001). This change was group dependent (p = 0.003), with the largest decrease in the prostate cancer group. Large interindividual differences were observed. Storage at 4C for 7 days of sera of 39 patients with localized and disseminated prostate cancer (total PSA 1.7 to 298 micro g./l.) gave a more pronounced decrease in free PSA than in total PSA. Adjustment of serum pH to 5.5 had a stabilizing effect on free PSA and on the free-to-total PSA ratio, giving a significantly smaller change in both values (p <0.0001). Conclusions In vitro instability of free PSA in serum and large interindividual differences should be considered when using the ratio of free-to-total PSA in evaluation of patients with suspected prostate cancer. Serum samples should be stored frozen if not analyzed immediately or acidified to pH 5.5. Interpretation of data from determination of free-to-total PSA ratio should be done with caution if the sampling and storage conditions are not known.