Johan Bjerner

Longitudinal Serum Testosterone, Luteinizing Hormone, and Follicle-Stimulating Hormone Levels in a Population-Based Sample of Long-Term Testicular Cancer Survivors

PURPOSE To assess longitudinal long-term alterations of testosterone, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) in testicular cancer survivors (TCSs). PATIENTS AND METHODS In all, 307 TCSs treated from 1980 to 1994 provided blood samples after orchiectomy but before further treatment, at Survey I (SI; 1998-2002), and Survey II (SII; 2007-2008). Levels of sex hormones were categorized according to quartiles and reference range (2.5 and 97.5 percentiles) of 599 controls for each decadal age group. TCSs were categorized according to treatment: surgery, radiotherapy (RT), or chemotherapy (CT). The risk of higher (LH) or lower (testosterone) levels was assessed with χ(2) test (FSH) or ordinal logistic regression analysis and expressed as odds ratios (ORs) with 95% CIs. RESULTS Risk of lower testosterone and higher LH and FSH levels was significantly increased for TCSs at all time points after RT or CT. At SII, ORs were 3.3 (95% CI, 2.3 to 4.7) for lower testosterone categories and 5.2 (95% CI, 3.5 to 7.9) for RT and CT. ORs for increased LH and FSH were 4.4 (95% CI, 3.1 to 6.5) and 18.9 (95% CI, 11.0 to 32.6) for RT, respectively, and 3.6 (95% CI, 2.4 to 5.3) and 14.2 (95% CI, 8.3 to 24.4) for CT, respectively. The cumulative platinum dose was significantly associated with risk of higher LH levels at both surveys and higher FSH at SI. In total, half the TCSs had at least one of three sex hormone levels outside the reference range at SII. CONCLUSION Long-term TCSs are at risk of premature hormonal aging. Our findings may pertain to cancer survivors in general, underlining the importance of extended follow-up.

Reference intervals for serum testosterone, SHBG, LH and FSH in males from the NORIP project

Abstract Reference intervals were calculated for male testosterone, SHBG, FSH and LH in serum from 599 individuals in the NORIP study. At 30 years of age, reference limits were calculated to 10.4–32.6 nmol/L testosterone, 13.5–57.4 nmol/L SHBG, 1.93–9.7 IU/L LH and 1.5–10.3 IU/L FSH, at 50 years, 9.3–31.3 nmol/L (testosterone), 18.4–75.6 nmol/L (SHBG), 2.01–10.4 IU/L (LH) and 2.04–12.4 IU/L (FSH), and at 70 years 8.6 to 30.7 nmol/L (testosterone), 27.8–101 nmol/L (SHBG), 2.22–11.2 IU/L (LH) and 2.71–14.2 IU/L (FSH). All age-+related changes were statistically significant. Reference intervals were also calculated for indices derived from testosterone, SHBG and albumin. Free androgen index, simply the ratio between testosterone and SHBG, returned results differing from the other elaborate indices, and the study thus favors use of a more elaborate index such as calculated free testosterone (CFT).

Reference intervals for carcinoembryonic antigen (CEA), CA125, MUC1, Alfa‐foeto‐protein (AFP), neuron‐specific enolase (NSE) and CA19.9 from the NORIP study

Objective. Adhering to current IFCC recommendations, we calculated upper 97.5 % reference limits for serum tumor markers. Material and methods. Serum samples from 498 healthy individuals from the Nordic reference interval project (NORIP) were investigated for carcinoembryonic antigen (CEA), CA125 and MUC1 (episialin, CA15.3) using in‐house immunofluorometric assays and, for α‐foetoprotein (AFP), a PerkinElmer Life Sciences assay, neuron‐specific enolase (NSE) using an in‐house immunoradiometric assay and CA19.9 using a Beckman Access assay. All assays participate in external quality assessment programs. Results. CEA concentrations increased with age and smoking. Upper reference limits for non‐smokers were 3.59 µg/L at 50 years and 4.12 µg/L at 70 years. CA125 concentrations were age‐independent and the upper reference limit was 35.8 kU/L. MUC1 increased with age and body mass index (BMI). Upper reference limits were 31.7 kU/L at 40 years and BMI 24, 37.5 kU/L at 70 years and BMI 24, and 33.7 kU/L at 40 years and BMI 30. AFP increases with age, and the upper reference limits were 3.82 kU/L at 20 years and 8.70 kU/L at 60 years. An upper reference limit for NSE was 8.91 µg/L in non‐smokers; smokers exhibited significantly lower levels. The upper reference limit for individuals expressing CA19.9 was 28.3 kU/L. Conclusions. For AFP, CA125 and CA19.9, the reference levels obtained were close to previously reported reference ranges. Smoking and age were confirmed as covariates for CEA. The associations between MUC1 with age and BMI and between NSE and smoking have not been reported previously.

Heterophilic antibody interference in commercial immunoassays; a screening study using paired native and pre-blocked sera

Abstract Background: Heterophilic antibodies are still an important source of interference in immunoassays. We have conducted a screening study for interference in a panel of commercially available assays using two sera known to contain high titer Fc-reactive heterophilic antibodies. Methods: The sera were distributed to laboratories participating in the Nordic External Quality Assessment cooperation (EQANord). Duplicate samples pre-blocked with aggregated murine monoclonal MAK33 were also supplied. Discrepancies (>50%) between the results for native and blocked samples were used to classify the tested assays as susceptible to interference. A total of 170 different assay kits covering 91 analytes were tested. Results: We found that 21 assays, covering 19 different analytes, were susceptible to interference from the heterophilic antibodies in the two sera. Many of these are clinically and commercially important assays. Some of the false results were grossly elevated and could have been detrimental to patient care in a clinical setting. Conclusions: Heterophilic antibodies with Fc-reactivity remain a threat. A more widespread use of antibody fragments and aggregated immunoglobulin could potentially improve the heterophilic antibody resistance of assays intended for clinical use.

Progastrin-Releasing Peptide: Stability in Plasma/Serum and Upper Reference Limit

Background: Progastrin-releasing peptide (proGRP) is a promising tumor marker for small cell lung cancer (SCLC). Here we study the stability of proGRP in serum and plasma, as well as proGRP levels in healthy individuals, to provide a framework for clinical studies. Methods: Serum, with and without protease inhibitors, and plasma from SCLC patients and healthy individuals were assayed for proGRP immediately after collection and following various storage conditions. Results: No degradation was observed in serum or plasma after storage for 4 weeks at –30°C. Serum proGRP levels were stable for up to 3 days at 4°C, but decreased at room temperature. Addition of protease inhibitors to patient serum did not markedly improve stability. In EDTA plasma, proGRP concentrations increased upon storage in some samples at room temperature and 4°C. When assayed immediately after collection, no significant variations in proGRP concentrations were observed between serum and EDTA plasma (n = 171). A 97.5-percentile reference limit of 58.9 ng/l was calculated from data from 806 individuals. However, proGRP levels were significantly correlated with age, sex, creatinine concentrations, body mass index and smoking. Conclusion: Serum is the preferred material for measuring proGRP, provided it is stored at 4°C and assayed within 3 days.

Automated Time-Resolved Immunofluorometric Assay for Progastrin-Releasing Peptide

BACKGROUND Small cell lung cancer accounts for approximately 20% of new cases of lung cancer, and advanced disease is prevalent at the time of diagnosis. Neuron-specific enolase (NSE) has been the primary tumor marker in small cell lung cancer but it has relatively low sensitivity in early-stage disease. Progastrin-releasing peptide (proGRP) is a promising alternative or complementary marker for NSE. We have previously described a time-resolved immunofluorometric assay (TR-IFMA) for proGRP that lacked the necessary sensitivity and robustness for use in the routine clinical laboratory. Herein we describe the development of an improved assay using a novel monoclonal antibody pair. METHODS Mice were immunized with different conjugated proGRP peptides, including residues 31-98, 1-98, and preproGRP(-23-125). Pair combinations of the resulting monoclonal antibodies (mAb) were tested. The improved TR-IFMA was compared with the only other available proGRP assay, the proGRP ELISA (IBL). RESULTS A panel of 12 high-affinity mAbs was produced. The best assay combination was between our original E146 mAb as solid-phase antibody and the new mAb M16 as tracer. The new TR-IFMA had a linear dose-response curve, a wide dynamic range (13-13 500 ng/L), and a limit of detection of 2.8 ng/L. Total CV was <5.6% over the whole measuring range. Bland-Altman difference analysis indicated a significant positive bias between the IFMA and the ELISA. CONCLUSIONS We describe a sensitive and robust mAb-based TR-IFMA for proGRP. The assay is fully automated and displays high quality performance.

Chronic fatigue in 812 testicular cancer survivors during long-term follow up: increasing prevalence and risk factors

BACKGROUND Chronic fatigue (CF) has been reported to be slightly more prevalent in testicular cancer survivors (TCSs) than in the general population. In this study, we wished to explore possible determinants of CF in TCSs median 12 (survey I) and 19 years (survey II) after treatment, in particular the relation to late effects after treatment. PATIENTS AND METHODS Overall, 812 TCSs treated between 1980 and 1994 provided blood samples (testosterone and luteinizing hormone) and completed questionnaires at survey I (1998-2002) and survey II (2007-2008). Hormone levels were categorized according to quartile thresholds for decadal age groups of controls. Associations between CF and possible risk factors, including the Hospital Anxiety and Depression Scale (HADS), treatment, physical activity, hormone levels, neurotoxicity, and comorbidity, were analyzed by logistic regression. RESULTS Prevalence of CF increased from 15% at survey I to 27% at survey II (P < 0.001). At survey II, risk for CF was increased three- to four-fold for high levels of neuropathy compared with no neuropathy, and two- to three-fold for high levels of Raynaud-like phenomena, and having testosterone levels in the lowest quartile, while being moderately and highly physically active, had a protective effect. Risk for CF in TCSs with higher levels of HADS-Anxiety and HADS-Depression was increased two- to five-fold, respectively. CONCLUSIONS The increasing prevalence of CF in TCSs is a novel finding. Lifestyle interventions, early detection and treatment of depression and anxiety, and possibly testosterone substitution might reduce the risk of CF. Extended long-term follow-up seems to be important.

MUC1 Serum Assays in Breast Cancer: Tumor Specificities and Reference Levels

The aim of the study was to establish reference ranges and to explore the tumor specificities of two automated assays for MUC1. Sera from 124 female blood donors, 144 patients with benign disease of the breast, 69 patients with stage I, 75 with stage II, 89 with stage III and 38 patients with stage IV breast cancer were analyzed for MUC1 levels using two automated immunometric assays employing assay antibody pairs Ma695/Ma552 and BC2/GP1.4, respectively. All subjects were female. The Ma695/Ma552 assay yielded means of 13.86 (SD 6.55) kU/l for blood donors, 15.98 (SD 8.31) kU/l for benign disease, 15.83 (SD 7.92) kU/l for stage I, 15.01 (SD 8.03) kU/l for stage II, 33.80 (SD 66.53) kU/l for stage III and 469.22 (SD 906.61) kU/l for stage IV breast cancer. The BC2/GP1.4 assay gave means of 12.00 (SD 6.41) kU/l for blood donors, 14.68 (SD 9.33) kU/l for benign disease, 14.13 (SD 8.12) kU/l for stage I, 12.10 (SD 6.61) kU/l for stage II, 19.80 (SD 29.05) kU/l for stage III and 191.04 (SD 527.16) kU/l for stage IV breast cancer. Patients with benign diseases of the breast had slightly higher values than female blood donors with both assays leading to correspondingly different reference ranges. The Ma695/Ma552 assay had higher specificity for tumor MUC1 than the BC2/GP1.4 assay for all stages, and the study thus confirms the differences in tumor specificity for MUC1 assays.

Serum levels of soluble transferrin receptor correlate with severity of disease but not with iron stores in patients with malignant lymphomas.

Soluble transferrin receptor levels in serum (s-sTfR) may be useful in differentiating between iron deficiency anemia and anemia of chronic disease. However, there is both theoretical and clinical evidence for elevated s-sTfR levels in patients with various hematological malignancies. In the present study, routine bone marrow aspirations were performed in 82 patients with malignant lymphomas (63 with non-Hodgkin’s lymphoma and 19 with Hodgkin’s disease). Smears were stained for evaluation of iron stores and graded. Patients were also given a disease score based on bone marrow morphology, erythrocyte sedimentation rate and LDH. s-sTfR levels correlated better with disease score [partial Spearman rank correlation coefficient (rs) controlled for iron stores was 0.51 (95% confidence interval 0.39–0.65); p < 0.001] than with iron stores [partial rs controlled for disease score was –0.25 (95% confidence interval –0.44 to –0.03); p = 0.027]. This study showed elevated s-sTfR levels in patients with malignant lymphomas without any signs of iron deficiency anemia. The diagnosis of iron deficiency anemia should not be established upon the basis of s-sTfR alone in this group of patients.

Testing and validating a homogeneous immunometric assay for interference.

Abstract 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 μg/l for Kryptor vs. 13.3 μg/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 inhouse 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.