Sanja Mandić

Falsely elevated serum oestradiol due to exemestane therapy

In this study, we present a case of falsely elevated oestradiol (E2) concentration, determined by two immunoassays, in a breast cancer patient receiving exemestane therapy. The positive bias of immunochemical measurements was revealed using liquid chromatography tandem mass spectrometry which showed undetectable E2 concentration. The discrepancy is expected to be a consequence of the structural resemblance of E2 and exemestane sharing the same steroidal backbone. Inaccurate laboratory findings in therapy monitoring, as in this case, may lead to unnecessary changes of therapy.

STEROID HORMONES PROFILE DURING AN OVARIAN SYNCHRONIZATION PROCEDURE IN DIFFERENT AGE CATEGORIES OF RED DEER HINDS (Cervus elaphus L.)

The objective of the present study was to compare estradiol/progesterone ratios of different age categories of red deer hinds and use it as a predictor of estrus synchronization success and consequently conception rate. To accomplish this we used 38 red deer hinds to establish serum progesterone and estradiol levels in young (21 animals), mature (10 animals) and old (7 animals) hinds during the estrus synchronization procedure (transvaginal/cervical AI). The following estrus synchronization was used: at the start of the experiment each hind received a controlled intravaginal drug-releasing device (CIDR, Pharmacia&Upjohn, New Zealand) containing 0.3 g of progesterone. The device was removed on day 11, simultaneously with an application of 250 IU of Pregnant Mare Serum Gonadotropin (PMSG, Folligon® Intervet International, Boxmeer, Holland). Transvaginal/cervical AI (artificial insemination) was performed 48 hours after CIDR withdrawal (day 13). Blood samples were obtained from the jugular vein using a Venoject® vacutainer without an anticoagulant for hormonal tests on the same experimental day (0, 11th and 13th day). A statistically (p<0.01) higher progesterone level was found in young hinds on the 11th day after controlled intravaginal drug-releasing device insertion. A significantly higher (p<0.01) estrogen level was observed in the young in regard to mature and old hinds on the expected day of estrus (13th day). Estradiol/progesterone ratios showed a statistically significant difference (p<0.01) on insemination day (13th day) between old and young hinds (98.67 : 46.59) and between old and mature hinds (98.67 : 51.79). Out of a total of 38 hinds only 9 had their offspring, 6 of the young and 3 of the mature hinds.

Differentiation of acute pyelonephritis from other febrile states in children using urinary neutrophil gelatinase-associated lipocalin (uNGAL)

Abstract Background: Acute pyelonephritis is a severe disease which is sometimes difficult to recognize based on clinical symptoms and routinely available diagnostic tests, especially in young children. The aim of this study was to assess the diagnostic value of urinary neutrophil gelatinase-associated lipocalin (uNGAL) as a biomarker of acute pyelonephritis. Methods: In this case-control study we analyzed 134 children (median age 2.5 years) who were admitted to the Pediatric Clinic of University Hospital Centre Osijek, Croatia. Eighty of them had acute pyelonephritis, while 54 children had febrile state of different etiology including cystitis and they represented the control group. uNGAL, white blood cells, C-reactive protein, urinanalysis, urine culture, kidney ultrasound and a dimercaptosuccinic acid scintigraphic scan were done for each child. uNGAL was measured using chemiluminiscent microparticle immunoassay on ARHITECT i1000SR (Abbott Diagnostics, IL, USA). Results: uNGAL values were significantly higher in children with acute pyelonephritis compared to the control groups (113.6 ng/mL vs. 10.2 ng/mL, p<0.001). A receiver operating characteristic curve comparison was done for tested parameters and encouraging results were obtained for uNGAL (AUC=0.952). A cut-off value of 29.4 ng/mL had 92.5% sensitivity and 90.7% specificity. We showed that uNGAL can also serve in differentiating acute pyelonephritis from cystitis (cut-off 38.5 ng/mL), and for differentiation of cystitis from febrile states with etiology other than urinary tract infection (UTI) (cut-off 20.4 ng/mL). Conclusions: uNGAL can be a useful diagnostic biomarker in acute pyelonephritis in children, but also in differentiating cystitis from febrile states other than UTI.

Serum carbohydrate sulfotransferase 7 in lung cancer and non-malignant pulmonary inflammations

Abstract Background: Carbohydrate sulfotransferases (CHST) were shown to be involved in carcinogenesis. The aim of the study was to assess the diagnostic value of serum CHST7 concentration in differentiation between lung cancer and non-malignant pulmonary inflammations. Methods: Clinical case-control study involving 125 participants was conducted: the control group containing cases of pneumonia and chronic obstructive pulmonary disease was compared to the lung cancer group composed of primary and metastatic cancers. Serum concentrations of CHST7 and routinely used markers including carcinoembryonic antigen (CEA), cytokeratin fragment 21-1 (CYFRA 21-1) and neuron-specific enolase (NSE) were determined for each participant using immunochemical methods. Statistical association, receiver operating characteristic (ROC) analysis and cross-validation were used for the evaluation of CHST7 either as a standalone biomarker or as a part of a biomarker panel. Results: In comparison to the control group, serum CHST7 was elevated in lung cancer (p<0.001), but no differences between the overall stages of primary cancers were detected (p=0.828). The differentiation performance in terms of ROC area under curve (AUC) was 0.848 making CHST7 superior biomarker to the NSE (p=0.031). In comparison to CEA and CYFRA 21-1, the performance differences were not detected. CHST7 was not correlated to other biomarkers, and its addition to the routine biomarker panel significantly improved the cross-validated accuracy (85.6% vs. 75.2%) and ROC AUC (p=0.004) of the differentiation using a machine learning approach. Conclusions: Serum CHST7 is a promising biomarker for the differentiation between lung cancer and non-malignant pulmonary inflammations.

Statistical learning confirms the diagnostic significance of the anemia panel in breast cancer.

Abstract Background: Diagnostic value of available tumor markers, such as cancer antigen CA 15-3 and carcinoembryonic antigen (CEA) in breast cancer is limited. There is an ongoing search for additional, potentially better diagnostic blood markers with improved clinical utility. The aim of this study is to evaluate performance of the approach based on routine blood tests accompanied by a statistical learning tool to the diagnosis of breast cancer. Methods: Blood was collected from total of 104 subjects which were divided into two groups: breast cancer patients and a control group that consisted of asymptomatic volunteers and patients who had benign breast lesions at the time of blood collection. Random forest statistical learning method and the external method validation have been applied to evaluate diagnostic performance of 31 routine blood tests. Results: The applied statistical learning approach assigned the highest diagnostic importance to the anemia panel among all analyzed blood tests that also included CA 15-3. External validation has shown utility of selected statistical approach – we were able to select tests that provide a diagnostic accuracy comparable to some diagnostic tools described in literature and based on more demanding laboratory techniques, such as gene expression microarrays. Conclusions: Inclusion of tests for anemia significantly improves diagnostic accuracy for the breast cancer in comparison to the diagnostic accuracy of the CA 15-3 alone. Application of the random forests also enables the reduction of number of laboratory tests needed for the establishment of diagnosis. Differences in relevant test values between the cancer and control group are small but application of multiparametric statistical learning ensured diagnostic accuracy of 72.0% associated by a sensitivity of 64.7% and specificity of 84.9%.

INFLUENCE OF EXTRACTION TECHNIQUES ON GC-MS SCREENING RESULTS

Gas chromatography coupled with mass spectrometry (GC-MS) is often used as a confirmatory technique for positive results obtained with immunoassay screening tests, but it can also be used as a screening method in clinical and forensic toxicology. Because of different reasons (amount of sample available, urgency, cost of analysis, etc.), laboratory is often in position to perform just one extraction and screening per sample. Influence of different extraction procedures on results is usually overlooked, but it can have great impact on obtained results, both quantitatively and qualitatively. We prepared four different levels of commercial urine toxicology control samples Liquichek (BIO-RAD), containing 12 drugs-ofabuse using three different extraction procedures: liquid– liquid extraction system TOXI-TUBES A (VARIAN), liquid–liquid extraction system Chem Elut (VARIAN) and solid phase extraction system based on Amberlite XAD2 polymeric adsorbent columns (Sigma–Aldrich). All extracts were analyzed with GC-MS (SHIMADZU GCMS-QP5050A). Obtained results are presented and influence of extraction procedure on GC-MS screening results is evaluated for every extraction procedure.