G. C. Guidi

HE4 in ovarian cancer: from discovery to clinical application.

Despite the relatively low prevalence, ovarian cancer is the fifth leading cause of death from cancer among women. As such, an early diagnosis for establishing a timely surgical and/or chemotherapeutic treatment is essential for improving the outcome. The most reliable, but not always straightforward, approach to diagnose ovarian cancer relies on multiple, time-consuming and expensive investigative tools. These typically include clinical presentation (i.e., pelvic or abdominal pain, urinary frequency or urgency, increased abdominal size or bloating) with pelvic examination, transvaginal ultrasonography (US), and measurement of carbohydrate antigen 125 (CA125). Although the conventional pathway to develop and market a clinically useful biomarker is challenging, recent advances in genomic and proteomic technologies have led to the identification of previously unknown candidate markers of ovarian cancer. Some of these are currently under clinical validation. The human epididymis protein 4 (HE4) has recently been approved by the Food and Drug Administration for monitoring recurrence or progression of epithelial ovarian cancer. Nevertheless, reliable clinical evidence demonstrates that HE4, used alone or in combination with CA125, substantially improves the accuracy of screening and/or disease monitoring. This chapter will review the current knowledge on biologic and clinical applications of ovarian cancer biomarkers, with particular emphasis on the newly proposed marker, HE4.

Acute variation of biochemical markers of muscle damage following a 21-km, half-marathon run.

Objective. Although there is information on biochemical markers of muscle and cardiac damage following strenuous exercise, little is known about the kinetics of these markers in athletes performing sub‐maximal exercise. Material and methods. Fifteen healthy, trained, Caucasian males took part in a 21‐km run. Blood samples were collected before the run, immediately after (post), and 3u2005h, 6u2005h and 24u2005h thereafter. Biochemical markers of muscle and cardiac damage were evaluated on the Modular System, employing proprietary reagents. In no case did the concentration of troponin T increase by >0.03u2005ng/mL. The values of aspartate aminotransferase (AST), creatine kinase (CK), CK MB, lactate dehydrogenase (LDH) and myoglobin increased significantly immediately after the run and remained elevated 24u2005h thereafter. Results. The number of subjects with values above the upper limit of the relative reference ranges did not vary throughout the study period for AST and LDH, while it increased significantly for CK, CK MB and myoglobin. The major variation over the pre‐run value was recorded for myoglobin (3‐fold increment), whereas AST and LDH increased 1.1 and 1.3‐fold, respectively. Conclusions. The results suggest the hypothesis that sub‐maximal exercise influences the concentration of several biomarkers of muscle damage for up to 24u2005h with no biochemical signs of myocardial damage.

Influence of temperature and time before centrifugation of specimens for routine coagulation testing

The accurate standardization of the preanalytical phase is of pivotal importance for achieving reliable results of coagulation tests. Because information on the suitable storage conditions for coagulation testing is controversial, we aimed at investigating the sample stability with regard to the temperature and time before centrifugation. The activated partial thromboplastin time (aPTT), prothrombin time (PT), fibrinogen and D‐dimer were assayed in specimens collected from 26 consecutive patients on antivitamin K therapy on the ACL TOP analyzer. Three primary 3.6‐ml siliconized evacuated tubes containing 0.109u2003mol/l buffered trisodium citrate were sequentially collected from each patient. These three tubes were mixed, pooled and divided into seven identical aliquots. The first aliquot was immediately centrifuged according to the standard protocol [1500u2003g for 15u2003min at room temperature (RT)] and analyzed. The other aliquots were left for 3, 6 and 24u2003h, respectively, at RT or 4u2003°C, and then centrifuged and analyzed. Test results were compared with those obtained on the reference specimen. Statistically significant prolongations were observed for aPTT in all the samples. Such differences exceeded the analytical quality specifications for desirable bias in the samples stored for 24u2003h. A significant reduction, yet comprised within the desirable bias, was observed for PT and fibrinogen in uncentrifuged specimens stored at RT for 3 and 6u2003h. No significant biases could be recorded in D‐dimer. In conclusion, a 6‐h storage of uncentrifuged specimens at either RT or 4u2003°C may still be suitable to achieve results of routine coagulation testing comprised within the analytical quality specifications for desirable bias.

Impact of different inhibitor reactivities with commercial factor VIII concentrates on thrombin generation

Summary.u2002 In order to describe the haemostatic role of a variation in inhibitor reactivity with different factor VIII (FVIII) concentrates, we have compared inhibitor titres against a panel of FVIII concentrates and correlated titre with the capacity to inhibit thrombin generation. Three plasma‐derived concentrates were tested in vitro in mixing experiments with inhibitor plasmas from 11 patients with severe haemophilia A: Fanhdi, which contains von Willebrand factor (VWF) with a final ratio of approximately 1:1 (VWFu2003IU per IU FVIII:C); Haemate‐P with a ratio of 2.5:1 and Hemofil‐M containing only trace amounts of VWF. In addition, the recombinant FVIII concentrate Kogenate Bayer containing no VWF was included. Inhibitor titres and the capacity to generate thrombin were measured. A statistically significant difference in measured titres was found with the highest titres recorded against Hemofil‐M. The inhibitor titres needed to inhibit 50% maximum thrombin generation were the lowest for Kogenate Bayer and the highest and similar for Fanhdi and Haemate‐P with intermediate titres needed for inhibition of Hemofil‐M. In this study, the thrombin generation assay provides additional indications for the role of VWF in the treatment of patients with inhibitors. The VWF‐containing concentrates Fanhdi and Haemate‐P, added to FVIII‐deficient plasma with the presence of inhibitor, generate more thrombin than do the purified concentrates Hemofil‐M and Kogenate Bayer.

Acute Variation of Estimated Glomerular Filtration Rate Following a Half-Marathon Run

The accurate estimation of glomerular filtration rate (GFR) is pivotal in sports medicine. However, there is controversial information on the acute influence of physical exercise on kidney function in healthy athletes. The estimated GFR (EGFR) was assessed by the recommended Modification of Diet in Renal Disease (MDRD) equation before a 21-km half-marathon, at the end, and 3, 6, 24 hrs thereafter on 17 trained, middle-aged males. Results were corrected for plasma volume changes. The mean EGFR at the baseline was 76 mL/min/1.73 m (2); it decreased at the end of the run (62 mL/min/1.73 m (2)) and for the following 3 hrs (68 mL/min/1.73 m (2)) and 6 hrs (70 mL/min/1.73 m (2)), though statistical significance was only achieved immediately after the run (mean decrease 16 %, p < 0.01). The frequency of athletes with EGFR below the normal threshold was higher than the baseline immediately after the race and for the following 6 hrs. Twenty-four hours after the run, the EGFR had returned to values similar and nonsignificantly different from those recorded at the baseline. These results attest that medium to high strains of running in healthy, middle-aged, trained individuals do not cause renal damage, but a limited and temporary decline in renal function.

Evaluation of platelet turnover by flow cytometry

The number of circulating newly produced platelets depends on the thrombopoietic capacity of bone marrow as well as platelet removal from the bloodstream. Flow cytometric analysis with thiazole orange (TO), a fluorescent dye that crosses platelet membranes and binds intracellular RNA, has been used to measure circulating reticulated platelets (RPs) with high RNA content as an index of platelet turnover. We first assessed the specificity of TO flow cytometry and then applied this method in the diagnosis of thrombocytopenia caused by impaired platelet production or increased destruction. We also explored the utility of TO flow cytometry to predict thrombocytopoiesis after chemotherapy-induced bone marrow aplasia. Venous blood, anticoagulated with K2EDTA, was incubated with 0.6u2009µg/ml TO plus an anti-GPIIIa monoclonal antibody. The mean percentage of RPs in control subjects (nu2009=u200923) was 6.13u2009±u20093.09%. RPs were 10.41u2009±u20099.02% in patients (nu2009=u200910) with hematological malignancies during aplasia induced by chemotherapy and a significant increase in RPs (35.45u2009±u20096.11%) was seen in the recovery phase. In 10 patients with idiopathic thrombocytopenic purpura, the percentage of TO positive platelets was 67.81u2009±u200918.79 (Pu2009<u20090.001 vs. controls). In patients with thrombocytopenia associated with hepatic cirrhosis (nu2009=u200921; 21.04u2009±u200916.21%, Pu2009<u20090.001 vs. controls) or systemic lupus erythematosus (nu2009=u20096, 29.08u2009±u200915.57%; Pu2009<u20090.001 vs. controls) increases in TO-stained platelets were also observed. Measurement of TO positive platelets may be a reliable tool for the laboratory identification of platelet disorders, with a higher sensitivity than measurement of platelet volume. Measurement of RPs may also prove useful to recognize the underlying pathogenetic mechanisms in thrombocytopenia.

Right or wrong sample received for coagulation testing? Tentative algorithms for detection of an incorrect type of sample

Inappropriate blood collection potentially comprises the major pre‐analytical problem for coagulation testing. Inappropriate samples are most difficult to detect when received as secondary aliquots, common for referred tests. This study aimed to identify a simple, quick and inexpensive process to help laboratories distinguish the type of sample, should there be suspicion of inappropriate collection. Samples from 15 patients [selected on the basis that four different primary tubes were available: serum, citrated plasma, ethylene diamine tetraacetic acid (EDTA) plasma, lithium‐heparin plasma], were tested for common electrolytes that might substantially differ according to the type of sample. In citrated plasma, potassium, chloride, calcium and magnesium were significantly decreased compared with serum and lithium‐heparin plasma, while sodium was markedly increased. In EDTA plasma, sodium and chloride were significantly decreased compared with both serum and lithium‐heparin plasma, potassium was always >14u2003mmol/l, whereas magnesium and calcium were virtually undetectable. These data allowed development of two algorithms for differential identification of citrated plasma vs. other samples with 100% sensitivity and specificity, the former based on the sequential measurement of potassium, calcium and sodium, the latter on potassium and sodium. These simple assays can supplement classical coagulation test methods to identify most inappropriate blood collections and validate sample rejection.

Epigenetic alteration: new insights moving from tissue to plasma – the example of PCDH10 promoter methylation in colorectal cancer

Background:Tumour-released DNA in blood represents a promising biomarker for cancer detection. Although epigenetic alterations such as aberrant promoter methylation represent an appealing perspective, the discordance existing between frequencies of alterations found in DNA extracted from tumour tissue and cell-free DNA (cfDNA) has challenged their practical clinical application. With the aim to explain this bias of agreement, we investigated whether protocadherin 10 (PCDH10) promoter methylation in tissue was associated with methylation pattern in matched cfDNA isolated from plasma of patients with colorectal cancer (CRC), and whether the strength of concordance may depend on levels of cfDNA, integrity index, as well as on different clinical–pathological features.Methods:A quantitative methylation-specific PCR was used to analyse a selected CpG site in the PCDH10 promoter of 67 tumour tissues, paired normal mucosae, and matched plasma samples. The cfDNA integrity index and cfDNA concentration were assessed using a real-time PCR assay.Results:The PCDH10 promoter methylation was detected in 63 out of 67 (94.0%) surgically resected colorectal tumours and in 42 out of 67 (62.7%) plasma samples. The median methylation rate in tumour tissues and plasma samples was 43.5% (6.3–97.8%) and 5.9% (0–80.9%), respectively. There was a significant correlation between PCDH10 methylation in cfDNA and tumour tissue in patients with early CRC (P<0.0001). The ratio between plasma and tissue methylation rate increases with increasing cfDNA integrity index in early-stage cancers (P=0.0299) and with absolute cfDNA concentration in advanced cancers (P=0.0234).Conclusion:Our findings provide new insight into biological aspects modulating the concordance between tissues and plasma methylation profiles.

No influence of a butterfly device on routine coagulation assays and D-dimer measurement.

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Usefulness of serum HE4 in endometriotic cysts.

Sir, n nHuhtinen et al (2009) recently reported about the usefulness of Human Epididymal Protein 4 (HE4) in discriminating ovarian tumours from ovarian endometriotic cysts, which is of pivotal importance as CA125 is often increased in benign gynaecological diseases such as endometriosis (Muyldermans et al, 1995). Therefore, we performed a similar study on 46 patients affected by ovarian cancer and 21 patients affected by endometriosis (12 ovarian endometrioma and 9 non-ovaric endometriosis). Serum levels of CA125 were determined using a chemiluminescent enzyme immunoassay on the Liaison (DiaSorin, Saluggia, Italy). Serum levels of HE4 were determined using an ELISA kit developed by Fujirebio Diagnostic Inc. (Malvern, PA, USA). n nThe mean serum concentration of HE4 in patients with ovarian cancer was significantly higher than that observed in patients with endometriosis (810.0 vs 49.4u2009pM, P<0.0001). The mean serum concentrations of HE4 in our patients with ovarian endometrioma were similar to those observed in the study of Huhtinen et al (2009) (47.5 vs 46.0u2009pM). However, in our investigation, 10 out of 12 patients with ovarian endometrioma showed CA125 values higher than 35u2009Uu2009ml−1, but only one patient showed an HE4 serum concentration higher than 70u2009pM, which is the upper limit of the reference range (Moore et al, 2008). More importantly, receiver operator characteristic curve analysis showed that HE4 has a significantly higher area under the curve as compared with CA125 (0.85 vs 0.77, P<0.0001) for differentiating ovarian cancer from ovarian endometrioma. The results of our investigation confirm that HE4 is a promising marker for the early differentiation of ovarian cancer from ovarian endometriotic cysts, showing better diagnostic performances than CA125.