G. L. Salvagno

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 3 h, 6 h and 24 h 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 >0.03 ng/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 24 h 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 24 h 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 0.109 mol/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 [1500 g for 15 min at room temperature (RT)] and analyzed. The other aliquots were left for 3, 6 and 24 h, respectively, at RT or 4 °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 24 h. A significant reduction, yet comprised within the desirable bias, was observed for PT and fibrinogen in uncentrifuged specimens stored at RT for 3 and 6 h. No significant biases could be recorded in D‐dimer. In conclusion, a 6‐h storage of uncentrifuged specimens at either RT or 4 °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.  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 (VWF IU 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.

Transillumination: a new tool to eliminate the impact of venous stasis during the procedure for the collection of diagnostic blood specimens for routine haematological testing

Introduction:  The collection of diagnostic blood specimens for routine haematological testing (RHT) is traditionally performed with tourniquet. However, the transillumination devices based on cold near‐infrared LEDs have been formerly proposed as a valuable tool for identifying reliable venous accesses, especially in patients with difficult or small veins, such as children. This study was aimed to evaluate whether a transillumination device can advantageously replace the use of the tourniquet during the procedure for collection of blood specimens for RHT and thereby eliminating the discomfort and risk of spurious results caused by excessive or prolonged venous stasis.

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.

Elimination of the venous stasis error for routine coagulation testing by transillumination.

The preanalytical phase is responsible for more than two-thirds of all errors attributed to the clinical laboratory [1–4] and there are only a few routine procedures for the detection of nonconformities in this field of activity [5,6]. In this phase the procedures involving phlebotomy, critical to the obtainment of diagnostic blood specimens, are poorly studied as regards the major sources of errors and the procedures related to quality control process [7,8]. The collection of diagnostic blood specimens for routine coagulation tests are traditionally performed by phlebotomists using a tourniquet [9]. The Clinical and Laboratory Standards Institute (CLSI) recommends the use of the tourniquet for localizing suitable veins for ≤60 s. When performing specimen collection for diagnostic purposes such an interval of time both allows easy localization of vein paths and concomitantly circumvents possible problems due to excess venous stasis [10–12]. Although the venous stasis can influence the concentration and/or the activity of several blood analytes, the tourniquet time is rarely regarded as a potential source of laboratory variability [13–17]. Reportedly, the mean tourniquet application times by phlebotomists were 98 s in public laboratories and 70 s in private laboratories respectively, thus raising some issues about proper specimen collection [18]. The use of transillumination devices, based on cold near infrared light-emitting diodes (LEDs) whose lightsare absorbed by intra-erythrocyte hemoglobin flowing along the veins, has been initially proposed in order to ease the vein puncture in children [19]. The efficacy of palm transillumination for establishing venous access in small infants has already been assessed [20]. Moreover, the transillumination has been proposed for mapping veins to be cannulated prior to ambulatory phlebotomy because it allows accurate visualization of the vein course [21]. Reliability in coagulation testing is pivotal to the appropriate diagnosis and treatment of patients with hemostasis disturbances [17,22]. In this context some preanalytical details/procedures appear critical such as a) adequate fasting time before blood collection [23], b) use of appropriate tubes [24–26] and additives [27], c) appropriateness of blood collection, storage and centrifugation[28–30], and d) strict conformity to the recommendations regarding tourniquet time. Clinical laboratory results are estimated to be able to influence 60% to 70% of medical decisions and thus affect diagnostic outcome and/or patient treatments [31], such as oral anticoagulant therapy employed in patients at risk of thrombosis [32,33] or blood transfusion components prescription [34] recommended in bleeding patients with disseminated intravascular coagulation (DIC) and prolonged

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 0.6 µg/ml TO plus an anti-GPIIIa monoclonal antibody. The mean percentage of RPs in control subjects (n = 23) was 6.13 ± 3.09%. RPs were 10.41 ± 9.02% in patients (n = 10) with hematological malignancies during aplasia induced by chemotherapy and a significant increase in RPs (35.45 ± 6.11%) was seen in the recovery phase. In 10 patients with idiopathic thrombocytopenic purpura, the percentage of TO positive platelets was 67.81 ± 18.79 (P < 0.001 vs. controls). In patients with thrombocytopenia associated with hepatic cirrhosis (n = 21; 21.04 ± 16.21%, P < 0.001 vs. controls) or systemic lupus erythematosus (n = 6, 29.08 ± 15.57%; P < 0.001 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 >14 mmol/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.

1 Ameri A, Bousser MG. Cerebral venous thrombosis.Neurol Clin 1992; 10: 87–111. 2 Einhaupl KM, Villringer A, Meister W, Mehraein S, Garner C, Pellkofer M, Haberl RL, Pfister HW, Schmiedek P. Heparin treatment in sinus venous thrombosis. Lancet 1991; 338 (8767): 597–600. 3 Yamini B, Loch Macdonald R, Rosenblum J. Treatment of deep cerebral venous thrombosis by local infusion of tissue plasminogen activator. Surg Neurol 2001; 55: 340–6. 4 Bates SM, Grand’Maison A, Johnston M, Naguit I, Kovacs MJ, Ginsberg JS. A latex D-dimer reliably excludes venous thromboembolism. Arch Intern Med 2001; 161: 447–53. 5 Keeling DM,Wright M, Baker P, Sackett D. D-dimer for the exclusion of venous thromboembolism: comparison of a new automated latex particle immunoassay (MDA D-dimer) with an established enzymelinked fluorescent assay (VIDAS D-dimer).Clin LabHaematol 1999; 21: 359–62. 6 Wildberger JE, Mull M, Kilbinger M, Schon S, Vorwerk D. [Cerebral sinus thrombosis: rapid test diagnosis by demonstration of increased plasma D-dimer levels (SimpliRED)]. Rofo Fortschr Geb Rontgenstr Neuen Bildgeb Verfahr 1997; 167: 527–9. 7 Tardy B, Tardy-Poncet B, Viallon A, Piot M, Garnier P, Mohamedi R, Guyomarc’h S, Venet C. D-dimer levels in patients with suspected acute cerebral venous thrombosis. Am J Med 2002; 113: 238–41. 8 Lalive PH, de Moerloose P, Lovblad K, Sarasin FP, Mermillod B, Sztajzel R. Is measurement of D-dimer useful in the diagnosis of cerebral venous thrombosis? Neurology 2003; 61: 1057–60. 9 Talbot K,WrightM, Keeling D. Normal D-dimer levels do not exclude the diagnosis of cerebral venous sinus thrombosis. J Neurol 2002; 249: 1603–4.