Autoimmune disease and sickle cell anaemia: 'Intersecting pathways and differential diagnosis'.

Sickle cell disease (SCD) is an inherited disorder, which occurs due to a single gene mutation. It has multisystemic manifestations, affecting millions of people worldwide. The effect of SCD on joints and musculature can overlap with clinical features of autoimmune disease (AD). It is therefore difficult for clinical haematologists and physicians treating SCD patients to discriminate between these two conditions clinically. A delay in diagnosis leads to untreated symptoms and treatment differs considerably. An accurate knowledge of clinical findings and laboratory results of AD and SCD can help physicians avoid this. In the review that follows, we examine the existing literature on SCD and AD, and describe the features that may distinguish SCD and autoimmune disease such as systemic lupus erythematosus and rheumatoid arthritis. We aim to guide clinical haematologists and physicians towards a more rapid diagnosis of AD in sickle cell anaemia patients, by correct interpretation of the clinical assessment and commonly available diagnostics.

Effect of a liberal versus a restrictive pre-donation blood pressure policy on whole-blood donor adverse reactions.

BACKGROUND AND OBJECTIVES: The role of pre-donation blood pressure (BP) as independent contributor to post-donation vasovagal reactions (VVRs) is still debated. Differences between a liberal (i.e., inclusion of hypotensive donors) and a restrictive policy (i.e., not accepting hypotensive donors) should be investigated. This study aims to investigate the consequences of a liberal policy in development of VVRs after whole-blood donations. MATERIALS AND METHODS: We compared the incidence of VVRs between 2015 (restrictive policy) and 2016 (liberal policy) and the associated risk factors. We evaluated respectively 22 789 vs. 21 676 blood donations obtained from 18 001 blood donors (12 501 donated in both years). RESULTS: Comparing the results we obtained between 2015 and 2016, donations showed an overlap of the cohorts. Two hundred fifteen VVRs (incidence rate 0·48%) were observed, 104 (0·46%) of which in 2015, and 111 (0·51%) in 2016. A preliminary univariate analysis showed that donors with systolic BP <110 mm Hg had a two-fold risk of VVRs compared to normotensive donors (VVR/donation rate of 0·99% vs. 0·46%; P = 0·001). The subsequent multivariable logistic regression model showed that VVRs were highly associated with weight, site of collection, age and number of donations, excluding a role for systolic and diastolic BP. CONCLUSION: A liberal pre-donation BP policy seems to be safe for blood donors. Our analysis confirms that older donors with higher body-weight who already had donated blood are unlikely to experience VVRs.

Insight into the complex pathophysiology of sickle cell anaemia and possible treatment.

Sickle cell anaemia (SCA) is the consequence of abnormal haemoglobin production due to an inherited point mutation in the ß-globin gene. The resulting haemoglobin tetramer is poorly soluble when deoxygenated, and when this is prolonged, intracellular gelation of sickle haemoglobin occurs, followed by haemoglobin polymerisation. If many cycles of sickling and unsickling occur, the red cell membrane will be disrupted leading to haemolysis and vaso-occlusive events. Recent studies have also shown that leucocyte adhesion molecules and nitric oxide (NO) depletion are involved in endothelial damage. New insights in SCA pathophysiology and vascular biology have shown that cell-derived microparticle (MP) generation is also involved in the vaso-occlusion. Endothelial damage is perpetuated by impaired production or increased consumption of protective modulators such as protein C, protein S and NO. New therapeutic interventions should address these aspects of SCA pathogenesis. To date, the only US-FDA-approved therapy to prevent painful vaso-occulsive episodes is hydroxyurea that reduces haemoglobin polymerisation in sickle cells by increasing the production of foetal haemoglobin and L-glutamine. However, several new drugs have been tested in the last years in randomised clinical trials. We here report an update on the current status of knowledge on SCA.

Validity and reliability of serologic immunophenotyping of multiple blood group systems by ORTHO Sera with fully automated procedure.

CONCLUSIONS: The increase of immunization against blood group antigens has reinforced the need for automated extensive blood typing. The aim of this study was to assess both the validity and reliability of red blood cell (RBC) automated agglutination technology in testing for antigens of Kidd (Jk), Duffy (Fy), and MNS (Ss) blood systems. ORTHO Sera (Ortho Clinical Diagnostics, Raritan, NJ) anti-Jka, anti-Jkb, Anti-Fya, anti-Fyb, anti-S, and anti-s reagents were each tested on RBC samples previously typed. Replicates were performed on three separate testing sessions with three consecutive repetitions within each session, thus obtaining 486 test results. Accuracy was assessed by aggregate analysis of sensitivity, specificity, and area under the receiver operating characteristics curve (AUC). Reliability was estimated by a cross-classified mixed-effect logistic model. All reagents tested yielded optimal accuracy (100% for sensitivity and specificity, and 1.00 for AUC), except for anti-S, for which performance was slightly lower (98%, 100%, and 0.99, respectively), owing to misclassification of one sample in a single replicate. Anomalous automated measurements were recorded in 38 of 486 tests (7.8%), which then needed additional manual interpretation. Different sessions and samples were the major contributors to measurement failures (38% and 18%, separately). Order of repetitions and antigen specificity across replicates did not contribute to the risk of failures, although weak evidence of enhanced risk was observed with Jk testing. Automated RBC typing with ORTHO Sera reagents against antigens in the Kidd, Duffy, and MNS blood group systems displayed nearly 100 percent accuracy. However, a sizable number of replicates needed additional ad hoc interpretation, thus suggesting that the reliability could still be improved. Automated agglutination technology represents a viable option for phenotyping large volumes of samples.

Autoantibody Profiling in a Cohort of Pediatric and Adult Patients With Autoimmune Hepatitis.

BACKGROUND: Autoimmune hepatitis (AIH) is a rare condition characterized by the presence of autoantibodies distinctive of type 1 AIH (AIH-1) and type 2 AIH (AIH-2). The aim of this study was to evaluate the autoantibody profile in a cohort of pediatric and adult AIH patients, using both indirect immunofluorescence (IIF) and a new multiplexed line-blot assay. METHODS: Sera from 63 pediatric and 53 adult AIH patients were tested for antinuclear (ANA), antismooth muscle (SMA), anti-liver kidney microsome 1 (anti-LKM1), anti-liver cytosol 1 (anti-LC1) autoantibodies using IIF methods; for anti-LKM1, anti-LC1, and soluble liver antigen/liver-pancreas (anti-SLA/LP) autoantibodies using the line-blot; for anti-F-actin autoantibodies using IIF both on VSM47 cell-line and on rat intestinal epithelial cells. RESULTS: AIH-1 was the most common type of AIH in the adult cohort (73.6%), while AIH-2 was the most common AIH in the pediatric cohort (61.9%). Both in adult and pediatric AIH-2 anti-LKM1 were the prevalent autoantibodies. In pediatric AIH-2 anti-LC1 autoantibodies were more frequent than in adult AIH-2 (59 vs. 28.6%), and in 35.9% of cases they were present alone. In 17 patients anti-LC1 autoantibodies were detected only with the line-blot assay. The levels of anti-LKM1 and of anti-LC1 were not different between adult and pediatric AIH, and the overall agreement between the results obtained with the two IIF methods for F-actin detection was 98.8% (CI 95%: 94.4-99.7%). CONCLUSIONS: The line-blot assay showed a higher sensitivity than IIF for anti-LC1 detection. Anti-LKM1 and anti-LC1 autoantibody levels are not different in adults and children. An almost perfect agreement between the two IIF methods for anti-F-actin detection has been observed.

Ten years differences in recently onset atrial fibrillation and flutter incidence and management.

ABSTRACT: Ten years differences in recently onset atrial OBJECTS AND BACKGROUND: Atrial fibrillation (AF) and atrial flutter (AFl) are the most common arrhythmias in day-life clinical practice. Purpose of our study was to verify differences occurred in the last ten years in AF and AFI incidence and treatment in the emergency room (ER). METHODS: from the 17th January to the 15th February 2000 and from the 18th January to the 16th February 2010 all the consecutive patients with AF or AFl referring to the ER of our hospital were included in the study. Epidemiological data were collected along with information about treatment, admission to hospital wards, days of hospital stay and therapy. Data from the year 2000 were compared to these collected ten years later. RESULTS: incidence of AF and AFl has increased in the years (50%), patients are older (73.5 vs. 65.2 years; p 0.029) and refer late to the ER (45.6% in 2010 and 23.7% in 2000 with a delay of > 48 hours from arrhythmias onset; p 0.054). In 2010 only a minority of these patients is directly discharged from the ER (15.8% vs 14.4%) and there is an increased admission rate due to AF or AFl (67.5%; p 0.026), if compared to the whole admissions of the hospital. The median duration of hospital stay decreased from 6 days to 4.5 days in the year 2010 (NS). CONCLUSION: AF and AFl incidence is still increasing and account for a high admission rate from the ER to the hospital wards. Costs are consequently continuously increasing.

Differential reactivity to IMPDH2 by anti-rods/rings autoantibodies and unresponsiveness to pegylated interferon-alpha/ribavirin therapy in US and Italian HCV patients.

PURPOSE: Autoantibodies to cytoplasmic structures called rods and rings (RR) are primarily specific to patients with hepatitis C virus (HCV) infection treated with pegylated interferon-alpha/ribavirin (IFN/R). Our aim is to examine anti-RR antibodies specificity and correlation with the response to IFN/R therapy in two independent cohorts (US and Italy) of HCV patients. METHODS: Sera from the US cohort (n = 47) and the Italian cohort (n = 46) pre-selected for anti-RR antibodies were analyzed by immunofluorescence and radioimmunoprecipitation. The prevalence and titers of anti-RR were analyzed for correlation with the response to IFN/R therapy. RESULTS: In the US cohort, anti-RR antibodies were more frequently non-responders to IFN/R (71 % vs 29 % responders). Titers in responder patients (n = 11) were ≤1:3200, whereas titers in non-responder patients (n = 27) reached 1:819,200 (p = 0.0016). In the Italian cohort, anti-RR titers ranged from 1:200 to >1:819,200 and only relapsers had the highest anti-RR titers. Radioimmunoprecipitation demonstrated that anti-RR autoantibodies were mainly anti-inosine monophosphate dehydrogenase 2 (IMPDH2) - 96 % in the Italian cohort vs. 53 % in the US cohort. CONCLUSIONS: In the two cohorts analyzed, the anti-IMPDH2 response as a component of the anti-RR response is much more prominent in the Italian cohort. The reason for the difference between the US and Italian cohorts is unclear but it possibly illustrates the heterogeneity in response and the overall negative correlation between the production of these autoantibodies and response to IFN/R therapy. Patients with high titer anti-RR antibodies are either relapsers (Italian) or non-responders/relapsers (US).

The Importance of Being Aware of Intrinsic Methods' Limitation in Low-Density Lipoprotein Cholesterol Determination to Correctly Identify Cardiovascular Risk: Is Direct Determination Obtained with the Roche System Systematically Overestimating LDL in Very High-Risk Patients with Triglycerides Concentration of Less than 2.25 mmol/L?

BACKGROUND: low-density lipoprotein cholesterol (LDL-C) is a strong cardiovascular risk factor, but the methods used to correctly determine it are is still questioned. The aim of this study was to compare the direct determination of LDL-C levels, obtained with the Roche cobas c system, with LDL-C values calculated through Sampson's and Friedewald's equations in very high-risk patients with triglycerides concentrations of less than 2.25 mmol/L (<200 mg/dL). METHODS: in 127 consecutive patients with a recent diagnosis of acute coronary syndrome and triglycerides of less than 2.25 mmol/L, plasma LDL-C was measured directly and calculated with Sampson's and Friedewald's equations before hospital discharge, and the data were compared. RESULTS: median LDL values obtained with the Friedenwald and Sampson equations and with direct determination were 2.31 (IQR 1.59-3.21), 2.36 (IQR 1.66-3.26) and 2.64 (IQR 1.97-3.65) mmol/L, respectively. Direct measurements were higher by 0.35 and 0.40 mmol/L when compared to the levels calculated with Sampson's or Friedewald's equations, respectively (p < 0.01). The correlation between the two equations was almost perfect (rho 0.997) but decreased when the directly determined data were compared to those calculated with Sampson's equation (rho 0.954) or Friedewald's method (rho 0.939). CONCLUSION: direct determination generated higher values of LDL-C concentration through a probable systematic overestimation.

Liver Disease and Sickle Cell Disease: Auto-Immune Hepatitis more than a Coincidence; A Systematic Review of the Literature.

In patients with SCD, chronic liver damage is a common manifestation. More than 50% of SCD patients have elevated liver enzymes. Common underlying aetiologies include sickle cell hepatic crisis, viral hepatitis, sickle cell intrahepatic cholestasis and hepatic sequestration in the acute setting, and cholelithiasis and iron overload in the chronic setting. Autoimmune hepatitis (AIH) is a rare disease that appears to occur more commonly in the sickle cell disease (SCD) population than in the general population. There are many schools of thought as to why this is the case, including the phosphatidylserine hypothesis, the heme inflammatory hypothesis, the complement generation hypothesis, and the transfusion alloimmunization hypothesis. Due to the natural history of the two illnesses, SCD is almost always diagnosed first in cases of dual pathology. Symptoms such as jaundice, fatigue, and abdominal pain are common in SCD, as are abnormal liver function tests (LFTs). These abnormalities, attributed to the other more frequent liver involvements in SCD, can lead to delays in AIH diagnosis in this population. Corticosteroids, sometimes with other immunosuppressive agents, such as azathioprine, are the cornerstone of acute AIH treatment. However, corticosteroid use in the SCD population has been shown to carry an increased risk of vaso-occlusive crises, providing a treatment dilemma. The following is a review of AIH in the SCD population, where we explore the pathophysiology behind the association between the two disorders, discuss an approach to investigating abnormal LFTs in SCD, and examine treatment options in this population with co-existing diseases.

Influence of hemolysis on routine laboratory cardiac marker testing.

BACKGROUND: Reliable information on the potential bias arising from processing in vitro hemolysis specimens referred for conventional cardiac biomarker testing is scarce and controversial as yet. The present investigation was designed to assess the influence of low levels of in vitro hemolysis on cardiac biomarker testing. METHODS: Three aliquots, prepared by serial dilutions of homologous hemolysed samples collected from 14 different subjects and containing final concentrations of plasma hemoglobin of 0, 0.3, and 0.6 g/L were tested for the following parameters: cardiac troponin I (cTnI) and T (cTnT), myoglobin (Myo), creatine kinase isoenzyme-MB (CK-MB), brain natriuretic peptide (BNP) and NT-prohormone-brain natriuretic peptide (NT-pro BNP). RESULTS: No statistically significant differences were observed in any of the parameters tested nor did the bias achieve clinical significance. CONCLUSIONS: The results of this study show that moderate hemolysis, as low as 0.6 g/L, has no influence on the reliability of cardiac biomarker testing.

An unusual case of a primary blood collection tube with floating separator gel.

We describe an unusual case of a primary evacuated blood collection tube with floating separator gel, which has been collected from a 50-year-old man submitted to a percutaneous coronary intervention (PCI). The sample was collected from the femoral artery in a primary evacuated blood collection tube containing lithium-heparin. After centrifugation of the specimen, an unusual positioning of the separator gel was observed, which migrated at the topmost layer, whereas the packed blood cells remained in the middle and the plasma at the bottom. The potential interfering substance was found to be a contrast dye, 140 ml of which were administered to the patient during a revascularization procedure for acute myocardial infarction. The potential aspiration of the gel inappropriately positioned at the top of the tube by laboratory instrumentation can produce several technical and clinical problems, when not reliably detected. First, the needle of the instrument might be partially or completely obstructed by the gel, thus jeopardizing the integrity and correct functioning of the instrument. The aspiration of gel along with the sample matrix might also spuriously modify the test results, since an inappropriate amount of serum or plasma would be analyzed.

Utility of serum indices in a particular case of serum protein electrophoresis.

Screening and measurement of monoclonal (M) proteins are commonly performed using capillary zone electrophoresis (CZE). The identification of M-protein or monoclonal component (CM) is an essential requirement for diagnosis and monitoring of monoclonal gammopathies. The detection of CM has been largely improved by CZE. Capillary electrophoresis estimates CM more accurately, because absence of variation due to different dye binding affinities of proteins as instead seen with agarose gel electrophoresis. However, interferences can be present in CZE. This occurs because all substances absorbing at 200 nm can be identified. Recognition and handling of specimens exhibiting such interferences is essential to ensure accurate diagnostic and patient safety. We herein report on an unusual case of serum protein electrophoresis, to highlight that laboratory staff must be aware of and familiarise with the information provided by laboratory instruments. For example, in the case of serum indices, about specimen quality.

Blood cells characteristics as determinants of acute myocardial infarction.

BACKGROUND: The aim of this study is to analyse the relation between red blood cells, platelets morphology and acute myocardial infarction (AMI), and to assess whether they could supplement the role of traditional cardiac biomarkers in the early identification of patients with AMI. METHODS: All consecutive patients admitted to our emergency department between the 1st January and the 31st August 2009 due to chest pain of suspected cardiac origin were included in the study. All the patients underwent physical examination, a 12-lead ECG, blood sampling for determination of cardiac troponin I and a complete blood count. RESULTS: A percentage of 6.7% of the 1971 patients had a confirmed diagnosis of AMI. Mean corpuscular volume (MCV), red blood cells distribution width (RDW) and platelets count (Plt) did not differ between patients with and without AMI. However, the mean platelet volume (MPV) was significantly higher in AMI patients (7.9 vs. 7.7 fL; p=0.0457). After stratification for gender, men with AMI displayed a lower RDW (p=0.0464) and a higher MPV (p=0.0062) as compared with those without AMI. The MCV and Plt were not significantly different. Women with AMI had a higher RDW (p=0.0079) as compared with those without AMI, while the MCV, Plt and MPV were not significantly different. CONCLUSIONS: Our study partially confirms previous data on the association between MPV or RDW and AMI. The inclusion of these parameters along with other conventional cardiac biomarkers might be a valuable perspective when evaluating patients with suspected AMI, although gender differences should be taken in account.

Comparative evaluation of capillary zone electrophoresis and HPLC in the determination of carbohydrate-deficient transferrin.

BACKGROUND: Transferrin (Tf) glycoform lacking one or two complete or incomplete glycan chains (i.e., asialo-monosialo- and disialo-Tf) typically appear in blood after chronic alcohol consumption, though recently it was reported that monosialo-Tf is associated with trisialo-Tf but not with alcohol consumption. These glycoforms are collectively known as carbohydrate-deficient transferrin (CDT). Since samples from alcoholic patients are characterized by decreased sialic acid content in serum transferrin, the assessment of CDT is thereby widely used for laboratory evaluation of chronic alcohol abuse. METHODS: CDT analysis has been performed in 6011 consecutive subjects undergoing national mandatory testing after the confiscation of driving license for driving under the influence of alcohol. Out of the 6011 specimens, 539 (9%) displayed values exceeding the specific cut-off (>1.3%) on multicapillary electrophoresis (MCE) (Capillarys2 Sebia, France), and were further analyzed with a routine high-pressure liquid chromatography (HPLC) technique. RESULTS: The overall correlation between the methods in the total 539 samples was satisfactory, displaying a correlation coefficient (r) of 0.960. Nevertheless, the correlation was lower in the group with CDT values comprised between 1.3% and 1.9% (group 1; r=0.60) than in those with CDT values >2.0% (group 2; r=0.98). Moreover, the discordance between values exceeding the method-specific threshold in the former group of samples was also remarkably high (62% of samples in group 1 vs. 0.6% in group 2). Finally, a significant difference of CDT values was observed in group 1 (p<0.001), and in group 2 (p<0.0001) by Wilcoxon test. CONCLUSIONS: The MCE is characterized by a high throughput and it seems a suitable approach for laboratory monitoring of alcohol abuse when CDT is used as medical parameter in the diagnosis and follow-up of heavy drinking. However, CDT measured by screening techniques must be confirmed by a confirmatory technique, in particular for forensic purpose.

Comparison of Freelite and N-Latex serum free light chain assays: a critical review.

INTRODUCTION: The measurement of serum free light chain (FLC) represents a fundamental aspect on the assessment of patients with monoclonal gammopathies (MG). Different analytical methods for FLC have become available with the possibility to obtain different value with a substantial impact on the assessment of patients with MG. This study aimed to evaluate FLC results obtained with two different assays and how the difference value obtained can impact in the patient's assessment. MATERIALS AND METHODS: Ninety-three patient serum samples that underwent analysis for FLC with two different methods, Serum Freelite (The Binding Site, Birmingham, UK) and N-Latex FLC (Siemens, Marburg, Germany), were included in this retrospective study. Statistical analysis was performed to evaluate correlation, difference, and the grade of concordance between the results obtained with the two methods. RESULTS: Significant statistical differences between the results obtained from the two methods were found (P < 0.05). A good correlation was found (0.99 for κ FLC, 0.95 for λ FLC, and 0.94 for the κ/λ ratio, respectively). We found a weighted kappa value of 0.65 for κ/λ ratio, 0.65 for λ FLC and 0.90 for κ FLC. A positive bias found with the Bland-Altman plot mirrors overestimation of κ FLC and κ/λ ratio with Freelite compared to N-Latex, whilst a negative bias underscores underestimation of λ FLC by Freelite compared to N-Latex. CONCLUSION: Although in general the concordance between Freelite and N-Latex appears satisfactory, several discrepancies could be evidenced and consequently the two assays are not interchangeable.

Preliminary assessment of the new Sysmex XN parameter Iron-Def for identifying iron deficiency.

BACKGROUND: Although the clinical assessment of iron status is usually based on iron stores, a rapid and accurate diagnosis of iron deficiency is challenging since ferritin is often unavailable as an urgent test and its value is frequently increased in acute phase conditions. This study was therefore aimed at evaluating the diagnostic performance of the new Sysmex XN "Iron Deficiency?" (Iron-Def) parameter for identifying patients with iron deficiency. MATERIALS AND METHODS: The study population consisted of 688 consecutive patients (median age: 71 years; 341 women and 347 men), referred for routine diagnostics to the Laboratory of Clinical Pathology of Lecco Hospital, Italy. A complete clinical chemistry profile and haematological testing were performed for identifying iron deficiency anaemia. RESULTS: A significant negative correlation was found between Sysmex XN Iron-Def and ferritin, serum iron, mean cell haemoglobin concentration, mean cell haemoglobin, mean corpuscular volume and age, while a positive correlation was noted with transferrin, percentage of microcytic red cell, red blood cell count and red blood cell distribution width. The diagnostic accuracy of Iron-Def for identifying patients with a percentage of saturation of transferrin <15% (n=104) was 84%, with a sensitivity of 0.952 and specificity of 0.538. A sub-analysis of 71 patients with ferritin <20 ng/dL yielded an even better diagnostic performance (86%, with a sensitivity of 0.935 and specificity of 0.620). DISCUSSION: Although additional confirmatory investigations would be needed, the preliminary findings of our study attest that Iron-Def may be an easy, inexpensive, rapid and reliable parameter for screening iron deficiency anaemia.

Intravenous ferric carboxymaltose is effective and safe in patients with inflammatory rheumatic diseases.

BACKGROUND: The aim of this study was to evaluate the efficacy and safety of ferric carboxymaltose in rheumatic patients with iron deficiency anaemia. MATERIALS AND METHODS: The study retrospectively evaluated a cohort of 34 patients with iron deficiency anaemia affected by inflammatory rheumatic diseases that are refractory or intolerant to oral iron therapy. They were treated with ferric carboxymaltose for a total of 56 cycles of treatment. The primary end point was to evaluate the increase of haemoglobin after ferric carboxymaltose treatment. The secondary end point was safety, including the occurrence of disease flare. RESULTS: Median age of the cohort was 60 years (range 31-91 years), with a male/female ratio of 4/30. Nine (26.5%) were affected by rheumatoid arthritis, 10 (29.4%) by spondyloarthritis, and 15 (44.1%) by other autoimmune connective tissue diseases. Median time from diagnosis was 7 years (IQR 2-12). At time of treatment (T0), median haemoglobin was 9.3 g/dL (IQR 8.2-10.3), transferrin saturation 6.2% (IQR 3.8-9.8), and ferritin 8.5 ng/mL (IQR 6.0-12.8). Median ferric carboxymaltose dose was 1,000 mg. At 6 weeks from T0, median haemoglobin was 12.3 g/dL (IQR 11.6-13.3), with a mean increase of 3.0 g/dL (p<0.01). Twelve (35.3%) patients needed re-treatment with ferric carboxymaltose for recurrence of iron deficiency anaemia. Four (4.3%) patients developed mild grade side effects. One suspected flare reaction has been observed. DISCUSSION: In patients affected by inflammatory rheumatic diseases, ferric carboxymaltose is safe and effective in correcting iron deficiency anaemia.

Different collection tubes in cardiac biomarkers detection.

The role played by different collection tubes is one of the more frequently forgotten parameters, which influences the quality of laboratory results.We aimed to determine whether there is a significant difference in assay of myoglobin, cardiac troponin I, and CK-MB depending on the presence of anticoagulant and gel separator in the collecting tubes.Blood samples were collected in 40 consecutive patients admitted for acute coronary syndrome. The samples were taken at the same moment in three different tubes (tubes without anticoagulant and with gel separator, heparin tubes with gel separator and heparin tubes without gelseparator;Venosafe, Terumo Europe, Leuven Belgium). Myoglobin, CK-MB and cardiac troponin I were measured with a double side (sandwich) chemiluminescent immuenzymatic assay (Access Myoglobin, Access CK-MB, Access AccuTnI).Cardiac troponin I was not influenced by the type of collection tube used. On the contrary, myoglobin and CK-MB showed a statistical difference depending on the test tube. In particular, there was a significant difference between tubes without anticoagulant and with gel separator and tubes with heparin and without gel.