Serum cystatin C in renal transplant patients

BACKGROUND: Waiting temperature before centrifugation and anticoagulants used, markedly effect total homocysteine concentrations. The aim of this study was to investigate the effect of different anticoagulants and temperature on plasma homocysteine levels. METHODS: We studied total homocysteine concentrations in 23 healthy subjects. Blood was drawn in K(3)EDTA, sodium citrate- or sodium fluoride-containing tubes, and kept at 0 degrees C or 22 degrees C for 3 h. Total homocysteine measurements were performed with fluorescence polarization immunoassay (FPIA) method. We compared all results with baseline EDTA values (samples put on crushed ice and centrifuged immediately) recommended in literature for reference handling. RESULTS: At 22 degrees C, the tubes containing sodium citrate and sodium fluoride showed significantly higher total homocysteine concentrations than their respective baseline values (p=0.000). However, sodium fluoride tubes were not significantly different than baseline EDTA levels. Waiting 3 h at 0 degrees C did not effect sodium citrate and EDTA plasma total homocysteine concentrations when compared to baseline EDTA, but sodium fluoride-containing plasma levels were significantly decreased (p=0.000). CONCLUSIONS: According to our results, the most available and practical temperature and anticoagulant for total homocysteine determination is sodium fluoride at room temperature up to 3 h.     

Heparin plasma sampling as an alternative to EDTA for BNP determination on the Access-Beckman Coulter--effect of storage at -20 degrees C

To determine BNP, EDTA plasma is the only suitable specimen recommended by the manufacturer. Since many laboratories, especially in Europe, use heparin plasma rather than EDTA plasma for many or most of their clinical assays and in particular for determination of cardiac markers (cTnI, myoglobin), it appeared critical to evaluate the use of heparin plasma samples, in comparison to EDTA plasma, for BNP determination on a automated immunochemiluminescent analyzer. The aim of this study was first, to evaluate the use of heparin plasma samples for Biosite BNP testing on the Beckman Coulter Access Immunoassay System (n=88) and second, to evaluate the effect of storage at -20 degrees C, without protease inhibitors, on the Biosite BNP assay. We obtained acceptable imprecision results with CVs ranging from 1.7 to 11.7% regardless of the anticoagulant used. The linearity of EDTA samples was good and comparable to the results observed with heparin plasma. The concentration of BNP was categorized according to the classification of the New York Heart Association (NYHA). With EDTA fresh samples as reference anticoagulant, 90% vs. 89% of subjects were classified as "concordant" with heparin fresh vs heparin frozen plasma samples, respectively. After storage at -20 degrees C, only 86% of the values of EDTA frozen were concordant with values of EDTA fresh. No subject varied by two NYHA classes. Heparin plasma is an attractive alternative to the established EDTA samples which can be used for BNP determination. This flexibility allows the simultaneous determination of CK, CK-MB, cTnI and BNP on a single heparin specimen, which facilitates blood collection for clinicians and nursing staff in an emergency unit. In addition, our results suggest that BNP could be stored at -20 degrees C for at least one month in order to perform retrospective studies.     

Multicenter evaluation of the Roche NT-proBNP assay and comparison to the Biosite Triage BNP assay

BACKGROUND: Brain natriuretic peptides (BNPs) are useful in the assessment of heart failure, left ventricular dysfunction, and acute coronary syndromes. METHODS: We performed a multicenter evaluation of the automated Roche NT-proBNP assay and compared its performance to the Biosite Triage BNP assay. RESULTS: The N-terminal (1-76) pro brain natriuretic peptide (NT-proBNP) method is precise (CV2-fold higher CV, and plasma samples are more labile when stored at room temperature and 4 degrees C. Comparison studies showed a reasonable correlation between NT-proBNP and BNP assays, with a substantially higher slope bias of 6-20 for the NT-proBNP assay. CONCLUSIONS: The automated Roche NT-proBNP assay has good analytical performance and better precision than the Biosite BNP assay. Unlike BNP, NT-proBNP is stable in EDTA plasma for 3 days at room temperature or longer at 4 degrees C. The Roche NT-proBNP is fully automated and will accommodate the testing of large numbers of clinical samples for assessing cardiac dysfunction.     

不同处理和保存条件下体外HCV RNA稳定性研究

对献血者或病人进行HCV核酸检测时，如果标本的采集、处理、保存不当，会造成病毒核酸降解，从而影响检测结果的真实性。本研究的目的是对不同抗凝剂、不同温度、不同保存时间下的HCV RNA病毒稳定性进行研究，以考察常规采供血过程中，标本采集及保存方式对NAT检测的影响。采集7例HCV RNA阳性献血者的血样，采用不同的抗凝剂、经不同的温度和不同的时间保存后，采用荧光定量PCR方法测定HCV RNA病毒含量，考察HCV RNA的稳定性。结果表明：①不同抗凝剂抗凝的全血于4℃保存48小时过程中，病毒含量下降至原滴度的42．7％；EDTA抗凝组各时间点的滴度均低于其它3组（分别相当于其它3组的67．6％-25．1％）。②ACD抗凝的全血在4、25和37℃保存48小时．病毒含量分剐下降到原滴度的53．8％、72．5％、29．8％。③ACD抗凝的全血离心分离出血浆，4℃或25℃继续保存7天，病毒含量分别下降至原滴度的70．9％和25．1％。④ACD抗凝的全血分离的血浆，反复冻融4次，病毒含量下降到原滴度的38．9％。结论：①用于核酸检测的标本应该用无菌采血管采样；②在无菌采血管采样的前提下，核酸检测标本用未抗凝血、EDTA、ACD、CPDA抗凝血均可；③采集的全血应避免放置37℃以上，ACD抗凝全血在4℃、25℃保存48小时内、37℃保存14小时内，HCV RNA病毒仍较为稳定；④分离后的血浆应避免放置25℃以上，ACD抗凝全血分离后的血浆在4℃保存7天，25℃保存3天，HCV RNA病毒仍比较稳定；⑤血浆标本应避免多次冻融，但冻融3次的血浆HCV RNA病毒含量仍然较为稳定；⑥无菌采样对维持病毒的稳定性非常重要；单纯的机械性溶血并不会明显导致病毒的降解；只要注意无菌的问题和有合适的核酸提取方法去除血红素，HCV RNA病毒实际上比以前认为的要稳定得多。     

Osteocalcin concentrations in plasma prepared with different anticoagulants

We investigated the effects on plasma osteocalcin concentrations of different anticoagulants used to collect the blood samples. Plasma osteocalcin concentrations measured by enzyme immunoassay and radioimmunoassay are influenced by the nature of the anticoagulants used. The most significant difference between concentrations found in plasma and serum was seen with oxalate/fluoride anticoagulant, which reduced osteocalcin concentrations to 37.3% of serum values. This is probably related to increased hemolysis with this anticoagulant compared with osteocalcin concentrations in plasma prepared with other anticoagulants. Samples prepared with sodium citrate (0.105 mol/L) or lithium heparin gave values 92.4% and 83.6% of those obtained with matched serum samples. Osteocalcin concentrations were relatively stable in plasma and serum at -20 degrees C for two freeze/thaw cycles. In blood from 100 patients there was a good correlation between osteocalcin concentrations in serum and plasma (lithium heparin) (r2 = 0.831); the slope and intercept (+/- SE) were 0.924 +/- 0.04 and 4.92 +/- 1.25 micrograms/L, respectively. However, in 10 patients, serum osteocalcin concentrations were two- to threefold higher than those in matched plasma samples.     

Alpha 1-antitrypsin: the effect of anticoagulants on the trypsin inhibitory capacity, concentration and phenotype

The effects of anticoagulants on the determination of both trypsin inhibitory capacity and the concentration of alpha 1-antitrypsin measured by radial immunodiffusion, and on the alpha 1-antitrypsin phenotype were investigated. These results were compared with those obtained for serum. The following anticoagulants were investigated: sodium citrate; sodium oxalate; buffered citrate; potassium oxalate/sodium fluoride; sodium heparin; and potassium EDTA. It was found that plasmas from all of the anticoagulants, except sodium heparin, resulted in apparently significant decreases of both trypsin inhibitory capacity and concentration of alpha 1-antitrypsin measured by radial immunodiffusion, relative to serum. These decreases were not simply due to dilution by anticoagulants. Using both acid starch gel electrophoresis followed by immunofixation and isoelectric focusing in agarose, no interference was found in the phenotype determination. It is concluded that serum should be used to measure the trypsin inhibitory capacity or the concentration of alpha 1-antitrypsin by radial immunodiffusion, although plasma is also suitable provided that sodium heparin is used as the anticoagulant. The alpha 1-antitrypsin phenotype can be determined with either serum or any of the plasma. None of the anticoagulants employed in this study was present in excess.     

Influence of sample matrix and storage on BNP measurement on the Bayer Advia Centaur

The assessment and management of congestive heart failure relies increasingly on the measurement of B-type natriuretic peptide (BNP). However, the effective contribution of this biochemical test in the clinical decision making is influenced by reliability of the measure, which also depends on several preanalytical issues. Since there is controversy on the influence of the matrix and the storage conditions on BNP measurement, we compared results of BNP in serum, K2 ethylene diamine tetra-acetic acid (EDTA) plasma and lithium heparin plasma fresh samples and in matching samples stored at -20 and -80 degrees C for 1 week. BNP measured on the Bayer Advia Centaur was systematically underestimated in heparin plasma (-47%) and serum (-62%) when compared to K2 EDTA plasma. According to the established 100 ng/L cutoff value, 25% and 37% of the fresh samples collected in heparin plasma or serum were misclassified from the reference K2 EDTA fresh specimen, respectively. When compared to the fresh specimens, the mean and interindividual bias observed for samples stored at either -20 degrees C or -80 degrees C was, overall, modest for K2 EDTA plasma (-2%) and heparin plasma (+6% and -4%, respectively), though it appeared clinically meaningful in serum (+47% and +28%, respectively). Although we can not rule out that other BNP assays using different antibodies may be not affected from degradation during storage to the same extent, results of our investigation demonstrate that K2 EDTA plasma is the most suitable specimens for BNP testing on fresh and frozen samples stored at either -20 degrees C or -80 degrees C for up to 1 week.     

Effect of collection tube type and preanalytical handling on myeloperoxidase concentrations

BACKGROUND: Myeloperoxidase (MPO) has shown potential as a marker for cardiovascular disease. Limited studies have been published with a variety of sample types, resulting in a wide range of MPO values. Little is known or understood about the impact of collection tube type and preanalytical handling of specimens for MPO determination. METHOD: MPO concentration was determined by use of the ARCHITECT(R) MPO research use assay, which is currently under development. Samples were collected into multiple anticoagulant collection tubes from donors and patients presenting to the emergency department with symptoms of acute coronary syndromes. Whole blood was stored on ice or at room temperature for predetermined time periods. We also evaluated serum and plasma after centrifugation followed by storage at room temperature, 2-8 degrees C, and below -10 degrees C. RESULTS: Baseline sample concentrations were dependent on collection tube type as well as handling conditions. MPO concentrations were consistently higher in samples collected in serum and heparin plasma tubes than in samples in EDTA or citrate tubes. Spike recovery was acceptable in all sera and plasma tested, indicating that the increased MPO concentrations were not due directly to an anticoagulant interference. CONCLUSIONS: The collection tube type and preanalytical handling are critical for accurate and consistent MPO measurement. The preferred anticoagulant and tubes are the EDTA or EDTA plasma preparation tube. MPO concentrations in samples collected in these tubes are stable before centrifugation as whole blood as well as plasma after processing.     

Hormone stability in human whole blood

OBJECTIVE: To determine whether significant changes in the plasma concentrations of 17 hormones occur when human whole blood is held at 4 or 24 degrees C for up to 24 h before separation of the plasma fraction. DESIGN AND METHODS: Blood samples (EDTA) from healthy human volunteers were held at 4 degrees C or 24 degrees C for 0.5, 6 or 24 h before separation. Plasma concentrations of ACTH, aldosterone, gonadotrophin alpha-subunits, AVP, C-peptide, estradiol, FSH, GH, glucagon, IGF-1, IGFBP3, insulin, leptin, LH, prolactin, PTH and VIP were measured and the results compared to baseline values. Nonlinear regression was used to test for a significant mean rate of change. The time interval for median concentrations to change by 10% was determined. RESULTS: Significant changes were observed for ACTH (decrease at 18.6 hr, 4 degrees C; 17.5 hr, 24 degrees C); AVP (increase at 2.6 h, 24 degrees C); insulin (decrease at 16.8 hr, 4 degrees C; 16.9 hr, 24 degrees C) and VIP (increase at 18.6 h, 24 degrees C). No changes were detected for the remaining analytes.B CONCLUSIONS: The measurement of some hormones is compromised by a delay in plasma separation from normal human blood. While many hormones appear stable in normal whole blood, we recommend that processing occurs without delay.     

The Quick and Owren prothrombin time methods for oral anticoagulant therapy do not agree well using the International Normalized Ratio (INR) units

Abstract

Natriuretic peptides are a laboratory tool with significant implications for the diagnosis and prognosis of heart failure (HF). The International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) recommended that assays must be examined for sample stability because there appears to be assay dependent. We aimed to evaluate the in vitro stability of B-type natriuretic peptide (BNP) under different handling conditions and using a BNP assay from Fujirebio Diagnostics (Tokyo, Japan). BNP concentrations were measured in plasma EDTA samples from 11 subjects to evaluate the in vitro stability at room temperature and at 4?°C and in 10 subjects to check the in vitro stability of samples stored at –20?°C during 1 and 3 months. Stability limit was defined according to Spanish Society of Laboratory Medicine (SEQC-ML) recommendations. At room temperature and 4?°C, BNP concentrations decreased progressively in samples collected in both groups, remaining stable within four hours from collection. BNP concentrations also were stable within four hours from collection in whole blood at room temperature. Finally, at –20?°C, BNP concentrations remained stable in both groups at 1 and 3 months, respectively. According to our results, BNP, stored at room temperature or at 4?°C, should be assayed in the first four hours after collection. Besides, BNP was shown to be stable in whole blood for at least four hours at room temperature. If the testing cannot be performed within the first four hours, the plasma should be frozen and kept at –20?°C for up to 3 months.     

Influence of anticoagulants on the measurement of S100B protein in blood

OBJECTIVE: Evaluate anticoagulants influence on plasma S100B levels. DESIGN AND METHODS: Blood were collected from 18 healthy adult subjects using: no anticoagulants, EDTA, heparin, and citrate. S100B levels were determined using LIA-mat assay. RESULTS: Heparin plasma and citrate increased plasma S100B levels (p < 0.001), whereas EDTA had no effect (p = 0.24). Heparin plasma samples were highly (r2 = 0.97, p < 0.001), citrate samples were moderately (r2 = 0.49, p < 0.001), and EDTA samples were not (r2 = 0.22, p = 0.059) correlated with serum samples. CONCLUSIONS: When anticoagulant is required, heparin plasma should be the primary choice for measurement of S100 B levels.     

Evaluation of the UV-340 spectrophotometric determination for pseudocholinesterase activity (EC 3.1.1.8) in human serum

A pseudocholinesterase catalytic activity assay using p-hydroxybenzoylcholine as substrate and measuring the decrease of NADPH at 340 nm was compared with a colorimetric method using acetylthiocholine as substrate. The assay is simple, uses 50 microliters serum and is performed at 37 degrees C. Precision of the UV-340 method was good except at low ranges. The catalytic activity was depressed by the anticoagulants citrate and fluoride but not by EDTA or heparin. The reference values obtained with the evaluated UV-340 method are somewhat higher than those with the colorimetric method. As the results with both methods are comparable, the choice of procedure will depend on the local facilities.     

Comparison of Abbott AxSYM and Roche Elecsys 2010 for measurement of BNP and NT-proBNP

BACKGROUND: B-type natriuretic peptide (BNP) and N-terminal pro-brain natriuretic peptide (NT-proBNP) are small cardiac hormones released from the heart. They can be used as an important aid to diagnose congestive heart failure (CHF). METHODS: We compared the performances of the Abbott AxSYM and Roche Elecsys 2010 for the measurement of BNP and NT-proBNP. The first method uses a microparticle enzyme-linked immunoassay, whereas the other uses chemiluminescent immunometric assay. RESULTS: The CVs using pooled sera ranged from 3.7% to 12.7% for the AxSYM and 0.9% to 2.2% for the Elecsys 2010. The Passing and Bablok regression was Elecsys 2010 NT-proBNP=7.23xAxSYM BNP+2.53. The BNP in EDTA plasma was more stable than in serum. The immunoreactivity difference of NT-proBNP in serum or EDTA plasma was within 10% when stored at 4 degrees Celsius or 25 degrees Celsius for 72 h. Receiver operating characteristic (ROC) curves were different for both assays, and the areas under the curves were 0.704 and 0.841 for the AxSYM and Elecsys 2010 method, respectively. CONCLUSIONS: Both assays were not entirely specific for heart failure. The precision and stability for NT-proBNP was better than for BNP in serum. It is important to use method-appropriate reference ranges (or cutoff) for the BNP and NT-proBNP, respectively, in the assessment of CHF.     

Long-term stability of endogenous B-type natriuretic peptide (BNP) and amino terminal proBNP (NT-proBNP) in frozen plasma samples.

The aim of the present study was to assess the long-term stability of endogenous B-type natriuretic peptide (BNP) and amino terminal proBNP (NT-proBNP) in plasma samples stored at -20 degrees C without addition of protease inhibitors (e.g., aprotinin). Stability of BNP and NT-proBNP was tested in 60 EDTA plasma samples with BNP values between 30 and 420 pg/ml. Initial BNP and NT-proBNP plasma concentrations were determined within four hours after blood collection using the AxSYM BNP and the Elecsys NT-proBNP assays. Subsequently, all samples were stored at -20 degrees C and were thawed for the second BNP and NT-proBNP determination on the two instruments after one day, 30 days, 60 days, 90 days and 120 days, respectively. Mean recovery (i.e., residual immunoreactivity) of BNP and NT-proBNP expressed in percent of the initial value for the given time interval of storage was calculated. Mean recovery of BNP was less than 70% after one day of storage at -20 degrees C and decreased to less than 50% after two to four months of storage (e.g., recovery of endogenous BNP after three months of storage at -20 degrees C ranging from 0% to 71%). In contrast, mean recovery of NT-proBNP was generally greater than 90%, irrespective of the duration of storage at -20 degrees C (e.g., recovery of endogenous NT-proBNP after three months of storage at -20 degrees C ranging from 91% to 112%). In conclusion, the determination of endogenous BNP with the AxSYM assay using frozen plasma samples may not be valid under the conditions tested. In contrast, NT-proBNP as measured by the Elecsys assay may be stored at -20 degrees C for at least four months without a relevant loss of the immunoreactive analyte.     

不同标本类型间髓过氧化物酶水平的检测分析

目的探讨不同标本类型间髓过氧化物酶(MPO)检测结果的差异性、抗凝剂的选择及检测结果的比对。方法同时采集165例健康体检人群含乙二胺四乙酸二钾(EDTA-K2)、肝素钠两种抗凝剂的血浆标本及不含抗凝剂的普通生化管的血清标本,分别检测3种标本类型中MPO水平,并对各组检测结果进行统计学分析。结果同一例体检者不同抗凝剂的血浆标本间及与不含抗凝剂的血清标本间MPO检测结果差异有统计学意义(P0.05)。结论不同标本类型对血液标本中MPO水平检测结果差异较大,建议各个实验室检测时根据不同抗凝剂制订相应的参考区间;由于EDTA-K2抗凝血浆不受体外白细胞中MPO释放的影响,推荐采用EDTA-K2抗凝血作为检测MPO水平的首选。     

Influence of the sample matrix on the stability of beta-CTX at room temperature for 24 and 48 hours

The measurement of beta-C-telopeptides of type I collagen (beta-CTX) reflects the rate of bone resorption in a variety of metabolic bone disorders and it is increasingly used to assist diagnosis and follow-up of these pathologies. Since preanalytical biases in the results of this marker can decrease its clinical usefulness, specific stability studies should be developed to prevent that inconsistent results of laboratory testing might affect patient health and waste economical resources. Three blood samples were simultaneously collected without venous stasis into evacuated tubes containing no additives, K2 EDTA or lithium heparin, from 23 out-patients referred to our phlebotomy service for routine laboratory testing. After centrifugation and separation of the specimens, a first aliquot was immediately analyzed, whereas a second and third aliquot was processed after a 24- and 48-hour storage at room temperature (21 degrees C). Beta-CTX was assayed on the automated electrochemiluminescence analyzer E170. A modest and clinically irrelevant underestimation was observed in lithium heparin plasma when compared with either K2 EDTA (-7.1%; 95% C.I. -2.0 to -12.3%; p < 0.001) or serum (-7.8%; 95% C.I. -3.2 to -12.4%; p < 0.001), but not between serum and K2 EDTA (+0.8%, 95% C.I. -5.3 to +6.9%; p = 0.260). Storage at room temperature in K2 EDTA plasma introduced a modest and clinically negligible decay in immunoreactivity (-4.4% and -5.7% at 24 and 48 hours, respectively), whereas storage at room temperature in both serum (-17.6% and -28.6% at 24 and 48 hours, respectively) and lithium heparin plasma (-29.1% and -44.0% at 24 and 48 hours, respectively) was associated with a substantial decay and a larger inter-individual variability in the measurable concentration of the analyte. In conclusion, the results of our investigation demonstrate that EDTA plasma is the most suitable sample matrix for the storage of beta-CTX at room temperature after centrifugation.     

Assay values for thiamine or thiamine phosphate esters in whole blood do not depend on the anticoagulant used

We compared the whole blood, plasma, and erythrocyte (red blood cell (RBC)) concentrations of thiamine and thiamine phosphate esters in the presence of heparin or EDTA as anticoagulants. Three blood specimens were collected from each of 24 healthy volunteers into evacuated collection tubes containing the following anticoagulants: heparin, Na2EDTA, or K2EDTA. The concentrations of nonphosphorylated free thiamine (T), thiamine monophosphate (TMP), thiamine diphosphate (TDP), and thiamine triphosphate (TTP) were determined by the NH2-column HPLC method. The anticoagulant used had no effect on the concentrations obtained in whole blood and plasma of thiamine or any of the above thiamine compounds (P>0.05). RBCs were isolated by centrifugation and washed with isotonic saline, and the cell counts of the washed cells were adjusted to their whole blood values. In the washed RBCs with any anticoagulant, the concentrations of T, TMP, and TDP expressed either as nmol/L of whole blood or a ratio to hemoglobin were significantly lower (P<0.05) than those in whole blood.     

Plasma ascorbic acid: measurement, stability and clinical utility revisited

Aims: To compare plasma ascorbic acid results by the colorimetric FRASC (Ferric Reducing/Antioxidant and Ascorbic Acid) assay and a reference HPLC method; to re-examine plasma ascorbic acid stability, and anticoagulant effect.

Design and methods: For method comparison, 31 plasma samples were tested by both methods. For stability, matching EDTA, heparin, citrate and fluoride/oxalate plasma, stored under different conditions of time and temperature, was measured.

Results: FRASC is an acceptable alternative to HPLC for plasma ascorbic acid: precision, limit of detection and recovery were similar, and results by the two methods were indistinguishable: mean (95% CI) difference:1.8 (−1.1–4.6; n = 31) μmol/L. Ascorbic acid was most stable in heparinized plasma. Marked loss (p < 0.05) in EDTA plasma occurred within 30 min of blood collection.

Conclusions: FRASC offers a speedy and reliable alternative to HPLC for plasma ascorbic acid. Heparin is proposed as the anticoagulant of choice; loss of ascorbic acid is rapid in EDTA plasma ex vivo.     

Effects of storage conditions on lymphocyte phenotypes from healthy and diseased persons

Anticoagulant and room temperature storage of peripheral blood was evaluated in healthy and diseased persons (AIDS and CLL) for its effect upon lymphocyte phenotyping by flow cytometry. Whole blood was stored in either Li heparin, EDTA, or ACD for up to 96 hours. Aliquots at 5 time intervals were evaluated using CD3, CD2, CD4, CD8, and CD19 monoclonal antibodies. Blood from healthy donors stored in EDTA produced an apparent increase in T cells and decrease in B cells most evident by 96 hours storage. Blood from diseased persons stored in Li heparin up to 96 hours showed insufficient changes in phenotype to warrant a different clinical interpretation. We conclude that all three anticoagulants are adequate for short term (less than 24 hr) room temperature transport and storage of peripheral blood for flow cytometry. Li heparin or ACD are more appropriate if prolonged transport times are anticipated.     

EDTA-dependent pseudothrombocytopenia: further insights and recommendations for prevention of a clinically threatening artifact

Ethylenediaminetetra-acetic acid (EDTA) is widely used as anticoagulant in laboratory medicine. EDTA-dependent pseudothrombocytopenia is a rare phenomenon (i.e., around 0.1% in the general population), which is mostly due to the presence of EDTA-dependent antiplatelet antibodies that react optimally between 0°C and 4°C, recognize the cytoadhesive receptors gpIIb-IIIa, stimulate the expression of activation antigens, trigger activation of tyrosine kinase, platelet agglutination and clumping in vitro, which finally lead to a spuriously decreased platelet count. The reliable and timely identification of this artifact is essential, since there a high chance that it may be confused with other life-threatening platelet disorders, or otherwise lead to inappropriate clinical and therapeutic decision-making. Five basic criteria should be fulfilled to raise the clinical suspicion of EDTA-dependent pseudothrombocytopenia, i.e., (i) abnormal platelet count, typically <100×10(9)/L; (ii) occurrence of thrombocytopenia in EDTA-anticoagulated samples at room temperature, but to a much lesser extent in samples collected with other anticoagulants and/or kept warmed at ~37°C; (iii) time-dependent fall of platelet count in the EDTA specimen; (iv) evidence of platelet aggregates and clumps in EDTA-anticoagulated samples with either automated cell counting or microscopic analysis; (v) lack of signs or symptoms of platelet disorders. Several remedies have been proposed, such as warming the sample to 37°C or using additives or specific formulations of anticoagulants including buffered sodium citrate, heparin, ammonium oxalate, β-hydroxyethyltheophylline, sodium fluoride, CPT (trisodium citrate, pyridoxal 5'-phosphate and Tris), antiplatelet agents, potassium azide, amikacin, kanamycin or other aminoglycosides, and calcium replacement with the simultaneous addition of calcium chloride/heparin. According to available evidences, the most suitable and practical approach so far for most clinical laboratories seems, however, the recollection of blood samples using sodium citrate, CPT or calcium chloride/heparin as additives, maintaining the specimen at 37°C until the platelet count has been completed.