EDTA-anticoagulated Samples Research Articles

Alterations due to dilution and anticoagulant effects in hematologic analysis of rodent blood samples on the Sysmex XT-2000iV.

Clinical pathology of rodents is hindered by sample volume limitations. A single whole heparinized blood sample is often submitted for hematologic and clinical chemistry analysis in exploratory research settings, and sample dilution may be required. Published information on the potential impact of sample dilution and heparin use on hematology variables in rodents is sparse. The purpose of the study was to evaluate the effects of sample dilution and of anticoagulant on hematologic analysis of mouse and rat blood samples on the Sysmex XT-2000iV. Mice and rats were obtained from various ends of study research projects, and whole blood was collected via terminal cardiocentesis in lithium heparin, and additionally in EDTA when paired samples were obtained from rats. Hematology analytes were measured on the Sysmex XT-2000iV straight and diluted from ×2 to ×5. Significant differences between heparinized samples analyzed straight vs diluted were found for MCV and MCHC, with a bias for several additional variables. Significant differences between paired heparinized and EDTA-anticoagulated samples at each dilution point were found for most variables, with the largest differences found in platelet count. Evidence of platelet clumping presumably due to heparin exposure was noted in numerous samples. Dilution-induced changes occur in some hematologic variables and may render dilution unacceptable in the exploratory research environment. Many variables, most notably platelet count, differ based on the anticoagulant used, and values from heparinized vs EDTA-anticoagulated samples should not be directly compared.

Monocytes circulate in constant reversible interaction with platelets in a [Ca2+]-dependent manner

We aimed to provide evidence that blood monocytes belonging to all subsets predominantly circulate in constant and usually reversible interactions with platelets, which are predominantly [Ca2+] dependent. The proportions of monocyte–platelet aggregates (MPAs) attributable to individual monocyte subsets in fresh and promptly processed heparin-anticoagulated blood from 10 healthy subjects (median age 35 years, 50% male) were analysed by flow cytometry and compared to samples anticoagulated with a potent [Ca2+] chelator, ethylenediaminetetraacetic acid (EDTA). Additional experiments with [Ca2+] depletion or supplementation were also performed. Monocytes subsets were defined as CD14++CD16–CCR2+ cells (Mon1), CD14++CD16+CCR2+ cells (Mon2) and CD14+CD16++CCR2− cells (Mon3). Vast majority of monocytes showed aggregation with platelets in heparinised samples, but most monocytes were free of platelets when EDTA was used (p < 0.001 for all subsets). Addition of the heparinised blood to EDTA-containing vacutainers reduced the proportion of MPAs to values seen in the directly EDTA-anticoagulated blood (p = 0.005 for all subsets). Supplementation with CaCl2 resulted in dose-dependent increase in MPAs (p < 0.001 for all subsets). Although the overall trend for the monocyte–platelet interactions was applicable to all monocyte subsets, the proportion of MPAs in heparinised samples was lowest for Mon3 (p < 0.0001). In contrast, Mon3 showed the highest proportion of MPAs in EDTA-anticoagulated samples (p = 0.004). In healthy subjects monocytes circulate in constant, but predominantly reversible and [Ca2+]-dependent aggregation with platelets. These observations may reflect a complex involvement of platelets in regulation of monocyte activity.

Decreased Mean Platelet Volume in Patients With Amylodiosis

We have read the article published by Erdem et al with a great interest. They have examined whether red cell distribution width and mean platelet volume (MPV) values differ between patients with reactive amyloid A (AA) amyloidosis due to chronic inflammatory disease and healthy controls. They have found red cell distribution width, erythrocyte sedimentation rate, and platelet count levels to be significantly higher in patients with AA amyloidosis compared to the controls and MPV was significantly lower in patients with amyloidosis. This is the first study in this subject. On the other hand, we want to mention minor criticism about this study from the methodological aspect. First, they did not mention about the time between blood sampling and analysis. They used EDTA-anticoagulated tubes for sample collection. However, MPV increases over time in EDTA-anticoagulated samples and this increase was shown to be proportional with the delay in time between sample collection and laboratory analysis. With impedance counting, the MPV increases over time as platelets swell in EDTA, with increases in 7.9% within 30 minutes having been reported and an overall increase in 13.4% over 24 hours, although the majority of this increase occurs within the first 6 hours. The recommended optimal measuring time of MPV is maximum 120 minutes after venipuncture. For reliable MPV measurement, the potential influence of anticoagulant on the MPV must be carefully controlled by standardizing the time delay between sampling and analysis (less than 2 hours). This situation is not clear in study. Second, there are significant associations of MPV with smoking, obesity, coronary artery disease, metabolic syndrome, statin use, and atrial fibrillation. They did not mention about the body mass index of patients and controls, smoking status, coronary heart disease, proportion of patients and controls with metabolic syndrome, rhythm status, and proportion of patients and controls using statin. If we look at the low-density lipoprotein cholesterol levels, there are high values such as 373 mg/dL in the patient group and such as 196 mg/dL in the controls. So, there can be patients and controls using statin. Higher MPV values were reported in smokers and in patients with obesity, coronary artery disease, metabolic syndrome, and atrial fibrillation. On the other hand, statin treatment decreases MPV. These situations are not clear in the study.

Platelet volume in cervicocephalic artery dissections

We have read the article published by Kamisli et al. [1] with a great interest. They have examined the mean platelet volume (MPV) and platelet counts in patients with vertebral artery dissections and carotid artery dissections and they compared them with controls. They found a significantly higher MPV values only in the vertebral artery dissection group. On the other hand, they did not find a significant difference in platelet count between the dissection and control groups. Although the sample volume is small, this is the first study in this subject. On the other hand, we want to offer minor criticism of this study from a methodological perspective. First, the authors did not indicate the time which elapsed between blood sampling and analysis. They used ethylenediaminetetraacetic acid (EDTA) anticoagulated tubes for sample collection. However, MPV increases over time in EDTA-anticoagulated samples and this increase was shown to be proportional with the delay in time between sample collection and laboratory analysis. With impedance counting, the MPV increases over time as platelets swell in EDTA, with increases of 7.9 % within 30 min having been reported and an overall increase of 13.4 % over 24 h, although the majority of this increase occurs within the first 6 h [2]. The recommended optimal measuring time of MPV is maximum 120 min after venipuncture. For reliable MPV measurement, the potential influence of anticoagulant on the MPV must be carefully controlled by standardizing the time delay between sampling and analysis (\2 h). This situation is not clear in study. Second, there are significant associations of MPV with smoking, obesity and metabolic syndrome [3]. They did not mention about the body mass index of patients and controls, smoking status, proportion of patients and controls with metabolic syndrome. These situations are not clear in the study. Recently, it has been demonstrated that blood flow through the false lumen is a powerful activator of the coagulation and fibrinolytic systems. Because, there is intense thrombin generation and fibrinolysis and platelet activation in acute aortic dissection patients in the acute phase [4]. This appears to be major mechanism of increased MPV, an indicator of platelet reactivity. Platelet volume is mainly determined in the bone marrow. It is supposed that the large platelets are caused by a reduced fragmentation of megakaryocytes. MPV has been shown to inversely correlate with the total platelet count, which could even suggest the consumption of small platelets and a compensatory production of larger reticulated platelets [5]. Although, they did not find a significant difference in platelet count between the dissection and control groups, this may be due to small sample size. Activated platelets make thrombi at the injury site in dissections [6]. As a result, consumption of small platelets in dissection site and a compensatory production of larger reticulated platelets may be another reason of higher MPV.

Potential Clinical Impact of Inaccurate Automated Platelet Counts in the Setting of Severe Thrombocytopenia

AbstractAbstract 3428Severe thrombocytopenia is not an uncommon finding in the setting of hematologic malignancies. The key to a successful outcome often lies in the ability to support the patient through periods of cytopenia by adopting an aggressive transfusion strategy for platelets. Therefore, an accurate assessment of the platelet count is of utmost importance particularly as it relates to various levels which “trigger” prophylactic platelet transfusion. However, the accuracy and precision of conventional platelet counting methods have been shown to be suspect in severely thrombocytopenic samples. We investigated the accuracy of several commercial hematology analyzers currently available in clinical practice which use optical, impedance and immunologic platelet determination methods and assessed whether biased results could have an impact on decisions regarding platelet transfusion. Four-hundred three (403) EDTA-anticoagulated samples collected from patients with hematologic malignancies and a platelet count of < 50 × 10e3/uL were selected from the standard workflow processed in the hematology laboratories at MSKCC. Samples were split and tested within 8 hours of collection using optical based methods on the Advia 2120i (Siemens, Tarrytown, NY) and CELL-DYN Sapphire (Abbott Diagnostics, Santa Clara, CA). During the study, an additional analyzer became available, the XE-2100 (Sysmex, Kobe, Japan) which enumerates platelets by impedance and optical technology. One hundred twenty seven (127) of the 403 samples were split and tested by the Sysmex analyzer. Platelet counts from each method were then compared using the CD61 immunoplatelet determination (Abbott, Santa Clara, CA) as the reference value. The CD61 immunoplatelet method was chosen as the reference standard because we found excellent correlation in linear regression between this method and phase microscopy (r =0.99, y = −0.96 + 0.88x; n = 37). Also, the International Counsel for Standardization in Haematology and the International Society for Laboratory Hematology recommend the counting of specifically labelled platelets (CD41, CD61) by flow cytometry as a reference method for the enumeration of platelets. Bland-Altman difference plots were used to visualize the agreement between the reference and test methods and the paired t-test evaluated the statistical significance of the difference between methods. We then compared the number of platelet transfusion indications (at various platelet thresholds) as determined by all methods with potential transfusion decisions made using the reference method. There was a statistically significant positive bias among the optical and impedance methods compared with CD61 enumeration. Using various platelet transfusion decision points, the number of patients who were at risk for under transfusion (platelet count above threshold when reference result is < threshold) ranged from 1–2 % at a threshold of 50k/uL to 30–40 % when the threshold was lowered to 10k/uL. These results highlight the limitations in the accuracy of optical and impedance methods, particularly in the setting of severe thrombocytopenia. Since prophylactic platelet transfusions are by and large based on standard “triggers,” an overestimation of the platelet count may lead to under-transfusion, especially in patients at the highest risk for hemorrhage. Clinicians should be aware of the limitations of automated platelet determinations and consider this when making medical decisions regarding supportive transfusions. Disclosures:Marionneaux:Abbott Diagnostics: Consultancy.

Influence of in vitro hemolysis on nucleated red blood cells and reticulocyte counts

Nucleated red blood cells (NRBCs) and reticulocytes are early and important measures of red blood cells' (RBCs) turnover, but little is known on how spurious hemolysis may affect the reliability of these parameters. Ten EDTA-anticoagulated samples were divided into three aliquots. The first was immediately tested, where-as the others (defined A and B) were mechanically hemolyzed by aspiration 5 and 10 times through a small-gauge needle. RBC, NRBC, and reticulocyte counts were performed on Sysmex XE-2100. An increasing amount of hemolysis was produced in hemolyzed aliquots A and B. The RBC and reticulocyte counts progressively decreased from the nonhemolyzed sample to hemolyzed aliquots 'A' and 'B'. The NRBC count increased in 3 of the 10 samples and decreased in the remaining seven. Hemolysis of venous blood samples may seriously jeopardize NRBC and reticulocyte counts.

Increased Thrombopoietin and Mean Platelet Volume in Patients With Ischemic Stroke

I read the article published by Balcik et al with great interest. They examined both the mean platelet volume (MPV) and the thrombopoietin (TPO) values in patients with ischemic stroke and compared this with healthy controls. Both TPO and MPV values were significantly higher in patients with stroke. Although previous studies have reported higher MPV values in ischemic stroke, the mechanism of this is still not clear. This is the first study demonstrating both increased TPO and MPV in the same patient group with ischemic stroke. Since TPO is considered the primary physiological regulator of megakaryopoiesis, increased TPO levels may increase both platelet counts and platelet size, resulting in hemostatically more active platelets. Senaran have reported that MPV and TPO values were found to be elevated in patients with acute myocardial infarction and unstable angina pectoris when compared with control participants. They have reported similar results in ischemic stroke. This is an interesting study from this aspect. On the other hand, we want to mention minor criticism about this study from methodological aspect. First, they used ethylenediaminetetraacetic acid (EDTA) anticoagulated tubes for sample collection. However, MPV increases over time in EDTA-anticoagulated samples and this increase was shown to be proportional with the delay in time between sample collection and laboratory analysis. With impedance counting, the MPV increases over time as platelets swell in EDTA, with increases of 7.9% within 30 minutes and an overall increase of 13.4% over 24 hours having been reported, although the majority of this increase occurs within the first 6 hours. The recommended optimal measuring time of MPV is 120 minutes maximum, after venipuncture. For reliable MPV measurement, the potential influence of anticoagulant on the MPV must be carefully controlled by standardizing the time delay between sampling and analysis (less than 2 hours). This situation is not clear in this study. Second, there are significant associations of MPV with type 2 diabetes mellitus, prediabetes, smoking, hypertension, hypercholesterolemia, obesity, coronary artery disease, metabolic syndrome, statin use, and atrial fibrillation. In patients group, there were more patients with hypertension and diabetes mellitus. In patients group 22 patients had coronary artery disease and none in control group. Higher MPV values were reported in patients with hypertension, diabetes mellitus, and coronary artery disease. As a result, increased MPV values might be due to these factors. The factors must have been adjusted in 2 groups to say that the increase in MPV is solely dependent on the stroke. Third, they did not mention the rhythm of the patients. Atrial fibrillation is major risk factor for stroke. The MPV values were reported to be higher in patients with paroxysmal atrial fibrillation. Moreover, Ha et al reported that MPV was a predictive marker for stroke; its predictive power for stroke was independent of age, gender, and other CHADS(2) score components in patients with AF. They had to mention the presence or absence of atrial fibrillation or how many people were in atrial fibrillation. Moreover, laboratory findings in Table 2 do not seem to be consistent with the findings stated in results and discussion section (platelet count, MPV, and age). They also did not mention the physical examination and echocardiographic findings of patients to search the cardioembolic source of stroke. In all patients with stroke echocardiographic examination was performed to determine the cardiac source of emboli. For example, rheumatic mitral stenosis is an important source of ischemic stroke. The MPV has been shown to be significantly elevated in patients with mitral stenosis who were in sinus rhythm compared to control participants. As a result, presence of mitral stenosis or other cardiac sources of emboli may have a role in higher MPV values in patients with stroke.

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.

Apparent pseudohyperkalemia in a Chinese Shar Pei dog

Whole blood in a serum clot tube and EDTA-anticoagulated samples from an 8-year-old spayed female Chinese Shar Pei dog were submitted by an external clinic to the diagnostic laboratory at Atlantic Veterinary College for routine biochemical and hematologic analysis prior to entropion surgery. Laboratory abnormalities included mild hyperkalemia (6.3 mmol/L, reference interval 3.6-6.0 mmol/L), mild normocytic, hypochromic, nonregenerative anemia (HCT 0.31 L/L, reference interval 0.37-0.55 L/L; MCHC 290 g/L, reference interval 320-360 g/L), and increased red cell distribution width (RDW; 26.2%, reference interval 11-14%). A small subpopulation of macrocytic, slightly hypochromic erythrocytes was noted on Wright's-Giemsa-stained blood smears. Biochemical and hematologic data obtained from this patient over the previous 7.5 years indicated that serum (and in 1 case, heparinized plasma) potassium concentration was increased (range, 6.3-10.9 mmol/L) in 5 of 8 samples (HCT ranged from 0.31-0.43 L/L, Hgb 91-124 g/L, MCHC 280-312 g/L, and RDW 18.2-26.9%). Clinical signs suggestive of hyperkalemia were not observed at any time, suggesting pseudohyperkalemia as the cause of the increased potassium concentrations. An erythrocyte lysate prepared from a heparinized blood sample had a high potassium concentration (16.8 mmol/L) compared with that of a clinically healthy, non-Shiba control dog (6.7 mmol/L). An osmotic fragility test of the patient's erythrocytes showed 50% hemolysis at 0.57% NaCl, compared with 0.48% NaCl for the control dog, indicating increased fragility. On scanning electron microscopy, a small subpopulation of erythrocytes were large, flattened, and had a tendency to fold. These findings supported the provisional diagnosis of pseudohyperkalemia due to increased intracellular RBC potassium concentration. High-potassium erythrocytes have been reported in Akitas, Shibas, Jindos, other East Asian dog breeds, and occasionally, in mixed-breed dogs. Pseudohyperkalemia should also be considered when an otherwise unexplained elevation in serum or plasma potassium concentration is observed in Chinese Shar Pei dogs, and may be accompanied by increased RDW, low MCHC, and increased osmotic fragility with or without mild anemia.

A convenient method of DNA extraction from blood anticoagulated with EDTA.

Surplus blood often remains after routine clinical tests using EDTA-anticoagulated samples. To use this blood for DNA analysis, we isolated white cells by adherence to polyethylene terephthalate fibers, which could be stored for several weeks transported by mail after methanol fixation. DNA yield was sufficient and correlated with white cell count. Extracted DNA was free of hemoglobin contamination and durable to polymerase chain reaction and enzyme digestion, which yielded products visualized as well-separated bands on electrophoresis. We found our method to be practical for the routine clinical laboratory.

Technical influences on immunophenotyping by flow cytometry The effect of time and temperature of storage on the viability of lymphocyte subsets

The typing of lymphocyte subsets may be influenced by a variety of technical influences including the duration and temperature of sample storage and the method used for staining samples. We have extended a previous study examining the effect of storage conditions on the baseline values of a number of lymphocyte subsets. EDTA-anticoagulated samples from 13 HIV-1-positive and 15 healthy laboratory controls were analyzed for a number of lymphocyte subsets (CD3+, CD4+ /CD3+, and CD8+ /CD3+ T cells and CD19+ B cells) (whole blood lysis method, Becton-Dickinson FACScan flow cytometer and reagents) at 0, 24, 48, 72, and 96 h after storage at 4°C, 17°C or 21°C. During storage at both 4°C and 21°C, there were significant changes in baseline values of the majority of lymphocyte subsets and some of these were related to the HIV status of the donor. The optimum temperature for storage in our system appeared to be around 17°C in both our study groups. We have also used propidium iodide in order to discriminate between viable and non-viable cells during flow cytometry of lymphocytes from eight HIV-1-positive and five control subjects. The results show that for both HIV-positive and control samples stored at 4°C, and for control subjects at 21°C, the changes in baseline values of lymphocyte subsets observed were not due to selective loss of particular subsets arising from cell death during storage. However, there was substantial loss of cells from all three subsets in HIV-positive subjects during storage at 21°C, with loss of CD8+ and CD3+ T cells being more significant than loss of CD4+ T cells.

Neutrophil integrin assay for clinical studies

The level of expression of neutrophil adhesion molecules may be a useful marker for neutrophil activation in clinical studies. We therefore determined neutrophil integrin expression under various experimental conditions using a Fluorescence Activated Cell Sorter (FACS) after the cells had been labelled with fluorescent conjugated antibodies to the integrin subunits CD11a, CD11b and CD18. Levels of labelled CD11b and CD18 increased after activation with the chemotactic peptide formyl-methionyl-leucyl phenylalanine (fMLP) in a dose- and time-dependent manner, but CD11a did not, indicating that CD11a would not be a useful marker of neutrophil activation. The baseline expression of CD11b and CD18 on unstimulated neutrophils was similar in heparin and EDTA anti-coagulated blood but the response to activation with fMLP was significantly less for the EDTA anti-coagulated samples (p < 0.01 in paired t-test). The labelling of integrins was significantly higher in unfixed whole blood samples compared to samples fixed with 1 per cent paraformaldehyde. However, the increase in labelling induced by fMLP was similar whether or not the samples were fixed after activation. Labelling of CD11b and CD18 was greater for preparations of isolated neutrophils than for neutrophils in whole blood, and the response to fMLP stimulation tended to be lower for the isolated cells. Our results indicate that heparin should be used as anti-coagulant in clinical studies utilizing whole blood if subsequent activation of neutrophils is planned (e.g. to detect in vivo priming), although EDTA may be used if baseline expression alone is to be measured. Fixation of blood samples should not affect the ability to detect neutrophil activation.

Effects of anticoagulants and incubation time on neutrophil nitroblue tetrazolium score.

We studied the effect of anticoagulants and incubation time on neutrophil nitroblue tetrazolium (NBT) scores. Heparin (10 IU/ml) and EDTA (1.5 mg/ml) were used, and the preparations were incubated for periods ranging from 20 to 180 min. Heparin, in contrast to EDTA, was found to be associated with cell destruction, cell aggregates and higher NBT scores; the effects were more profound with endotoxin stimulation. The duration of incubation significantly affects NBT scores in both the heparin- and EDTA-anticoagulated samples, and the effect is more marked with heparin. Cell destruction and cell clumping are both enhanced with incubation, again more so with heparin. It is concluded that when performing NBT tests, blood samples would best be anticoagulated with EDTA to ensure more consistent results.