Magnesium Sulfate as an Alternative In Vitro Anticoagulant for the Measurement of Platelet Parameters?

OBJECTIVES: There are conflicting reports on the reliable measurement of platelet count and mean platelet volume (MPV) using EDTA or citrate. The anticoagulant properties of magnesium sulfate (MgSO4) are known from the literature. The aim of this study was to evaluate MgSO4 as an in vitro anticoagulant for platelet count, MPV, platelet distribution width, and platelet activation. METHODS: Whole blood from volunteers was anticoagulated by EDTA, citrate, or MgSO4 Platelets were counted by the XE 5000 (Sysmex, Norderstedt, Germany) impedance and fluorescence optical technique. RESULTS: The mean impedance platelet counts were 227.7, 197.0, and 201.1 × 10(9)/L in EDTA-, citrate-, or MgSO4-anticoagulated blood, respectively. The counts were 4.7% higher (EDTA) after 3 hours of storage but 4% lower in citrate-anticoagulated blood. The counts in magnesium samples remained stable. The MPV was 10.4 fL (EDTA), 9.5 fL (citrate), and 9.3 fL (MgSO4). EDTA samples showed cell swelling within the first 3 hours. This was lower in citrate and only marginal in magnesium samples. High activation of platelets was observed only in EDTA samples. CONCLUSIONS: Magnesium anticoagulation might be advantageous for more reliable MPV measurements. Although platelet count is underestimated when the impedance method is used, the platelet count reveals similar results when measured by the fluorescent optical method.

Performance of the XN-2000 WPC channel-flagging to differentiate reactive and neoplastic leukocytosis.

BACKGROUND: The distinction between reactive and neoplastic leukocytes, especially atypical lymphocytes suspected to be reactive or neoplastic, is a particular challenge in automated hematological cell differentiation. The aim of the study was to evaluate the performance of the XN analyzer supplemented with the WPC channel for differentiating between reactive and neoplastic leukocytosis. METHODS: Blood samples of 253 patients with viral infections, lymphoma or leukemia were analyzed by the Sysmex XN-2000 analyzer equipped with the WPC channel. The results were compared to routine leukocyte differentiation using the routine Sysmex XE-2100 analyzer and automated digital microscopy (DM96). The combined information from standard morphology, immune phenotyping and clinical diagnosis served as a reference. RESULTS: The XN WPC channel demonstrated an excellent performance for differentiating neoplastic (AUC=0.933) and reactive leukocytosis (AUC=0.900) as compared to morphological smear examination (AUC=0.949 and AUC=0.968, respectively) or to the differentiation results of our routine hematology analyzer (AUC=0.630 and AUC=0.635, respectively). CONCLUSIONS: Our data show that the combined WDF/WPC of the Sysmex XN-Series analyzer is advantageous in the automated differentiation of neoplastic and reactive leukocytosis, thus supporting the correct diagnostic decision in the daily laboratory routine.

Measurement of Platelet Counts and Volume Using Magnesium Sulfate as an Anticoagulant: Comparison of Impedance and Light-Scatter Technology.

OBJECTIVES: Magnesium sulfate (MgSO 4 ) was recently reported as an alternative in vitro anticoagulant in pseudo-thrombocytopenia. Its suitability as an anticoagulant for the determination of reliable platelet parameters is the subject of this study. METHODS: Platelet count and mean platelet volume were measured in blood samples anticoagulated with EDTA and MgSO 4 and compared. The platelet parameters were determined by impedance (XE 5000 [Sysmex, Norderstedt, Germany]; DxH 800 [Beckman-Coulter, Krefeld, Germany]) and laser light-scatter technology (Advia 120 [Siemens Healthcare Diagnostics, Eschborn, Germany]). RESULTS: MgSO 4 anticoagulation underestimated platelet counts compared with EDTA. Mean platelet volume (MPV) in magnesium-anticoagulated blood was lower when measured by impedance but higher when light-scatter technology was used. Storage of the differently anticoagulated blood led to differently lower platelet counts after 24 hours, independent of the anticoagulant. In EDTA blood, the mean platelet volume increased moderately when measured by impedance but markedly when measured by laser light scatter. In MgSO 4 -anticoagulated blood, the MPV increase was negligible. CONCLUSIONS: Impedance technology and magnesium anticoagulation might be advantageous for standardizing MPV measurements, although the mean platelet count is slightly underestimated by both technologies.

Exclusion of thrombocytopenia as a contraindication for invasive radiofrequency ablation in a patient with paroxysmal atrial fibrillation by using magnesium anticoagulation instead of EDTA: another case of anticoagulant-induced pseudo-thrombocytopenia.

Thrombocytopenia might be an exclusion criterion for invasive radiofrequency catheter ablation; therefore it is necessary to differentiate between pseudo-thrombocytopenia and a low platelet count due to other etiologies.A 69-year-old female presented to the cardiology department with recurrent atrial fibrillation that was resistant to conventional drug treatment. The initial laboratory findings were within the normal ranges, except for low platelet counts that occurred without a specific bleeding history. The reason for thrombocytopenia was anticoagulant-induced in vitro aggregation of platelets in the presence of EDTA as well as in citrated blood samples. As recently communicated, magnesium anticoagulated blood samples prevent platelet aggregation in individuals with anticoagulant-associated pseudo-thrombocytopenia. Although its aggregation-inhibiting effect is known from previous clinical observations, magnesium sulphate has not been introduced as an anticoagulant in analytical medicine.Based on our observations, blood anticoagulated with magnesium sulphate is recommended to verify low routine platelet counts before final clinical decisions are made.

Effective estimation of correct platelet counts in pseudothrombocytopenia using an alternative anticoagulant based on magnesium salt.

Pseudothrombocytopenia remains a challenge in the haematological laboratory. The pre-analytical problem that platelets tend to easily aggregate in vitro, giving rise to lower platelet counts, has been known since ethylenediamine-tetra acetic acid EDTA and automated platelet counting procedures were introduced in the haematological laboratory. Different approaches to avoid the time and temperature dependent in vitro aggregation of platelets in the presence of EDTA were tested, but none of them proved optimal for routine purposes. Patients with unexpectedly low platelet counts or flagged for suspected aggregates, were selected and smears were examined for platelet aggregates. In these cases patients were asked to consent to the drawing of an additional sample of blood anti-coagulated with a magnesium additive. Magnesium was used in the beginning of the last century as anticoagulant for microscopic platelet counts. Using this approach, we documented 44 patients with pseudothrombocytopenia. In all cases, platelet counts were markedly higher in samples anti-coagulated with the magnesium containing anticoagulant when compared to EDTA-anticoagulated blood samples. We conclude that in patients with known or suspected pseudothrombocytopenia the magnesium-anticoagulant blood samples may be recommended for platelet counting.

Role of lipid apheresis in changing times.

During the last decades, LDL-apheresis was established as an extracorporeal treatment option for patients with severe heterozygous or homozygous familial hypercholesterolemia (FH) that is resistant to conventional treatment strategies such as diet, drugs, and changes in lifestyle. Nearly half a century ago, the first LDL-apheresis treatment was performed by plasma exchange in a child with homozygous FH. At the beginning of the 1970s, the clinical advantage of regular extracorporeal LDL-elimination was demonstrated in siblings suffering from homozygous FH. These findings encouraged researchers especially from Germany and Japan to develop extracorporeal devices to selectively eliminate LDL-cholesterol in the 1980s. Although the selectivity of the currently available LDL-apheresis devices is different, the efficacy of LDL-elimination during a single treatment is rather similar and ranges between 55 and 65 % of the pretreatment LDL plasma concentration.In the 1990s, the patients regularly treated by extracorporeal LDL-elimination, diet, and drugs were included in regression studies assessed by angiography. It was shown that the combined treatment with LDL-apheresis, diet, and drugs resulted in less progression of coronary lesions than drugs and/or diet alone. However, although a tendency was evident, results did not reach criteria for significance. During the last decade, apheresis registries were established to collect data on efficiency, safety, and clinical outcome of regular long-term LDL-apheresis. The evaluation of registry data will certainly permit further insights in the therapeutic benefit of this expensive and time-consuming therapeutic approach. Furthermore, the future of LDL-apheresis will depend upon the availability of highly efficient new drugs and molecular genetic approaches such as RNA silencing of the apoB gene, whereas the liver transplantation and gene therapy of the LDL-receptor deficiency will not replace LDL-apheresis in severe familial hypercholesterolemia in the near future.

Intra coronary freshly isolated bone marrow cells transplantation improve cardiac function in patients with ischemic heart disease.

BACKGROUND: Autologous bone marrow cell transplantation (BMCs-Tx) is a promising novel option for treatment of cardiovascular disease. In this study we analyzed whether intracoronary autologous freshly isolated BMCs-Tx have beneficial effects on cardiac function in patients with ischemic heart disease (IHD). RESULTS: In this prospective nonrandomized study we treated 12 patients with IHD by freshly isolated BMCs-Tx by use of point of care system and compared them with a representative 12 control group without cell therapy. Global ejection fraction (EF) and infarct size area were determined by left ventriculography.Intracoronary transplantation of autologous freshly isolated BMCs led to a significant reduction of infarct size (p < 0.001) and an increase of global EF (p = 0.003) as well as infarct wall movement velocity (p < 0.001) after 6 months follow-up compared to control group. In control group there were no significant differences of global EF, infarct size and infarct wall movement velocity between baseline and 6 months after coronary angiography. Furthermore, we found significant decrease in New York Heart Association (NYHA) as well as significant decrease of B-type natriuretic peptide (BNP) level 6 months after intracoronary cell therapy (p < 0.001), whereas there were no significant differences in control group 6 months after coronary angiography. CONCLUSIONS: These results demonstrate that intracoronary transplantation of autologous freshly isolated BMCs by use of point of care system is safe and may lead to improvement of cardiac function in patients with IHD. REGISTRATION NUMBER: ISRCTN54510226.

Relation between the frequency of CD34⁺ bone marrow derived circulating progenitor cells and the number of diseased coronary arteries in patients with myocardial ischemia and diabetes.

BACKGROUND: Bone marrow-derived circulating progenitor cells (BM-CPCs) in patients with coronary heart disease are impaired with respect to number and mobilization. However, it is unknown whether the mobilization of BM-CPCs depends on the number of diseased coronary arteries. Therefore, in our study, we analysed the correlation between the diseased coronary arteries and the frequency of CD34/45+ BM-CPCs in peripheral blood (PB) in patients with ischemic heart disease (IHD). METHODS: The frequency of CD34/45+ BM-CPCs was measured by flow cytometry in 120 patients with coronary 1 vessel (IHD1, n = 40), coronary 2 vessel (IHD2, n = 40), coronary 3 vessel disease (IHD3, n = 40) and in a control group of healthy subjects (n = 40). There was no significant difference of the total number of cardiovascular risk factors between IHD groups, beside diabetes mellitus (DM), which was significantly higher in IHD3 group compared to IHD2 and IHD1 groups. RESULTS: The frequency of CD34/45+ BM-CPCs was significantly reduced in patients with IHD compared to the control group (CD34/45+; p < 0.001). The frequency of BM-CPCs was impaired in patients with IHD3 compared to IHD1 (CD34/45+; p < 0.001) and to IHD2 (CD34/45+; p = 0.001). But there was no significant difference in frequency of BM-CPCs between the patients with IHD2 and IHD1 (CD34/45+; p = 0.28). In a subgroup we observed a significant negative correlation between levels of hemoglobin AIc (HbAIc) and the frequency of BM-CPCs (CD34/45+; p < 0.001, r = -0.8). CONCLUSIONS: The frequency of CD34/45+ BM-CPCs in PB is impaired in patients with IHD. This impairment may augment with an increased number of diseased coronary arteries. Moreover, the frequency of CD34/45+ BM-CPCs in ischemic tissue is further impaired by diabetes in patients with IHD.

Expression of functional protease-activated receptor 1 in human prostate cancer cell lines.

Functional protease-activated receptors (PAR) are expressed by a variety of malignant cells. In the present study, RT-PCR assays demonstrated the expression of the thrombin receptor PAR-1 mRNA in human prostate cancer cell lines DU 145, LnCAP, and SV40-immortalized human prostate epithelial cell line PNT1A. In contrast, the additional thrombin receptors PAR-3 and PAR-4 were not detected. PAR-1 protein localized to the cellular surface was detected by flow cytometry in all three cell lines. To demonstrate the functional importance of the PAR-1, the effects of different concentrations of thrombin on cell proliferation kinetics were assessed. The treatment of growth-arrested cells with varying concentrations of thrombin demonstrated dose- and time-dependent effects. At low concentration (<0.5 U/ml), thrombin induced proliferation of all prostate-derived cell lines. Thrombin at higher concentration (1.0 U/ml) initially stimulated PNT1A and LnCAP cells to proliferate (time of thrombin application 24 h and 48 h) followed by inhibited growth when assessed after 72 h of incubation. In contrast, 1.0 U/ml thrombin caused earlier inhibition of DU 145 proliferation starting at 48 h of incubation. Our results suggest that PAR-1 mediates the proliferation-modulating effects of thrombin on prostate cancer cells.

Comparison of two filter combinations for low-density lipoprotein apheresis by membrane differential filtration: a prospective crossover controlled clinical study.

Membrane differential filtration is an accepted procedure for the extracorporeal removal of low-density lipoprotein (LDL). Reduction rates largely depend on the nature of the membranes and are ideally evaluated in a crossover study design. Four patients who had been treated by LDL apheresis for at least 6 months were included. Six consecutive weekly sessions (40 ml plasma/kg body weight) were scheduled per system (Plasmacure PS06/Evaflux Eval 5A [Kuraray] versus Plasmaflo OP05W/Cascadeflo AC1770 [Asahi]). Laboratory measurements indicated reductions of plasma concentrations for fibrinogen (37% [Kuraray] versus 44% [Asahi]), IgG (15% versus 20%), IgA (24% versus 28%), IgM (63% versus 53%), and total protein (11% versus 16%). Total cholesterol was eliminated by 52% versus 49%, LDL by 67% versus 66%, triglycerides by 56% versus 41%, and high-density lipoprotein by 10% versus 20%. Three therapies employing the Asahi filter combination were terminated prematurely due to saturation of the plasma fractionator. In conclusion, despite similar physical properties, the membranes differ significantly concerning selectivity and sensitivity to saturation.