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- Hansson, Kenny, 1972-, et al.
(författare)
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Surface plasmon resonance (SPR) analysis of coagulation in whole blood with application in prothrombin time assay
- 1999
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Ingår i: Biosensors & bioelectronics. - 0956-5663 .- 1873-4235. ; 14:8-9, s. 671-682
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Tidskriftsartikel (refereegranskat)abstract
- It is previously shown that surface plasmon resonance (SPR) can be used to study blood plasma coagulation. This work explores the use of this technique for the analysis of tissue factor induced coagulation, i.e. prothrombin time (PT) analysis, of whole blood and plasma. The reference method was nephelometry. The prothrombin time analysis by SPR was performed by mixing two volumes of blood/plasma, one volume of thromboplastin, and one volume of CaCl2 solution directly on a sensor surface. The measurements show good agreement between nephelometry and SPR plasma analysis and also between SPR plasma and whole blood analysis. The effect of anticoagulant treatment on the clotting times was significant both quantitatively and qualitatively. The impact on the SPR signal of different physiological events in the coagulation process is discussed, and tentative interpretations of the sensorgram features are given. The major advantage of the SPR method compared to nephelometry is the possibility to perform analysis on whole blood instead of plasma. In conclusion, SPR is a promising method for whole blood coagulation analysis.
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- Ramström, A Sofia, et al.
(författare)
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A flow cytometric assay for the study of dense granule storage and release in human platelets
- 1999
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Ingår i: Platelets. - : Informa Healthcare. - 0953-7104 .- 1369-1635. ; 10:2-3, s. 153-158
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Tidskriftsartikel (refereegranskat)abstract
- The clinical manifestations of platelet dense (δ) granule defects are easy bruising, as well as epistaxis and bleeding after delivery, tooth extractions and surgical procedures. The observed symptoms may be explained either by a decreased number of granules or by a defect in the uptake/release of granule contents. We have developed a method to study platelet dense granule storage and release. The uptake of the fluorescent marker, mepacrine, into the platelet dense granule was measured using flow cytometry. The platelet population was identified by the size and binding of a phycoerythrin-conjugated antibody against GPIb. Cells within the discrimination frame were analysed for green (mepacrine) fluorescence. Both resting platelets and platelets previously stimulated with collagen and the thrombin receptor agonist peptide SFLLRN was analysed for mepacrine uptake. By subtracting the value for mepacrine uptake after stimulation from the value for uptake without stimulation for each individual, the platelet dense granule release capacity could be estimated. Whole blood samples from 22 healthy individuals were analysed. Mepacrine incubation without previous stimulation gave mean fluorescence intensity (MFI) values of 83±6 (mean ± 1 SD, range 69–91). The difference in MFI between resting and stimulated platelets was 28±7 (range 17–40). Six members of a family, of whom one had a known δ-storage pool disease, were analysed. The two members (mother and son) who had prolonged bleeding times also had MFI values disparate from the normal population in this analysis. The values of one daughter with mild bleeding problems but a normal bleeding time were in the lower part of the reference interval.
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- Rånby, Mats, et al.
(författare)
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Isocitrate as Calcium Ion Activity Buffer in Coagulation Assays
- 1999
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Ingår i: Clinical Chemistry. - 0009-9147 .- 1530-8561. ; 45:8, s. 1176-1180
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Tidskriftsartikel (refereegranskat)abstract
- Background: Ca2+ activity close to the physiological concentration of 1.3 mmol/L is essential in blood coagulation. Is this also true for the performance of global diagnostic coagulation assays? We searched for compounds that would buffer Ca2+ activity at ∼1.3 mmol/L without disturbing coagulation reactions and investigated whether such Ca2+ buffering improves diagnostic efficacy in global diagnostic coagulation tests.Methods: Buffering was investigated by mixing CaCl2 and 11 candidate compounds and determining Ca2+ activity. The best candidates were added to mixtures of plasma and thromboplastin to detect interference with coagulation reactions. The best of these candidates, isocitrate, was used to modify an activated partial thromboplastin time (APTT), buffering final Ca2+ activity to ∼1.3 mmol/L. Plasma samples from 22 healthy individuals and 120 patients were analyzed with original and modified APTT to determine whether diagnostic efficacy was improved.Results: Two suitable Ca2+ buffers, citrate and isocitrate, were found. Isocitrate was preferred as being less coagulation inhibitory, a better Ca2+ buffer, and possibly a better anticoagulant. The isocitrate-modified APTT showed a final Ca2+ activity of 1.60 ± 0.07 mmol/L, compared with 2.73 ± 0.20 mmol/L for the original APTT. The means and SDs for the healthy individuals were determined for both procedures, and the values were used to express patient deviation from normality (difference from mean divided by SD). The deviation was greater for the modified APTT; 4.3 ± 5.7, compared with 3.6 ± 5.0 (P <0.005) for the original APTT.Conclusions: Isocitrate can be used to buffer Ca2+ activity at physiological concentrations and can serve as an anticoagulant. APTT with isocitrate-buffered Ca2+ activity shows signs of improved diagnostic efficacy.
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