| 1. |
- Lindahl, Tomas, et al.
(författare)
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INR calibration of Owren-type prothrombin time based on the relationship between PT% and INR utilizing normal plasma samples
- 2004
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Ingår i: Thrombosis and Haemostasis. - 0340-6245 .- 2567-689X. ; 91:6, s. 1223-1231
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Tidskriftsartikel (refereegranskat)abstract
- Prothrombin time (PT) is clinically important and is used to monitor oral anticoagulant therapy. To obtain PT results in international normalized ratio (INR), the current standardization procedure is complex and involves reference reagents. The PT of diluted plasma samples can be determined with a combined thromboplastin (the Owren-type procedure), but not necessarily with a plain thromboplastin (the Quick-type procedure). Owren-type PT procedures can therefore, as an alternative to the INR calibration, be calibrated with diluted normal plasma to give PT results in percent of normal PT activity (PT%). The present study explored if a plasma-based calibration of an Owren-type PT procedure can be used to obtain results in INR. The approach was to establish a relationship between PT% and INR by multi-center analysis of 365 samples from healthy individuals and patients on warfarin treatment. INR values were obtained by manual Quick-type reference procedure and PT% values by various automated Owren-type procedures. A relationship INR = (I/PT% + 0.018)10.028 was found. A calibration procedure, based on the relationship, was investigated. Calibrators were the median PT of 21 normal plasma at dilutions representing 100%, 50%, 25%, 12.5% and 6.25% of normal PT activity. These were assigned INR values of 1.00, 1.36, 2.07, 3.05 and 6.36. Calibration of various Owren-type assays was repeatedly performed by 5 expert laboratories during 3 consecutive years. The INR values of certain lyophilised or frozen control plasmas were determined. The frozen control plasmas had externally assigned INR values according to WHO guidelines. Within the laboratory, CV was typically below 3%. No appreciable difference among the results of the different laboratories or the three assay occasions was found. Externally assigned and INR values were essentially identical to those found. These and other results indicated that the calibration procedure was reproducible, precise and accurate. Thus, an Owren-type PT assay can be calibrated with normal plasma samples to give results in INR and the investigated calibration procedure can be proposed for this purpose.
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| 2. |
- Edlund, T, et al.
(författare)
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Isolation of cDNA sequences coding for a part of human tissue plasminogen activator.
- 1983
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - 0027-8424 .- 1091-6490. ; 80:2, s. 349-52
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Tidskriftsartikel (refereegranskat)abstract
- We have isolated a cDNA sequence coding for a part of human tissue plasminogen activator. mRNA coding for tissue plasminogen activator was partially purified, copied into double-stranded cDNA, and cloned into Escherichia coli. Two sets of partially overlapping oligodeoxynucleotide mixtures corresponding to all possible coding sequences for a known portion of the tissue plasminogen activator gene were prepared. One set was used as a probe to screen cDNA containing bacterial clones and both were used as probes in hybridization against purified plasmid DNA. Of 4,200 bacterial clones examined, 1 carried a plasmid that hybridized to both sets of oligonucleotides. This plasmid contained a 370-base-pair cDNA insert, which was shown by nucleotide sequence analysis to code for the cleavage site region in the one-chain form of the human tissue plasminogen activator.
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| 3. |
- Hansson, Kenny M., et al.
(författare)
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Abnormalities in coagulum lysis and structure are associated with deep venous thrombosis
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- The present study aimed to investigate the relationship between deep venous thrombosis (DVT) and fibrinolytic susceptibility of plasma coagulum, including the possible role of this property in laboratory diagnosis. From 276 patients consecutively admitted to hospital for suspected deep venous thrombosis (DVT), 75 patients and 47 controls were selected. With certainty, the patients and controls either had or did not have DVT. Fibrinolytic susceptibility was assayed by reacting plasma with thromboplastin and tissue plasminogen activator and recording a nephelometric signal. Coagulation time (CT), coagulum lysis time (CLT) and maximal increase in coagulum light scatter (CLS) were determined. Increase in D-dimer levels caused by coagulum lysis was also determined. This was viewed as a fibrinogen measure. CL T and CLS were interpreted as measures of fibrinolytic susceptibility and coagulum structure, respectively. CL T and CLS for patients and controls differed, p<0.025 and p<0.001, respectively. Compared to 5% for controls, 24% and 43% of the patients showed CL T and CLS outside the reference range. High fibrinogen levels could not explain the findings, since these were normal in most patients with abnormal CL T and CLS. Abnormal coagulum lysis and abnormal coagulum structure were thus found to be associated with DVT. Possible laboratory diagnostic role of CL T and CLS was investigated with bivariate reference ranges that excluded 5% and 0.3%. These ranges excluded significantly (p<0.0001) more patients, 47% and 27%, respectively. Tests for abnormal fibrinolytic susceptibility and coagulum structure may thus have a role in laboratory diagnosis of thrombotic disorders.
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| 4. |
- Hansson, Kenny M., et al.
(författare)
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Surface plasmon resonance and free oscillation rheometry in combination : A new approach forstudies on haemostasis and biomaterials
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- In haemostasis and biomaterial research it is important to be able to study biological processes at surfaces and in the bulk. Surface plasmon resonance (SPR) is sensitive to changes at surface and free oscillation rheometry (FOR) probes the bulk. The present work demonstrates the usefulness of the combination of the techniques for simultaneous real-time measurements on coagulation and fibrinolysis of blood plasma, as well as coagulation of whole blood. SFLLRN stimulated coagulation of native whole blood presented a higher SPR signal with a different appearance than for plasma coagulation, while the FOR signals corresponding to plasma and whole blood coagulation were similar. This result indicated that the SPR technique was more sensitive to cell-surface interactions than to fibrin formation in whole blood, while the FOR technique were equally sensitive to coagulation in whole blood and plasma. Spontaneous coagulation of native whole blood in contact with methyland hydroxyl-terminated self-assembled monolayers on gold and gold surfaces regenerated after coagulation by degradation of adsorbed proteins with trypsin and SOS were also studied. The regenerated gold surfaces displayed the shortest coagulation times, although the contact-activation of blood coagulation was found to be low. The methylated and hydroxylated surfaces were comparable in terms of coagulation activation, while the hydroxylated surfaces presented FOR signals that indicated difficulties for the coagulum to attach to the surface. The combination of SPR and FOR may be suited for studies of cell-surface interactions, and may find applications in studies of blood cell defects in patients and testing of medical substances.
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| 5. |
- 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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| 6. |
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| 7. |
- Wallén, P, et al.
(författare)
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Purification and characterization of a melanoma cell plasminogen activator.
- 1983
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Ingår i: European Journal of Biochemistry. - 0014-2956 .- 1432-1033. ; 132:3, s. 681-6
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Tidskriftsartikel (refereegranskat)abstract
- The plasminogen activator from a human melanoma cell line was purified with immunoadsorption as a major step. The cells were cultured in the presence of aprotinin in order to avoid proteolysis. A three-step purification involved adsorption on antibodies to porcine tissue plasminogen activator before chromatographies on arginine-Sepharose and Sephadex G-150. All solvents contained Tween-80 (0.01%) and, except for the last step, aprotinin. The final product had a specific activity of about 220000 IU/mg measured against the WHO urokinase standard. The activator obtained has an apparent Mr of 72000 and consists of single-chain molecules. Evidence was obtained that four different types of activator variants occur. First and known previously, the one-chain form can be proteolytically cleaved into a two-chain form. Secondly, both the one-chain and two-chain molecules exhibit two forms with molecular weight differences of about 3000 (possibly due to carbohydrate differences). Thirdly, the one-chain preparations contain two variants, each constituting about 50% of the material and differing in length by three N-terminal amino acids. Finally, a possible positional microheterogeneity was detected. Digestion with plasmin yields the two-chain form with disulfide-bonded polypeptide chains, 'A' and 'B' (from the N-terminal and C-terminal parts, respectively). At the same time, the variability of the original N terminus is removed. The A chain keeps the two Mr variants (now about 40000 and 37000, respectively). The B chain (Mr about 33000) contains the active site of the molecule, as demonstrated by labelling with [3H]diisopropyl phosphofluoridate, and is homologous to the enzymatically active chains of thrombin, plasmin and other serine proteases. In contrast to these enzymes, the plasminogen activator is enzymatically active in the one-chain form. A speculative explanation for this activity may possibly be the presence of an epsilon-amino group of a lysine residue at a position close to the bond cleaved in the two-chain form.
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| 8. |
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