Special clamp for rapid solvent extraction of a large number of plasma samples for radioimmunoassay

Abstract. To determine optimal conditions for blood collection during clinical trials, where sample handling logistics might preclude prompt separation of erythrocytes from plasma, healthy subjects (n=8, 6 M/2F) were recruited and non-fasting blood samples were collected into tubes containing different anticoagulants (ethylenediaminetetra-acetic acid (EDTA), Li-heparin or Na-heparin). We hypothesized that heparin, but not EDTA, would effectively protect plasma tocopherols, ascorbic acid, and vitamin E catabolites (α- and γ-CEHC) from oxidative damage. To test this hypothesis, one set of tubes was processed immediately and plasma samples were stored at −80°C, while the other set was stored at 4°C and processed the following morning (~30 hours) and analyzed, or the samples were analyzed after 6 months of storage. Plasma ascorbic acid, as measured using HPLC with electrochemical detection (LC-ECD) decreased by 75% with overnight storage using EDTA as an anticoagulant, but was unchanged when heparin was used. Neither time prior to processing, nor anticoagulant, had any significant effects upon plasma α- or γ-tocopherols or α- or γ-CEHC concentrations. α- and γ-tocopherol concentrations remained unchanged after 6 months of storage at −80°C, when measured using either LC-ECD or LC/mass spectrometry. Thus, refrigeration of whole blood at 4°C overnight does not change plasma α- or γ-tocopherol concentrations or their catabolites. Ascorbic acid is unstable in whole blood when EDTA is used as an anticoagulant, but when whole blood is collected with heparin, it can be stored overnight and subsequently processed.

Download Full-text

Factor XII Determinations in the Presence and Absence of Phospholipid Antibodies

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614225 ◽

1998 ◽

Vol 79 (01) ◽

pp. 87-90 ◽

Cited By ~ 16

Author(s):

D. W. Jones ◽

M. Winter ◽

M. J. Gallimore

Keyword(s):

Lower Limit ◽

Lupus Anticoagulant ◽

Factor Xii ◽

Chromogenic Substrate ◽

Plasma Samples ◽

Presence And Absence ◽

Lower Limit Of Normal

SummaryFactor XII (FXII) levels were determined in plasma samples from 29 normal donors, 10 patients with inherited FXII deficiency (all lupus anticoagulant [LA] negative) and 67 LA positive patients, using clotting (FXIIct), chromogenic substrate (FXIIcs) and immunochemical (FXIIag) assays. Excellent correlations were obtained in the three FXII assays with the LA negative samples and between the FXIIcs and FXIIag assays in the LA positive samples. Correlations between both the FXIIcs and FXIIag with FXIIct in the LA positive patients were poor. Of 67 LA positive samples studied, 25 (37.3%) showed lower values in the FXIIct assay; 13 (19.4%) of these patients were pseudo FXII deficient with values of FXII below the lower limit of normal.These results indicate that a diagnosis of FXII deficiency can be made inappropriately in the presence of phospholipid antibodies and that such a diagnosis should not be made by FXIIct assay alone.

Download Full-text

Fibrinogen-Fibrin-Related Antigen Pattern in Human Blood

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647697 ◽

1974 ◽

Vol 32 (02/03) ◽

pp. 266-276

Author(s):

Carl D. Jacobsen ◽

John C. Hoak ◽

Kenneth K. WU ◽

Glenna L. Fry

Keyword(s):

Human Blood ◽

Plasma Samples

SummaryIn serum from patients with DIC at least 3 different FR-antigenic components could be found. It was difficult to demonstrate these components in the corresponding plasma samples. It is possible that a portion of these antigens formed as a result of in vitro clotting despite the presence of proteolytic inhibitors. These results suggest that the interpretation of “increased split products in serum” may be more complex than current concepts indicate.

Download Full-text

In Vitro Effects of Urokinase — Prevention by Different Inhibitors

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647327 ◽

1990 ◽

Vol 64 (03) ◽

pp. 402-406 ◽

Cited By ~ 5

Author(s):

M D Oethinger ◽

E Seifried

Keyword(s):

In Vitro Study ◽

Thrombin Time ◽

Plasma Samples ◽

Temperature Dependent ◽

Chloromethyl Ketone ◽

Control Value ◽

Dose Time ◽

In Vitro Effects ◽

Full Protection

SummaryThe present in vitro study investigated dose-, time- and temperature-dependent effects of two-chain urokinase plasminogen activato(u-PA, urokinase) on normal citrated plasma. When 10 μg/ml u-PA wereadded to pooled normal plasma and incubated for 30 min at an ambient temperature (25° C), α2-antiplas-min decreased to 8% of the control value. Incubation on ice yielded a decrease to 45% of control,whereas α2-antiplasmin was fully consumed at 37° C. Fibrinogen and plasminogen fell to 46% and 39%, respectively, after a 30 min incubation at 25° C. Thrombin time prolonged to 190% of control.Various inhibitors were studied with respect to their suitability and efficacy to prevent these in vitro effects. Aprotinin exhibited a good protective effect on fibrinogen at concentrations exceeding 500 KlU/ml plasma. Its use, however, was limited due to interferences with some haemostatic assays. We could demonstrate that L-Glutamyl-L-Glycyl-L-Arginyl chloromethyl ketone (GGACK) and a specific polyclonal anti-u-PA-antibody (anti-u-PA-IgG) effectively inhibited urokinase-induced plasmin generation without interfering with haemostatic assays. The anti-u-PA-antibody afforded full protection ofα2-antiplasmin at therapeutic levels of u-PA.It is concluded that u-PA in plasma samples from patients during thrombolytic therapy may induce in vitro effects which should be prevented by the use of a suitable inhibitor such as GGACK or specific anti-u-PA-antibody.

Download Full-text

Immunoreactivity of Tissue Plasminogen Activator and of Its Inhibitor Complexes

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646605 ◽

1989 ◽

Vol 61 (03) ◽

pp. 409-414 ◽

Cited By ~ 70

Author(s):

M Rånby ◽

G Nguyen ◽

P Y Scarabin ◽

M Samama

Keyword(s):

Plasminogen Activator ◽

Tissue Plasminogen Activator ◽

Degradation Products ◽

Gel Filtration ◽

Free Form ◽

Enzyme Linked Immunosorbent Assay ◽

Plasma Samples ◽

Significant Difference ◽

Tissue Plasminogen ◽

Pai 1

SummaryAn enzyme linked immunosorbent assay (ELISA) based on goat polyclonal antibodies against human tissue plasminogen activator (tPA) was evaluated. The relative immunoreactivity of tPA in free form and tPA in complex with inhibitors was estimated by ELISA and found to be 100, 74, 94, 92 and 8l% for free tPA and tPA in complex with PAI-1, PAI-2, α2-antiplasmin and C1-inhibitor, respectively. Addition of tPA to PAI-1 rich plasma resulted in rapid and total loss of tPA activity without detectable loss of ELISA response, indicating an immunoreactivity of tPA in tPA/PAI-1 complex of about l00%. Three different treatments of citrated plasma samples (acidification/reneutralization, addition of 5 mM EDTA or of 0.5 M lysine) prior to determination by ELISA all resulted in increased tPA levels. The fact that the increase was equally large in all three cases along with good analytical recovery of tPA added to plasffi, supported the notion that all tPA antigen present in plasma samples is measured by the ELISA. Analysis by ELISA of fractions obtained by gel filtration of plasma from a patient undergoing tPA treatment identified tPA/inhibitor complexes and free tPA but no low molecular weight degradation products of tPA. Determinations of tPA antigen were made at seven French clinical laboratories on coded and randomized plasma samples with known tPA antigen content. For undiluted samples there was no significant difference between the tPA levels found and those known to be present. The between-assay coefficient of variation was 7 to 10%. In conclusion, the ELISA appeared suited for determination of total tPA antigen in human plasma samples.

Download Full-text

Effect of Aspirin on the Thrombelastogram of Human Blood

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649127 ◽

1973 ◽

Vol 30 (03) ◽

pp. 494-498 ◽

Cited By ~ 1

Author(s):

G de Gaetano ◽

J Vermylen

Keyword(s):

Oral Dose ◽

Single Oral Dose ◽

Platelet Function ◽

Human Blood ◽

Platelet Rich Plasma ◽

Plasma Samples ◽

Release Reaction ◽

Platelet Release

SummaryThrombelastograms of both native blood and re-calcified platelet-rich plasma samples taken from subjects given a single oral dose of aspirin (1 gram) were not significantly different from the pretreatment recordings. Aspirin also did not modify the thrombelastogram when preincubated in vitro with platelet-rich plasma at concentrations inhibiting the platelet “release reaction” by collagen. Thrombelastography therefore cannot evaluate the effect of aspirin on platelet function.

Download Full-text

Inactivation of Factor XIa in Vivo: Studies in Chimpanzees and in Humans

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650621 ◽

1996 ◽

Vol 76 (04) ◽

pp. 549-555 ◽

Cited By ~ 16

Author(s):

Walter A Wuillemin ◽

C Erik Hack ◽

Wim K Bleeker ◽

Bart J Biemond ◽

Marcel Levi ◽

...

Keyword(s):

Antithrombin Iii ◽

Life Time ◽

In Vivo Studies ◽

Clinical Samples ◽

Plasma Samples ◽

C1 Inhibitor ◽

Elimination Kinetics ◽

Factor Xia ◽

Best Parameter

SummaryC1-inhibitor (C1Inh), antithrombin III (ATIII), α1-antitrypsin (a1AT), and α2-antiplasmin (a2AP) are known inhibitors of factor XIa (FXIa). However, their precise contribution to FXIa inactivation in vivo is not known. We investigated FXIa inactivation in chimpanzees and assessed the contribution of these inhibitors to FXIa inactivation in patients with presumed FXI activation.Chimpanzees were infused with FXIa and the various FXIa-FXIa inhibitor complexes formed were measured. Most of FXIa was complexed to C1Inh (68%), followed by a2AP (13%), a1AT (10%), and ATIII (9%). Analysis of the plasma elimination kinetics revealed a half-life time of clearance (t1/2) for the FXIa-FXIa inhibitor complexes of 95 to 104 min, except for FXIa-a1AT, which had a t1/2 of 349 min. Due to this long t1/2, FXIa-a1AT complexes were predicted to show the highest levels in plasma samples from patients with activation of FXI. This was indeed shown in patients with disseminated intravascular coagulation, recent myocardial infarction or unstable angina pectoris. We conclude from this study that in vivo C1Inh is the predominant inhibitor of FXIa, but that FXIa-a1 AT complexes due to their relatively long t1/2 may be the best parameter to assess FXI activation in clinical samples.

Download Full-text