Rapid Radioimmunoassay For Fibrinopeptide A In Human Plasma

Sequential Samples

The originally-described method for assay of fibrino- peptide A (FPA) in human plasma includes alcohol precipitation and dialysis steps which are complex, time consuming, and limit the applicability of the assay. The purpose of this work was to devise an assay for FPA which could be more easily applied to clinical studies, and to use this assay to study some of the factors which might cause artefactual results on blood samples obtained from patients. A rapid method for FPA assay has been developed which is simple, robust and sensitive and allows results to be obtained within 2.5 hrs. of blood collection. The assay depends on the use of bentonite to absorb fibrinogen from plasma, and gives a normal range for plasma FPA (0.3 - 1.5 pmol/ml) which is similar to that measured using the originally-described method. There is an equally good correlation between results obtained by the two methods on samples obtained from patients with various levels of circulating FPA and/or fibrinogen/fibrin degradation products (FDPs). Following venepuncture, FPA levels in sequential samples of blood drawn through 19-gauge ‘Butterfly’ needles became progressively lower in successive samples, indicating that a larger than usual discard of blood is necessary if basal levels are to be accurately assessed. Different antisera showed differences in affinity for FPA and cross reaction with des amino tyrosyl FPA, but such differences are unlikely to cause problems when assaying clinical samples. Generation of FPA from whole blood in vitro is completely suppressed by heparin/Trasylol anticoagulant in conventionally-used concentration, and we therefore find no evidence in favour of the suggestion that this anticoagulant mixture is unsuitable for sample collection for FPA assay.

RISTOMYCIN (AGGRISTIN) PRECIPITATION TEST: A NEW, RAPID AND RELIABLE METHOD FOR THE DETECTION OF INTRAVASCULAR CLOTTING

10.1055/s-0038-1643133 ◽

1987 ◽

Author(s):

G Pfliegler ◽

J Arnout ◽

J Vermylen

Keyword(s):

Reliable Method ◽

Degradation Products ◽

Laboratory Diagnosis ◽

Blood Collection ◽

Vein Thrombosis ◽

Acid Citrate Dextrose ◽

Citrate Dextrose ◽

Precipitation Test

The rapid and specific detection of fibrin monomers (fm) and fibrin degradation products (fdp) is of major importance in the laboratory diagnosis of disseminated intravascular coagulation, deep vein thrombosis or pulmonary embolism. Most methods in use are either time-consuming, needing special techniques, or insensitive and poorly specific. Some time ago, Watanabe and Tullis described a simple and rapid, semiquantitative test to detect fm and fdp in plasma, based on the finding that ristocetin in low concentrations (1.0-1.5 mg/ml) can specifically precipitate fm and fdp. To 0.4 ml ACD plasma, 0.1 ml ristocetin (2.5 mg/ml) is added and vortexed. The mixture is then incubated for 30 min at 20°C and centrifuged at 50xg for 5 min. The test is considered to be positive when fibrin-like strands or small or large pellets are observed on the bottom of the tube. More recently, Pfliegler et al. reported that ristomycin (AGGRISTIN), a structural analogue of ristocetin, can replace ristocetin in this test.Here we report on further results with the ristomycin (AGGRISTIN) precipitation test in 138 patients with various intravascular thrombotic events. The results of this test, performed on ACD plasma, were compared to the serum fdp values detected by immunoelectrophoresis (IEF) and by the haemagglutina-tion inhibition test (HIT). In all 30 cases with serum fdp above 30 ug/ml (HIT) or 28 pg/ml (IEF), the precipitation test was positive; at lower fdp concentrations, as detected by HIT or IEF, the test still was positive in 70 per cent of these thrombosis patients, suggesting a superior sensitivity. In 16 patients with elevated fibrinogen levels (but no evidence of thrombosis), the test was positive in only 3. No false positive results were detected in 16 healthy controls. Preliminary results show that the minor disadvantage of the test (blood collection on acid citrate dextrose) may readily be overcome by the in vitro adjustment of the pH of citrate plasma, commonly used for other haemostatic tests, to between 7.0 and 7.4.On the basis of our results we suggest that the AGGRISTIN (ristomycin) precipitation test is a simple, rapid and reliable method for the laboratory diagnosis of intravascular clotting.

D-Phe-Pro-Arg-Chloromethylketone: Its Potential Use in Inhibiting the Formation of In Vitro Artifacts in Blood Collected During Tissue-Type Plasminogen Activator Thrombolytic Therapy

10.1055/s-0038-1661632 ◽

1986 ◽

Vol 56 (02) ◽

pp. 160-164 ◽

Cited By ~ 47

Author(s):

M A Mohler ◽

C J Refino ◽

S A Chen ◽

A B Chen ◽

A J Hotchkiss

Keyword(s):

Plasminogen Activator ◽

Degradation Products ◽

Significant Loss ◽

Tissue Type ◽

Blood Samples ◽

Tissue Type Plasminogen Activator ◽

Type Plasminogen Activator ◽

Potential Use

Summary In vitro artifacts due to proteolysis may occur in blood samples containing recombinant tissue-type plasminogen activator (rt-PA) due to continued activation of plasminogen to plasmin by rt-PA. The aim of this study was to identify a rapid inhibitor of rt-PA that would not interfere in assays designed to monitor thrombolytic events.When rt-PA was added at 5 μg/ml to whole blood and incubated at 25° C, fibrinogen decreased 50 percent, plasminogen levels decreased 90 percent and α2-antiplasmin decreased below detectable levels. If D-Phe-Pro-Arg-chloromethylketone (PPACK) or aprotinin were added before the addition of rt-PA there was no significant loss of fibrinogen. Only PPACK completely inhibited changes in fibrin degradation products, plasminogen and α2-antiplasmin. PPACK was also found to inhibit the binding of rt-PA to plasma protease inhibitors in vitro.Rhesus monkeys were infused with rt-PA and blood samples were taken with either PPACK or aprotinin in the collection syringe. There was a significant increase in the recovery of immunoreactive rt-PA and consistent measures of fibrinogen, FDPs, plasminogen, and α2-antiplasmin in the PPACK group as compared to the aprotinin group which indicates that PPACK will prevent the in vitro formation of artifacts due to the presence of active rt-PA

Importance of Preanalytical Factors in Measuring Cr and Co Levels in Human Whole Blood: Contamination Control, Proper Sample Collection and Long-Term Storage Stability

Journal of Analytical Toxicology ◽

10.1093/jat/bkaa062 ◽

2020 ◽

Author(s):

Yuliya L Sommer ◽

Cynthia D Ward ◽

Joaudimir Castro Georgi ◽

Po-Yung Cheng ◽

Robert L Jones

Keyword(s):

Stainless Steel ◽

Whole Blood ◽

Consumer Products ◽

Blood Collection ◽

Sample Collection ◽

Blood Samples ◽

Contamination Control ◽

Human Whole Blood

Abstract A number of errors with potentially significant consequences may be introduced at various points in the analytical process, which result in skewed, erroneous analytical results. Precautionary procedures such as contamination control, following established sample collection protocols, and having a complete understanding of the long-term stability of the elements of interest can minimize or eliminate these errors. Contamination control is critical in the quantification of Cr and Co in human whole blood. Cr and Co levels in most biological samples are low, but these elements occur naturally in the environment and are often found in commercial and consumer products, which increases the risk of contamination. In this paper, we demonstrated that lot screening process in which we pre-screen a sub-set of manufactured lots used in collecting, analyzing and storing blood samples is a critical step in controlling Cr and Co contamination. Stainless steel needles are often utilized in blood collection but are considered as a potential source of introducing metal contamination to the patient sample. We conducted two studies to determine if there is a possibility of Cr or Co leaching into the human whole blood from the needles during blood collection. We analyzed blood collected from 100 donors and blood collected in vitro in the laboratory from designated vessel containing spiked blood with higher levels of Cr and Co. Two blood tubes were consecutively collected through one needle. In both studies, Cr and Co concentration levels in the two consecutively collected tubes were compared. Based on the results from donor and in vitro blood collection studies, we concluded that there was no Cr and Co leaching from the limited sets of stainless steel needles used in these studies. Furthermore, we demonstrated that Cr and Co human whole blood samples are stable for 1 year stored at temperatures of −70, −20 and 4°C and 6 months at room temperature.

A Monoclonal Antibody-Based Enzyme Immunoassay for Fibrin Degradation Products in Plasma

10.1055/s-0038-1642777 ◽

1988 ◽

Vol 59 (02) ◽

pp. 310-315 ◽

Cited By ~ 64

Author(s):

P W Koppert ◽

E Hoegee-de Nobel ◽

W Nieuwenhuizen

Keyword(s):

Enzyme Immunoassay ◽

Degradation Products ◽

Blood Clot ◽

Tissue Type ◽

Type Plasminogen Activator ◽

Strong Positive Reaction ◽

Sandwich Type ◽

Capture Antibody

SummaryWe have developed a sandwich-type enzyme immunoassay (EIA) for the quantitation of fibrin degradation products (FbDP) in plasma with a time-to-result of only 45 minutes.* The assay is based on the combination of the specificities of two monoclonal antibodies (FDP-14 and DD-13), developed in our institute. FDP-14, the capture antibody, binds both fibrinogen degradation products (FbgDP) and FbDP, but does not react with the parent fibrin(ogen) molecules. It has its epitope in the E-domain of the fibrinogen molecule on the Bβ-chain between amino acids 54-118. Antibody DD-13 was raised using D-dimer as antigen and is used as a tagging antibody, conjugated with horse-radish peroxidase. A strong positive reaction is obtained with a whole blood clot lysate (lysis induced by tissue-type plasminogen activator) which is used as a standard. The EIA does virtually not detect FbgDP i. e. purified fragments X, Y, or FbgDP generated in vitro in plasma by streptokinase treatment. This indicates that the assay is specific for fibrin degradation products.We have successfully applied this assay to the plasma of patients with a variety of diseased states. In combination with the assay previously developed by us for FbgDP and for the total amount of FbgDP + FbDP (TDP) in plasma, we are now able to study the composition of TDP in patients plasma in terms of FbgDP and FbDP.

Influence of Acute Myocardial Infarction and rt-PA Therapy on Circulating Fibrinogen

10.1055/s-0038-1651605 ◽

1993 ◽

Vol 69 (04) ◽

pp. 321-327 ◽

Cited By ~ 3

Author(s):

E Seifried ◽

M Oethinger ◽

P Tanswell ◽

E Hoegee-de Nobel ◽

W Nieuwenhuizen

Keyword(s):

Myocardial Infarction ◽

Acute Myocardial Infarction ◽

Thrombolytic Agent ◽

Degradation Products ◽

Maximum Plasma ◽

Normal Range ◽

Fibrinogen Degradation Products ◽

Rebound Phenomenon ◽

The Mean

SummaryIn 12 patients treated with 100 mg rt-PA/3 h for acute myocardial infarction (AMI), serial fibrinogen levels were measured with the Clauss clotting rate assay (“functional fibrinogen”) and with a new enzyme immunoassay for immunologically intact fibrinogen (“intact fibrinogen”). Levels of functional and “intact fibrinogen” were strikingly different: functional levels were higher at baseline; showed a more pronounced breakdown during rt-PA therapy; and a rebound phenomenon which was not seen for “intact fibrinogen”. The ratio of functional to “intact fibrinogen” was calculated for each individual patient and each time point. The mean ratio (n = 12) was 1.6 at baseline, 1.0 at 90 min, and increased markedly between 8 and 24 h to a maximum of 2.1 (p <0.01), indicating that functionality of circulating fibrinogen changes during AMI and subsequent thrombolytic therapy. The increased ratio of functional to “intact fibrinogen” seems to reflect a more functional fibrinogen at baseline and following rt-PA infusion. This is in keeping with data that the relative amount of fast clotting “intact HMW fibrinogen” of total fibrinogen is increased in initial phase of AMI. The data suggest that about 20% of HMW fibrinogen are converted to partly degraded fibrinogen during rt-PA infusion. The rebound phenomenon exhibited by functional fibrinogen may result from newly synthesized fibrinogen with a high proportion of HMW fibrinogen with its known higher degree of phosphorylation. Fibrinogen- and fibrin degradation products were within normal range at baseline. Upon infusion of the thrombolytic agent, maximum median levels of 5.88 μg/ml and 5.28 μg/ml, respectively, were measured at 90 min. Maximum plasma fibrinogen degradation products represented only 4% of lost “intact fibrinogen”, but they correlatedstrongly and linearly with the extent of “intact fibrinogen” degradation (r = 0.82, p <0.01). In contrast, no correlation was seen between breakdown of “intact fibrinogen” and corresponding levels of fibrin degradation products. We conclude from our data that the ratio of functional to immunologically “intact fibrinogen” may serve as an important index for functionality of fibrinogen and select patients at high risk for early reocclusion. Only a small proportion of degraded functional and “intact fibrinogen”, respectively, is recovered as fibrinogen degradation products. There seems to be a strong correlation between the degree of elevation of fibrinogen degradation products and the intensity of the systemic lytic state, i.e. fibrinogen degradation.

Influence of Certain Drugs on the Adhesiveness of Human, Rat, and Rabbit Blood Platelets in Vitro

10.1055/s-0038-1656242 ◽

1965 ◽

Vol 13 (02) ◽

pp. 428-438 ◽

Cited By ~ 2

Author(s):

K Reber ◽

A Studer

Keyword(s):

Sodium Citrate ◽

Blood Platelets ◽

Drug Application ◽

Normal Range ◽

Blood Samples ◽

Serotonin Antagonist ◽

Thrombocyte Count ◽

Particle Count ◽

Rabbit Blood

SummaryThis is a comparative study of the methods described by H. P. Wright and O’Brien for determining the adhesiveness of thrombocytes. An attempt is made to characterize and statistically correlate both techniques. With the aid of a Coulter Counter for thrombocyte counts, a normal range is presented for human, rat, and rabbit blood. Anticoagulants used are sodium citrate and Heparin.The influence of Cocaine and the Serotonin antagonist Ro 3-0837 was studied on these same substrates, to determine a pharmacological interference with results of either Wright’s test or O’Brien’s. Both drugs are found to induce a statistically significant increase in the “thrombocyte count” as compared to the corresponding controls. These effects are not real but to be attributed to an increase in particle count due to thrombocyte fragmentation as a consequence of drug application. There is no evidence for the claim that these drugs decrease the adhesiveness of thrombocytes.Numerical results of both tests often show a high and statistically significant correlation, especially following the addition of Ro 3-0837. Such is not true of individual blood samples to which no drug has been added. Evidentally, both tests are not specific for the same characteristic of normal blood platelets. But, when Ro 3-0837 is added, the breakdown of unstable platelets is induced; and the corresponding increase in count of thrombocyte fragments is expressed by both tests in the same fashion.

Identification of Crosslinked Fibrin Degradation Products in Plasma of Patients During Fibrinolytic Therapy Using a Heat Extraction/SDS-Polyacrylamide Gradient Gel Electrophoretic Technique

10.1055/s-0039-1684842 ◽

1979 ◽

Author(s):

C.W. Francis ◽

V. J. Marder ◽

S.E. Martin

Keyword(s):

Degradation Products ◽

Fibrinolytic Therapy ◽

Heat Extraction ◽

Molecular Weights ◽

Normal Plasma ◽

Electrophoretic Technique ◽

Gradient Gel Electrophoresis ◽

Derivatives Of

To quantitate plasmic degradation products of crosslinked fibrin in plasma, a technique has been developed which employs heat precipitation, SDS-polyacrylamide gradient gel electrophoresis of the dissolved, reduced heat precipitate, and quantitation by densitometrie analysis of γ-γ derivatives identified in the stained get. When studied with this sensitive electrophoretic technique, plasmic digests of purified crosslinked fibrin were found to contain a heterogeneous group of γ-γ chain derivatives with molecular weights between 76,000 and 100,000 daltons. In samples of normal plasma to which digests of crosslinked fibrin had been added, this heat extraction/ge1 electrophoretic technigue allowed the detection of γ-γ derivatives with a sensitivity of 20 µg/ml. Derivatives of γ-γ chains with molecular weights of 82,000 and 86,000 daltons have been identified in the plasma of patients with DIC and during fibrinolytic therapy but were not found in normal plasma or in normal plasma treated in vitro with urokinase. This quantitative assay can be performed in 24 hours and appears to be of value in judging the efficacy of thrombolytic therapy.

Identification of D-Dimer-E Complex in Disseminated Intravascular Coagulation (DIC)

10.1055/s-0039-1684840 ◽

1979 ◽

Author(s):

A.N. Whitaker ◽

E.A. Rowe ◽

P.P. Masci ◽

P.J. Gaffney

Keyword(s):

Gel Electrophoresis ◽

Degradation Products ◽

Polyacrylamide Gel Electrophoresis ◽

Stable Complex ◽

Pneumococcal Sepsis ◽

D Antigen ◽

D Dimer

D-dimer (D2), a product of the plasmin lysis of cross-linked (XL) fibrin, but not of non-XL fibrin or fibrinogen, has been identified in the plasma of patients with DIC due to amniotic fluid embolism. In vitro, D is involved with fragment E as a stable complex (D2-E) but D2 -E has not been identified in vivo before. Fibrin degradation products (FDP) were studied in a patient having fulminant postsplenectomy pneumococcal sepsis and DIC, by immunoprecipitation with anti-fibrinogen (f) and anti-fragment E and characterization by SDS polyacrylamide gel electrophoresis (PAGE). With both antisera soluble HMW fibrin complexes, D2 and E but no X, Y or D were obtained from serum. D2 and E were identified in the supernatant after removing partially XL HMW complexes and fibrinogen from plasma with 2.5 M β-alanine. The presence D antigen in the D2-E complex precipitated by monospecific anti-E was confirmed by crossed Ag-Ab electrophoresis. Crossed Ag-Ab electrophoresis of serum in agarose gave E peaks of slow mobility and no fast-moving free E was found. Thus, D2-E complex exists in vivo and its easy identification, proving the lysis of XL fibrin, would be of value in studying thrombosis. D2-E complex has been identified in other patients with sepsis but at lower concentrations than described above.

Porcine reproductive and respiratory syndrome virus RNA detection in different matrices under typical storage conditions in the UK

Veterinary Record ◽

10.1136/vr.105312 ◽

2019 ◽

Vol 185 (1) ◽

pp. 21-21 ◽

Cited By ~ 1

Author(s):

Jinghui Fan ◽

Priscilla F Gerber ◽

Ana Cubas Atienzar ◽

Lysan Eppink ◽

Chong Wang ◽

...

Keyword(s):

Storage Condition ◽

Early Morning ◽

Respiratory Syndrome Virus ◽

Blood Collection ◽

Practical Reasons ◽

Sample Type ◽

Sample Collection ◽

Blood Samples ◽

Fta Cards ◽

The Uk

In the UK, approximately 40 per cent of the pig breeding herds are outdoors. To monitor their porcine reproductive and respiratory syndrome virus (PRRSV) status, blood is collected commonly from piglets around weaning. Sample collection in British outdoor pigs often occurs during the early morning hours when the piglets tend to accumulate inside sheltered areas. For practical reasons, dry cotton swabs are occasionally used for blood collection and stored at room temperature until arrival in the laboratory. Detection of PRRSV RNA is a function of viral concentration, sample type and storage condition. To evaluate a possible impact of the sampling protocol on PRRSV1 detection, experimentally spiked blood samples using three dilutions of a representative PRRSV1 strain were prepared. In addition, blood samples from pigs naturally infected with PRRSV were obtained from a PRRSV-positive British herd. Spiked blood and blood from infected pigs were used to obtain sera, dry or wet (immersed in saline) polyester or cotton swabs and FTA cards. The different samples were stored for 24 hours, 48 hours or 7 days at 4°C or 20°C and tested by a real-time reverse transcriptase PRRSV PCR assay. Under the study conditions, the best matrix was serum (96.7 per cent), followed by wet swabs (78 per cent), dry swabs (61.3 per cent) and FTA cards (51 per cent). Polyester swabs (76 per cent) showed a better performance than cotton swabs (63.3 per cent). The reduction in sensitivity obtained for swabs and FTA cards was particularly high at low viral concentrations. The results indicate that wet polyester swabs should be used whenever possible.

A NEW QUANTITATIVE ENZYME-IMMUNOASSAY FOR FIBRIN DEGRADATION PRODUCTS (FbDP) IN PLASMA, BASED ON MONOCLONAL ANTIBODIES

10.1055/s-0038-1643128 ◽

1987 ◽

Author(s):

P W Koppert ◽

E Hoegee-de Nobel ◽

W Nieuwenhuizen

Keyword(s):

Monoclonal Antibodies ◽

Enzyme Immunoassay ◽

Degradation Products ◽

Blood Clot ◽

Tissue Type ◽

Type Plasminogen Activator ◽

Strong Positive Reaction ◽

Sandwich Type

We have developed a sandwich-type enzyme immunoassay (EIA) for the quantitation of fibrin degradation products (FbDP) in plasma with a time-to-result of only 45 minutes. The assay is based on the combination of the specificities of two monoclonal antibodies (FDP-14 and DD-13), developed in our institute. FDP-14, the catching antibody, binds both fibrinogen degradation products (FbgDP) and FbDP. It has its epitope in the E-domain of the fibrinogen molecule on the BB-chain between amino acids 54-118 (Blood 68, 437, 1986). Antibody DD-13 was raised using D-dimer as antigen and was used as a tagging antibody, conjugated with horse-radish peroxidase.A strong positive reaction is obtained with a whole blood clot lysate (lysis induced by tissue-type plasminogen activator) which is used as a standard.The EIA does not detect FbgDP i.e. purified fragments X, Y, D:E complexes or FbgDP in plasma treated in vitro with streptokinase. This indicates that the assay is specific for fibrin degradation products.We have successfully applied this assay to the plasma of patients with a variety of diseases. In combination with the assays previously developed by us for FbgDP (Thromb. Haemostas. 1987, in press) and for the total amount (TDP) of FbgDP + FbDP in plasma (J. Lab. Clin. Med. 1987, in press), we are now able to study the composition of TDP in terms of FbgDP and FbDP in patients.