Red Cell Hemagglutination Inhibition Immunoassay Research Articles

Determination of plasma antithrombin III by tanned red cell hemagglutination inhibition immunoassay

Concentrations of antithrombin III have been evaluated in normal and pathological plasmas by a new immunochemical technique, the tanned red cell hemagglutination inhibition methods that analyze the biological activities of antithrombin III.

Evaluation of New Latex FDP Assay

The Dade latex FDP (fibrin/fibrinogen degradation products) reagents, recently introduced to the market, were evaluated against the Thrombowellco latex FDP reagents and the tanned red cell hemagglutination inhibition immunoassay (TRCHII). The Dade reagents gave positive results in the 10-40 µg/mL range in four of 20 normal sera; none of these samples was positive (ie,⩾10 µg/mL) with Thrombowellco or TRCHII reagents.With 54 patient samples, almost 60% showed FDP titers in the range of 20-80 µg/mL with the Dade assay; only about 30% of the same samples were shown to be in that range with the other two methods. In separate experiments,the agitation time was prolonged from two to 2.5 minutes. Five of eight normal sera turned positive with the Dade method, while none did so with the other two methods. The results indicate an unusual sensitivity of the Dade product toward serum FDP.

Localizaton of fibrin converted by thrombin from released platelet fibrinogen

Treatment of platelets (10(9) cells/ml) with thrombin (1 U/ml) resulted in rapid disappearance of fibrinogen from the system as measured by the tanned red cell hemagglutination inhibition immunoassay (TRCHII). Plasmin digestion of individual pellet and supernatant fractions that had been previously separated from thrombin-treated platelet suspensions by centrifugation resulted in recovery of TRCHII-detectable material in platelet pellets. To elucidate the specific association of fibrin to platelet membranes, control and thrombin-treated platelets were homogenized by a modified glycerol-loading and nitrogen decompression technique. Ultracentrifugation of homogenates through 27% sucrose cushions yielded three subcellular fractions: supernatant, small membrane vesicles, and a particulate fraction for controls; and supernatant membrane vesicles, and aggregated membrane “ghosts” for thrombin preparations. Ultrastructurally identifiable fibrin was noted only in the thrombin fraction containing membrane ghosts. Fibrinogen recovered from 3 thrombin fractions was markedly decreased (3% of the control). Plasmin digestion produced 23% and 46-fold increase in TRCHII- detectable material from 3 subcellular fractions of control and thrombin preparations, respectively. More than 97% of TRCHII material recovered from thrombin preparations was in the fraction containing aggregated membrane fractions. Results suggest that platelet plasma membranes function as surfaces for fibrin deposition.

Localization of Fibrin Converted by Thrombin From Released Platelet Fibrinogen

Treatment of platelets (10(9) cells/ml) with thrombin (1 U/ml) resulted in rapid disappearance of fibrinogen from the system as measured by the tanned red cell hemagglutination inhibition immunoassay (TRCHII). Plasmin digestion of individual pellet and supernatant fractions that had been previously separated from thrombin-treated platelet suspensions by centrifugation resulted in recovery of TRCHII-detectable material in platelet pellets. To elucidate the specific association of fibrin to platelet membranes, control and thrombin-treated platelets were homogenized by a modified glycerol-loading and nitrogen decompression technique. Ultracentrifugation of homogenates through 27% sucrose cushions yielded three subcellular fractions: supernatant, small membrane vesicles, and a particulate fraction for controls; and supernatant membrane vesicles, and aggregated membrane "ghosts" for thrombin preparations. Ultrastructurally identifiable fibrin was noted only in the thrombin fraction containing membrane ghosts. Fibrinogen recovered from 3 thrombin fractions was markedly decreased (3% of the control). Plasmin digestion produced 23% and 46-fold increase in TRCHII-detectable material from 3 subcellular fractions of control and thrombin preparations, respectively. More than 97% of TRCHII material recovered from thrombin preparations was in the fraction containing aggregated membrane fractions. Results suggest that platelet plasma membranes function as surfaces for fibrin deposition.

Evaluation of a New Anti-Fibrinogen-Coated Latex Particle Agglutination Test in the Measurement of Serum Fibrin Degradation Products

A new latex particle agglutination test (FIBROTEX) has been compared with the Thrombo-Wellcotest and the tanned red cell hemagglutination inhibition immunoassay (TRCHII) for sensitivity to purified fibrinogen and plasmic degradation products and for reactivity with 31 normal sera and 170 sera from patients with thrombotic disorders. The latex particles in the FIBROTEX are coated with antibodies against fibrinogen and fragment E (anti-FE), in contrast with the anti-fragment D and anti-fragment E antibodies which coat the latex particles of the Thrombo-Wellcotest (anti-DE). The relative sensitivities of the TRCHII and the anti-FE and anti-DE latex tests were, respectively, 0.5, 2.5 and 7.5 microgram/ml for fibrinogen; 0.5, 0.5 and 4.0 microgram/ml for fragment X; and 1.25, greater than 30 and 8.0 microgram/ml for fragment D. Compared with anti-DE, the anti-FE particles demonstrated fewer false-positive results with sera and urines of normals and higher titers, fewer false negatives and better overall correlation with the TRCHII results (r = 0.89 vs. 0.75) on pathologic sera. The FIBROTEX is simple and rapid (5 minutes) and provides a more sensitive and accurate determination of serum fibrin degradation products than the Thrombo-Wellcotest.

Intravascular coagulation in hyperlipidemia

Activation of the coagulation system was evaluated in 30 patients with familial hyperlipoproteinemia (types II and IV), by measurement of high molecular weight fibrin(ogen) derivatives (HMWFD). These HMWFD can be separated from fibrinogen and its derivatives X, D, and E by gel filtration of plasma on 4% agarose columns. Type II patients had a higher plasma concentration of HMWFD (31.6% of total plasma fibrinogen-like material) than type IV patients (19.8%) or normal subjects (3.2%). The HMWFD were shown to consist at least in part of crosslinked fibrin by polyacrylamide gel electrophoresis. Serum fibrin(ogen) degradation products (FDP) were assayed by staphylococcal clumping test and tanned red cell hemagglutination inhibition immunoassay and were found to be normal in type II and type IV patients. Our data show that type II patients have increased circulating HMWFD, while type IV patients have lesser increases in these soluble fibrin complexes.

Comparison of a Direct Latex-agglutination Technic with the Tanned Red Cell Hemagglutination Inhibition Immunoassay (TRCHII) for Semiquantitation of Fibrinogen/Fibrin Degradation Products

A new latex-agglutination kit for rapid screening for fibrinogen/fibrin degradation products (FDP/fdp) has been compared with a standard tanned red cell hemagglutination inhibition immunoassay (TRCHII). The latex-agglutination test results agreed with the TRCHII results for 489 of 588 samples (83%). FDP/fdp values were elevated by the latex-agglutination kit when normal by the TRCHII for 79 (13%) samples, and the reverse was true for 20 (3%) samples. Serial dilution of serum sample allowed reliable semiquantitation of FDP/fdp levels compared with TRCHII results (r=less than .001). The latex-agglutination test gave FDP/fdp values of larger than or equal to 10 mug. per ml. for sera from 11 of 13 patients who had acute pulmonary embolism and for only one of 24 normal control samples. The direct latex-agglutination kit for FDP/fdp appears to have appropriate sensitivity to serve as a screening test for acute pulmonary embolism in patients not receiving heparin therapy.

Characterization of elevated fibrin split products following thermal injury

These studies establish that the staphylococcal clumping test is superior to the tanned red cell hemagglutination inhibition immunoassay for monitoring fibrin split product concentration in burn sera. It is strongly suggested that the principal circulating degradation product is a complex of soluble fibrin monomer with fragment D. Finally, there does not appear to be any effect on measured fibrin split product concentration in sera of burn patients receiving prophylactic heparin or aspirin.

A tanned red cell hemagglutination inhibition immunoassay (TRCHII) for the quantitative estimation of thrombin-antithrombin III and plasmin-alpha 1-antiplasmin complexes in human plasma

A tanned red cell hemagglutination inhibition immunoassay (TRCHII) for the quantitative estimation of thrombin-antithrombin III and plasmin-alpha 1-antiplasmin complexes in human plasma

Fibrin-fibrinogin degradation products in cardiovascular surgery.

Two hundred fifty-five determinations of the protamine sulfate test, the staphylococcal clumping test, and the tanned red blood cell hemagglutination inhibition immunoassay were performed on 30 patients undergoing cardiovascular surgery. The level of serum fibrin-fibrinogen degradation products rose in all patients and reached the highest levels between the fifth and ninth postoperative day. After the first postoperative day, a fibrin-fibrinogen degradation product increase of greater than 10μg/ml within 24 to 48 hours was associated with clinically recognized thrombotic complications in only two out of five patients. Isolated levels of serum fibrin-fibrinogen degradation products as high as 67μg/ml could not be correlated with clinically evident thrombotic disease or serious complications of surgery. Increased excretion of urinary fibrin-fibrinogen degradation products was associated with, but not predictive of, acute ischemic renal failure.

Detection of fibrinogen antigens with two latex techniques applied to urine concentrates.

Fibrinogen antigens were measured either with an agglutination inhibition method (using latex particles coated with fibrinogen; Diagen test) or with a direct agglutination technique (using latex particles coated with a mixture of anti-D and anti-E antibodies; Thrombo-Wellcotest). Both methods were compared with the tanned red cell haemagglutination inhibition immunoassay (TRCHII) during progressive degradation of fibrinogen with plasmin and using purified fibrinogen fragments or urine concentrates from chronic glomerulonephritis or transplanted patients. Due to the different sensitivity of the two latex techniques to fibrinogen and its plasmin derivatives, their combined use may be helpful to distinguish the nature of the fibrinogen-like material excreted in urine.

Serial estimation of urinary fibrin fibrinogen degradation products in kidney transplantation.

The urinary excretion of fibrin/fibrinogen degradation products (F.D.P.) of 81 human cadaver kidney transplants has been measured serially by the techniques of tanned red cell haemagglutination inhibition immunoassay and immunonephelometry. Acute rejection episodes in functioning transplants have been associated with increased F.D.P. excretion which in 80% of cases has preceded clinical diagnosis by periods of one to seven days. Recovery from these episodes has been associated with a rapid fall of F.D.P. excretion to undetectable levels. The level of F.D.P. excretion during a rejection episode is a guide to its ultimate outcome. Irreversibly rejected kidneys excrete high levels of F.D.P. for long periods. Viable kidney transplants with prolonged oliguric phases can be distinguished, while still oliguric, from rejected kidneys by their low F.D.P. excretion. F.D.P. cannot usually be detected in the urine of well-functioning transplants. Episodes of raised F.D.P. excretion in the absence of acute clinical rejection, however, occur occasionally and may be associated with permanent impairment of renal function.

Highly sensitive method for the assay of plasminogen.

Abstract An assay of plasminogen based upon the tanned red cell hemagglutination inhibition immunoassay was developed. The method proved highly sensitive and was suitable for the determination of plasminogen in plasma, urine, and spinal fluid.

Identification of intrarenal fibrin deposition.

The site and extent of intrarenal fibrin deposition has been examined by routine histological staining, immunofluorescence, and electron microscopy in 109 patients with a wide variety of renal diseases. The findings have been correlated with the amount of urinary fibrin/fibrinogen degradation product (FDP) excretion as measured by the tanned red cell haemagglutination inhibition immunoassay. The results show that routine histological stains (Martius Scarlet Blue and Picro Mallory) are unreliable, particularly where there is mesangial accumulation of material or where the amount of fibrin deposited is small and confined to a subendothelial position. Similarly the electron microscope may overassess the degree of fibrin deposition, particularly if this is associated with the deposition of immunoglobulins and complement. There is a close relationship between the site and extent of fibrin, as detected by immunofluorescence, and the maximal urinary FDP excretion.

Detection of serum fibrinogen and fibrin degradation products: Comparison of six technics using purified products and application in clinical studies

Six assay systems for serum fibrin degradation products (tanned red cell hemagglutination inhibition immunoassay (TRCHII), staphylococcal clumping test, Fi test, flocculation, immunodiffusion and anticoagulant assay) were compared for reactivity with plasmin digests of fibrinogen and fibrin, with purified degradation products and with serum obtained from patients suspected of having circulating degradation products. The TRCHII reacts best with undigested fibrinogen and with the intermediate degradation fragments X and Y. Although less reactive with purified fragments, the staphylococcal clumping test is as reliable for the assay of test serums, and both react with about 90 per cent of test serums. This suggests that circulating complexes of degradation products with fibrin monomer or with fragment X may be responsible for the positive results of test serums. One patient with defibrination syndrome and one with therapeutic thrombolysis were studied in detail with respect to the usefulness of the six assay systems in reflecting the presence of a hemorrhagic diathesis. The flocculation test is probably of the greatest value for emergency clinical situations, as it is rapid and simple, yet consistently sensitive to levels of degradation products that cause abnormal bleeding.

Comparison of the Tanned Red Cell Hemagglutination Inhibition Immuno-assay and radial immunodiffusion for the quantitation of fibrinogen-related antigen in urine concentrates.

Scandinavian Journal of HaematologyVolume 8, Issue S13 p. 225-228 29. COMPARISON OF THE TANNED RED CELL HEMAGGLUTINATION INHIBITION IMMUNO-ASSAY AND RADIAL IMMUNODIFFUSION FOR THE QUANTITATION OF FIBRINOGEN-RELATED ANTIGEN IN URINE CONCENTRATES+ Ayesha Molla, Ayesha Molla Laboratory of Blood Coagulation (Head: Prof. Dr. M. Verstraete) and Section of Nephrology (Head: Prof. Dr. P. Michielsen), Academisch Ziekenhuis St. Rafaël, University of Leuven, Belgium Research worker from Pakistan, awarded a postgraduate Fellowship by the University of Leuven.Search for more papers by this authorM.B. Donati, M.B. Donati Laboratory of Blood Coagulation (Head: Prof. Dr. M. Verstraete) and Section of Nephrology (Head: Prof. Dr. P. Michielsen), Academisch Ziekenhuis St. Rafaël, University of Leuven, Belgium On leave of absence from the Laboratory of Blood Coagulation (Prof. B. Bizzi) Department of Internal Medicine (Prof. R. Breda), Università Cattolica, Roma, Italy. Fellowship of the Ministero della Pubblica Istruzione, Roma.Search for more papers by this authorG. Dotremont, G. Dotremont Laboratory of Blood Coagulation (Head: Prof. Dr. M. Verstraete) and Section of Nephrology (Head: Prof. Dr. P. Michielsen), Academisch Ziekenhuis St. Rafaël, University of Leuven, BelgiumSearch for more papers by this authorJ. Vermylen, J. Vermylen Laboratory of Blood Coagulation (Head: Prof. Dr. M. Verstraete) and Section of Nephrology (Head: Prof. Dr. P. Michielsen), Academisch Ziekenhuis St. Rafaël, University of Leuven, BelgiumSearch for more papers by this author Ayesha Molla, Ayesha Molla Laboratory of Blood Coagulation (Head: Prof. Dr. M. Verstraete) and Section of Nephrology (Head: Prof. Dr. P. Michielsen), Academisch Ziekenhuis St. Rafaël, University of Leuven, Belgium Research worker from Pakistan, awarded a postgraduate Fellowship by the University of Leuven.Search for more papers by this authorM.B. Donati, M.B. Donati Laboratory of Blood Coagulation (Head: Prof. Dr. M. Verstraete) and Section of Nephrology (Head: Prof. Dr. P. Michielsen), Academisch Ziekenhuis St. Rafaël, University of Leuven, Belgium On leave of absence from the Laboratory of Blood Coagulation (Prof. B. Bizzi) Department of Internal Medicine (Prof. R. Breda), Università Cattolica, Roma, Italy. Fellowship of the Ministero della Pubblica Istruzione, Roma.Search for more papers by this authorG. Dotremont, G. Dotremont Laboratory of Blood Coagulation (Head: Prof. Dr. M. Verstraete) and Section of Nephrology (Head: Prof. Dr. P. Michielsen), Academisch Ziekenhuis St. Rafaël, University of Leuven, BelgiumSearch for more papers by this authorJ. Vermylen, J. Vermylen Laboratory of Blood Coagulation (Head: Prof. Dr. M. Verstraete) and Section of Nephrology (Head: Prof. Dr. P. Michielsen), Academisch Ziekenhuis St. Rafaël, University of Leuven, BelgiumSearch for more papers by this author First published: March 1971 https://doi.org/10.1111/j.1600-0609.1971.tb02012.x + This investigation was supported by grant N° 1216 of the Fonds voor Geneeskundig Wetenschappelijk Onderzoek, Brussel. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Volume8, IssueS13March 1971Pages 225-228 RelatedInformation

Fibrinogen degradation products in serum and urine of patients with systemic lupus erythematosus. Relation to renal disease and pathogenetic mechanism.

AbstractSerum and concentrated urine from blood donors, patients with rheumatoid arthritis and SLE patients with and without renal disease were tested for the presence of fibrinogen degradation products (FDP), using the tanned red cell hemagglutination inhibition immunoassay. The majority of blood donors had serum titers of 1:2 to 1:4, while the majority of patients with rheumatoid arthritis and systemic lupus erythematosus had titers of 1:8 to 1:32. FDP were detected in low titers in urine of nearly all blood donors, patients with rheumatoid arthritis and SLE patients without renal disease, whereas elevated titers were found in SLE patients with active renal disease. Several possible sources of the urinary FDP are discussed, and the role of fibrin deposition in glomerular injury is reviewed.

An Accelerated Red-Cell Hemagglutination Inhibition Immunoassay for Measuring Fibrinogen Degradation Products in Human Serum

SummaryA further modification of Merskey’s hemagglutination inhibition assay for measurement of fibrinogen degradation products (FDP) in human serum was developed. Blood is clotted in the presence of protamine chloride (25 u/ml), in addition to thrombin and s-aminocaproic acid, and at room temperature. Defibrination is complete within 5 min even in blood obtained from patients on heparin treatment. The hemagglutination immunoassay was also considerably accelerated by immediate addition of sensitized red cells to the mixtures of antiserum and antigen dilutions at 37° C. Accurate readings of FDP titers may thus be obtained within 20 min following venipuncture.

Quantitation of secondary fibrinolysis in cyanotic heart disease

<h2>Summary</h2> A sensitive quantitative method for measurement of FDP was used to restudy the sera of patients with cyanotic heart disease. Cyanotic patients with polycythemia had levels of FDP clearly above the normal limits of tanned red cell hemagglutination inhibition immunoassay, but too low to be detected by less sensitive methods. This same group has been previously shown to have levels of coagulation factors consistent with low-grade disseminated intravascular coagulation.

Letter To The Editor

I would like to comment on the report, "Split Products of Fibrin in Newborns," by Drs. Stiehm and Clatanoff. We have examined over 50 specimens of cord serum from babies delivered normally or by uncomplicated cesarean section, and have been unable to detect increased levels of fibrin degradation products (F.D.P.) in any of the samples. The technique used is a modification of the Tanned Red Cell Hemagglutination Inhibition Immunoassay (TRCHII) described by Merskey, Kleiner and Johnson.1