Tanned Red Cell Hemagglutination Inhibition 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.

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.

Fibrinogen/Fibrin Degradation Products in Serum of Patients with Idiopathic Thrombocytopenic Purpura: Elevated Levels during Severe Thrombocytopenic Phase of the Disease

Sera from 23 patients with idiopathic thrombocytopenic purpura (ITP), 14 patients with aplastic anemia with severe thrombocytopenia and healthy control subjects were tested for the presence of fibrinogen/fibrin degradation products (FDP), using the tanned red cell hemagglutination inhibition immunoaassay. The concentrations of circulating FDP of ITP patients (mean 12.01 mug/ml) were significantly higher than those of the patients with aplastic anemia (mean 4.01 mug/ml, p less than 0.05) or normal controls (mean 3.10 mug/ml, p less than 0.001). The patients with untreated ITP with very low platelet counts had higher levels of FDP than those of the treated group (p less than 0.01). Serum FDP and a battery of other coagulation-fibrinolysis tests were serially carried out over a period of 10 weeks in two patients with ITP. The initially high FDP promptly decreased as circulating platelets increased in response to steroid in both patients, while plasma fibrinogen, euglobulin lysis time, prothrombin time and partial thromboplastin time remained essentially normal during the course of observation. The exact source of the increased serum FDP in ITP was not established, but a few possible mechanisms responsible for this abnormality were discussed.

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

Estimation of fibrinogen-fibrin related antigen by continuous flow analysis

A precise method for the determination of fibrinogen-fibrin related antigen (FR antigen) in an automated tanned red cell haemagglutination inhibition system is described. In a continuous flow system aliquots of serum are mixed with a dilution of rabbit anti-human fibrinogen serum. The FR antigen neutralizes the anti-fibrinogen serum and on addition of tanned human red cells sensitized with human fibrinogen, the cells agglutinate according to the remaining anti-fibrinogen activity. The agglutinates are removed and the remaining free cells are haemolysed with saponin and the colour measured at 550 nm. The density of the colour formed is in proportion to the amount of FR antigen present. The range in 102 normal males was 4.8±2.4 μg/ml. A significant increase in the levels of FR antigen throughout pregnancy was found, the highest levels being in the latter half of the third trimester. The levels of FR antigen in 31 normal urines was 0.46±0.44 μg/ml.

The Use of Immunoelectro-Osmophoresis in the Laboratory Diagnosis of Disseminated Intravascular Coagulation

The method of immunoelectroosmophoresis used for the detection and titration of degradation products of fibrinogen and fibrin has been demonstrated to be a sensitive and relatively quick procedure. Comparison with other well-established methods such as tanned red cell haemagglutination inhibition, agglutination of anti-fibrinogen sensitized latex particles, and immunodiffusion has shown that it could serve as a useful complementary test in the laboratory diagnosis of disseminated intravascular coagulation. The method of immunoelectroosmophoresis used for the detection and titration of degradation products of fibrinogen and fibrin has been demonstrated to be a sensitive and relatively quick procedure. Comparison with other well-established methods such as tanned red cell haemagglutination inhibition, agglutination of anti-fibrinogen sensitized latex particles, and immunodiffusion has shown that it could serve as a useful complementary test in the laboratory diagnosis of disseminated intravascular coagulation.

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.

Methods of examining fibrinolysis in plasma.

IMMUNOLOGICAL methods have been used to estimate the levels of fibrinogen and fibrin degradation products in serum samples during thrombolytic therapy and in various clinical situations. A recent report1 has suggested that methods such as the tanned red blood cell haemagglutination inhibition immunoassay2 and latex aggregation3 measure mainly the products of fibrin degradation. We wish to report on techniques which have proved useful in differentiating between the degradation products of fibrinogen and fibrin clots in whole plasma systems in vitro and which have helped in confirming results obtained4,5 using purified fibrinogen and fibrin clots.

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.

Fibrin Split Products in the Severely Burned Patient

Fibrin split products were measured by a tanned red cell hemagglutination inhibition immuno-assay in 77 patients with burns and 40 normal individuals who were the controls. The severely burned patient showed only a modest elevation of fibrin degradation products. These results did not correlate well with traditional coagulation measurements which may be affected by a number of mechanisms. The level of fibrin split products did not correlate with the percentage of surface body burns, nor was it of predictive value.

Immunologically reactive equivalents of fibrinogen in sera and urine of children with renal disease.

Fibrin degradation products (FDP) were assayed by the tanned red cell haemagglutination inhibition technique in the sera and urine of children with renal disease, and their correlation with other parameters of proteinuria was examined. Urinary FDP were present in greater quantities in subjects with heavy and unselective proteinuria, and were not correlated with serum levels of FDP. FDP may derive from fibrinogen filtered through leaky glomeruli or from intravascular coagulation restricted to the kidney: the data presented are compatible with either hypothesis.

Plasmin lysis of fibrinogen and fibrin and the antigenic properties of their degradation products.

Summary. Purified fibrinogen was lysed by plasmin and the degradation products identified by gel filtration, immunoelectrophoresis and the tanned red cell haem‐agglutination inhibition (TRCHI) test. After 15 min of lysis, fractions with molecular weight (mol wt) 270 000 (fragment X), 110 000 (containing both fragments D and E) and 15 000 were present. After 6 hr lysis, only fractions of mol wt 80 000 (fragments D and E) and 15 000 were present. Fibrin was lysed by plasmin for 30 min, 4, 8 and 24 hr. In each case fractions of mol wt 150 000 (fragment Y), 80 000 (fragments D and E) and 15 000 were produced. It was shown that early and late products of fibrin lysis are similar to those of fibrinogen, and that the tanned red cell haemagglutination inhibition test using anti‐fibrinogen antibody provides a reliable estimate of fibrin lysis both in vitro and in vivo.