Tanned Red Cell Research Articles

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.

Thyroglobulin and Microsomal Antibodies in Diabetes Mellitus

In the course of studies on the occurrence of antithyroid antibodies in diabetes mellitus, serum antibodies to microsomes of thyroid epithelial cells, as well as circulating antibodies to thyroglobulin, were demonstrated by tanned red cell hemagglutination.These thyroglobulin and microsome-coated tanned red blood cells can be efficiently made with a commercially prepared reagent. (Fuji Zoki Co. Tokyo) The sera of 507 diabetics and 2,350 normal subjects was tested using these thyroid autoantibodies tests.In diabetics, the percentage of sera positive for antithyroglobulin antibodies was found to be 2.5% compared with 2.3% in controls. And the percentage of sera positive for antimicrosomal antibodies was found to be 6% compared with 2.5% in controls.In diabetics treated with insulin, the percentage of antithyroglobulin antibodies and antimicrosomal antibodies were found to be 5% and 16% respectively.In diabetics not treated with insulin, the percentage of thyrid antibodies were 2% and 3% respectively.The incidence of antithyroglobulin and antimicrosomal antibodies was higher in diabetics treated with insulin.The incidence of thyroid antibodies in normal subjects was found to be increased with aging, but in diabetics the incidence of thyroid antibodies was found to be decreased with aging. There was no clear relationship between the incidence of these antibodies with the duration of diabetes.

Incidence of thyroglobulin and microsomal antibodies in normal subjects (author's transl)

In the course of studies on the occurrence of antithyroid antibodies in various thyroid disorders, serum antibodies to microsome of thyroid epithelial cells, as well as circulating antibodies to thyroglobulin, are demonstrated by tanned red cell hemaggulutination. These thyroglobulin and microsome-coated tanned red blood cells can be efficiently demonstrated with a commercially prepared reagent. (Fuji-Zoki Co.) The sera of 2,350 normal subjects were tested by these thryoid autoantibodies tests. Fifty-one (2.3 per cent) of the sera of 2,350 normal subjects showed a positive reaction for thyroglobulin antibodies, and fifty-nine (2.5 per cent) persons showed a positive reaction for microsomal antibodies. The incidence of thyroglobulin and microsomal antibodies in males and females were progressively greater with age, particularly between ages 60 to 69.

The clinical and immunological association of insulin antibodies and thyroid antibodies in diabetics (author's transl)

We have already reported a high rate of occurrence of antimicrosomal antibodies in diabetes mellitus. Thirteen of 507 diabetics (2.5%) were positive with antithyroglobulin antibodies and thirty-one (6.1%) were positive with antimicrosomal antibodies compared to 2.3% and 2.5% respectively in normal controls. Two of 34 insulin dependent diabetics (5.9%) were positive with antithyroglobulin antibodies and ten (29.4%) were positive with antimicrosomal antibodies compared to 2.3% and 4.4% respectively in 473 insulin independent diabetics. To clarify the association of insulin antibodies and thyroid antibodies in diabetics, antithyroid antibodies in 507 diabetics were tested by tanned red cell hemagglutination test and insulin antibodies were demonstrated by using a method descrived by Wright in a modified form. Twelve of 482 diabetics negative-insulin antibody (2.5%) were positive with antithyroglobulin antibodies and thirty(6.2%)were positive with antimicrosomal antibodies. Only one of 25 positive-insulin antibody (4%) was positive with antithyroid antibodies respectively. No evident correlation was observed between antithyroid antibodies and insulin antibodies.

Clinical studies on thyroidal autoantibodies.

In an attempt to clarify autoimmune nature of Grave's disease and Hashimoto's thyroiditis, thyroidal autoantibodies have been studied on 86 cases of Grave's disease, 54 cases of Hashimoto's thyroiditis, 31 cases of simple goiter, 11 cases of primary hypothyroidism a-d 22 cases of thyroid neoplasia as well as on 364 healthy subjects. Two kinds of tanned red cell hemagglutination tests were carreid out on each case. One method using refined human thyroglobulin for sensitization of sheep red cells has been found to be specifice for anti-thyroglobulin antibody and to be frequently positive in Hashimoto patients. However, in another method using human thyroidal microsomes (purified by ultracentrifugation) as sensitizing antigen, some heterogenous antibodies including anti-thyroglobulin antibody might be detected together with the antibody against the antigen proper to thyroidal micorsome (HF antigen)which is derived from hyperfunctioning thyroidal follicular cells of Grave's patients. hAnti-HF antibody is frequently detected both in Hashimoto and Grave's patients. In other thyroid diseases the titers of both thyroidal autoantibodies were generally low, though higher than in healthy subjects. The effect of aging on thyroidal autoantibodies in healthy subjects was clearly observed in females but not in males. From the results of these two test, it is possible to speculate that high=columnar and hyperfunctioning thyroidal follicular cells might exist focally in most cases of Hashimoto's thyroiditis.

Radioimmunologic assay for antithyroglobulin antibodies

Abstract A double-antibody radioimmunologic assay for antithyroglobulin antibodies (RATA) in serum is described. The assay employs human thyroglobulin labeled in vitro. The reaction product of antithyroglobulin antibodies and labeled human thyroglobulin is measured after precipitation with rabbit antihuman gamma-globulin serum. This test has a high degree of positivity in autoimmune thyroid disorders. In chronic lymphocytic thyroiditis, 100 per cent of sera from patients with surgically proved disease were positive and 83.8 per cent of those in whom this disease was clinically suspected were positive. In Graves' disease, 87.5 per cent of untreated patients' sera and 57.7 per cent of treated patients' sera were positive. In primary hypothyroidism, 94.4 per cent of patients' sera were positive. The sera from many patients with autoimmune thyroid disorders which were negative or had low titers to the commonly used tanned red cell agglutination test (TRCA) were positive with the RATA test. Four per cent of sera from normal control subjects, 15 per cent of sera from patients with other autoimmune disorders, and 8 per cent of sera from patients with thyroid disorders not considered to be of an autoimmune etiology were positive to the RATA. The RATA is simple and quantitative with sensitivity and specificity equal to or exceeding present tests for circulating antithyroglobulin antibodies.

The fibrous variant of Hashimoto's thyroiditis

The fibrous variant of Hashimoto's thyroiditis presents a clinicopathologic picture quite distinct from that of typical Hashimoto's disease. Fifty-six patients with this entity are described, and these represent 12.5 per cent of all cases with a pathologic diagnosis of Hashimoto's thyroiditis. The lesion is characterized by a marked fibrous replacement of one-third to the majority of the parenchyma and changes typical of Hashimoto's disease in the remaining tissue. In contrast to Riedel's struma, there is no extension of the fibrotic process beyond the thyroid capsule, and the dense hyaline form of connective tissue differs from the active proliferative fibrosis seen in Riedel's thyroiditis. The lesion may be mistaken for cancer with islands of metaplastic epithelium in the fibrotic tissue simulating foci of carcinoma cells. Distinctive clinical features include a very firm goiter, often with a striking recent enlargement, severe neck pressure symptoms, frequent diagnostic confusion with malignant disease, and a markedly elevated tanned red cell antibody titer to thyroglobulin. These titers often tend to parallel a history of recent thyroid growth and the degree of fibrosis on sequential needle biopsy studies. The similarities and possible relationship between the fibrous variant of Hashimoto's thyroiditis and idiopathic primary myxedema are discussed.

The tanned red cell hemagglutination-inhibition immunoassay of fibrinogen-fibrin degradation products methodological and statistical aspects

The accuracy and sensitivity of the tanned red cell hemagglutination-inhibition immunoassay (TRCHII) applied to fibrinogen-fibrin degradation products were investigated on venous serum and menstrual “serum”. Limiting the number of serial dilutions and the use of freshly thawn blood cells for each assay were means by which to increase the accuracy. The sensitivity was set at 0.5 fibrinogen/ml by varying the antiserum concentration. Factors influencing the TRCHII levels were investigated. The temperature and time during which the plasma samples were clotted were of minor importance, whereas the pH of the clotting solution influenced the levels markedly. Using 125I-labelled fibrinogen and degradation products derived both from fibrinogen (FDP) and from fibrin (fdp), the trapping of the products was estimated. Increased TRCHII levels at low pH were probably caused both by a decreased trapping of FDP/fdp and by residual fibrinogen, soluble fibrin and/or non-clottable complexes. When the fibrinogen concentration was decreased, the FDP/fdp were generally trapped to a lesser extent. FDP were always trapped more than fdp. The correctness of presenting results from TRCHII in terms of weight/volume is questioned. The results are presented as the reciprocal value of the highest dilution that inhibits agglutination. This value is called the dilution factor. The necessity to use non-parametric statistical tests, such as the median test, is stressed.

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.

Determination of fibrinogen degradation products using antifibrinogen serum and anti-fragment E serum

The level of purified fibrinogen degradation products was estimated by tanned red cells hemagglutination immuno assay with the use of anti-fibrinogen serum and anti-fragment E serum. The use of anti-fragment E serum is of significance in the late stages of diffuse intra-vascular coagulation since it permits to reveal products of fibrinogen degradation non detectable with fibrinogen anti-serum.

Differentiation of chronic lymphocytic thyroiditis and simple goiter in pediatrics

Eighty-one children with goiter were studied. Thirty-five had open biopsies and 23 of these patients had one or more simultaneous needle biopsies. In the remainder, the diagnosis was made by clinical and laboratory findings. Of 35 biopsied patients, the histologic diagnosis was chronic lymphocytic thyroiditis (CLT) in 22 and simple goiter in 13. Needle biopsy proved inadequate for diagnosis in 40 per cent. In biopsied patients, thyroid antibody studies by the tanned red cell technique were positive with a titer of 1:16 or greater in only a few cases of CLT. An immunofluorescent technique revealed antibodies in 100 per cent of cases of CLT and was negative in all cases of simple goiter. A difference between protein-bound iodine and thyroxine >2.0 μg per cent was found in almost as many cases of simple goiter as in CLT. Clinical and laboratory findings are reported for the entire group of 81 patients.

Evaluation of a tanned red cell technique for thyroid microsomal antibodies.

A haemagglutination test for thyroid microsomal (cytoplasmic) antibodies, using formolized tanned sheep red cells, is described and evaluated. The technique is essentially similar to the thyroglobulin tanned red cell test. It has been shown to be more sensitive and easier to use than the complement-fixation test. A combined screening test to detect both thyroglobulin and microsomal antibodies is described.

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.

Properties of Radiolabeled Rabbit C1q

Abstract Rabbit C1q was purified from fresh serum through two successive euglobulin precipitation steps in low ionic strength EDTA buffers. The purified protein showed one band on acrylamide electrophoresis and had a specific hemolytic activity of 2 ng N/SFU (site-forming units). Functional integrity was also tested by agglutination of tanned red cells coated with aggregated γ-globulin. Inhibition of this agglutination reaction was observed with γ-globulin, DNA and endotoxin. Labeling with 125I was performed with chloramine-T in a 1:50 ratio followed by filtration on Sephadex G-25 to remove unreacted 125I and chloramine-T. The labeled protein (C1q*) had a specific radioactivity of 21.7 µCi/µg N, as determined by RID (radioimmunoassay). About 89% of the radioactivity was protein bound as determined by precipitation in 10% TCA. The labeled material retained only 20% of its original hemolytic activity. Incubation of C1q* with EA 1 × 109/ml in EDTA buffer, RSC 0.07 resulted in minimal binding of the label to the cells, while 25% binding was observed in the presence of 10-3 M Ca++.

Anterior uveitis in Bantu children.

Full blood count and erythrocyte sedimentation rate, antistreptolysin titre, albumin/ globulin ratio, blood sugar estimation, gonococcal complement-fixation test, Wassermann and Treponema pallidum immobilization tests, Waaler-Rose latex-fixation test, Toxoplasma complement-fixation test, Tine test, histoplasmosis complement-fixation test. Urine and stool examination for parasites and bacteria. Xray of the chest, teeth, sinuses, and lumbosacral spine. Lymphocytes from the peripheral blood were also cultured and grown and karyotyping done. Peripheral bloodwas also utilized in searching for antiuveal antibodies using the tanned red cell technique. Studies involving blast transformation in peripheral lymphocytes stimulated with uveal antigen was also begun in a few cases.

Differences in clinical and convalescent-phase antibodies of Rhesus monkeys infected with monkey pox, tanapox, and Yaba poxviruses.

Complement-fixing and complement-fixing inhibiting (CFI) antibodies were demonstrated in the clinical and convalescent stages, respectively, of rhesus monkeys infected with either monkey poxvirus, Tanapoxvirus, or Yaba poxvirus. Specificity of the CFI antibody was confirmed by its failure to cross-react with heterologous poxvirus antigens and by experiments demonstrating the CFI test as being antigen dependent. Serum containing CFI antibody neutralized homologous poxvirus but failed to agglutinate antigen-coated, tanned red blood cells. The application of CFI test as a seroepidemiologic tool for studies of poxvirus infection of man and simian monkeys and the biologic role of CFI antibody in pathogenesis were discussed.

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.