Studies on the platelet radioactive anti-immunoglobulin test.

A platelet radioactive antiimmunoglobulin test (PRAT) has recently been introduced. In the present report, a detailed analysis the influence of varying test conditions (i.e. efficiency of iodination of anti-IgG, cell number, degree of sensitization of platelets, times and temperatures of incubation, frequency of washings) on the results of this technique is presented. A standard procedure based on these findings is outlined. It is shown that the PRAT is two to four times more sensitive than platelet complement fixation for the detection of HLA antibodies.

Catabolism of fibrinogen in pregnant rabbits before and after delivery.

16 non-pregnant female and 19 pregnant rabbits were injected with purified rabbit 125I-fibrinogen in order to study the catabolism of fibrinogen before and after delivery. The half-life time (T 1/2) of the clottable 125I-radioactivity in pregnant rabbits during the last third of gestation was 27.3 +/- 5.3 hr in comparison to 54.4 +/- 3.7 hr in non-pregnant rabbits. After delivery, T 1/2 returned to normal values of 47.9 +/- 10.9 hr. The fractional catabolic rate (FCR) of the clotabble 125I-radioactivity was 67.1% X d-1 +/- 8.6 before and 41.6% X d-1 after delivery whereas FCR in non-pregnant rabbits amounted to 44.7% X d-1 +/- 4.8. These figures demonstrate a pronounced increase in fibrinogen catabolism during the last third of gestation in pregnant rabbits and a normalization immediately after delivery. Although in pregnant rabbits the elimination of fibrinogen from the circulating blood was pronouncedly increased, the plasma fibrinogen concentration did not change. Only after delivery did the fibrinogen concentration increase when the fibrinogen catabolism had already normalized.

Crosslinking of soluble fibrin and fibrinogen.

Monomeric 125I-desAA-fibrin and 125I-desAABB-fibrin were prepared by treating 125I-fibrinogen with thrombin. Preactivated factor XIII was added to 125I-fibrin/131I-fibrinogen mixtures, and after incubation for 2 and 6 hours, samples were investigated by SDS polyacrylamide gel electrophoresis under reducing conditions. The electrophoresis pattern showed a gamma-gamma band containing both 125I-fibrin and 131I-fibrinogen. These experiments indicate that a fibrin molecule polymerizes with a fibrinogen molecule in a similar way as a fibrin molecule polymerizes to a second fibrin molecule. This type of polymerization results in conformational changes of the molecules involved thus enabling FXIIIa for specific crosslinking reaction. The polymerization of fibrin and fibrinogen molecules as well as the crosslinking of fibrin to fibrinogen molecules seem to represent a mechanism for interrupting the process of further fibrin polymerization.

Studies on catabolism of 125I-labelled fibrinogen in normal rabbits and in rabbits with indwelling intravenous catheters: methodologic aspects.

The catabolism of rabbit fibrinogen labelled with radioactive iodine was studied over a period of 7-9 days in normal rabbits and in rabbits with indwelling intravenous catheters. The labelled fibrinogen was highly clottable and homogeneous on immunoelectrophoresis and disc electrophoresis. The protein-bound 125I-activity amounted to at least 99%. Normal rabbits with a weight between 1.6 and 2.2 kg exhibited a mean plasma volume of 41.65 ml/kg. A mean half-life time of 1.69 days and a mean fractional catabolic rate of 59.8% of the plasma fibrinogen pool per day was computed by tracing labelled fibrinogen. The absolute catabolic rate revealed a mean value of 58.7 mg/kg body weight/day. Comparable results were obtained for all three labelled fibrinogen batches. A siliconized polyvinyl tubing with an outer diameter of 2.0 mm inserted into a jugular vein up to a length of 10-12 cm and indwelling for more than 10 days did not significantly influence the parameters studied for controlling the fibrinogen catabolism except for the absolute catabolic rate of 112.8 mg/kg/day which differed significantly from that of the control group. Thus, the indwelling intravenous catheter had no or only a minor effect on the activation of intravascular coagulation.

Studies on circulating soluble fibrin: separation of 125I-Des-AB fibrin and 131I-fibrinogen by gel filtration.

The behaviour of labelled des-AB fibrin in plasma was studied by gel filtration after it had been injected into rabbits. Purified rabbit [125I]des-AB fibrin was prepared by clotting of [125I]fibrinogen by thrombin and solubilizing the formed clot in buffered 3 M urea. Gel filtration of this material on urea-equilibrated columns showed a single peak identical to the elution profile of fibrinogen. This indicated the existence of monomeric des-AB fibrin. When plasma from rabbits injected with monomeric [125I]des-AB fibrin and [131I]fibrinogen was gel-filtered through plasma-equibrated columns, two separate peaks of radioactivities were obtained. The first peak eluted mainly with the void volume and contained [125I]des-AB fibrin whereas the second peak eluting within the fractionation range contained [131I]fibrinogen. Identical elution profiles were obtained in in vitro studies when monomeric [125I]des-AB fibrin was mixed with plasma containing [131I]fibrinogen and gel-filtered through plasma-equilibrated columns. We conclude from these studies that monomeric des-AB fibrin formed high-molecular weight aggregates or changed its conformation posing as a larger molecule than fibrinogen when injected into rabbits. No complex formation between des-AB fibrin and fibrinogen was observed as [131I]fibrinogen was not incorporated into des-AB fibrin aggregates. Thus, soluble des-AB fibrin can circulate in the blood without forming fibrin-fibrinogen complexes.

Fibrinogen turnover in pregnant rabbits during the first and last thirds of gestation.

Although thrombin-mediated fibrinogen derivatives have been observed in normal pregnancy, an increased turnover of fibrinogen has been suspected but has not yet been demonstrated. Fibrinogen turnover was studied in 14 pregnant and 10 nonpregnant female rabbits by use of purified rabbit 125I-labeled fibrinogen. In the last third of gestation the half life of fibrinogen was shortened by 45% and the fractional catabolic rate increased by 44% in comparison to the values for the first third of gestation obtained in the same rabbits. The distribution volume of the injected fibrinogen representing the plasma volume increased by a mean of 18% during gestation. Fibrinogen concentration did not change during gestation. From a comparison of the measured data from the first and last thirds of gestation, it can be calculated that the synthesis rate of fibrinogen increased by about 80%. Furthermore, a positive correlation was found between the number of young per litter and the acceleration of fibrinogen elimination, indicating that a local process of intravascular coagulation in the placenta is responsible for the accelerated turnover of fibrinogen during gestation.

[Immunological aspects of syngenesioplastic bone transplantation in treatment of infantile bone tumors (author's transl)]. / Immunologische Aspekte syngenesioplastischer Knochentransplantation bei der Behandlung kindlicher Knochentumoren.

Bone transplantations are made often in the treatment of benign bone tumours or tumour like affections. Especially at the appropriate infantile illness the use of an autologous transplant may prove impossible. Homologous and heterologous osseous transplants are largely unsuitable, because of the induction of immunological reactions. So called syngenesioplastic transplants let expect a tolerance of the recipient organism. This is a report of the results of clinical, radiological and especially immunological follow-up-examinations of 11 patients treated from August 1974 to October 1976 with syngenesioplastic spongiosa transplantation.

Assessment of red cell autoantibodies in autoimmune hemolytic anemia of warm type by a radioactive anti-IgG test.

To evaluate the applicability of a radioactive 125I-anti-IgG test (RIAT) for the detection of small amounts of IgG antibodies on red blood cells (RBC) of patients with autoimmune hemolytic anemia of warm type (AIHA), RBC of 125 patients were studied (AIHA, n = 53; Coombs'-negative AIHA, n = 6; chronic cold agglutinin disease, n = 7; non-immune anemias, n = 59). It was found that the RBC of all cases (33/33) with a positive direct IgG antiglobulin test (DAT-IgG), but also 13 out of 20 patients with a negative DAT-IgG, but detectable complement (C3/C4), and 4 out of 6 cases with Coombs'-negative AIHA gave positive results in the direct RIAT. RBC-associated IgG was higher in the DAT-IgG positive group (n = 33; -x = 8.1%) than in the DAT-IgG negative group (n = 26; -x3.4%). There was no correlation between hypergammaglobulinemia and RBC-associated IgG. The sensitivity of the indirect RIAT was not remarkable better as compared to the indirect antiglobulin test. The RIAT is valuable in the serology of borderline cases of AIHA.

125 I-anti-immunoglobulin test: a new tool for the detection of drug-allergic platelet antibodies.

It is shown that the 125I-anti-immunoglobulin test with platelets is as sensitive and reproducible for the detection of quinidine-associated antibodies as platelet complement fixation. Because of its independence from complement activation, this test might prove most valuable for the demonstration of non-complement-fixing, drug-associated antibodies.

Behaviour of 125I-fibrinogen and 131I-albumin in experimental galactosamine-induced hepatitis.

The turnover of 125I-labelled fibrinogen and 131I-labelled albumin was studied in the course of galactosamine-induced hepatitis in rabbits. In addition to galactosamine, some animals were treated with epsilon-aminocaproic acid (EACA) to inhibit the activation of the fibrinolytic system. The infusion of galactosamine and EACA caused generation of fibrin-rich microclots in the renal glomerular capillaries in seven out of 12 rabbits. Correspondingly, the incorporation of 125I-radioactivity into liver, spleen, and kidneys was pronounced in galactosamine- and EACA-treated rabbits compared with control animals treated with EACA. An acceleration of the 125I-fibrinogen elimination from the plasma was observed between eight and 12 hours after the start of the galactosamine infusion. The administration of heparin in addition to galactosamine and EACA prevented the occurrence of intravascular coagulation, but shortened the survival times of the animals because of bleeding into visceral organs. The elimination of 131I-albumin in plasma as well as the distribution of 131I-radioactivity in organs were similar in all the rabbits independent of the treatment with galactosamine, EACA, or heparin. The experiments indicate that, in addition to diminished synthesis of coagulation factors, disseminated intravascular coagulation is involved in galactosamine-induced hepatitis and contributes to the haemostatic disorder.

The role of alloimmunization in platelet survival studies.

The role of platelet alloimmunization in the survival of 51Cr-labeled allogeneic platelets was investigated in 89 patients with severe thrombocytopenias. The serological analysis included HLA typing of patients, screening of their sera in the lymphocytotoxicity test (LCTT), the platelet complement fixation test (PCFT), and the platelet radioactive anti-IgG test (PRAT; N = 38). Platelet donors were selected according to the best available HLA match and crossmatch in LCTT. Alloantibodies against HLA antigens were found in the sera of 17 patients (19.1%). No platelet-specific alloantibodies were detected. The use of compatible, allogeneic platelets permitted the discrimination of diminished platelet production from increased platelet turnover in thrombocytopenic patients with proven alloimmunization. Our results stress the necessity of a serological workup prior to platelet survival studies.

In vivo behaviour of homologous urea-soluble 131I-fibrin and 125I-fibrinogen in rabbits: the effect of fibrinolysis inhibition.

The in vivo behaviour of urea-soluble fibrin monomer (FM) was compared with that of fibrinogen in rabbits. Purified rabbit 125I-fibrinogen was injected into 36 unanaesthetized rabbits. Three days later the rabbits received either purified rabbit 131I-FM I mg/kg body weight, which corresponds to cI% of the circulating plasma fibrinogen pool 131I-fibrinogen, or buffered urea only. The distribution volume of FM was 43.4 +/- 6.9 ml/kg and of fibrinogen 43.5 +/- 7.7 ml/kg (mean +/- SD). The elimination curve of urea-soluble FM as represented by the clottable 131I-radioactivity. plotted on a semi-logarithmic paper, consisted of an initial steep decay within the first 6 h and a slow flattening of the slope 6 to 24 h after injection of FM. Although a terminal single-exponential slope, as observed in fibrinogen elimination, could not be computed for the 24 h following FM injection the mean half-life time of the last segment of the clottable-radioactivity curve, between 12 h and 24 h, was 12 h. Within 24 h a mean of 83-5% of the injected FM were removed from the circulating blood. The elimination characteristics of fibrinogen were not influenced by the injected FM. Control experiments showed that buffered 3.0 M urea, the solvent of FM, does not influence distribution volume and elimination of 125I-fibrinogen. The distribution of 131I as well as 125I-radioactivities in organs representing FM and fibrinogen respectively did not differ from each other. Elevated levels of 131I-radioactivity, however, were found in the urine after FM injection suggesting an accelerated elimination of FM in comparison to fibrinogen. Fibrinolysis inhibition with high doses of aprotinin did not significantly reduce the urinary excretion of 131I-radioactivity representing breakdown of FM. Furthermore, inhibition of the fibrinolytic system had no influence on the elimination characteristics of either fibrinogen or FM. In order to explain the results obtained in this study the following theory is proposed. Intravenously injected FM forms complexes with fibrinogen and fibrinolytic degradation products. The complexes continuously increase in size until they dissociate into fibrin oligomers and carrier proteins. The oligomers are eliminated whereas the carrier proteins recycle.

Lack of efficacy of high-dose intravenous immunoglobulin in autoimmune haemolytic anaemia: a clue to its mechanism.

5 patients with autoimmune haemolytic anaemia (AIHA) of warm type (4 idiopathic, 1 associated with systemic lupus erythematosus and thrombocytopenia) were treated with high doses of i.v. immunoglobulin (IgG; Sandoglobulin; 2.0 g/kg body weight). IgG therapy was ineffective in all 5 cases as indicated by a lack of clinical improvement, continuous signs of accelerated red blood cell (RBC) destruction, and an unchanged survival rate of 51Cr-labelled autologous RBC in 4 patients studied. IgG infused at equivalent doses into 5 healthy volunteers led to an increase of IgG coating of autologous RBC (irrespective of the ABO blood groups) without concomitant changes of haemoglobin, haematocrit or reticulocytes, increase of serum IgM in 3/5, a decrease of serum C4 in 5/5, and a decrease of serum haptoglobin in 2/5 individuals. We conclude that IgG at a dose of 2.0 g/kg body weight is ineffective in AIHA. This may be caused by an increased, though clinically inapparent, interaction of IgG-coated autologous RBC with the mononuclear phagocyte system.