I. Mahn

Detection of platelet autoantibodies by a radioactive anti-immunoglobulin test.

Abstract. A direct and an indirect version of a platelet radioactive anti‐immunoglobulin test (PRAT) has been developed. It is shown that this assay is sensitive, reproducible and quantitative for the demonstration of HLA as well as incomplete P1A1 antibodies. Furthermore, platelet autoantibodies were searched for in sera of 105 thrombocytopenic patients. Positive results were obtained in 15 out of 45 patients with chronic idiopathic thrombocytopenic purpura (ITP), in 1 of 17 cases with acute ITP and in 3 of 4 patients with ITP‐like syndrome of systemic lupus erythematosus (SLE). The platelet autoantibodies mostly had low titers, reacted with autologous platelets and did not show any specificity. An elevated concentration of IgG on autologous platelets was detected by the direct PRAT in 11 of 18 patients with ITP and in 4 of 5 patients with thrombocytopenia and suspected SLE. 4 of the 7 negative ITP cases were in clinical remission or had platelet counts of over 50,000/μl. There appeared to be an inverse correlation between the concentration of surface‐bound IgG and the platelet number in patients with ITP and SLE. It is concluded that the PRAT is a new and valuable tool for the detection of various types of platelet antibodies and therefore a true alternative to existing serological techniques in platelet immunology.

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.

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.

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.

IgG therapy in autoimmune haemolytic anaemia of warm type.

SummaryThree patients with autoimmune haemolytic anaemia of warm type were treated with high doses of intravenous immunoglobulin (Sandoglobulin®). The therapy was ineffective in all three cases. The possible reasons for this therapeutic failure are discussed.

Formation and dissociation of soluble fibrin complexes in plasma at 20°C and at 37°C

Abstract Small amounts of thrombin were added to human plasma containing 125 I-fibrinogen to generate fibrin. Thereafter, thrombin was quenched with hirudin and 131 I-fibrinogen added for internal calibration. Gel filtration of this mixture on sepharose CL-6B columns equilibrated with plasma revealed two peaks, one (peak A) eluting with the void volume, the other (peak B) with the volume corresponding to the fibrinogen peak. At 20°C, a small amount of 131 I-fibrinogen (4.5%) was eluted together with the thrombin-treated 125 I-fibrinogen in peak A, whereas at 37°C, no 131 I-fibrinogen was recovered in peak A. If isolated fractions of peak A eluted at 20°C were rechromatographed at 20°C, they were eluted again in the void volume. At 37°C, however, this material was mainly eluted in peak B corresponding to the fibrinogen peak. If isolated fractions of peak B at 37°C were rechromatographed at 37°C, they were eluted with the same volume as in the original chromatography. At 20°C, however, the material was eluted in peak A as well as in peak B. These data show that at 20°C fibrin aggregates generated in a plasma medium from monomeric fibrin without incorporating fibrinogen. From these experiments, we conclude that so-called fibrin-fibrinogen complexes not stabilized by factor XIIIa generate only in vitro at 20°C, but do not exist at 37°C.

Gel filtration of 125I-fibrin and 131I-fibrinogen at 20°C and 37°C

Abstract Purified rabbit 125I-fibrin was prepared in monomeric form and mixed with rabbit plasma containing 131I-fibrinogen. Using gel filtration, 125I-fibrin could be separated from 131I-fibrinogen indicating two new findings. 1. Fibrinogen was not eluted together with fibrin at 37°C and only in a small amount of 3% at 20°C. 2. Approximately 90% of the applied fibrin could not be eluted from the column at 37°C, whereas about 50% were retained on the column at 20°C. The experiments demonstrate that fibrin partially precipitated in the column when separated from fibrinogen. Thus, fibrinogen and/or other plasma proteins are necessary for keeping fibrin in solution, but fibrin does not form complexes with fibrinogen without factor-XIII action at 37°C. Furthermore, the precipitation of fibrin in the columns during gel filtration may pose problems in the quantification of fibrin in plasma.

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.

125I-Anti-Immunoglobulin Test: A New Tool for the Detection of Drug-Allergic Platelet Antibodies

Abstract. 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.

The role of alloimmunization in platelet survival studies

ZusammenfassungBei 89 Patienten mit hochgradigen Thrombozytopenien wurde der Einfluß einer möglichen Alloimmunisierung gegen Thrombozytenantigene auf die Bestimmung der Überlebenszeit von51Cr-markierten, allogenen Thrombozyten untersucht. Die serologische Analyse stützte sich auf die HLA-Typisierung der Patienten, Antikörpersuche im Lymphozytotoxizitätstest (LCTT), Plättchen-Komplementfixationstest (PCFT) und Plättchen-Radioimmun-Anti-IgG-Test (PRAT; N=38). Die Spenderauswahl erfolgte nach bestmöglicher Übereinstimmung der HLA-Antigene und dem crossmatch im LCTT. Bei 17 Patienten (19,1%) wurden Alloantikörper gegen HLA-Antigene, in keinem Fall thrombozytenspezifische Alloantikörper nachgewiesen. Durch HLA-kompatible Spenderauswahl war es möglich, auch bei nachweisbar vorimmunisierten thrombozytopenischen Patienten Bildungsvon Umsatzstörungen abzugrenzen. Diese Befunde weisen auf die Notwendigkeit serologischer Voruntersuchungen bei Überlebenszeitstudien mit allogenen Thrombozyten hin.SummaryThe role of platelet alloimmunization in the survival of51Cr-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.