bluuebunny/crossref_metadata_2025 huggingface dataset:
Usefulness Of Determining Coagulation Abnormalities For Early Diagnosis Of Deep Vein Thrombosis And Pulmonary Embolism
Authors: Rodzynek J J , Schoenfeld P L , Martin T , Léautaud P , Wettendorff P , Delcourt A
Published: July 2018

Citrated whole blood thrombo-elastogram (TEG) and its several parameters (r, k, am, IPT), Ethanol gel test (EGT), Fibrinogen degradation products (FDP) and Raby’s transfer test (RTT) were studied in 52 healthy volunteers acting as normal controls, in 211 consecutive patients admitted in a general hospital for non thrombotic pathologies (group 1), in 59 consecutive patients admitted for deep vein thrombosis (group 2) and in 45 consecutive patients addmitted for pulmonary thrombo embolism, confirmed at scintigraphy (group 3).

In conclusion: l. TEG (r, k, am, IPT) offers little help in the early diagnosis of deep vein thrombosis as well as pulmonary thrombo embolism. 2. EGT although offering a high specificity (6.6% false positive), lacks of sensitivity for the diagnosis of deep vein thrombosis (56.6% false negative). 3. RTT appears as a highly sensitive detector of deep vein thrombosis (positive in 96.6% of cases) and of pulmonary embolism (positive in 97.7% of cases) as well as reasonably specific (12.3% false positive tests). 4. Negativity of FDP and RTT appears as a strong argument against the diagnosis of thrombotic disease.
Diagnostic Value Of Ethanol Gelation Test (EGT) For Detection Of Early Deep Vein Thrombosis (DVT) In Patients With Stroke
Authors: Hamborg T , Godal H C
Published: July 2018

In the present study, EGT was applied for detection of fibrinaemia in patients with early DVT, as diagnosed by the 125-I-fibrinogen uptake test (FUT). The study included 25 patients with completed stroke and upper motor paralysis of at least one extremity. Patients who were critically ill, or with duration of symptoms for more than one week, were not included.

The isotope was injected at admission and FUT became definitely positive (FUT+) 2-12 (mean 5) days later in 17 patients (68%). In the remaining 8, FUT was negative (FUT−) for 11-14 days. FUT recordings were taken for up to 2 weeks, and EGT was analyzed thrice a week, or at least until DVT was diagnosed. The two groups were comparable in respect with age and sex distribution, mortality and frequency of serious impairment of consciousness.

Results: During the study period, EGT was positive at least once in 13 of the 17 FUT+ patients, against only 1 of the 8 FUT− patients (P< 0.02). EGT was positive in 34 (37%) of totally 97 specimens from the FUT+ group, but in only 1 (2%) of 50 specimens from the FUT− group (P<0.001). In the FUT+ group, the frequency of positive EGT increased from about 10 to 70%, with a marked rise three days (day −3) before the first positive FUT (day 0).

During the period from day −11 to −4, the average frequency was 13% (3/24), while it was 46% (33/72) in the last period from day −3 to +9). This increase in correlation to the time of first positive FUT, was statistically significant (P<0.01).

Conclusion: A positive EGT in moderately ill stroke patients, necessitates further examinations in respect with DVT, as some of these may need anticoagulants to prevent serious embolic complications.
Detection Of 111-Indium Labelled Platelets In Experimental Venous Thrombosis
Authors: Grimley R P , Fejfar J , Hawker R J , Drolc Z
Published: July 2018

Successful detection of Indium-lll-oxine labelled platelets in venous thrombi offers a new meth od of diagnosis of venous thrombosis. Femoral vein thrombosis was established in 20 dogs by a combination of partial venous occlusion and vein electrification. In this model, thrombosis is first detectable on venography after 48 hours. Autologous labelled platelets were re-injected into each dog; a sham operation having been performed on the opposite femoral vein. Bilateral venography was performed 2 days and 7 days post-operatively and each dog was sacrificed after removal of femoral veins. Isotopic detection of venous thrombosis was determined by comparison of scintillation counts over each femoral vein daily for one week.

Ratios of isotope counts between abnormal and normal legs indicated thrombus formation in 8 out of 10 dogs with known thrombi;two false negative results were due to hematoma formation. The mean duration of detection was 2.5 days. The counts were higher over venous thrombi in 3 out of 4 dogs who received platelet labelling prior to thrombus formation. Here, the mean duration of detection was 4 days. In 6 control animals, where veins were partially occluded only, counts were similar in each leg.
Problematic Aspects Of Synthetic Peptide Substrate Assays For Coagulation Parameters
Authors: Messmore Hary L , Fareed Jawed , Walenga Jeanine M , Kniffin Judith M , Bermes Edward W
Published: July 2018

With the advent of synthetic chromogenic and fluorogenic peptide substrates, coagulation testing has entered a new era. We have evaluated amidolytic assays for prothrombin, factor X, prekallikrein; prothrombin time (PT) and partial thromboplastin time (PTT) equivalent assays. Factor X is quantitated after activating with Russell’s viper venom with Xa specific substrates. In normals a good correlation is obtained between the coagulant and amidolytic assays (r=0.91, n=50), a poor correlation is reported in patients with liver disease and those treated with oral anticoagulants (r=0.76, n=50). Prothrombin is quantitated using ecarin, staphylocoagulase and phospholipid/Ca+2 as activators. The decarboxy glutamic forms of factor X and II should be compared in the coagulant and amidolytic assays to establish their equivalency. Although PT and PTT equivalent assays using amidolytic assays have been proposed, substrate inhibition of Xa generated during activation and other serine protease have not been fully explored, Dextran activated prekallikrein assay requires adequate level (≥ 50%) of Hageman factor. Heparin is quantitated in absolute terms and do not reflect clinical anticoagulation. In the antiplasmin assay, plasmin preparations cannot be used unless standardized in biochemical terms. Thrombins are standardized in terms of NIH U/ml and contain non-clotting molecular variants with amidolytic activity; therefore thrombin be best standardized in amidolytic terms for these assays. Certain serine protease-inhibitor complexes retain the amidolytic activity while losing their biologic activity, therefore a study on the amidolytic properties of these complexes is warranted. Standardization of the amidolytic method is almost nonexistent and claims for their bioequivalency to the existing clotting assays require support data. While synthetic substrate offers certain advantages, their application should be assessed critically and their limitations determined.
Non-Thrombogenic Polymer Surfaces
Authors: Merrill E W , Salzman E W , Sa da Costa V , Brier-Russe D
Published: July 2018

Activation of the intrinsic clotting system, and platelet adsorption and activation by a foreign surface are preceeded by adsorption of plasma proteins, often accompanied by change of conformation (denaturing). Protein molecules are modelled as particles having heterogeneous surface characteristics, i.e., separate “patches” of lipophilic (hydrophobic) groups, of H-bonding groups,of fixed aniomic groups, and of fixed cationic groups. Any of these “patches” can anchor the protein to the “foreign” surface if it finds a receptive “patch” on the surface of sufficient area. When thus stabilized on the surface, and in sufficiently close proximity, proteins like fibrinogen can then bind platelets which can become activated. We postulate that protein adsorption is minimized, if the molecular segments of polymer molecules which create the synthetic surface (1) are disordered (amorphous) rather than ordered (crystalline) (2) undergo Brownian fluctuations rather than being fixed in the glassy state (3) are mixed in chemical composition rather than being all of the same type (4) are such as to minimize the bonding energy per segment to any of the receptive sites on the protein (anionic, cationic, H-bonding, hydrophobic) (5) are “diluted” by swelling in water, to reduce the surface density. Using the above model and postulates, we review currently used polymers (vinyls, silicones, polyurethanes etc). Surfaces containing polyethers such as polyethylene oxide (PEO) that are water absorbing and non-crystalline, rank highest in the list of non-thrombogenic polymers. We suggest that this arises from the alternation of hydrophobic CH2-segments with hydrophilic ether oxygen, and the total absence of ionic groups and bonding hydrogens. Our observations on the blandness of hydrophilic cross-linked polyethers is supported by evidence from the literature concerning nearly total phase exclusion of protein from homogeneous PEO solutions, and the non-adsorption of proteins on surfaces coated by PEO.
A Methodological Investigation On The One-Stage Factor VIII Assay
Authors: Over J , van Mourik J A , van den Brink-Zantingh P , Smit-Jansen R
Published: July 2018

Assay of Factor VIII coagulant activity (VIII: C) in Factor VIII concentrates has since long met difficulties, such as l) non-paralleility of dose-response curves of plasma standard and Factor VIII concentrate, 2) spuriously low values of VIII: C in concentrates as revealed by abnormally high in vivo recoveries after transfusion, and 3) large interlaboratory variation in assay results. In an attempt to analyze the cause of these problems several parameters of the one-stage assay system were varied systematically and their effect on the parallellity of dose-response curves and on the final VIII: C value was analyzed. Nonparallel1ity was partially corrected with a protein-rich dilution medium, and almost always completely with undiluted instead of 1:1 diluted hemophilic substrate plasma. In both conditions apparently higher VIII:C values were found.

A number of assay systems used by different producers of Factor VIII concentrates were compared. The standard and, in some cases, the phospholipid reagent seemed to contribute for the largest past to the inter1aboratory variation, but also other, as yet unidentified, factors exerted some influence. These findings initiated a cooperative study by five Red Cross Blood Transfusion Services in Europe on standardization of the one-stage assay for VIII:C. This resulted in a better correspondence between these institutes (CV 13%) compared to the previous situation (CV 23%).

It is concluded that 1) substrate plasma should not be diluted, especially when Factor VIII concentrate is to be tested against a plasma standard, 2) the standard should be of the same type as the testmaterial, and 3) this standard should be properly calibrated against the International Standard for Factor VIII.
Four Cases Of Acquired Von Willebrand's Syndrome Associated With Monoclonal Gammapathy
Authors: Horellou M H , Baumelow E , Sitbon N , Conard J , Marie J P , Fine J M , Gorin N C , Zittoun R , Samama M
Published: July 2018

Acquired Von Willebrand syndrome is reported in four patients with monoclonal lgG : benign gammapathy in three cases, multiple myeloma in one case ; to our knowledge, this last association has not been previously reported.

Coagulation abnormalities included a borderline bleeding time, a low platelet retention on glass beads, decreased levels of factor VIII coagulant activity (VIII: c), factor VIII related-antigen (VIII R: Ag) and ristocetin-induced agglutination cofactor (VIII R: Cof).

The late clinical onset, the negative family history and the immunological abnormality suggest an acquired Von Willebrand syndrome. After cryoprecipitate infusion the patients did not show the expected rise and there was no secondary increment in factor VIII: c.

Time-dependent inhibition of factor VIII R: Cof was found in one case only and was associated with qualitative abnormality of factor VIII R: Ag demonstrated by crossed-immunoelectrophoresis. It was not possible to interpretate this last test in the other cases, due to the very low level of factor VIII R: Ag. The inhibitor activity was neither found in the IgG fraction nor in the monoclonal IgG but disappeared after IgG adsorption on staphylococcal protein A.

The factor VIII abnormalities might be related to the binding and/or destruction of factor VIII by a circulating antibody, or to the adsorption of this factor on the malignant lymphocytes.
Study On Long Term Tolerance And Recovery Of Cold Sterilized Ppsb In Chimpanzees
Authors: Stephan W , Prince A M , Kotitschke R
Published: July 2018

Recent experiments have shown, that PPSB (factor IX-concentrate) derived from ß-propiolactone/ultraviolet (ß-PL/UV)-treated (cold sterilized)plasma is not infectious in chimpanzees in respect to hepatitis B and Non A-Non B. To answer the question whether the B-PL/UV treatment influences the tolerance and efficacy of the cold sterilized PPSB- concentrate, long term application of PPSB-Biotest was performed in chimpanzees.

After 12 applications of 25 units factor IX/kg in weekly intervals no signs of intolerance were observed by means of skintesting and observation of blood pressure during i.v. application. Determination of coagulation factor activity during the application period shows the same factor IX- recovery at the beginning and at the end of the study.
Thrombogenicity Results Of Cold Sterilized (ß-Propiolactone/Ultraviolet Irradiation) PPSB In Chimpanzees
Authors: Kotitschke R , Stephan W , Prince A M , Brotman B
Published: July 2018

In vitro tests were demonstrated to be insufficient for the determination of the thrombogenicity of PPSB preparations with peptide substrates or the TGt50 and the NAPTT. Unequivocal determinations of the thrombogenicity of PPSB preparations are possible only up to now in in vivo models. As an alternative to the up to now proposed dog or hemophilia B dog models we have determined the thrombogenicity of cold sterilized PPSB in chimpanzees. PPSB isolated from ß-propiolactone treated and UV irradiated plasma was injected into the chimpanzees at a dose of approximately 100 units/kg body weight. An FDA licensed PPSB preparation served as a control.

15 minutes, 1 h, 4 h, and 24 h after the PPSB application the following parameters were determined in the chimpanzee blood: factors II, VII, IX, X, VIII, fibrinogen, AT III, thrombin coagulase, Quick value, APTT and platelet count.

Neither the untreated control preparation nor the PPSB from ß-propiolactone treated and UV irradiated plasma showed signs of a thrombogenic effect in this chimpanzee model.
Assessment Of FVIII Functional Activities With Quantitative Electrophoresis
Authors: Seghatchian M J
Published: July 2018

Electrophoretically separated fractions of FVIII concentrates were quantitatively assayed to assess heterogeneity in FVIII related proteins. The Method was successfully applied to the characterisation of multiple forms of FVIII in normal, pathological, haemophilia A, and v. WD. post-infused samples. Similarly, FVIII:C inactivation properties of several FVIII antibodies, which differed from one another on the basis on their complex formations, were investigated by: i) identifying the molecular forms of the Residual FVIII and ii) by monitoring the electrophoretic distribution of the complexes using a radiolabelled VIII:C IgG. 200 ul samples were applied to rectangular wells (22 × 3mm) cut in a 2.5mm thick agarosegel (1-2%). Electrophoresis in the first dimension was performed in 0.023M barbital buffer (pH8.2). The gels were then cut into 20-30 equal slices, eluted with 0.025M Tris-saline buffer (pH7.4) and assayed by currently used methods.

The results indicate that FVIII concentrates are highly heterogeneous, consisting of several subpopulations with differing functional and inhibitor neutralization activities. Most classical assays (including IRMA) were sensitive to the larger forms but methods based on the optimal Fxa generations (VIII:C 2 stage & VIII:C amidolytic) measured smaller forms of FVIII more sensitively. In the post-infused samples a transient complex was observed which reflected the rate of clearance of FVIII from the circulation. Furthermore the novo synthesis of FVIII in v. WD patients appeared to be quantitatively indistinguishable from circulating FVIII present in these patients. Some populations of FVIII were totally and specifically inactivated by some FVIII:C inhibitors but partially by the others. The inhibitor which inactivate totally FVIII:Cam also failed to produce high molecular weight radiolabelled complexes. On the basis of these results, inhibitor patients could be treated specifically with an appropriate subpopulation of FVIII.
Enzyme-Linked Immunosorbent Assay For The Detection Of Alloantibodies To Factor IX In Hemophilia B
Authors: Örstavik K H , Örstavik I
Published: July 2018

A solid phase enzyme-linked immunosorbent assay (ELISA) was developed for the detection and quantitative determination of acquired inhibitors to factor IX. Wells of polystyren Micro-ELISA plates were coated with the IgG fraction of a sheep antiserum to human factor IX. After incubation with pooled normal plasma as a factor IX source, the wells were incubated with test plasma. The binding of alloantibodies to the factor IX-sheep-anti-factor IX complexes was then detected by incubation with alkaline phophatase conjugated antiserum to human IgG. As substrate was used p-nitrophenyl phosphate.

Plasma samples from five patients with severe hemophilia B and acquired inhibitors to factor IX were examined. All samples gave a positive reaction in the ELISA. The titers as determined in the ELISA were in good agreement with the titers as determined in a coagulation assay (0.1-800 U/ml). Plasma from 13 patients with hemophilia B and no detectable inhibitor in a coagulation assay all gave a negative reaction in the ELISA. A negative reaction was also found in plasma from four patients with hemophilia A and acquired inhibitors to factor VIII, and in plasma from 15 healthy persons.

It is concluded that the ELISA is a simple and sensitive technique for the determination of acquired inhibitors to factor IX in hemophilia B.
Investigation of the binding of calcium to prothrombin by two dimensional immuno electrophoresis
Authors: Charles Gray , David Perry , Pamela Riches , David Timlin
Published: July 2018

The reported binding constant of calcium to purified prothrombin based on equilibrium dialysis is K<104 . This study investigated the ability of EDTA, EGTA, to remove calcium from the normal prothrombin, in the plasma of patients treated with warfarin.

Coumarin plasma was obtained from freshly drawn venous blood of patients on warfarin. This plasma contains prothrombin with no GLA’s, which does not bind calcium, and prothrombin with GLA’s, which does bind calcium. The plasma anti coagulated with EDTA or EGTA was subjected to two dimensional immuno electrophoresis (2DIE). In parallel experiments the plasma was dialysed against normal saline containing EDTA 2mM for 24 hours. The dialysis was performed at pH 8,9 and 10, and the dialysed plasmas, subiected to 2DIE

RESULTS All plasmas, irrespective of the anti coagulant or pH of dialysis exhibited the same two prothrombin peaks on 2DIE, which indicates that the normal prothrombin had retained its bound calcium. This suggests that native prothrombin binds calcium with a K assos>l010 the binding constant for EDTA and calcium at pHl0.0.
Blood Coagulation Disturbances During Oliguria And Polyuria Of Acute Renal Failure In Man
Authors: Schrader J , Köstering H , Kaiser H , Kramer P , Scheler F
Published: July 2018

The blood coagulation system makes a significant contribution to renal damage in many disease processes. Intrarenal coagulation appears to occur in a wide variety of diseases as a primary or secondary event. As there is evidence that intraglomerular coagulation is a significant factor in the development and maintenance of oliguria in acute ischemic renal failure, blood coagulation investigations were performed in 20 patients with acute renal failure of varied etiology. The investigations were done on a daily basis from the onset of oliguria (urine flow <20 ml/h)until serum creatinine declined to less than 2,0 mg%. Thus, we were able to detect changes in blood coagulation during oliguria and polyuria. We found an enhanced thrombin generation in both oliguria and polyria. Fibrin monomer complexes were significantly increased in both states, but more predominantly in polyuria. Factor VIII and alpha-1 antitrypsin activities were also elevated. PTT and r- and k-time in TEG were shortened more in polyuria than in oliguria, whereas fibrinogen was elevated more in oliguria than in polyuria. Factor XIII activity and prothrombin complex activity (Quick’s test) were lowered in both states, the lowest values of the former being found in polyuria, the lowest values of the latter in oliguria with a normalizing tendency in the following days. Fibrinolytic activity was also decreased. No significant changes were found in plasminogen, antithrombin III, alpha-2 macroglobulin, factor V and thrombin time. In summary, we found a hypercoagulability in these patients with acute renal failure, which was more predominant during polyuria and which correlated with the tendency to thrombosis and to shorter indwelling periods of i.v. catheters in this state. Consequently, the changes in blood coagulation of 3 patients with acute postrenal failure were not as significant as those found in the other patients. The treatment with anticoagulants in patients with acute renal failure will be discussed.
Improvement Of Plasmatic Coagulation In Patients With Liver Disease To Enable Necessary Diagnostic And Therapeutic Steps
Authors: Vogel G E , Tuchtenhagen S , Komm Ch , Oberdorfer A
Published: July 2018

Specific plasmatic concentrations of coagulation factors are necessary in endoscopic treatment e.g. slerosis of esophageal varices. Thrombotest of 35 to 40% is necessar. To correct with factor concentrates and fibrinogen is dangerous in cause of pushing DIC (disseminated intravascular coagulation). The substitution of the inhibitor AT III which is succeeded from prothrombin concentrates and fibrinogen avoid fearful consumption reactions. The AT III plasma activity was measured by chromogenic substrates in citric plasma. In 5 patients (2 female, 3 male- age 19-67) the diminished AT III-levels were increased to the normal value of 80%. After normalization of the inhibitors prothrombin complexes and if necessary fibrinogen were substituted. It was seen an assessable improvement of the plasmatic coagulation which depends on the unit concentration of the substituted factors. In the improved coagulation situation 2 laparoscopies and 3 slerosis of esophageal varices could be done without any bleeding complication.

Patients with liver diseases and coagulation disorders who were unsuitable for endoscopic and surgical treatment can be treated with this concept.
Evaluation Of The Dudley Clotting Test In Human Volunteers
Authors: O’Connell J , Rumaks A , Williams J L , Griggs T R
Published: July 2018

We have evaluated four materials (polyurethane, silicone rubber, Teflon, and heparin-coated polyurethane) for compatibility with blood in six human volunteers. The test method employs a standard indwelling intravenous catheter (16 ga, 2 1/2") through which 100 cm lengths of small-bore (0.5 mm inner diameter) tubes made from the materials to be tested are passed. The tubes are filled with saline before they are inserted, and the distal end of the tube is immersed in sterile mineral oil. The rate and duration of flow through these tubes is determined by counting drops of blood as they emerge into the mineral oil. The four materials were tested in each of six volunteers, in random order during a single day.

The testing produced no complications in the subjects. The average blood loss after 5 hours of testing was 25 ml.

A safe and simple method for testing artificial materials for reactivity with native human blood is described. The major characteristics of the test are that the blood is in contact only with the test material and that multiple samples can be tested in a single subject on a single day.
Isolation, Pasteurization, And Lyophilization Of Human Plasma Fibronectin
Authors: Miekka Shirley I , Busby Thomas F , Ingham Kenneth C , Menache Doris
Published: July 2018

The goal of this research is to develop a suitable method for purification of plasma fibronectin for potential replacement therapy in patients with acquired deficiencies. Using a modification of published affinity procedures, a simplified one-step method has been achieved. Plasma or cryoprecipitate is chromatographed on gelatin-agarose and the unadsorbed and loosely adsorbed proteins are removed by washing with pH 7.5 buffer (0.05 M Tris HC1, 0.05 M ∈-amino caproic acid, 0.02 M sodium citrate) followed by 1 M NaCl. The bound fibronectin is eluted simply by decreasing the buffer pH to 5.5 (0.02 M sodium citrate). The eluate can then be neutralized and further processed as desired. Sedimentation velocity measurements suggest that the elution may be related to a conformational change in the fibronectin molecule below pH 7.

Studies of thermal stability have been initiated with the objective of defining conditions under which the protein can withstand pasteurization (10 h, 60°C) to minimize the risk of hepatitis. Using an increase in the fluorescence of added 1,8-anilinonaphthalene sulfonate to monitor thermal denaturation, a variety of potential stabilizers was tested. No protection was afforded by gelatin, heparin, citrate, phosphate, EDTA, CaCl2, glycine, glycerol, or PEG; however, neutral sugars increased the midpoint of the denaturation curve to well above 60°C. Lyophilization was found to have no detectable deleterious effect on fibronectin. Freeze-dried samples were readily soluble in water, showed no change in sedimentation behavior, and retained the ability to augment the uptake of gelatin-coated particles by rat liver slices.
Easma Fibrinopeptide A And β-Thrombogloblin In The Patients With Thrombotic Disorders
Authors: Murakoshi T , Takei H , Seya T , Oguma Y , Yamauchi M , Hasegawa H
Published: July 2018

Fibrinopeptide A(FPA) is a sensitive indicator of the thrombin action and β-thrombogloblin(β-TG) is a useful marker of the platelet activation. In order to know the coagulation disturbances in thrombotic disorders, the measurement of the two indicaters is very important. However, relationship between these two indicators is not well studied. We measured plasma FPA and β-TG levels in the patients with decompensated DIC, lung cancer, gastric cancer, leukemia, thrombosis, and ARDS. In addition, influences of heparin and urokinase on-plasma FPA and β-TG levels were investigated. Plasma FPA and β-TG levels were eleveted in decompensated DIC(mean FPA: 14.0 ng/ml, mean β-TG:73.9 ng/ml), but in some cases, these two indicators didn’t change in the same manner. This difference was thought to be due to the concentration of plasma fibrinogen and a number of platelets. In the patients with lung cancer, elevated FPA and β-TG levels were found, and the former was elevated with progress of clinical stage. In some patients with leukemia, reduced β-TG levels due to hypoproduction of platelet were found in contrast to elevated FPA levels. On the other hand, in the patients with gastric cancer complicated with DIC, FPA levels elevated very slightly possibly due to extremely low concentration of fibrinogen, and β-TG levels showed a significant elevation. These results suggest that the measurement of both FPA and β-TG lead us to precise and accurate comprehension of coagulation abnormality. In the patients with overcompensated DIC, elevated FPA and β-TG levels(mean FPA:11.7, mean β-TG:98.0 ng/ml) were found, indicating hypercoagulability. Additionaly, we tried to use the ultrafiltration apparatus instead of dialysis tubing to shorten a time required for FPA assay, because dialysis tubing for plasma dialysis requires a lot of time, and is inappropriate for clinical investigation, especially in an emergency.
Problems Raised By The Measurement Of Coagulation Factors On Chromogen Substrates By Automatic Methods
Authors: Droulle C , Lafaurie M , Remy M G , Florent B , Potron G
Published: July 2018

The use of chromogen substrates has permitted a new approach of the factors responsible for the regulation and equilibrium of hemostasis.

Automation has allowed to have access to the problems raised by enzymatic reactions in non-purified medium. The specificity and sensibility of substrates, the specificity of activators, the role of inhibitors and physiologic substrates in different technical conditions (pH, ionic forces, temperature, dilution) modifying the maximal velocity, have been studied for a standardizing approach of enzymatic kinetics in hemostasis. Measurements of antithrombin III and fast α2 antiplasmin, factor X, prekallikrein and plasminogen and heparinemies have been analyzed by an automatic apparatus (Kern O Mat 2-Coultronics) with different chromogen substrates (Kabi-Stago).

Optimum technical conditions, units in which results are expressed, interpretation of normal and pathological values are systematically studied.
Isotopic And Immunological Half-Life Determination Of Antithrombin III In Various Types Of Antithrombin III Deficiency
Authors: Sas G , Kremmer T , Spät B , Pető I
Published: July 2018

Using the modified crossed immunoelectrophoresis technique (Heparin in agarose), different patterns were obtained characterizing the various types of antithrombin III (AT-III) deficiency. 125J labelled and purified commercial (KABI, Stockholm) AT-III preparates were administered to patients of different types in order to determine the biological half-life of AT-III. Various methods, including crossed immunoelectrophoresis and disc electrophoresis as well as heparin-Sepharose 4B chromatography were applied for the characterization of the AT-III administered.

These techniques revealed the molecular heterogeneity of AT-III preparates, especially those labelled by 125J. In the light of these observations it became obvious that among the AT-III determinations the most reliable and most specific method is the quantitative measurement of the fast migrating AT-III component (I-AT-III1) obtained by the modified method of crossed immunoelectrophoresis. This component corresponds to the “natural” AT-III of fresh normal plasma.

Applying this technique, one of our propositi (B.I.) displayed a significant shortening of the biological half-life of AT-III (26 hrs) while the two others (M.L. and B.J.) had approximately normal time courses (55 and 52 hrs, respectively). The different biological half-life times of AT-III in these patients also support the assumption of the heterogeneity of the congenital AT-III deficiency.
Crossed Immunoelectrofocusing Of Antithrombin III In Healthy And In Congenital And Acquired Deficiency
Authors: Leone G , Valori V M , Traisci G , Bizzi B
Published: July 2018

Isoelectrofocusing was carried out in LKB Multiphor apparatus with pH 4-6.5 carrier ampholites using poly- acrilamide gels. Specimens of human purified antithrombin, normal plasma pool, plasma with congenital and acquired AT- III deficiency were isoelectric focused. Purified Antithrombin showed two peaks. The isoelectric points of these antithrombin peaks were 4.6 and 4.9. Next the poly- acrilamide gel slabs were placed on glass plates coated with agarose containing 4% anti human antithrombin antibody; crossed electrophoresis was then carried out. Normal pool plasma exhibits two peaks, which correspond to the isoelectric focusing position of the purified AT-III. Plasmas of two families with congenital AT-III deficiency exhibit only one peak at pH lower. On the contrary plasmas of patients with acquired AT-III deficiency (hepatic cirrhosis, asparaginase therapy) exhibit two peaks. Considering that microheterogeneity is fundamentally due to a difference in glycosylation (Borsodi and Nerasimhan, Brit. J. Haemat. 1978, 39,121) it is possible to hypothesize a defect in glycosylation in congenital defect.
Antithrombin III (At III) In Acute Liver Failure (ALF): Experimental Data After Intoxications Of Dogs
Authors: Heirnburger N , Ronneberger H , Hein B
Published: July 2018

Vogel, G., Bottermann, P. et al. [Verh. Dtsch. Ges. inn. Med. 85, 477 (1979)] observed a benefit therapeutical effect of AT III in patients with ALF. With respect to the fact that AT III has been used as one drug in a combination therapy the effect of AT III itself had still to be proven.

ALF was induced in Beagle-dogs by intoxications with carbon tetrachloride (CCl4, 0.4 ml/kg, given orally over 2 days) and Galactosamine (Gain, 200 mg/kg, i. v.). First toxic symptoms were observed after 24 h: Elevated transaminases (GOT, GPT), alkaline phosphatase, serum bilirubin; the dogs developed severe coagulation disorders triggered by a DIC characterized by a fall of platelet counts and clotting factors AT III included. After Gain 5 of 6 untreated dogs died between 48 and 72 h. AT III concentrate prepared from human plasma (Behringwerke) and given i. v. in 3 different concentrations over a period of 3 days following intoxication reduced the toxicity: Associated with an inhibition of the consumption reaction there was a reduction of the death rate. The minimal effective dose was determined to be 3 × 50 U AT III/kg.

From the experimental data it can be concluded that AT III has a benefit effect on ALF. The results are in good agreement with the clinical observations by Vogel, Bottermann et al.
Pasteurization Of Antithrombin III: Stabilization By Heparin And Lyotropic Anions
Authors: Busby Thomas F , Atha Donald H , Ingham Kenneth C
Published: July 2018

Pasteurization of Antithrombin III (AT III) for 10 hours at 60°C is necessary to reduce the risk of transfusion hepatitis. Addition of appropriate stabilizers can largely prevent the loss of antithrombin activity which otherwise occurs during pasteurization. Studies of the mechanism of denaturation and stabilization have been facilitated by the use of 1,8-anilinonaphthalene sulfonate (ANS) which binds weakly to the inhibitor and whose fluorescence undergoes a sigmoidal response to increasing temperature as the protein unfolds. The extent of the increase in ANS fluorescence correlated roughly with the loss of antithrombin activity and with the extent of protein aggregation as determined by high pressure exclusion chromatography. The midpoint, Td, of the thermal denaturation curve increased by 13 and 19°C in the presence of 0.5 M and 1.0 M sodium citrate respectively. Phosphate, sulfate, and EDTA were also strong stabilizers while the chaotropic anions, iodidé and thiocyanate were potent destabilizers. Heparin, at 10 mg/ml, increased Td by 7°, presumbly through a direct binding mechanism. Reducing agents increased ANS fluorescence by an amount similar to that seen with thermally denatured samples, an effect which was inhibited by heparin but not by citrate. Furthermore, incorporation of 14C-iodoacetamide into AT III during thermal titration was coincident with the increase in ANS fluorescence suggesting that disulfide cleavage is the event which triggers the unfolding of the protein. Samples pasteurized for 10 hours at 60°C in the presence of 0.5 M and 1.0 M citrate retained full antithrombin activity but exhibited evidence of minor alterations in the ability to bind heparin.
Endogenous Heparin Activity In Normal Human Plasma
Authors: Engelberg Hyman , Lee Stephen
Published: July 2018

The scientific literature is contradictory as to the normal presence of heparin activity in human blood. The question has physiologic and clinical significance. The purpose of this study was to investigate whether biologic heparin activity was demonstrable in extracts of normal human plasma. The method involved initial precipitation of the plasma proteins by methanol-acetone, proteolysis of the precipitated proteins by papase or trypsin, dialysis of the supernatant, lyophilization, and then assay. The final extract showed heparin activity using the Kabi chromogenic substrate, the activated partial thromboplastin time test, and the anti factor Xa procedure. The level of heparin activity was 10-25 units % (app. 1-2 mg/L of plasma). We conclude that endogenous heparin activity is present in normal human plasma at physiologically significant levels, and that it is protein bound.
The Control Of Heparin Administration By The Activated Partial Thromboplastin Time(APTT)
Authors: Thomson Jean M
Published: July 2018

The UK National Quality Control Trials have previously shown that the various APTT methods differ in their ability to detect low levels of heparin (Poller et al 1980). UK and US proficiency surveys have also shown lack of linearity of some APTT methods over a range of heparin concentrations. A further, recent collaborative exercise, using lyophilised plasma from a heparinised donor, has confirmed that most of the commonly-used commercial reagents have a higher threshhold of sensitivity to heparin than the reference reagent provided by the National (UK) Reference Laboratory. Additional studies on fresh plasma samples obtained from heparinised patients, have demonstrated considerable variations in the detection of heparin by widely-used commercial APTT techniques.
The Effect of Partial Splenectomy on Platelet Production in Mice
Authors: Bessler H , Notti I , Djaldetti M
Published: July 2018

The effect of total and partial splenectomy on the number and production of circulating platelets was studied in mice. Five days after total and partial splenectomy the number of the peripheral blood platelets increased by 87% and 60%, respectively and the incorporation of 75selenium methionine (75Se-Met) into platelets was enhanced indicating that the thrombocytosis was due to increased platelet production. The results obtained by the two operative procedures were compared.

Since previous work from our laboratory has shown that a factor produced by splenic lymphocytes affects the platelet number in mice, it is suggested that the differences in the number of circulating platelets observed in animals after total and partial splenectomy may reflect a difference in the number of spleen lymphocytes removed.
Effect of Long-Term Aspirin Treatment on Platelet Adhesion to Chronically Damaged Canine Pulmonary Arteries
Authors: Schaub Robert G , Rawlings Clarence A , Keith James C
Published: July 2018

The effect of aspirin on platelet adhesion to chronically damaged pulmonary arteries was studied in 18 dogs. Chronic injury was produced in all dogs by infection with the canine heartwonn Dirofilaria immitis(DI). Ten dogs were subjected to 4 days of chronic injury. Eight dogs were subjected to 30 days of chronic injury. Five of the 4 day injury and 4 of the 30 day injury dogs received aspirin daily (325 mg/day orally). Aspirin was started 3 days prior to infection with DI. Pulmonary arteries were perfusion fixed in situ at physiologic pressure. The damaged pulmonary arteries were located by Evan’s blue staining (2ml/kg of 1% dye glven one hr prior to perfusion) and prepared for scanning electron microscopy. Both groups of dogs with 4 day DI infection had monolayers of platelets adhered to exposed subendothelium. Aspirin treated dogs had enhanced platelet adhesion to damaged arteries. Aspirin treatment for 33 days reduced platelet adhesion. The damaged arteries of treated dogs infected with DI for 30 days had very few platelets adhering to the damaged surface. However, non-treated dogs subjected to 30 days of infection had platelet adhesion equivalent to the 4 day non-treated infection group. These results suggest that although aspirin is ineffective in preventing platelet adhesion in short term therapy it is effective when given for longer time periods. This inhibitory effect may occur due to platelet membrane changes rather than because of aspirin inhibition of cyclooxygenase.
Factors Affecting the Size of Platelet Aggregates in Blood
Authors: Kennedy P S , Ware J , Horak J K , Solis R
Published: July 2018

Factors affecting the size distribution of platelet aggregates formed in whole blood were measured using an electronic particle counter. Lysis of red cells after dilution of blood for analysis allows detection of smaller aggregates without altering aggregate measurement. Differences in platelet response to increasing concentrations of ADP are detected by an increase in the mean size of platelet aggregates while the total volume of aggregates varies linearly with the volume of available platelets (r = .99). The mean aggregate size varies directly with the total volume of aggregates formed (r = .99). Mean aggregate size is inversely related to the packed red cell volume. As the reactivity of platelets is reduced by incubation with aggregation inhibitors at increasing concentrations, the mean size of aggregates becomes smaller, followed by a decrease in the total volume of aggregates. The total volume and mean size of aggregates formed after incubation with collagen and epinephrine, but not ADP, increases with time after venepuncture.
Prevention by a Glucocorticoid of the Thrombotic and Hemorrhagic Tendencies Consecutive to Shock and Surgical Intervention in Rat
Authors: Renaud S , Latouk J. G
Published: July 2018

Rats pretreated with acenocoumarin present a high incidence of hemorrhages from one to three days after an endotoxemic shock or a surgical intervention. The onset of hemorrhages is concomitant with a marked decrease in the platelet count and a considerable increase in the prothrombin time and the plasma clotting time. These hemorrhages as well as the changes in the parameters examined could be completely prevented by administration of triamcinolone at high doses (4 mg/kg), 2 hrs before and 2 hrs after the shock or the intervention.

Three days after the induction of a traumatic shock in hyperlipemic rats the count and adhesiveness of blood platelets was markedly increased. At this time, the intravenous injection of an endotoxin (S. typhosa lipopolysaccharide) induced a much higher incidence of large phlebothromboses in these traumatized rats than in the control animals. Here again, administration of high doses of triamcinolone could completely block the consequences of the traumatic shock, namely the changes in the platelet count and adhesiveness and the increased thrombotic tendency.
Thrombolytic Treatment with Tissue-type Plasminogen Activator (t-PA) Containing Liposomes in Rabbits: a Comparison with Free t-PA
Authors: Heeremans J L M , Prevost R , Bekkers M E A , Los P , Emeis J J , Kluft C , Crommelin D J A
Published: July 2018

In this study, we aimed at improving the therapeutic index of tissue- type Plasminogen Activator (t-PA) as thrombolytic agent in the treatment of myocardial infarction. Liposome-encapsulated t-PA was tested in a rabbit jugular vein thrombosis model: administration of free t-PA (t-PA) as a bolus injection in the ear vein was compared to a similar administration of liposomal t-PA (t-PA-lip), liposomal t-PA in plasminogen-coated liposomes (Plg-t-PA-lip), a mixture of free t-PA and empty liposomes (t-PA+empty lip) and a saline-blank (blank) in terms of thrombolytic activity and side effects.

Liposomal t-PA (t-PA-lip/Plg-t-PA-lip) showed a significantly better thrombolysis efficiency than equimolar doses of free t-PA (t-PA/ t-PA+ empty lip): about 0.24 mg/kg of liposomal t-PA practically equalled the lysis-activity of a dose of free t-PA of 1.0 mg/kg (t-PAlmg/kg). On the other hand, liposome encapsulation did not affect the systemic activation of alpha2-antiplasmin and plasminogen by t-PA.

We conclude that for this model an improvement in thrombolytic efficacy of t-PA is achieved by liposome encapsulation of t-PA. As t-PA-lip and Plg-t-PA-lip -treatment induced similar results, targeting of liposomal t-PA by coupled glu-Plg remains a topic to be optimized in future studies.
Calcium Ion-Dependent Monoclonal Antibody Against Human Fibrinogen: Preparation, Characterization, and Application to Fibrinogen Purification
Authors: Takebe Mikihiro , Soe Gilbu , Kohno Isao , Sugo Teruko , Matsuda Michio
Published: July 2018

We have produced a high-affinity monoclonal antibody classified as IgG1 with K-type light chains that recognizes the calcium ion(Ca2+)- dependent conformation of the D-domain of human fibrinogen. Binding of fibrinogen in solution to the insolubilized antibody increased in the presence of increasing concentrations of up to 2 mM Ca2+, the half-maximal binding being reached at 130 μM Ca2+. The dissociation constant was estimated to be 1.6 × 10-8 M at 2 mM Ca2+. The antibody was found also to be dependent on other divalent metal ions including Zn2+, Mn2+, Co2+ and Cu2+, but not Ba2+, Mg2+ or Sr2+. The synthetic Gly-Pro-Arg-Pro-amide peptide, which has recently been shown to bind to close proximity to the calcium binding site in the D-domain, was unable to elicit the conformation for the antigen to be recognized by this antibody. This antibody was found to be a suitable ligand for the immunoaffinity chromatography of normal and abnormal fibrinogens directly from citrated plasma depleted of the vitamin K-dependent proteins or heparinized plasma by eliminating the precipitation procedure widely adopted in conventional techniques of fibrinogen purification. Indeed, fibrinogen Marburg I with the Aa chains depleted of the carboxy-terminal Aα(461-610) residue segment has been purified by this technique, although this dysfibrinogen was difficult to purify by conventional precipitation techniques.
Über einen Fall kongenitaler Hypoprokonvertinämie (Faktor VII – Mangel)
Authors: Rák K , Waltner K
Published: July 2018

Als Ursache für die Bluterkrankheit eines 16jährigen Mädchens konnte kongenitale Hypoprokonvertinämie festgestellt werden. Die ähnlich schwere Blutungsneigung ihrer Schwester läßt familiäres Vorkommen vermuten. Es werden neben dem ausführlichen Hämostatus der Kranken auf mildere Gerinnungsstörungen der Eltern hinweisende Angaben mitgeteilt. Im Laufe der kurzen Besprechung des Falles wird hingewiesen:

1. auf diagnostische Fragen,

2. auf die Rolle des fehlenden Serumfaktors im Geritmungssystem,

3. auf die Notwendigkeit der Differenzierung von dem sog. Stuart-Faktor,

4. auf die wenigen von der Literatur akzeptierten echten Hypoprokonvertin-ämiefälle,

5. auf die Rolle des Prokonvertins in der Hämostase,

6. auf die Frage einer Möglichkeit zur Hebung des Faktor-VII-Spiegels bei der Behandlung der Krankheit mit Bluttransfusionen, sowie auf die Vitamin-K-Resistenz und endlich

7. auf die Vererbungsverhältnisse des Leidens.
Abdomino-perineal Resection of the Rectum in a Haemophilic
Authors: Christie T. H , Graham-Stewart C. W , Ingram G. I. C
Published: July 2018

A moderately affected haemophilic aged 69 years, with about 10% of the normal plasma level of AHG, underwent abdomino-perineal resection of the rectum for carcinoma, under cover of bovine and porcine AHG.

The post-operative course was complicated by the necessity of plugging the perineal wound to arrest operative haemorrhage from a vessel in the prostatic capsule; and considerable bleeding occurred on two occasions when the packs were removed. Otherwise, there was no abnormal bleeding.

The management of the case is described, and technical details are discussed from the points of view of the surgeon, the anaesthetist and the haematologist.

The Biggs’ AHG assay procedure was slightly modified to save time and stock haemophilic plasma. The importance of frequent assays is stressed.

The anaesthetist remained responsible for the intravenous drips throughout the post-operative course.