J. Heinrich Joist

Indium-111 labeled platelets: Studies on preparation and evaluation of in vitro and in vivo functions

Abstract Platelets have been labeled with a lipid soluble complex of the radionuclide indium-111. The complex is formed with 8-hydroxyquinoline and extracted in chloroform which is then evaporated to dryness. The residue is dissolved in 50 μl of ethanol and diluted to 200 μl with normal saline. Platelets are separated by differential centrifugation, washed and suspended either in Tyrodes-albumin solution or in normal saline. The solution of 111 In-complex is added to separated platelets and more than 95% of the radioactivity is incorporated with the platelets. The function of the labeled platelets has been studied by their aggregability using adenosine diphosphate and collagen as the stimulating agents. No adverse effects have been observed. The survival of the indium-labeled platelets was similar to that observed with chromium-51 labeled platelets. Labeled canine platelets have been administered to dogs in whom venous thrombi had been induced by alteration of the intima by an electric current. The thrombi were detected by imaging 50 minutes after administration of the labeled platelets. Twenty-four hours later the thrombi were removed and had 20–50 times the radioactivity of an equal weight of blood. Accumulation of large amounts of radioactivity in surgical wounds has also been observed. Damage of the intima of carotid arteries in animals by a balloon catheter demonstrated 8 to 20 times more activity in the damaged artery than in the normal artery. Results indicate that In-111 labeled platelets have potential both for platelet survival studies and for evaluation of vascular damage.

Acquired von Willebrand's disease. Evidence for a quantitative and qualitative factor VIII disorder.

Abstract To define further the factor VIII abnormality in acquired von Willebrands disease, we performed immunoelectrophoresis of factor VIII antigen, as well as quantitative measurements of the antigen, factor VIII procoagulant activity and von Willebrand factor activity on plasma from an affected 57-year-old man who also had a poorly differentiated lymphocytic lymphoma. No evidence for an inhibitor against factor VIII procoagulant activity or von Willebrand factor activity was detected, but immunoelectrophoresis showed none of the less anodic forms of factor VIII antigen. There were concomitant decreases in total antigen (0.19 U per milliliter) and von Willebrand factor levels (0.12 U per milliliter). Factor VIII-procoagulant activity was borderline low (0.45 U per milliliter). Correction of both the abnormal immunoelectrophoresis pattern and the quantitative abnormalities followed radiotherapy of the lymphoma. The factor VIII abnormalities might have resulted from binding or destruction of the less an...

Indium‐III: a New Radionuclide Label for Studying Human Platelet Kinetics

Summary. Indium‐III, when complexed with 8‐hydroxyquinoline (oxine), has been employed as a radioactive platelet label for thrombus imaging in animals and man. The short half‐life (2.8 d) and high yield of gamma photons of 111In make it ideal for in vitro counting and external imaging. To evaluate its suitability for studies of platelet turnover in man, platelet kinetic studies were carried out on 10 healthy volunteers using 111In‐and 51Cr‐platelets concurrently. For 111In labelling, platelets were harvested by differential centrifugation from 43 ml of whole blood drawn into acid‐citrate dextrose (ACD) solution. The platelets were washed and suspended in a mixture of ACD and isotonic saline and then incubated with 111In‐oxine, rewashed, and suspended in plasma for reinfusion. 51Cr labelling was performed using standard methods. Mean labelling efficiency was 73% with 111In and 6.5% with 51Cr. In vitro studies demonstrated minimal release, elution, and reutilization of the 111In label. There was no significant difference in the aggregation response of 111In‐ and 51Cr‐platelets to ADP and collagen. The in vivo recovery of 111In‐platelets was approximately 50% greater than that of 51Cr‐platelets whereas the platelet life spans were similar. These results indicate that 111In labelled platelets may be useful for thrombokinetic studies in man. The new method offers the advantages of reduced blood requirements, higher labelling efficiency, and the ability to perform external imaging of platelet distribution in vivo.

SCINTIGRAPHIC DETECTION OF ATHEROSCLEROTIC LESIONS AND VENOUS THROMBI IN MAN BY INDIUM-111-LABELLED AUTOLOGOUS PLATELETS

Accumulation of autologous platelets labelled with indium-111 at sites of atherosclerosis or venous thrombosis was demonstrated by scintigraphy in three patients. The lesions detected were bilateral ulcerated carotid-artery plaques, iliofemoral venous thrombosis, and renal-vein thrombosis. This technique shows promose as a non-invasive means of diagnosing focal atherosclerotic and thrombotic lesions.

Evaluation of a New Point-of-care Test that Measures PAF-mediated Acceleration of Coagulation in Cardiac Surgical Patients

Background This study was designed to evaluate a new point-of-care test (HemoSTATUS) that assesses acceleration of kaolin-activated clotting time (ACT) by platelet activating factor (PAF) in patients undergoing cardiac surgery. Our specific objectives were to determine whether HemoSTATUS-derived measurements correlate with postoperative blood loss and identify patients at risk for excessive blood loss and to characterize the effect of desmopressin acetate (DDAVP) and/or platelet transfusion on these measurements. Methods Demographic, operative, blood loss and hematologic data were recorded in 150 patients. Two Hepcon instruments were used to analyze ACT values in the absence (channels 1 and 2: Ch1 and Ch2) and in the presence of increasing doses of PAF (1.25, 6.25, 12.5, and 150 nM) in channels 3-6 (Ch3-Ch6). Clot ratio (CR) values were calculated with the following formula for each respective PAF concentration: clot ratio = 1 - (ACT/control ACT). These values also were expressed as percent of maximal (%M = clot ratio/0.51 x 100) using the mean CRCh6 (0.51) obtained in a reference population. Results When compared with baseline clot ratios before anesthetic induction, a marked reduction in clot ratios was observed in both Ch5 and Ch6 after protamine administration, despite average platelet counts greater than 100 K/micro liter. There was a high degree of correlation between clot ratio values and postoperative blood loss (cumulative chest tube drainage in the first 4 postoperative hours) with higher concentrations of PAF: CRCh6 (r = -0.80), %M of CRCh6 (r = -0.82), CRCh5 (r = -0.70), and %M of CRCh5 (r = -0.85). A significant (P < 0.01) improvement in clot ratios was observed with time after arrival in the intensive care unit in both Ch5 and Ch6, particularly in patients receiving DDAVP and/or platelets. Conclusions Activated clotting time-based clot ratio values correlate significantly with postoperative blood loss and detect recovery of PAF-accelerated coagulation after administration of DDAVP or platelet therapy. The HemoSTATUS assay may be useful in the identification of patients at risk for excessive blood loss and who could benefit from administration of DDAVP and/or platelet transfusion.

Antithrombin III during cardiac surgery: effect on response of activated clotting time to heparin and relationship to markers of hemostatic activation.

This study was designed to determine if, and to what extent, antithrombin III (AT) levels affect the response of the activated clotting time (ACT) to heparin in concentrations used during cardiac surgery, and to characterize the relationship between AT levels and markers of activation of coagulation during cardiopulmonary bypass (CPB).After informed consent, blood specimens obtained from eight normal volunteers (Phase I) were used to measure the response of the kaolin and celite ACT to heparin after in vitro addition of AT (200 U/dL) and after dilution with AT-deficient plasma to yield AT concentrations of 20, 40, 60, 80, and 100 U/dL. In Phase II, blood specimens collected before the administration of heparin and prior to discontinuation of CPB, were used to measure the response of the kaolin ACT to heparin (preheparin only), AT concentration, and a battery of coagulation assays in 31 patients undergoing repeat or combined cardiac surgical procedures. In Phase I, strong linear relationships were observed between kaolin (slope = 1.04 AT - 2, r2 = 0.78) and celite (slope = 1.36 AT + 6, r2 = 0.77) ACT slopes and AT concentrations below 100 U/dL. In the pre-CPB period of Phase II, only factors V (partial r = -0.49) and VIII (partial r = -0.63) were independently associated with heparin-derived slope using multivariate analysis; an inverse relationship was observed between AT and fibrinopeptide A levels (r = -0.41) at the end of CPB. Our findings indicate that the responsiveness of whole blood (ACT) to heparin at the high concentrations used with CPB is progressively reduced when the AT concentration decreases below 80 U/dL. Because AT is variably, and sometimes extensively, reduced in many patients before and during CPB, AT supplementation in these patients might be useful in reducing excessive thrombin-mediated consumption of labile hemostatic blood components, excessive microvascular bleeding, and transfusion of blood products. Implications: Heparin, a drug with anticoagulant properties, is routinely given to patients undergoing cardiac surgery to prevent clot formation within the cardiopulmonary bypass circuit. However, when levels are reduced, heparin is not as effective. Findings within this study indicate that administration of antithrombin III may help to preserve the hemostatic system during cardiopulmonary bypass. (Anesth Analg 1997;85:498-506)

Increased in vivo and in vitro platelet function in type II- and type IV-hyperlipoproteinemia☆

Abstract Platelet function in vivo and in vitro was examined in 10 patients with type IIa-, 8 patients with type IIb-, and 16 patients with type IV-hyperlipidemia (HLP) and 24 control subjects closely matched for age and sex. Patients with type IIb- and IV-HLP showed significantly shorter template bleeding times in the presence of similar blood platelet concentrations. Increased platelet factor 3-availability in intact platelet-rich plasma (PRP) and PRP exposed to collagen was observed in all patient groups. Plasma antiheparin activity was increased in patients with type IIa- and type IV-HLP. Platelet adhesiveness, platelet aggregation and 14 C-serotonin release in response to ADP, epinephrine and collagen, as well as plasma von Willebrand factor activity, were generally not increased in either patient group. Increased platelet function in endogenous HLP may be related to both increased platelet turnover secondary to premature atherosclerotic disease and abnormalities of platelet lipid composition induced by HLP-plasma.

EFFECTS OF PLATELETS AND WHITE BLOOD CELLS AND ANTIPLATELET AGENT C7E3 (ReoproTM) ON A NEW TEST OF PAF PROCOAGULANT ACTIVITY OF WHOLE BLOOD.

This study was designed to evaluate the effects of varying concentrations of platelets, white blood cells (WBC) and Fab fragments of a monoclonal antibody (c7E3, Reopro) directed at the platelet GpIIb-IIIa receptor complex on ACT-based clot ratio values (hemoSTATUS assay) in healthy volunteers. These measurements were made in heparinized whole blood from 10 normal volunteers in which either platelet or WBC concentrations had been varied by differential centrifugation. In addition, blood collected in either heparin or argatroban was incubated with varying concentrations of c7E3 (Reopro). Clot ratio values (%Maximal) in normal blood did not decrease until average platelet counts were less than 50,000. A marked reduction in clot ratios was observed when WBC concentration increased above or decreased below baseline clot ratios within each patient. Strong linear relationships were observed between white cell concentration and clot ratio values when white cell concentrations were either less or greater than baseline values. When argatroban was used as an anticoagulant, inverse relationships were demonstrated between clot ratio values and increasing c7E3 concentration (Ch 3: r = -0.33, Ch4: r = -0.84, Ch5: r = -0.87, Ch 6: r = -0.71). ACT-based clot ratio values determined in heparinized whole blood presumably reflecting PAF inducible platelet procoagulant activity, are affected by platelet concentration when counts are less than 50,000/microliter. The hemoSTATUS test was also found to be affected by WBC concentration since clot ratio values decreased when WBC counts were below 4,000/microliter or above 9,000/microliter. A dose-dependent reduction in clot ratio values was also observed with increasing concentrations of c7E3. This test can reliably detect platelet dysfunction only if the platelet count is > 50,000 and the WBC is normal.

Effect of aprotinin on activated clotting time whole blood and plasma heparin measurements

Twenty cardiac surgical patients requiring cardiopulmonary bypass were enrolled in this study designed to evaluate the effect of aprotinin on activated clotting time (kaolin and celite), whole blood, and laboratory-based plasma (anti-Xa) heparin measurements. Whole blood heparin measurements were not different (p = 0.98) between aprotinin-treated (3.2 +/- 2.8 U/mL) and control (3.2 +/- 3.0 U/mL) specimens. Plasma anti-Xa heparin measurements were also not different (p = 0.95) between aprotinin-treated (2.7 +/- 2.5 U/mL) and control (2.8 +/- 2.5 U/mL) specimens. The relationship between whole blood (plasma equivalent) and plasma heparin measurements was similar (p = 0.1) in the presence (slope, 1.04; r2 = 0.89) or absence (slope, 1.11; r2 = 0.89) of aprotinin. In contrast to weak correlations between celite (r = 0.50) or kaolin (r = 0.53) activated clotting time values, whole blood heparin measurements correlated well (r = 0.93) with plasma heparin measurements during cardiopulmonary bypass in the presence of aprotinin. These findings indicate that whole blood heparin measurements are unaffected by aprotinin and correlate well with plasma anti-Xa heparin measurements even in the presence of aprotinin. Therefore, the automated protamine titration assay can be used to monitor accurately heparin concentrations in patients receiving aprotinin.

Arachidonic acid-induced platelet aggregation independent of ADP-release in a patient with a bleeding disorder due to platelet storage pool disease.

Abstract Platelets from a patient with a bleeding disorder have been extensively studied. These platelets showed reduced levels of storage pool ADP, reversible aggregation induced by exogenous ADP, markedly reduced aggregation induced by collagen and absent epinephrine induced aggregation. Arachidonic acid, the prostaglandin - thromboxane precursor, induced normal aggregation with no release of enzymatically detectable ADP. Furthermore electron microscopy demonstrated that the aggregated platelets had not discharged their granules. In contrast, thrombin induced normal aggregation accompanied by complete discharge of the platelet granules. This patients platelets have uniquely demonstrated, a) that arachidonic acid can induce aggregation independent of the release reaction, b) that thrombin induced platelet aggregation and release by an independent mechanism.