Gastineau, D.A.

Abstract BACKGROUND: Coagulation factor XIII deficiency, in either its acquired or inherited form, is a rare cause of abnormal bleeding. In patients with Factor XIII (F XIII) deficiency, recommended means of factor replacement include infusion of fresh frozen plasma (FFP), cryoprecipitated plasma (Cryo), or F XIII concentrates1. Comparisons of F XIII concentration in FFP and Cryo are not well defined. To aid management of F XIII deficiency, we measured F XIII activity and antigen in FFP and Cryo. In addition, we determined concentrations of fibrinogen, factor VIII (F VIII) and von Willebrand factor (VWF), including antigen (VWF:Ag), ristocetin cofactor activity (VWF:RCo) and collagen binding activity (VWF:CB). STUDY DESIGNS AND METHODS: 10 bags each of FFP and of Cryo from blood group O donors obtained from the Mayo Clinic Division of Transfusion Medicine blood bank, were analyzed. F XIII activity was assayed by ELISA detecting activated F XIII-mediated amine incorporation into solid phase fibrinogen (Pefakit, Pentapharm). F XIII antigen was measured using radial immunodiffusion (The Binding Site). Fibrinogen was measured by Clauss kinetic clotting assay (Fibriquik, Biomerieux) and also by endpoint delta OD derived from the prothrombin time (ACL, Instrumentation Lab). F VIII was measured by 1-stage APTT-based assay; VWF:Ag by automated LIA (Diagnostica Stago); VWF:RCo by aggregometry of washed platelets; and VWF:CB by ELISA (Corgenix). RESULTS: Mean concentrations of F XIII activity were 60 U/bag (+/−30) in Cryo and 288 U/bag (+/− 77) in FFP. F XIII antigen concentrations paralleled the activity results and are depicted in Table 1, which also includes results for the other analytes. The mean fluid volumes of Cryo and FFP were 21.3 ml/bag (+/− 2.7) and 245 mL/bag (+/− 29), respectively. Table 1. Comparison of Hemostatic Components in Cryoprecipitated Plasma (Cryo) versus Fresh Frozen Plasma (FFP) Cryoprecipitate Fresh Frozen Plasma Component Mean (SD) Mean (SD) Factor XIII activity 60 U/bag (+/− 30) 288 U/bag (+/− 77) Factor XIII antigen 0.65 mg/bag (+/− 0.23) 2.36 mg/bag (+/− 0.76) Factor VIII 133 U/bag (+/− 36.5) 265 U/bag (+/− 83) Fibrinogen Clauss 184 mg/dl (+/− 44) 725 mg/dl (+/− 199) Fibrinogen ACL 319 mg/dl (+/− 76) 864 mg/dl (+/− 327) VWF:Ag 181 U/bag (+/− 53) 218 U/bag (+/− 70) VWF:RCo 168 U/bag (+/− 34) 221 U/bag (+/− 65) VWF:CB 165 U/bag (+/− 40) 208 U/bag (+/− 71) Bag volume 21.3 ml/bag (+/− 2.7) 245 ml/bag (+/− 29) CONCLUSION: In contrast to some of the other cryoprecipitable coagulation proteins, F XIII is only mildly enriched in Cryo when compared with FFP (about 2- to 3-fold). Although both products are practical means of F XIII replacement, and F XIII is somewhat enriched in Cryo, FFP may be a preferred product for F XIII replacement when infusion volume is not a major consideration (eg, in adults versus small children) and risks of exposure to multiple donors are considered. While Cryo is no longer recommended as the product of choice for VWF (or F VIII) replacement, our studies also demonstrate that Cryo is significantly enriched in VWF (which is not routinely assayed as a quality control measure for Cryo, in contrast to requirements for F VIII and fibrinogen content).

Diagnosis of Bernard-Soulier syndrome (BSS) is complicated by the difficulty of separating the giant platelets from other blood cells to pursue analyses of platelet function and structure. We report on the utility of three whole blood assay techniques for diagnosis of a patient with BSS. To our knowledge, these three techniques have not been simultaneously applied or compared for efficacy in laboratory diagnosis of BSS. (1) Whole blood platelet aggregation responses, studied with an electrical impedence aggregometer, were equivalent to those more laboriously obtained using platelet-rich plasma prepared by unit gravity sedimentation, studied with an optical light transmittance aggregometer. Platelet aggregation responses were normal with ADP or collagen stimulation, and absent with Ristocetin or bovine plasma stimulation. (2) Whole blood radioimmunoassay of platelet glycoprotein (GP) expression was performed using iodinated murine monoclonal antibodies HP1-1D (anti-GP IIb/IIIa) and 6D1 (anti-GPlb, kindly supplied by Dr. Barry Coller, Stony Brook, NY). After incubation with citrated whole blood, centrifugation was used to separate cell-bound antibody which was quantitated with a gamma counter. The patient’s whole blood had a normal level of cell-bound GP Ilb/IIIa, but a markedly reduced level of cell-bound GP lb (5% of normal mean; n = 20). (3) Whole blood smear immunocytochemical staining with the monoclonals (indirect immuno-alkaline phosphatase technique), and qualitative analysis by light microscopy, revealed a marked reduction of GP lb expression by the patient’s giant platelets, whereas GP Ilb/IIIa expression was normal. This latter technique might be especially valuable as a screening technique when the patient is not directly available for laboratory study. Together with the patient’s life-long history of thrombocytopenia and moderate bleeding diathesis, and other laboratory observations including markedly prolonged bleeding times and reduced whole blood prothrombin consumption, these data established diagnosis of BSS. We conclude that these three relatively simple assays of platelets in whole blood should be of particular value in the laboratory differential diagnosis of patients with congenital thrombocytopenias and giant platelet syndromes.