Margaret Karpatkin

Methods for the production of clinically effective intermediate- and high-purity factor-VIII concentrates.

Summary. An intermediate‐ and a high‐purity factor‐VIII concentrate for clinical use have been prepared on a large scale by cryoethanol precipitation, extraction of the precipitate with tris buffer, and fractionation with polyethylene glycol. With bench‐scale fractionation, the intermediate material is 22‐fold purified on the average and the mean yield is 63%, while the high‐purity factor VIII is 274‐fold purified with a mean yield of 62%. With fractionation of 100 1. or more of fresh frozen plasma, the intermediate material shows a 30% yield and 14‐30‐fold purification; the high‐purity factor VIII shows an 18% yield and 125–350‐fold purification using 5.8 g/100 ml polyethylene glycol (PEG). A yield of nearly 30% should be possible with PEG, 4–5 g/100 ml. Both factor‐VIII preparations are stable for over a year in the lyophilized state at 4°C. Other plasma proteins can be fractionated from the residual plasma by routine Cohn procedures.

Clinical Investigation of Intermediate- and High-Purity Antihaemophilic Factor (Factor VIII) Concentrates

Summary. Intermediate‐ and high‐purity antibaemophilic factor (factor‐VIII) concentrates developed by the American National Red Cross (ANRC) have undergone extensive clinical investigation. When administered to severely affected haemophiliacs, the recovery in vivo and the metabolic half‐disappearance time were similar to those achieved with plasma.

Prothrombin and protein C in early childhood: Normal adult levels are not achieved until the fourth year of life

Prothrombin and protein C are vitamin K--dependent proteins with important roles in hemostasis. During the neonatal period, levels of both proteins are lower than in the adult. TM However, the normal range in later infancy and the age at which adult levels are reached have not been established for either protein. We report values for protein C and prothrombin in 148 healthy children ranging in age from 1 month to 4 years. METHODS The children attended the Family Care Clinic at Belle- vue Hospital Center. All were born at term after uncom- plicated pregnancies, were well at the time of study, and had no significant previous illness. Capillary blood speci- mens were obtained by finger or heel stick into heparinized microhematocrit tubes. The tubes were centrifuged imme- diately, and the plasma stored at -70 ~ C until tested. from I 1 healthy adults obtained from venous blood drawn into dry heparin. Protein C level in this pool was 115.5%, and prothrombin was 116% expressed as a percentage of pooled plasma obtained from George King. Assuming a dilution factor of 1:6 in the George King plasma related to the anticoagulant, these values are 101.5% and 101%, respectively. RESULTS Mean protein C levels in plasma obtained from citrated blood of 28 healthy adults were 102% _+ 12.5% in venous samples and 118% + 14% in capillary samples. Assuming a dilution factor of 1:6 in the venous samples because of the anticoagulant, the mean level in the capillary samples was 103% + 14%. There was a significant correlation between the venous and capillary values in the individuals studied (r -- 0.995, P <0.001). The mean values for prothrombin in the same samples were venous 98.3%-+ 11.5% and capillary 113.0% _+ 13.0%. When dilution of the venous samples with antico- agulant was taken into account, the capillary values were 98.9% _+ 13.0%. The correlation between capillary and venous sample was also highly significant (r--0.995, P <0.001 ). Levels of protein C and of prothrombin in relation to age from 1 month to 4 years are shown in the Figure and the Table. At 36 months, mean values for both proteins were still significantly lower than the adult mean level, and did not reach adult values until sometime during the fourth year of life. The initial rise was relatively rapid, but became progressively slower (Figure). Levels of prothrom- bin were higher than those of protein C for the first 8 months, and the ratio of protein C to prothrombin was significantly lower than in the adult. By 1 year of age the ratio had reached the normal adult value of I, and remained there subsequently (Table).

Low protein C in the neonatal period

Summary. Protein C was measured by electroimmunoassay in 47 infants within 24 h of delivery. Gestational age ranged from 28 to 43 weeks. The mean level was 27% (range < 10–67%) of the normal adult mean. In the 22 infants who had no clinical problems, protein C levels correlated significantly with gestational age. In the 25 who were sick there was no correlation, and the mean level was significantly lower than that of the healthy infants. Postnatal rise was slow; on day 7 the mean was 32% and on day 28, 31%. Levels of protein C correlated significantly with prothrombin in both the healthy and sick infants. Crossed immunoelectrophoresis in the presence of calcium ions gave one protein C peak of the same electrophoretic mobility as is seen in plasma of healthy adults, indicating that the infants’ protein C is gamma carboxylated. It is concluded that: (1) Protein C in neonates is in or below the range associated with thromboembolism in patients congenitally deficient in this protein; (2) protein C levels correlate with gestational age; and (3) the low levels during the neonatal period are not due to decreased gamma carboxylation but may reflect decreased synthesis when compared to the older child or the adult.

Transient hemorrhagic diathesis associated with an inhibitor of prothrombin with lupus anticoagulant in a 1½-year-old girl: Report of a case and review of the literature

Acquired inhibitors of coagulation causing bleeding manifestations are rare in children, particularly without an associated underlying disorder such as autoimmune disease. We describe an otherwise healthy 1½‐year‐old girl who had extensive spontaneous bruising and prolonged bleeding from venipuncture sites. Prothrombin time (PT) and activated partial thromboplastin time (APTT) were prolonged, with evidence of an immediate‐acting inhibitor. Thrombin clotting time, fibrinogen, and platelets were normal. Biologic assay of factors II, V, VII, and X were all low, with increasing values at higher dilutions. However, by immunoassay and/or chromogenic assays, only factor II was reduced. An antibody which failed to neutralize prothrombin activity in vitro was detected against radiolabeled prothrombin. Coagulation studies normalized in parallel with clinical recovery and disappearance of the antibody. This case demonstrates acute hypoprothrombinemia‐lupus anticoagulant syndrome as a rare presentation of bleeding diathesis in a healthy young child.

Cooley anemia: High transfusion regimen and chelation therapy, results, and perspective

A tranfusion regimen aimed an maintaining hemoglobin levels above 9.5 gm/dl from the time of early diagnosis has permitted eight with Cooley anemia to liver normal, active lives, without hypersplenism or cardiac dysfunction (three to 13 years of observation). Echocardiography, however, has revealed increased thickness of the left ventricular wall. The amount of iron excreted in urine per 24 hours in response to daily administration of deferoxamine-B (20 mg/kg) was related to the estimated proportionate daily accumulation of iron from that administered by intermittent transfusion of erythocyutes, calculated from the amount of transfused RBCs The percentage of the dauily accumulated iron that was excreted after im injection of deferoxamine amounted to less than 30% in children up to five years of age; thereafter, it increased to 50%. When the same dose of the drug was given overnight by iv or sc infusion, the 24-hour excretion of iron increased threefold in the youngest and twofold in the oldest children. These data lead to the projection that deferoxamine-B, at the dose of 20 mg/kg, if given daily by im injection, may significantly reduce the iron overload and, if given daily at this dose by overnight infusion, may permit these children to approach iron balance, despite their large transfusion requirements.

An epidemic of maternal thrombocytopenia associated with elevated antiplatelet antibody: Platelet count and antiplatelet antibody in 116 consecutive pregnancies: Relationship to neonatal platelet count☆

Twenty-eight (24%) of 116 pregnant women studied prospectively during an 8-month period in 1983 had platelet counts of less than 150,000/mm3 at least once during pregnancy. Thirteen of these were thrombocytopenic in both the prenatal and the peripartum period. Eighteen were restudied 3 to 12 months after delivery. One woman, who was pregnant again, had a platelet count of 140,000/mm3. In the others, platelet counts were in the normal range. Platelet-associated immunoglobulin G and serum antiplatelet antibody levels were elevated in 79% and 61%, respectively, of these 28 women on at least one occasion. However, 59% of 73 pregnant nonthrombocytopenic women had increased platelet-associated immunoglobulin G levels and 59% had positive serum antiplatelet antibody test results. Twenty women who had increased platelet-associated immunoglobulin G levels and positive serum antiplatelet antibody test results were normal 6 to 10 months after delivery. Of 105 infants studied, 10 were thrombocytopenic. Neonatal thrombocytopenia was not predicted by maternal platelet count, platelet-associated immunoglobulin G, or serum antiplatelet antibody. By the fall of 1984, the incidence of thrombocytopenia had dropped to two in 280 consecutive pregnancies. We conclude that (1) epidemics of thrombocytopenia can occur in pregnant women and (2) if a women is found to be thrombocytopenic for the first time during pregnancy, she should not be subjected to the measures advocated for the management of pregnancy in women with autoimmune thrombocytopenic purpura.

Differentiation of autoimmune thrombocytopenia from thrombocytopenia associated with immune complex disease: systemic lupus erythematosus, hepatitis-cirrhosis, and HIV-1 infection by platelet and serum immunological measurements.

A method and approach are described to differentiate classic autoimmune thrombocytopenia (ATP) from immune complex‐associated thrombocytopenia in systemic lupus (SLE), hepatitis/chronic liver disease (LIV‐ITP) and HIV‐1 related thrombocytopenia (HIV‐1‐ITP). The platelet immunologic profile of IgG, C3C4 and IgM was measured with a solid‐phase ELISA, employing 125I‐staphylococcal protein A to detect indicator antibody binding. Polyethylene glycol was employed to precipitate immune complexes (PEG‐IC). Platelet‐associated IgG (PAIgG) was 2.8‐, 5.6‐ and 5.8‐fold higher in SLE, LIV‐ITP and HIV‐1‐ITP patients respectively compared to ATP patients; platelet C3C4 was 3.2‐, 4.8‐ and 4.5‐fold higher respectively; platelet IgM was 2.2‐, 3.7‐ and 3.8‐fold higher respectively; serum PEG‐IC levels were 4.2‐, 4.8‐ and 2.1‐fold higher respectively. With all parameters measured, there was no overlap between the 75th percentile for ATP patients and the 25th percentile for all three cohorts. The likelihood of having a platelet C3C4 level higher than the highest ATP level was 69% for SLE, 90% for LIV‐ITP and 94% for HIV‐1‐ITP respectively; with PEG‐IC measurements the likelihood was 83%, 100% and 100% respectively. Serum IgG, C3, C4, IgM and PEG‐IC were examined for a possible relationship with platelet measurements. Except for a positive correlation between serum and platelet IgM in ATP, r = 0.5, P < 0.04, there was no positive correlation with any of the parameters measured. An inverse correlation was noted between PEG‐IC level and platelet C3C4 in SLE, r = 0.7, P < 0.04. Thus platelet immunologic profile and serum PEG‐IC level measurements differentiated classic ATP from immune complex‐associated thrombocytopenias (SLE, LIV‐ITP, HIV‐1‐ITP). Except for IgM measurements in ATP, platelet measurements could not be attributed to their respective serum concentration.

Thrombocytosis after major lower extremity trauma: mechanism and possible role in free flap failure.

Microvascular thrombosis and free flap failure are complications of free tissue transfer for coverage of lower extremity soft-tissue and bony defects despite appropriate vessel selection and adherence to meticulous technique. Increased rates of flap failure have been associated with reconstruction performed between 3 days and 6 weeks after injury, as well as in patients with thrombocytosis. We have found that serum platelet levels rise significantly after lower extremity injury. It is our theory that a circulating mediator or cytokine is released in response to injury, inducing the thrombocytosis. Twenty-one patients with Gustilo grade IIIb and IIIc injuries were studied prospectively. Serum was collected throughout the postinjury period. Platelet count, leukocyte count, hemoglobin concentration, and hematocrit were determined. Samples were also subjected to a platelet aggregation study as well as enzyme-linked immunosorbent assay for interleukin-3, interleukin-6, interleukin-11, and granulocyte macrophage-colony-stimulating factor. Megakaryocyte growth and development factor enzyme-linked immunosorbent assay and a myleoproliferative leukemia virus-transfected cell line assay for thrombopoietin were performed. Bone marrow was studied with flow cytometric analysis. Mean initial platelet count was 196,000 per cubic millimeter. There was an initial 26% decline to 140,000 per cubic millimeter, followed by an increase to 361% of baseline on day 16. No significant variations in serum leukocyte count or hemoglobin concentration were seen. Spontaneous and induced platelet aggregation responses were normal, Interleukin-6 was detected at elevated levels. However, interleukin-3, interleukin-11, granulocyte macrophage-colony-stimulating factor, and thrombopoietin were not measurable. Marked megakaryocytosis was seen on bone marrow analysis. Interleukin-6 may, therefore, play a role in the mechanism of thrombocytosis. We suggest that because patients with complex bony injuries of the leg experience platelet elevations that peak approximately 2 weeks after injury, microvascular free flap reconstructions should be considered high risk during this time period.

Plasma Protease Inhibitors in Premature Infants: Influence of Gestational Age, Postnatal Age, and Health Status

Abstract In newborn infants, the influence of gestational age (GA), postnatal age (PA), and health status on the plasma protease inhibitors α2-macroglobulin (α2-M), α,-antitrypsin (α,-AT), C1 esterase inhibitor (C1E-INH), α2-antiplasmin (α2-AP), and antithrombin III (AT-III) was investigated. Inhibitor levels were measured by radial-immunodiffusion and expressed as a percentage of pooled plasma from adults (mean ± SEM). In total, 54 premature infants (28-36 weeks gestation) were classified at birth as healthy (N = 22) (IV fluids, antibiotics only) or sick (N = 32) (all other support, but excluding infants with disseminated intravascular coagulation (DIC) and studied on Days 1 and/or 7 of life. Healthy term infants (N = 18) and infants with DIC (N = 10) were studied on Day 1 only. All inhibitors except C, E-INH increased with increasing gestational age (P < 0.01). In healthy premature infants all inhibitor levels reached the normal adult range by 1 week of age. In contrast, at 1 week of age, sick infants had lower levels of α2-M and α2-AP, and higher levels of α,-AT compared to healthy infants (P < 0.01). The presence of DIC depressed all of the inhibitors on Day 1 except α,-AT when compared to healthy controls (P < 0.01). Thus, gestational age, postnatal age, and health status all significantly influenced the levels of these plasma protease inhibitors.