Outcomes among 562 recipients of placental-blood transplants from unrelated donors.

BACKGROUND: A program for banking, characterizing, and distributing placental blood, also called umbilical-cord blood, for transplantation provided grafts for 562 patients between August 24, 1992, and January 30, 1998. We evaluated this experience. METHODS: Placental blood was stored under liquid nitrogen and selected for specific patients on the basis of HLA type and leukocyte content. Patients were prepared for the transplantation of allogeneic hematopoietic cells in the placental blood and received prophylaxis against graft-versus-host disease (GVHD) according to routine procedures at each center. RESULTS: Outcomes at 100 days after transplantation were known for all 562 patients, and outcomes at 1 year for 94 percent of eligible recipients. The cumulative rates of engraftment among the recipients, according to actuarial analysis, were 81 percent by day 42 for neutrophils (median time to engraftment, 28 days) and 85 percent by day 180 for platelets (median, day 90). The speed of myeloid engraftment was associated primarily with the leukocyte content of the graft, whereas transplantation-related events were associated with the patient's underlying disease and age, the number of leukocytes in the graft, the degree of HLA disparity, and the transplantation center. After engraftment, age, HLA disparity, and center were the primary predictors of outcome. Severe acute GVHD (grade III or IV) occurred in 23 percent of patients, and chronic GVHD occurred in 25 percent. The rate of relapse among recipients with leukemia was 9 percent within the first 100 days, 17 percent within 6 months, and 26 percent by 1 year. These rates were associated with the severity of GVHD, type of leukemia, and stage of the disease. CONCLUSIONS: Placental blood is a useful source of allogeneic hematopoietic stem cells for bone marrow reconstitution.

Processing and cryopreservation of placental/umbilical cord blood for unrelated bone marrow reconstitution.

Clinical evidence of hematopoietic restoration with placental/umbilical cord blood (PCB) grafts indicates that PCB can be a useful source of hematopoietic stem cells for routine bone marrow reconstitution. In the unrelated setting, human leukocyte antigen (HLA)-matched donors must be obtained for candidate patients and, hence, large panels of frozen HLA-typed PCB units must be established. The large volume of unprocessed units, consisting mostly of red blood cells, plasma, and cryopreservation medium, poses a serious difficulty in this effort because storage space in liquid nitrogen is limited and costly. We report here that almost all the hematopoietic colony-forming cells present in PCB units can be recovered in a uniform volume of 20 ml by using rouleaux formation induced by hydroxyethyl starch and centrifugation to reduce the bulk of erythrocytes and plasma and, thus, concentrate leukocytes. This method multiples the number of units that can be stored in the same freezer space as much as 10-fold depending on the format of the storage system. We have also investigated the proportion of functional stem/progenitor cells initially present that are actually available to the recipient when thawed cryopreserved PCB units are infused. Progenitor cell viability is measurably decreased when thawed cells, still suspended in hypertonic cryopreservative solutions, are rapidly mixed with large volumes of isotonic solutions or plasma. The osmotic damage inflicted by the severe solute concentration gradient, however, can be averted by a simple 2-fold dilution after thawing, providing almost total recovery of viable hematopoietic progenitor cells.

Crystallographic analysis of the inhibition of porcine pancreatic elastase by a peptidyl boronic acid: structure of a reaction intermediate.

The crystal structure of porcine pancreatic elastase (PPE) complexed to carbobenzoxy-alanylisoleucine-boronic acid (ZAIB) is reported to 2.09-A resolution and refined to an R factor of 0.15. This is the first reported structural analysis of PPE with an isoleucine residu in the primary specificity pocket. The results include (1) marked displacement of the inhibitor out of the active site leading to (2) a close (2.2 A) direct contact between B (boron atom of the inhibitor) and N epsilon of His-57 and also (3) covalent bonding (1.5 A) to O gamma of Ser-195. A scheme for the mechanism of inhibition of PPE by ZAIB is proposed. A comparison with a peptidyl difluoromethyl ketone-PPE complex (Ki = 9.5 microns) is made to explain the strong inhibition of PPE by ZAIB (Ki = 0.3 micron). These results lead us to characterize this structure as a time- and space-averaged reaction intermediate, providing fresh insight into the cramped dimensions available in enzymatic catalyses.

X-ray diffraction analysis of the inhibition of porcine pancreatic elastase by a peptidyl trifluoromethylketone.

X-ray crystallographic data to 2.57 A resolution (1 A = 0.1 nm) have been measured for the complex of a peptidyl trifluoromethylketone inhibitor with porcine pancreatic elastase (PPE); R = 0.14. The inhibitor forms a stable complex with the enzyme by means of a covalent attachment to active site Ser195O gamma, resulting in a hemiketal moiety with tetrahedral geometry. The tripeptide protion binds as an antiparallel beta-sheet, with four hydrogen bonds augmenting the active-site covalent linkage, Ki = 9.5 microM. His57 exhibits a bifurcated H-bond to both Ser195O gamma and an F atom of the inhibitor. This study is one of a series which explores the binding geometry of a variety of small substrates and inhibitors to PPE. This peptidyl-PPE complex affords insight into the binding geometry of a novel trifluoromethylketone moiety to a serine proteinase.

A new human Duffy blood group specificity defined by a murine monoclonal antibody. Immunogenetics and association with susceptibility to Plasmodium vivax.

A new Duffy specificity, Fy6, defined by a murine monoclonal antibody of the IgG1 kappa class, is related to susceptibility to malarial invasion. In humans, Fy6 is present on the red cells of all persons except those of the Fy(a-b-) type, a distribution resembling that of Fy3. However proteolytic enzyme treatment of red cells enhances the reactivity of Fy3, whereas Fy6, like Fya and Fyb, is susceptible to degradation by this process. The number of Fy6 sites on human red cells was found to be 12,200 per cell, in close agreement with earlier estimates of the number of Fya sites. Anti-Fy6 reacted in western blots with a membrane glycoprotein of approximately 46,000 Mr, not significantly different from that of a molecule known to bear the Fya determinant. The Fy6 epitope is shown to be present on the red cells of some but not all nonhuman primate species, where it has a distribution not only distinctly different from Fya, Fyb, and Fy3, but in close accordance with susceptibility to penetration by Plasmodium vivax. Thus, the red cells of two species of macaques (Macaca mulatta and M. fascicularis), which are invaded by Plasmodium knowlesi but not by P. vivax are shown to have other Duffy antigens but to be devoid of Fy6. It appears, therefore, that the red cell epitopes used by these closely related species are distinct, and that susceptibility to P. vivax merozoite penetration is dependent on the presence of Fy6.

Low ionic antiglobulin tests.

Seven serologic procedures were studied to determine their respective value in compatibility and screening tests. All seven were significantly improved by the use of 4 volumes of serum, rather than 1, with 1 volume of red cell suspension, and a low-ionic antiglobin test (LIAGT) was distinctly superior to the other six procedures evaluated. In this test, during the incubation of serum and cells at 37 degrees for 20 minutes, ionic concentration was reduced 62 percent. However, after removal of all supernatant, the red cells were washed three times with an isotonic solution that provided 80 percent reduction in ionic concentration, and the washed cells were tested for their agglutinability with low-ionic (80% ionic reduction) anti-IgG antiglobin reagent. This modified LIAGT was usually more, and apparently never less, sensitive than a test described earlier and is expected to be associated with much less nonspecificity. The extreme sensitivity of LIAGT for many long-term frozen stored alloantiserums is a retained property of the modified test and has been associated with IgG aggregation during storage.

Monoclonal anti-K14 and anti-K2.

Mouse hybridoma clones have produced monoclonal antibodies directed against the K:14 and K:2 high-incidence antigens of the Kell blood group system. Two examples of anti-K14 were isolated, each arising from a separate fusion procedure. All three monoclonal antibodies are of the immunoglobulin class IgG1. Serological activity is consistent with that seen with human antibodies to high-incidence Kell system antigens, and their epitopes are destroyed, as usual, by 2-aminoethylisothiuronium bromide treatment. Specificity was further confirmed by adsorption and elution studies. Tests against nonhuman primate red cells demonstrated the expression of K:14 only by the great apes, whereas K:2 was present on all red cells tested. These findings emphasize the usefulness of monoclonal antibodies to elucidate the evolutionary patterns of blood group variants.

Two blood group M epitopes disclosed by monoclonal antibodies.

Two cloned mouse hybridomas, designated G8 and E3, produced anti-M of immunoglobulin classes IgG2b and IgG1, respectively. No discrepancies were observed in testing over 5,000 normal donor blood samples with appropriately diluted G8 and E3 culture supernatant fluids in parallel with rabbit anti-M and anti-N typing reagents. The specificity and titer of antibodies produced by G8 and E3 were minimally affected by changes in temperature (37 degrees C, 22 degrees C, 4 degrees C). G8 and E3 showed reduced activity with type MM red cells that had been treated with either neuraminidase or papain, but differences were observed in the susceptibility of the respective epitopes to treatment with neuraminidase. Furthermore, G8 and E3 exhibited different specificities when used to test the red cells of nonhuman primates and erythrocytes of the rare MgMg human blood type. These results indicate the existence of at least two M antigen epitopes.

Diagnosis and treatment of the immune hemolytic anemias.

Immune hemolytic anemia can be treated by blood transfusion, steroids, cytotoxic drugs, plasmapheresis, and splenectomy. However, the benefits of therapy are dependent upon the relationship of treatment to the etiology of disease. Thus, effective therapy requires sufficient diagnostic precision to distinguish between allogeneic and autologous antibodies, recognize the etiologic role of immunogenic drugs, and define the immunoglobulin classes of both cold and warm reactive autoantibodies along with their complement interactions.

Augmentation of hemagglutination by low ionic conditions.

Short incubation at 37 C, 80 per cent reduction in ionic concentration and removal of liquid phases after each reaction step, provided the basis for the construction of four new serologic tests for alloantibodies to human erythrocytes. In the first, the incubation fluid was replaced with protamine sulfate to aggregate intensely the evaluated red blood cells. After dispersal by phosphate buffer, residual antibody mediated agglutination could be discerned. As a second method, this low ionic polycation (LIP) test was followed by a normal ionic IgG antiglobulin test (LIP-AGT). A third method employed low ionic washing of erythrocytes and low ionic antiglobulin serum (LIAGT). Finally, a modified LIP test was conducted entirely under low ionic conditions and followed by a low ionic antiglobulin test (modified LIP-AGT). LIP, LIP-AGT and LIAGT were successfully employed for all routine blood bank serology tests. Their sensitivity and impact on blood bank performance are described.