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.

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.

Transfusion therapy for autoimmune hemolytic anemia.

Transfusion therapy for patients with autoimmune hemolytic anemia poses major problems to both the blood bank that is charged with providing blood and the clinician who must manage the patient successfully. Profitable discussions between the clinician and the blood bank are necessary before the patient can receive the most beneficial form of treatment. These problems are summarized in Table 1. Although not all of these problems apply to all patients, there are quite enough problems that are generated by every patient. The blood bank and the clinician are usually confronted by different aspects of problems posed by the same patient, but all aspects are related, and a meaningful dialogue between the blood bank and the clinician is absolutely essential to achieve optimal patient management.

Compatible transfusion therapy for paroxysmal cold hemoglobinuria.

Two children with paroxysmal cold hemoglobinuria (PCH) are described. Both cases were associated with cold-warm serum lysins having anti-Tja activity. Transfusion with compatible frozen-thawed red cells tided these children over the critical acute hemolytic phase of the syndrome. These are the first recorded examples of successful compatible transfusion therapy in PCH.

Fucosidosis type 2.

Two siblings, 9 and 4 1/2 years old, had alpha-L-fucosidase deficiency, angiokeratoma, progressive psychomotor retardation, neurologic signs, coarse facila features, and dysostosis multiplex. It appears that genetic heterogeneity is present in fucosidosis; there are at least two types. In type 1, patients have no vascular lesions, but have rapid psychomotor regression, severe and rapidly progressing neurologic signs, elevated sodium and chloride excretion in the sweat, and fatal outcome before the sixth year. In type 2, patients have angiokeratoma, milder psychomotor retardation and neurologic signs, longer survival, and normal salinity in the sweat. Quantitative studies on erythrocytes and in saliva disclosed severely increased expressions of Lea and Leb. Biopsies of skin and gingiva showed alterations as seen in angiokeratoma. There was also evidence of lysosomal storage in vascular endothelium, eccrine sweat gland epithelium, and fibroblasts of the skin.

Nonuniform distribution of occult blood in feces.

Inhibition of anti-Rh29 by erythrocytic stroma in feces was devised as a specific test for fecal occult blood. The sensitivity of this test was equivalent to that of a standard Hemoccult test, namely, 10(8) erythrocytes/g feces. Comparison of results of this test with results of Hemoccult tests of random stool specimens and of stools following ingestion of autologous blood revealed nonuniform distribution of occult blood in feces. The extent of nonuniformity was determined by testing samples of stool specimens following ingestion of 51Cr-labeled autologous blood. This allowed comparison of Hemoccult, inhibition of anti-Rh, and radioactivity, and showed that the three labels could separate in the feces and that some single small samples of feces could be relatively free of blood while blood was readily demonstrable in other portions. The variability of standard Hemoccult test was somewhat reduced by dispersing the feces in distilled water before performing the test.

Antigenic determinants of C3 and C4 complement components on washed erythrocytes from normal persons.

Well-washed erythrocytes from normal persons were agglutinated by antisera to C3, C3d, and C4, and this agglutination was specifically inhibited by the corresponding C3 or C4 protein. C3 and C4 antigenic determinants were present on the red blood cells of freshly shed blood promptly anticoagulated with EDTA, heparin, ACD, or CPD, and no significant changes in degree of agglutination were observed on storage of EDTA or CPD blood for two weeks at 4 C. Marked differences in degree of agglutination by anti-C3, anti-C3d, and anti-C4 were observed when erythrocytes of 16 normal persons were assayed, and significant correlations were obtained when the quantitative results with any two antisera were compared. Anti-C3c did not agglutinate erythrocytes from normal persons, suggesting that the C3 antigens detected on normal cells are carried by the C3d fragment. To avoid significant agglutination of the erythrocytes from some normal persons, very dilute preparations of anti-C3, -C3d, and -C4 had to be used for instrumented diagnostic direct antiglobulin tests. Stronger reagents could be used for indirect antiglobulin tests when the result of a suitable control could be subtracted.

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.

Quantitative Rh typing of r-Gr-G with observations on the nature of G (Rh 12) and anti-G.

Quantitative blood typing data on Mr. R. B. (r-Gr-G), his wife (R1r), a daughter (R1r-G), and a niece (r-Gr) strongly suggest that these Rh phenotypes are directly indicative of actual Rh genotypes. If so, the antigenic products of r-G are weak Rh2(rh' or C), normal Rh5 (hr" or e), an expression of Rh12 (rh-G) equal to that produced by R-1,2,-3 (r' or dCe) and half of what is produced by R1 (R or D) genes in either homozygous or heterozygous expression, very weak expression of Rh19 (hr-S), absence of Rh31 (hr-B), and slightly weakened expression of Rh17 (Hr-o or 'not D'). The LW status of r-Gr-G cells was equivalent to that of other Rh:-1 erythrocytes. Thus r-G resembles mutant r' in which only Rh5 is expressed normally. The weak Rh2 produced by r-G reacted much better with one Rh2 antiserum than with another. Rh21 (C-G) had been used to denote such additional reactivity, but one reagent that acted as anti-Rh2 in manual tests behaved like anti-Rh21 in instrumented tests. Therefore, anti-Rh21 may only indicate a more efficiently agglutinating anti-Rh2. Mr. R. B. showed no evidence of congenital stomatocytic hemolytic anemia characteristic of Rh-null or Rh-mod. Finally, anti-Rh12 eluates, recovered either sequentially from r'r followed by R2r or singly from r-Gr-G, agglutinated chimpanzee red cells more efficiently than did either anti-Rh1 (D) or anti-Rh4 (c), a result consistent with expectation for serological crossreactivity between Rh1 and Rh21.

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.