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To the Editor: To provide samples for neonatal mass screening, a few drops of blood are routinely collected from the heel of the newborn infant and allowed to dry on a thick filter paper called the Guthrie card.[1][1] Guthrie cards represent a valuable and comprehensive genetic repository because

The karyotypic abnormality t(14;18)(q32;q21) is reported to occur in 75% of follicular lymphomas. This translocation results in the rearrangement of a putative oncogene bcl-2, which resides at chromosome 18 band q21 (the 18q21 gene). Using two human genomic DNA fragments cloned from the chromosome 18 band q21 as probes, we analyzed 65 uncultured human lymphoma samples by the Southern blot technique. The 18q21 gene was rearranged in 18 of 26 (69%) follicular lymphomas, 3 of 5 (60%) follicular lymphomas transformed to large cell lymphomas, 8 of 20 (40%) diffuse large cell lymphomas (DLCLs), and 2 of 7 (29%) small noncleaved cell lymphomas (SNCs). Our analysis detected rearrangement of the 18q21 gene in 10 of 13 (77%) cases in which the t(14;18)(q32;q21) translocation was found by cytogenetic techniques. Our analysis also proved helpful in difficult karyotyping situations: (a) identifying the donor chromosome fragment as chromosome 18 band q21 in 4 of 9 (44%) cases that cytogenetically displayed a 14q+ chromosome of unknown origin, and (b) identifying a rearrangement of chromosome 18 band q21 in 12 of 18 (67%) cases that cytogenetically yielded no cells in metaphase. We also demonstrated three cases of submicroscopic rearrangement of the 18q21 gene. In our studies, patients with DLCLs and rearrangement of the 18q21 gene had a significantly higher incidence of extranodal involvement when compared with patients with DLCLs and no 18q21 gene rearrangement (P = 0.03).

A sensitive immunoblot technique for platelet glycoprotein IIIa (GPIIIa) was used to analyze the platelets of patients living in Israel who meet the diagnostic criteria for type I Glanzmann thrombasthenia. When reacted with solubilized normal platelets, a rabbit antiserum to GPIIIa identified a major band at molecular weight (mol wt) 90,000 and three additional minor bands at Mr 110,000, 81,000, and 64,000. The major band could not be detected, and the minor bands were either markedly reduced or absent in the platelet samples from 14 of the 15 patients from the Iraqi-Jewish population. In contrast, in all four Arab patients tested, the major band was detectable, although at markedly reduced levels, and the minor bands were either markedly reduced or absent; an additional minor band at mol wt 47,000 was also present in the platelets from these patients. One Iraqi-Jewish patient had a unique pattern in which two of the bands were present but reduced and two were undetectable. We conclude that the protein defect, and thus presumably the genetic defect, causing Glanzmann thrombasthenia in the majority of patients in the Iraqi-Jewish population differs from that in the Arab population, and we confirm that there is considerable biochemical heterogeneity among the patients who meet the criteria for type I Glanzmann thrombasthenia.

Posttransfusion purpura typically occurs in PLA1 negative blood recipients who have been previously immunized to the PLA1 antigen. Following transfusion, severe thrombocytopenia develops with the formation of anti-PLA1. Since the patients' platelets lack the PLA1 antigen, one would not expect this antibody to destroy autologous platelets. In this study we show that PLA1 antigen exists in stored blood and can absorb to PLA1 negative platelets making them PLA1 reactive. Incubating PLA1 (-) platelets with ultracentrifuged plasma from PLA1 (+) blood donors allowed anti-PLA1 to bind to PLA1 (-) platelets. Control plasma from PLA1 (-) blood donors did not lead to anti-PLA1 binding. Using an inhibition assay, we showed that stored blood contains PLA1 material that was not removed by ultracentrifugation. The material absorbing to PLA1 (-) platelets represented the PLA1 antigen, which was confirmed by Western blotting. After incubating plasma containing PLA1 antigen with PLA1 (-) platelets, reactivity at 95,000 D was observed. Native PLA1 (+) platelets showed a similar band. When PLA1 (-) platelets were incubated with plasma from a PLA1 (-) donor, this band was not present. These studies show that a soluble form of PLA1 antigen exists in stored blood that can absorb to PLA1 (-) platelets. Consequently, anti-PLA1 can bind to these platelets leading to thrombocytopenia. These observations may explain the autologous destruction of platelets in posttransfusion purpura.

To study the possible involvement of T lymphocytes in Philadelphia chromosome (Ph)-positive chronic myelocytic leukemia (CML) we analyzed the arrangement of the bcr gene in T cell and non-T cell samples of 12 CML patients. Although all the patients showed bcr rearrangements in non-T cell fractions, T cell populations lacked respective gene recombinations. Moreover, by Southern blot analyses using T cell receptor beta chain sequences our data indicate polyclonality of T cell samples from 11 of 12 cases; in one patient a clonal T cell population could be identified. These results suggest that T lineages of most Ph- positive CML patients are not derived from pluripotent stem cells involved in leukemogenesis and thus confirm previous investigations based on cytogenetic or glucose-6-phosphate dehydrogenase analyses. The demonstration of polyclonal T cell populations may reflect persistence of stem cells committed to differentiate only into T cells.

Human urinary erythropoietin has been highly purified by a combination of conventional purification methods and immunoadsorbent columns packed with hybridoma-produced antibodies against contaminants that seemed difficult to separate from erythropoietin by the usual means. By using the partially purified erythropoietin as an antigen, three hybridoma clones have been obtained that secrete monoclonal antibodies against erythropoietin. One of the clones has been quite stable, with a rapid growth rate and high production of antibody. Western blotting technique with monoclonal antibodies revealed occurrence of two species of erythropoietin. The monoclonal antibody will be useful as a probe for the purification of erythropoietin and for further studies of the hormone and its mechanism of action.

The binding of vitamin B12 by serum proteins was studied by separating Co57B12-enriched serum by Sephadex gel filtration, column chromatography with DEAE-cellulose, and paper electrophoresis. Each method of separation yielded two discrete B12-binding fractions. However, the analysis of each serum by all three separation technics indicated that one of the fractions was, in each case, bipartite. The "high" molecular weight B12-binding fraction defined by Sephadex gel filtration consisted of transcobalamin I and just part of the transcobalamin II fraction. The remaining portion of transcobalamin II was eluted from Sephadex gel in a "low" molecular weight fraction. Thus, transcobalamin II, equivalent to the β-globulin B12-binder, consisted of both "high" and "low" molecular weight components. This suggests that there are at least three serum proteins that can bind vitamin B12: two β-globulins, together comprising the transcobalamin II fraction and differing in molecular weight; and transcobalamin I.

Hereditary elliptocytosis is a heterogeneous disorder resulting from defects in the erythrocyte membrane skeleton. Although some cases of elliptocytosis result from defects in spectrin, the specific structural abnormality has yet to be identified in the majority of cases. Protein 4.1 plays an essential role in erythrocyte membrane physiology, and deficiencies have been implicated in only a few rare cases of elliptocytosis. By using 4.1 immunoblots and a 4.1 radioimmunoassay we identified distinct variants of protein 4.1 in 15 elliptocytic members of three US white families with the Rh-linked form of elliptocytosis. Elliptocytic members of family G were heterozygotes for a low-molecular weight (mol wt) 4.1 variant (65,000 to 68,000 daltons; normal, 80,000) inherited in linkage with the Rz phenotype. Elliptocytic members of family C expressed a simple partial deficiency of protein 4.1 (63% of the normal level) that was inherited in linkage with the r phenotype. Elliptocytic members of family N were heterozygotes for a high-mol wt 4.1 variant (100,000 daltons) also inherited in linkage with the r phenotype. These studies indicate that mutant forms of protein 4.1 are not uncommon in elliptocytosis among whites and that different kindreds probably express different mutations. The observed linkage of elliptocytosis and Rh blood type most likely results from the close proximities of the 4.1 gene (site of the mutation) and the Rh gene, which is located nearby on the short arm of chromosome 1.

twobands showed coagulant activity. Six murine monoclonal antibodies (Mabs) against HMWK were produced and purified. In immunoblotting studies. three Mabs bound to the isolated alkylated heavy chain and one to the alkylated light chain of HMWK. whereas the remainingtwo bound only to the single-chain or unreduced two-chain molecule. None of the Mabs inhibited the clotting activity of HMWK or its binding to kaolin. Two of the Mabs, one directed against the light chain and one against the heavy chain. were used as specific probes A POTENT vasoactive peptide, bnadykinin, is liberated in plasma from two precursor proteins, high- and low-mob wt kininogens (HMWK and LMWK), by the action of specific enzymes termed � Human HMWK and LMWK are single-chain glycoproteins, with mol wts of � I 1 0,000 to I 20,000 and 50,000 to 70,000, respectively. Each protein consists of three domains: an amino terminal heavy chain, a bradykinin moiety, and a carboxyb terminal bight chain.3’4 Based on cloning and sequence analysis of cDNAs for human high- and bow-mol-wt prekininogens, a complete identity in the amino acid sequence has been determined for a barge portion of the two molecules, including the heavy chain, the bradykinin sequence, and the first part of the carboxyl terminal region; the two proteins are transcribed from the same gene.5 The structural homology accounts for the recently discovered thiob protease inhibitory activity common to both proteins.� The unique structure of the light chain of HMWK, however, provides this protein with the function of accelerating the reactions of the contact phase of blood coagulation, whereby HMWK acts as a nonenzymatic cofacton by facibitating the proteobytic activations of factor XII, prekallikrein, and factor XI on a negatively charged surface.9”#{176} One of the activation products, kalliknein, potently cleaves HMWK at two specific sites to liberate bradykinin and to form a kinin-free protein.”’3 The cleavage occurs within a critical disulfide bridge so that single-chain HMWK is converted to a two-chain molecule, in which a heavy chain of -66,000 mob wt is disubfide-linked to a light chain of -55,000 mob wt. The light chain is further cleaved by kallikrein to a smaller chain of 45,000 mob wt.’3 The pathophysiologic role of plasma HMWK still remains largely unclear. The hereditary deficiency of this protein is not associated with a bleeding disorder.’4 Because bradykinin possesses a number of actions potentially involved in inflammation reactions, including vasodilation, increase in vascular permeability, and pain generation, a robe for HMWK and the other proteins of the contact activation system in the to study HMWK in plasma samples using an immunoblotting technique. The anti-light chain Mab identified two distinct bands (-120.000 and -.105.000 mol wt) in normal human plasma. but not in plasma from patients with hereditary HMWK deficiency. The anti-heavy chain Mab detected two additional bands (-60.000 and -54.000 mol wt) corresponding to low-mol-wt kininogen (LMWK) in normal plasma. A sensitive and specific quantitative immunoblotting assay of HMWK antigen in plasma was developed. Moreover, the immunoblotting technique with the anti-light chain Mab was used to detect the cleavage of HMWK in plasma samples after in vitro or in vivo activation of the contact system. The anti-light chain Mab demonstrated in vivo activation and cleavage of HMWK during an angioedema attack in a patient with hereditary angioedema and Cl -inhibitor deficiency. S 1986 by Grune & Stratton, Inc. pathogenesis of several pathological conditions has been suggested.’4 Evidence of HMWK cleavage in vivo, however, has been indirect only and has involved functional and/on immunological measurements of plasma levels of the protein or its released product, bnadykinin. We have prepared a series of monoclonab antibodies (Mabs) against HMWK and describe here their use as specific qualitative and quantitative probes for HMWK in plasma samples subjected to immunobbotting.