British Journal of HaematologyVolume 86, Issue 1 p. 1-5 SHOULD ALL PREGNANT WOMEN BE TESTED FOR THEIR PLATELET PLA (Zw, HPA-1) PHENOTYPE? Frances Flug, Frances Flug Departments of Pediatrics, New York University Medical School, New York, N.Y., U.S.A.Search for more papers by this authorMargaret Karpatkin, Corresponding Author Margaret Karpatkin Departments of Pediatrics, New York University Medical School, New York, N.Y., U.S.A.Dr Simon Karpatkin, Department of Medicine, New York University Medical School, 550 First Avenue, New York, NY 10016, USA.Search for more papers by this authorSimon Karpatkin, Simon Karpatkin Medicine, New York University Medical School, New York, N.Y., U.S.A.Search for more papers by this author Frances Flug, Frances Flug Departments of Pediatrics, New York University Medical School, New York, N.Y., U.S.A.Search for more papers by this authorMargaret Karpatkin, Corresponding Author Margaret Karpatkin Departments of Pediatrics, New York University Medical School, New York, N.Y., U.S.A.Dr Simon Karpatkin, Department of Medicine, New York University Medical School, 550 First Avenue, New York, NY 10016, USA.Search for more papers by this authorSimon Karpatkin, Simon Karpatkin Medicine, New York University Medical School, New York, N.Y., U.S.A.Search for more papers by this author First published: January 1994 https://doi.org/10.1111/j.1365-2141.1994.tb03244.xCitations: 37AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat References Blanchette, V.S., Chen, L., DeFriedberg, Z.S., Hogan, V.A., Trudel, E. & Decary, F. (1990) Alloimmunization to the PLA1 platelet antigen: results of a prospective study. British Journal of Haematology., 74, 209–215. Beer, J. & Coller, B.S. (1989) Evidence that platelet glycoprotein IIIa has a large disulfide-bounded loop that is susceptible to proteolytic cleavage. Journal of Biological Chemistry, 264, 17564. Bowditch, R.D., Tani, P.H., Halloran, C.E., Freilinger, A.L., III, McMillan, R. & Ginsberg, M.N. (1992) Localization of a PLA1 epitope to the amino terminal 66 residues of platelet GPIIIa. Blood, 79, 559–562. Burrows, R.F. & Kelton, J.G. (1988) Incidentally detected thrombocytopenia in healthy mothers and their infants. New England Journal of Medicine, 319, 142–145. Bussel, J.P., for the Neonatal Immune Thrombocytopenia Study Group (1988) Neonate alloimmune thrombocytopenia: a prospective case accumulation study. Pediatric Research, 23, 337A. Bussel, J.P., for the Neonatal Immune Thrombocytopenia Working Party (1987) Neonatal alloimmune thrombocytopenia (NAT): Information derived from a prospective international registry. Blood, 70, 336a. Bussel, J.P., Berkowitz, R.L., McFarland, J.G., Lynch, L. & Chitkara, U. (1988) Antenatal treatment of neonatal alloimmune thrombocytopenia. New England Journal of Medicine, 319, 1374–1378. Daffos, F., Capella-Pavlovsky, M. & Forestier, F. (1985) Fetal blood sampling during pregnancy with use of a needle guided by ultrasound: a study of 606 consecutive cases. American Journal of Obstetrics and Gynecology, 153, 655–660. Daffos, F., Forestier, F., Kaplan, C. & Cox, W. (1988) Prenatal diagnosis and management of bleeding disorders with fetal blood sampling. American Journal of Obstetrics and Gynaecology, 158, 939–946. Dancis, A., Ehmann, C., Ferziger, R., Grima, K. & Karpatkin, S. (1988) GPIIIa related PLA1 antigens with different molecular weights: studies in platelets, endothelial cells, and megakaryocytes. Blood, 71, 1056–1061. Decary, F. (1986) Is HLA-DR3 a risk factor in PLA1-negative pregnant women Current Studies in Haematology and Blood Transfusion, 52, 78–86. De Wall, L.P., Van Dalen, C.M., Engelfriet, C.P. & Von dem Borne, A.E.G.K. (1986) Alloimmunization against platelet specific Zwa antigen resulting in neonatal alloimmune thrombocytopenia or post transfusion purpura is associated with the supertypic DRw52 antigen including DR3 and DRw6. Human Immunology, 17, 45–53. Flug, F., Espinola, R., Liu, L-X, DaRossa, R., SinQuee, C. & Karpatkin, S. (1991) A 13-mer peptide straddling the leucine33/proline33 polymorphism in GPIIIa does not define the PLA1 epitope. Blood, 77, 1964–1969. Goldberger, A., Kolodziej, M., Poncz, M., Bennett, J.S. & Newman, P.J. (1991) Effect of single amino acid substitutions on the formation of the PLA and Bak allantigenic epitopes. Blood, 78, 681–687. Herman, J.H., Jumbelic, M.I., Ancona, R.J. & Kickler, T.S. (1986) In utero cerebral hemorrhage in alloimmune thrombocytopenia. American Journal of Pediatric Hematology, 8, 312–317. Kaplan, C., Daffos, F., Forestier, F., Cox, W.L., Lyon-Caer, D. & Dupuy-Montbrun, M.D. (1988) Management of alloimmune thrombocytopenia: antenatal diagnosis and in utero transfusion of maternal platelets. Blood, 72, 340–343. Kornecki, E., Chung, S-Y., Holt, J.C., Cierniewski, C.S., Tuszynski, G.P. & Niewiarowski, S. (1985) Identification of the PLA1 alloantigen domain on a 66 kD protein derived from glycoprotein IIIa of human platelets. Biochimica et Biophysica Acta, 818, 285–290. Kroll, H., Giers, G., Bald, R., Kiefel, V., Hoch, J., Hanland, P., Hansmann, M. & Mueller-Eckhardt, C. (1993) Intravenous IgG during pregnancy for fetal alloimmune thrombocytopenic purpura. Thrombosis and Hemostasis, 69, 1625A. Kunicki, T.J. & Aster, R.H. (1978) Deletion of the platelet specific alloantigen PLA1 from platelets in Glanzmann's thrombasthenia. Journal of Clinical Investigation, 61, 1225–1231. Kunicki, T.J. & Aster R.H. (1979) Isolation and immunologic characterization of the human platelet alloantigen, PLA1. Molecular Immunology, 16, 353–360. Liu, L-X. Nardi, M., Flug, F. & Karpatkin, S. (1992a) Development of a monoclonal antibody capable of differentiating platelet PLA1/PLA1/PL1/PL2 and PL2/PLA2. British Journal of Haematology, 81, 113–117. Liu, L-X., Nardi, M.A., Flug, F. & Karpatkin, S. (1992b) A monoclonal antibody (LK-4) which differentiaties PLA1 from PLA2 platelet extracts but not intact platelets. Thrombosis Research, 66, 309–320. Lynch, L., Bussel, J.B., McFarland, J.G., Chitkara, U. & Berkowitz, R.L. (1992) Antenatal treatment of alloimmune thrombocytopenia. Obstetrics and Gynecology, 80, 67–71. McFarland, J.G., Frenzke, M. & Aster R.H. (1989) Testing of maternal sera in pregnancies at risk for neonatal alloimmune thrombocytopenia. Transfusion, 29, 128–133. Mir, N. & Samson, D. (1988) Failure of antenatal high-dose immunoglobulin to improve fetal platelet count in neonatal alloimmune thrombocytopenia. Vox Sanguinis, 55, 188–189. Mueller-Eckhardt, C., Grubert, A., Weisheit, M., Mueller-Eckhardt, G., Kiefel, V., Kroll, H., Schmidt, S. & Santoso, S. (1989) 348 cases of suspected neonatal alloimmune thrombocytopenia. Lancet., i, 363–366. Mueller-Eckhardt, C., Mueller-Eckhardt, G., Willer-Ohff, H., Horz, A., Kuenzlen, E., O'Neill, G.J. & Schendel, D.J. (1985) Immunogenicity of an immune response to the human platelet antigen Zwa is strongly associated with HLA-B8 and DR3. Tissue Antigens, 26, 71–76. Muller, J.Y., Reznikoff-Eitebant, M.F., Patereau, C., Dangu, C. & Chesnel, N. (1985) Thrombopenies, neonates alloimmunes. Etude clinique et biologique de 84 cas. Presse Medicale, 14, 83–86. Murphy, M.F., Metcalfe, P., Waters, A.H., Ord, J., Hambley, H. & Nicolaides, K. (1993) Antenatal management of severe fetomaternal alloimmune thrombocytopenia: HLA incompatibility may affect responses to fetal platelet transfusions. Blood, 81, 2174–2179. Newman, P.J., Martin, L.S., Knipp, M.A. & Kahn, R.A. (1985) Studies on the nature of the human platelet alloantigen PLA1:localization to a 17, 000 dalton polypeptide. Molecular Immunology, 22, 719–729. Newman, P.J., Derbes, R.S. & Aster, R.H. (1989) The human platelet alloantigens, PLA1 and PLA2 are associated with a Leucine33/Proline33 amino acid polymorphism in membrane glycoprotein IIIa, and are distinguishable by DNA typing, Journal of Clinical Investigation, 83, 1778–1781. Niewiarowski, S., Norton, K.J., Eckhardt, A., Lukasiewicz, H., Holt, J.C. & Kornecki, E. (1989) Structural and functional characteristics of major platelet membrane components derived by limited proteolysis of glycoprotein IIIa. Biochimica et Biophysica Acta, 983, 91–99. Nicolini, V., Tannirandorn, Y., Gonzalez, P., Fisk, N.M., Beacham, J., Letsky, E.A. & Rodeck, C.H. (1990) Continuing controversy in alloimmune thrombocytopenia: fetal hyperimmunoglobulinemia fails to prevent thrombocytopenia. American Journal of Obstetrics and Gynecology, 163, 1144–1146. Pearson, H.A., Shulman, N.R., Marder, V.J. & Cone, T.E. (1964) Isoimmune neonatal thrombocytopenic purpura: clinical and therapeutic considerations. Blood, 23, 154–177. Reznikoff-Etievant, M.F., Dangu, C. & Lobet, R. (1981) HLA-B8 antigen and anti-PLA1 allo-immunization. Tissue Antigens, 18, 66–68. Reznikoff-Etievant, M.F., Kaplan, C., Muller, J.Y., Daffos, F. & Forestier, F. (1988) Alloimmune thrombocytopenias, definition of a group at risk; a prospective study. Current Studies in Hematology and Blood Transfusion, 55, 119–124. Reznikoff-Etievant, M.F., Muller, J.Y., Julien, F. & Patereou, C. (1983) An immune response linked to MHC in man. Tissue Antigens, 22, 312–314. Ryckewaert, J-J., Schweizer, B., Chapel, A. & Marguerie, G. (1992) Production of anti-PLA monoclonal antibodies. Journal of Laboratory and Clinical Medicine, 119, 52–56. Scott, J.R., Cruikshank, D.P., Kochenoor, N.K., Pitkin, R.M. & Warenski, J.C. (1980) Fetal platelet counts in the obstetric management of immunologic thrombocytopenic purpura. American Journal of Obstetrics and Gynecology, 136, 494–499. Shibata, Y., Matsuda, I., Miyaje, T. & Ichikaw, Y. (1986) Yuka, a new platelet antigen involved in two cases of neonatal alloimmune thrombocytopenia. Vox Sanguinis, 50, 177–180. Shulman, N.R. & Jordan, J.V. (1987) Platelet immunology. Hemostasis and Thrombosis (ed. by R. W. Colman, J. Hirsch, V. J. Marder and E. W. Salzman), pp. 476–483. Lippincott, Philadelphia . Shulman, N.R., Marder, V.J., Hiller, M.C. & Collier, E.M. (1964) Platelet and leukocyte isoantigens and their antibodies: serologic, physiologic and clinical studies. Progress in Hematology, 4, 222–304. Sia, C.G., Amigo, N.C., Harper, R.G., Farahani, G. & Kochen, J. (1985) Failure of cesarian section to prevent intracranial hemorrhage in siblings with isoimmune neonatal thrombocytopenia. American Journal of Obstetrics and Gynecology, 153, 79–81. Tanning, E., Antonsen, H., Petersen, S., Svejgaard, A. & Thomson, M. (1983) HLA antigens and maternal antibodies in alloimmune thrombocytopenia. Tissue Antigens, 21, 351–359. Van Loghem, J.J., Dorfmeyer, H., Van der Hart, M. & Schreider, F. (1959) Serological and genetical studies on a platelet antigen (Zw). Vox Sanguinis, 4, 161–169. Von dem Borne, A.E.G.K., Van Leeuwen, W.F., Von Riesz, L.E., Van Boxtel, C.J. & Engelfriet, C.P. (1981) Neonatal alloimmune thrombocytopenia: detection and characterization of the responsible antibodies by the platelet immunofluorescence test. Blood, 57, 649–656. Zalneraitis, E.L., Young, R.S.K. & Krishnamoorthy, K.S. (1979) Intracranial hemorrhage in utero as a complication of isoimmune thrombocytopenia. Journal of Pediatrics, 95, 611–614. Citing Literature Volume86, Issue1January 1994Pages 1-5 ReferencesRelatedInformation

A unique murine monoclonal antibody (LK-4) is described which differentiates PLA1/PLA1 platelet extracts from PLA2/PLA2 and PLA1/PLA2 platelet extracts on solid phase ELISA and immunoblot at the 100kD GPIIIa location, but not on intact platelets. LK-4 reacts equally with intact PLA1/PLA2 and PLA2/PLA2 platelets. Adsorbtion of LK-4 with PLA1/PLA1 platelets results in loss of reactivity for intact platelets as well as platelet extracts on ELISA or immunoblot. LK-4 inhibits platelet aggregation induced by ADP, epinephrine, collagen and thrombin, suggesting reactivity at or near the fibrinogen binding site on GPIIIa. It is suggested the LK-4 reacts with a conformation-induced common epitope for PLA1 and PLA2 on GPIIIa, with loss of this conformation for PLA2 GPIIIa following solubilization with Triton X-100.

A monoclonal antibody, LK-4, has been developed which distinguishes platelet PLA1/PLA1, PLA1/PLA2 and PLA2/PLA2 genotypes on platelet glycoprotein GPIIIa of Triton-solubilized platelet extracts. An ELISA assay has been developed which traps GPIIIa with Concanavalin A, enriching the platelet extract for the PLA antigens. A second monoclonal antibody, DEK-10, which reacts equally with GPIIIa of PLA1/PLA1 and PLA2/PLA2 platelet extracts is employed as an internal standard to correct for individual differences in GPIIIa content, GPIIIa extracted by Triton X-100 and GPIIIa trapped with Concanavalin A. This ELISA assay clearly differentiated 11 different PLA1/PLA1 subjects from eight PLA2/PLA2 women with a history of neonatal alloimmune thrombocytopenia as well as six unrelated obligate heterozygotes and should be useful in evaluating the PLA genotype of pregnant women and their families.

A monoclonal antibody, LK-4, has been developed which distinguishes platelet PLA1/PLA1, PLA1/PLA2 and PLA2/PLA2 genotypes on platelet glycoprotein GPIIIa of Triton-solubilized platelet extracts. An ELISA assay has been developed which traps GPIIIa with Concanavalin A, enriching the platelet extract for the PLA antigens. A second monoclonal antibody. DEK-10, which reacts equally with GPIIIa of PLA1/PLA1 and PLA2/PLA2 platelet extracts is employed as an internal standard to correct for individual differences in GPIIIa content, GPIIIa extracted by Triton X-100 and GPIIIa trapped with Concanavalin A. This ELISA assay clearly differentiated 11 different PLA1/PLA1 subjects from eight PLA2/PLA2 women with a history of neonatal alloimmune thrombocytopenia as well as six unrelated obligate heterozygotes and should be useful in evaluating the PLA genotype of pregnant women and their families.

HE PLA SYSTEM is a diallelic platelet antigen system T comprised of PLA' and PLA' that is most often implicated in two clinical syndromes. Neonatal alloimmune thrombocytopenia (NAIT) is a severe bleeding disorder in newborns that occurs when fetal platelets express PLA' while maternal platelets express PLA'.' Posttransfusion purpura (PTP) results in severe thrombocytopenia in a transfused patient based on a mismatch of the PLA antigen between donor and recipient platelet^.'^^ PLA' and PLA' are present on an integral platelet membrane glycoprotein (GP) called GPIIIa that has an apparent Mr of 100,000.4 Newman et al have recently demonstrated a T -+ C base change in the sequence of GPIIIa associated with the PLA'PLA' platelet allotypes, respectively.' This change would result in a leucine/proline polymorphism in the N-terminal portion of GPIIIa at amino acid position 33. In this report we confirm the above finding but demon- strate that a 13-mer polypeptide straddling this polymor- phism does not define the PLA' epitope. Two amino acid peptides inclusive of the leucine/proline polymorphism

We confirm the recent report (J Clin Invest 83:1778, 1989) of a polymorphism at amino acid 33 of platelet GPIIIa associated with the PLA1/PLA2 phenotype by using the polymerase chain reaction on cDNA derived from platelet RNA, using the base-pair primers 105–129 and 452- 428. Platelet cDNA from three PLA2-homozygous individuals, when digested with Nci I, gave two bands of 256 bp and 91 bp, whereas eight PLA1 cDNAs gave a single band of 347 bp. Two 13-mer amino acid peptides straddling the amino acid polymorphism: SDEALP (L/P) GSPRCD were synthesized for epitope studies. Two mouse polyclonal antibodies were raised: one against the PLA1-associated peptide, the other against the PLA2 peptide. Both antibodies react with either peptide, as well as with both PLA1 and PLA2 platelets. The PLA1 peptide did not block the binding of two different human anti-PLA1 antibodies to the 100-Kd GPIIIa band on immunoblot of platelet extracts; neither did it block the binding of the same antibodies to PLA1-platelet extracts in an enzyme-linked immunosorbent assay. Further studies were performed on the PLA1 epitope following subtilisin digestion of purified GPIIIa. A 55-Kd fragment was obtained that retained the PLA1 epitope as well as the first 13 N-terminal amino acids of GPIIIa. Reduction of the 55-Kd fragment resulted in loss of the PLA1 epitope with production of a 67- Kd, 21-Kd, and 10-Kd band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The 55-Kd band does not react with LK-2, a monoclonal antibody versus GPIIIa that inhibits adenosine diphosphate, collagen, epinephrine, and thrombin-induced aggregation. Thus, the PLA1 epitope is conformation-induced, resides on an N-terminal 55-Kd fragment composed of two or more peptides held together by -SH bonds, and is not required for platelet aggregation.

Publisher Summary This chapter reviews the physical and biological properties of thyroid hormone receptors and the relationship of the receptor to the avian erythroblastosis virus (AEV) v-erbA gene. The properties of thyroid hormone nuclear receptors derived from studies using GHi and GC cells are described in the chapter. The thyroid hormone receptor is related to the avian erythroblastosis virus v-erbA gene. The AEV—a defective leukemia retrovirus—induces sarcomas and erythroblastosis in vivo and induces the transformation of fibroblasts and erythroblasts to neoplastic phenotypes in vitro. The chapter also reviews the studies in which the rat growth hormone gene was used as a model to identify cis-acting DNA sequences and transacting regulatory proteins that are essential for cell-specific expression and transcriptional stimulation of the gene by the thyroid hormone. The thyroid hormone regulates the growth hormone gene expression at the transcriptional level. A detailed functional and protein-DNA footprint analysis of the elements that are involved in mediating thyroid hormone and cell-specific basal expression of the gene is also presented in the chapter.

In GC cells, a growth hormone-producing rat pituitary cell line, 3,5,3'-triiodo-L-thyronine (L-T3) rapidly stimulates the transcription rate of the growth hormone gene which parallels the level of chromatin-associated L-T3-receptor complexes (Yaffe, B. M., and Samuels, H. H. (1984) J. Biol. Chem. 259, 6284-6291). In this study we have functionally mapped the elements of the gene which are involved in mediating basal and hormone-regulated expression. Stable transformation studies indicate that transcriptional regulation of the gene by L-T3 is mediated by sequences in the 5'-flanking region. Transient expression studies were performed using a series of chimeric plasmids in which 5'-flanking DNA was ligated to the chloramphenicol acetyltransferase gene. Transient expression occurred only in cells which expressed the endogenous growth hormone gene. Sequences between -104 and +7 were found to be essential for basal expression. One of the most highly conserved regions (-105 to -145) contains elements which further enhance the level of basal expression but are not necessary for regulated expression by L-T3. DNA between -210 and -181 was found to be essential for stimulation by L-T3 and was shown to function most efficiently with the homologous rat growth hormone promoter (-104 to +7). Sequences from -206 to -198 show about 80% homology with a sequence in the 5'-flanking region of two other rat genes which are regulated by thyroid hormone. Glucocorticoid hormones, which also transcriptionally stimulate the rat growth hormone gene, elicited only minimal effects in both stable and transient expression studies. This suggests that the elements which mediate glucocorticoid regulation of the endogenous gene are found either upstream of the cloned 5'-flanking region (1800 base pairs) or 3' of the cap site.

We investigated the rearrangement patterns of the gene coding for the beta chain of the T cell receptor (T beta) in 11 patients with T-cell derived chronic lymphoproliferative disorders, including T-cell prolymphocytic leukemia (T-PLL) and T-cell chronic lymphocytic leukemia (T-CLL). We found that all five cases of T-PLL, and five of six cases of T-CLL, displayed T beta-gene rearrangements, clearly establishing their monoclonal nature. Clonality could not be determined in one case of T-CLL where the T beta gene was found unrearranged. Our results demonstrate that the majority of cases of both clinically aggressive T-PLL and clinically indolent T-CLL are monoclonal. These results suggest that the analysis of T beta gene rearrangements represents a valid tool for the differential diagnosis and clinical monitoring of T-cell derived chronic lymphoproliferative diseases.

Ig gene rearrangements represent markers of lineage, clonality, and differentiation of B cells, allowing a molecular diagnosis and immunogenotypic classification of B-cell neoplasms. We sought to apply a similar approach to the study of T-cell populations by analyzing rearrangements of the T-cell receptor beta-chain (T beta) gene. Our analysis, by Southern blotting hybridization using T beta-specific probes of DNAs from polyclonal T cells and from 12 T-cell tumors, indicates that T beta gene rearrangement patterns can be used as markers of (i) lineage, allowing the identification of polyclonal T-cell populations, and (ii) clonality, allowing the detection of monoclonal T-cell tumors. In addition, our data indicate that T beta gene rearrangements represent early and general markers of T-cell differentiation since they are detectable in histologically different tumors at all stages of T-cell development. The ability to determine lineage, clonality, and stage of differentiation has significant implications for future experimental and clinical studies on normal and neoplastic T cells.

We investigated the neoplastic cells obtained from 37 cases of 'non-B, non-T' (SIg-E-) acute lymphoblastic leukemia (ALL) for their expression of 13 distinct monoclonal antibody defined B lymphocyte associated differentiation antigens. We correlated the expression of these B cell antigens with terminal deoxynucleotidyl transferase (TdT), HLA-DR antigen, common ALL antigen (cALLa), and cytoplasmic mu heavy chain (Cu) expression by these neoplastic cells. In this way, we were able to describe a hierarchy of B lymphocyte associated differentiation antigens as well as the marked phenotypic heterogeneity of 'non-B, non-T' ALL. TdT and HLA-DR are expressed throughout the stages of B cell differentiation represented by 'non-B, non-T' ALL. The earliest B cell antigen appears to be Leu 12 (B4) followed by BA-2 and then BL2. OKB2, BL1 and BA-1 are acquired next, followed by B1, BL3, cALLa and Cu. BL7 appears just prior to SIg. OKB1, OKB4, OKB7 and BL4 appear at or after the time of SIg expression and hence are not expressed by 'non-B, non-T' ALL cells. This developmental hierarchy is supported by the results of phorbol ester (TPA) induction studies. Thus, cases of 'non-B, non-T' ALL constitute a useful model for probing the hierarchal expression of B cell antigens and delineating the B cell developmental pathway(s).

The authors performed immunophenotypic, functional, and molecular analysis of the neoplastic cells from 20 cases of SIg-, E-("null-cell") non-Hodgkin's lymphoma (NHL) in order to determine their lineage, better define this category of NHL, and evaluate the lineage specificity of selected phenotypic markers and the individual and collective utility of these approaches. They assigned 4 cases to the T-cell lineage, and 15 cases to the B-cell lineage, and 1 case remained indeterminant on the basis of immunophenotypic analysis. The cells from 2 cases assigned to the T-cell lineage expressed unusual phenotypes, but their T-cell derivation was confirmed by the demonstration of helper function in vitro. The 15 cases assigned to the B-cell lineage expressed a variety of B-cell-associated antigens, consistent with various stages of B-cell differentiation. Monoclonal antibodies OKT3, OKT4, OKT6, and OKT8 exhibited T-cell lineage restriction; and monoclonal antibodies OKB2, BL1, and B1 exhibited B-cell lineage restriction. Ia, TdT, cALLa, OKT9, and OKT10 exhibited lineage infidelity. Southern blot analysis for immunoglobulin heavy chain gene rearrangements confirmed 18 of the 19 lineage assignments made by immunophenotypic analysis and suggested that the 1 case of indeterminate phenotype was a B-cell neoplasm. One T-cell (OKT3+, T4+) neoplasm exhibited rearranged immunoglobulin heavy chain genes. Thus, neither immunophenotypic analysis nor the demonstration of rearranged immunoglobulin heavy chain genes alone permitted the satisfactory lineage assignment of every case of SIg-, E- NHL. However, combined immunophenotypic, functional, and genotypic analysis allowed us to assign every SIg-, E-NHL to the B- or T-cell lineage and to demonstrate that truly "null-cell" NHLs are probably very uncommon.