Monoclonal antibody (G10) to a common antigen of human squamous cell carcinoma: binding of the antibody to the H type 2 blood group determinant.

The IgM monoclonal antibody G10 was raised against the human squamous cell carcinoma (SCC) cell line UM-SCC-1. In initial screening against cultured cells, G10 bound to 2 SCC lines (UM-SCC-1 and UM-SCC-13) and 1 pancreatic carcinoma line (UM-PAd-1) but not to cultured fibroblasts (WI-38), ovarian carcinoma cells (SK-OV-3), or malignant melanoma cells (SK-MEL-28 and MeWo). In subsequent tests against cultured cell lines, G10 gave positive reactions with 30 of 33 SCC lines but only 4 of 29 non-SCC lines. The non-SCC lines that bound G10 were UM-PAd-1, 2 transitional cell carcinoma lines (T24 and RT4), and 1 melanoma line (SK-MEL-22). When tested against cultures derived from normal skin or mucosa, G10 was reactive with the epitheloid squamous cells but not with the fibroblasts in each culture. The antigen defined by antibody G10 was stable to fixation with Formalin, and its distribution in tissue sections was examined with the use of immunoperoxidase assays. All SCC biopsy specimens examined in this way were reactive with antibody G10. In similar tests against sections of fixed normal tissues, G10 stained the superficial squamous cells of the epidermis and the basal and suprabasal layers of mucosal squamous epithelial cells from the esophagus. All layers of the laryngeal epithelium were positive. Endothelial cells and certain glandular cells were also positive for G10 binding. G10 agglutinated human red blood cells of all blood groups except those from individuals of the Bombay group (Oh) who lack the H blood group determinant. Against defined oligosaccharides, G10 bound strongly only to the monofucosyl H type 2 structure and was slightly cross-reactive with the synthetic difucosyl H type 2 or Y structure. These results are consistent with previous reports of blood group antigen tissue distribution and indicate that the H type 2 determinant is expressed by all or nearly all mucosal squamous cancers. Less frequent expression by cells of other tumor types may correlate with tissue-specific activation of the H gene-specified fucosyltransferase.

Reactivity of FDA-approved anti-D reagents with partial D red blood cells.

Individuals whose RBCs are characterized as having a partial D phenotype may make anti-D if exposed to normal D+ RBCs; thus it is desirable that they be typed as D- should they require blood transfusion or Rh immune globulin (RhIG) prophylaxis. Further, use of different anti-D reagents by blood centers and transfusion services can account for FDA-reportable errors. For this study, anti- D reagents for use in tube tests were obtained from three U.S. manufacturers. They included three examples of IgM monoclonal anti-D blended with monoclonal IgG anti-D, one IgM monoclonal anti-D blended with polyclonal IgG anti-D, and two reagents formulated with human anti-D in a high-protein diluent. One anti- D formulated for use by gel column technology was also tested. Direct agglutination tests by tube or gel were strongly positive (scores 9-12), with partial D RBCs of types DII, DIIIa, DIIIb, and DIVa. No reagent anti-D caused direct agglutination of DVI type 1, DVI type 2, or DFR phenotype RBCs. One tube anti-D reagent formulated with an IgM monoclonal anti-D plus a polyclonal IgG anti-D failed to cause direct agglutination of DVa, DBT, and R(0)(Har) RBCs, while DVa RBCs reacted weakly with two high-protein reagents formulated with human IgG anti-D. In contrast, the anti-D used by gel column technology was strongly reactive (score 11) with DVa, DBT, and R(0)(Har) RBCs. The single monoclonal IgM-polyclonal IgG blended anti-D and the two high-protein reagents were also the only reagents that failed to react with R(0)(Har) RBCs by the IAT. Elimination of the test for weak D on all patient samples, using currently available FDA-licensed reagents, will ensure that partial D category VI (DVI) patients will type as D- for the purpose of RhIG prophylaxis and blood transfusion. However, RBCs of other partial D phenotypes will be classified as D+ in direct agglutination tests with some, if not all, currently available reagents. Testing donors for weak expression of D continues to be required, albeit that Rh alloimmunization by RBCs with a weak or partial D phenotype is uncommon. Further, because of differences in performance characteristics among FDA-approved reagents, conflicts between donor center D typing and transfusion service confirmatory test results are inevitable.

On a much higher than reported incidence of anti-c in R1R1 patients with anti-E.

A previous study involving tube IATs, untreated RBCs, and a low ionic-strength additive reagent revealed that approximately one-third of R(1)R(1) patients with anti-E have a concomitant anti-c. However, the current study finds a much higher incidence of anti-c in such patients, using gel technology in conjunction with ficin-pretreated RBCs. Results of antibody identification studies and transfusion records of 82 R(1)R(1) patients with anti-E were reviewed. Serologic test methods included a LISS wash solution for tube IATs (15 min at 37 degrees C, anti-IgG), ficin-tube IATs (30 min at 37 degrees C, anti-IgG + anti-C3), and gel IATs (untreated or ficin-treated RBCs or both, anti-IgG gels). LISS-tube or gel IATs with untreated RBCs revealed anti-c in 32 patients with anti-E. When gel-IAT and ficin-pretreated RBCs were used, 21 additional patients with anti-E were found to have anti-c. In samples from 26 R(1)R(1) patients with anti-E, anti-c was not demonstrable by ficin-gel IATs, and in 3 cases, the ficin-gel tests were inconclusive. In five cases in which E- RBCs not tested for c antigen were transfused to patients found by ficin-gel IAT to be without anti-c, all subsequently performed crossmatches with E-, c-untested RBCs were compatible. The incidence of anti-c in R(1)R(1) patients with anti-E in this study was 32 of 82 (39%) with untreated RBCs and 53 of 82 (65%) when the ficin gel data were included. The latter is significantly higher than the 32 percent incidence previously reported (p = 0.0001). Accordingly, all patients at our facility with an Rh antibody are now tested for those additional Rh antibodies they can make, as predicted from their Rh phenotype. The data from this study strongly support the selection of R(1)R(1) RBCs for all c- patients with anti-E.

Assay of anti-D using the Technicon Auto-Analyzer and the international standard anti-D typing serum.

A method is described for the automated quantitation of the anti-D content of sera. This procedure is far more reproducible than current manual techniques. The use of the International Standard anti-D typing serum in the calibration of a working standard enables results to be expressed in mug/ml of antibody protein.

Anti-Yta does not cause hemolytic disease of the newborn.

Anti-Yta has been reported to cause hemolytic disease of the newborn. We present a pregnancy complicated by anti-Yta alloimmunization. Based on reassuring monocyte monolayer assay results, the pregnancy was followed without invasive testing. The infant subsequently had a benign course in the nursery. We conclude that anti-Yta did not cause hemolytic disease of the newborn.

Peanut lectin agglutinin and alpha-lactalbumin. Binding and immunohistochemical localization in breast tissues.

We analyzed 31 breast lesions immunohistochemically for alpha-lactalbumin (ALA) production and T-antigen presence as defined by peanut lectin agglutinin (PNA). Most cases of well-differentiated infiltrating carcinomas (14 of 17) and of poorly differentiated carcinomas (two of four) showed positive immunoreactivity for ALA; ALA was also localized in three cases of normal breast tissues, in two fibroadenomas, and in five intraductal carcinomas. The PNA binding pattern was primarily along the luminal cytoplasmic cell membrane in normal breasts and in fibroadenomas. We saw both cell membrane and cytoplasmic binding in carcinomas. In seven cases of primary and metastatic carcinomas to axillary lymph nodes, both primary and metastatic tumors expressed T antigen and produced ALA. These studies indicate that ALA can be used as a diagnostic marker in breast carcinoma and that the PNA binding pattern in normal and benign breast tissues is primarily at the luminal cytoplasmic cell membrane, whereas both cytoplasmic and cell membrane binding are present in carcinomas. The production of ALA does not appear directly related to T-antigen expression or to metastatic disease.

PEG adsorption of autoantibodies causes loss of concomitant alloantibody.

Use of polyethylene glycol (PEG) to promote adsorption of autoantibodies is reported to give good recovery of concomitant alloantibodies. In initial experiments, PEG and ZZAP (Ficin and DTT) adsorption procedures were compared for removal of autoantibody and recovery of alloantibody. Postadsorption studies (n = 11) were performed and hemagglutination scores compared. In subsequent studies, equal volumes of alloantibody containing sera, PEG, and antigen-negative red blood cells (RBCs) were used in twofold adsorption experiments. Saline was substituted for PEG for control purposes. Postadsorption titers and immunoglobulin levels were determined. Autoantibodies were completely removed by both methods (n = 5); better by PEG (n = 3); better by ZZAP (n = 1); and not adsorbed (n = 1), and partially adsorbed by both (n = 1). Alloantibody recovery was comparable in three cases (E, K, Jka) but weaker by at least one reaction grade in four (K,E, Jka, and antibody to low-frequency antigen). The latter anti-Jka reacted 1+ with Jk(a+b+) RBCs after ZZAP adsorption but was nonreactive with the same RBCs following PEG adsorption. Titers of six alloantibodies adsorbed with antigen-negative RBCs in PEG were markedly weaker (range 2 to 8) compared to saline controls (range 4 to 32). IgG levels for PEG adsorbed (range 128 to 243 mg/dL) were 50% lower than controls (range 265 to 505 mg/dL). Although PEG adsorption is effective in removing autoantibody, the precipitation of immunoglobulin by PEG may result in failure to detect underlying alloantibody.

The gel test: use in the identification of unexpected antibodies to blood group antigens.

The IgG GEL test was compared with the LISS tube test (Löw and Messeter's low-ionic-strength saline) for antibody identification. The suitability of red blood cells (RBCs) pretreated with ficin, dithiothreitol (DTT), or chloroquine diphosphate (CDP) also was assessed for use in the GEL test. In addition, time-in-motion studies were performed comparing GEL (12 panels per batch) with polyethylene glycol (PEG) tube tests (3 panels per batch). In 57 antibody identification studies, there were 63 GEL+ LISS+, 2 GEL+ LISS-, and 6 GEL-LISS+ antibodies. Among the GEL+ LISS+ antibodies were 19 that yielded stronger reactions in GEL than in LISS; by virtue of their specificity, 14 of these are considered potentially significant: D, 5 E, 2 e, 2 Jka, 2 S, K, and Fya. There were 38 antibodies that yielded equivalent results by both methods, including 31 that are considered potentially significant. Of six antibodies with significantly greater reactivity in LISS, there were three anti-Rh and three that are considered harmless with respect to transfusion management. The two GEL+ LISS- antibodies (anti-Jkb) were potentially significant. GEL- LISS+ reactions involved only harmless antibodies. Of the 50 antibodies of potential significance, GEL yielded equivalent or superior results in 47 (94%) instances. Additionally, GEL failed to detect 6 of 21 harmless antibodies. Expected results were obtained with normal serum or plasma and antibodies of known specificity in tests with RBCs treated with ficin, DTT, or CDP. Hands-on-time required for each GEL panel was 2 to 21/2 minutes compared with 12 minutes for PEG. These data document the suitability of GEL for use in antibody identification studies.

The gel test: sensitivity and specificity for unexpected antibodies to blood group antigens.

The recently FDA-licensed anti-IgG gel test for pretransfusion antibody detection requires crossover validation before implementation. Six hundred coded samples sent for routine pretransfusion tests were used to compare GEL (ID-MTS, Ortho Diagnostic Systems Inc., Raritan, NJ) with Löw and Messeter's low-ionic-strength saline (LISS). There were 456 GEL-LISS-, 97 GEL+LISS+, 45 GEL-LISS+, and 2 GEL+LISS- tests. The 144 positive tests involved 157 antibodies; 67 of these (cold auto, anti-M, -Le, etc.) were considered harmless with respect to transfusion management. GEL-LISS+ tests included seven samples containing potentially significant antibodies (assumed from specificity): anti-K(4), -Jka, -Fyb, and -S. Two potentially significant antibodies (anti- C and -D) were GEL+LISS-. Sensitivity and specificity for potentially significant antibodies were 92% and 96% for GEL, and 98% and 90% for LISS, respectively. The seven GEL-LISS+ samples associated with potentially significant antibodies were from six patients. Five of these antibodies, all detected in immune-suppressed patients, reacted predominantly as agglutinins in LISS. None of these seven antibodies were detected reliably by polyethylene glycol and LISS-additive tube methods. In light of the immune status of the patients with GEL-LISS+ agglutinins with specificity normally considered potentially significant, and because other valid methods did not detect these antibodies, their clinical importance is questionable. Excluding these questionable antibodies, GEL has the same sensitivity and better specificity than LISS. GEL is a valid method for pretransfusion antibody detection.

State association-sponsored program for SBB candidates.

Concern about the closure of the only Specialist in Blood Banking (SBB) School in the State of Michigan prompted the Michigan Association of Blood Banks (MABB) to develop and administer a comprehensive series of lectures to meet the needs of technologists seeking SBB certification via the experiential route. The program consisted of more than 60 1(1/2)-hour lectures covering all aspects of blood banking (genetics, coagulation, immunology, blood group serology, blood collection and administration, and transfusion medicine) except infectious disease testing and management issues. The latter were presented during two free-standing one-day seminars (for which a fee was charged for those not registered for the lecture series). Educational techniques were covered in a self-study module. Two lectures were presented one morning each week from March through December, with no presentations during July. Lecturers were local volunteer experts, and detailed learning objectives were established for each topic. Each registrant was charged a nominal fee ($100 for MABB members; $150 for nonmembers). There were 32 registrants for the lecture series, with additional registrants for the seminars. Total revenue was $5,100, and expenses amounted to approximately $2,400, mostly for faculty honoraria and catering for the two seminars. The popularity of our program reflects the need for alternatives to formal SBB schools.

Biochemical characterization of a monoclonal antibody to the H type-2 antigen: comparison to other ABH antibodies.

Monoclonal and polyclonal antibodies to the ABH blood group antigens were tested for their specificity to glycoproteins with ABH activity on immunoblots of solubilized erythrocyte membranes. Immunoblots were stained with monoclonal antibody G10 to the H type-2 carbohydrate structure or with commercially prepared monoclonal and polyclonal antibodies to A, B, and H blood group antigens. G10 antibody specifically stained antigens in the regions that contain the erythrocyte membrane bands 3 and 4.5; the staining was proportional to the expected H content of the erythrocytes (O greater than A2 greater than B greater than A2B greater than A1 greater than A1B). No specific staining was observed with membranes derived from Oh (Bombay) erythrocytes which lack the H type-2 structure. A commercially prepared monoclonal anti-H did not specifically stain erythrocyte membrane antigens. Monoclonal and polyclonal anti-A specifically stained bands from A and AB but not O, B, or Oh erythrocytes (A1 greater than A1B greater than A2 greater than A2B). Polyclonal anti-B serum specifically stained bands from B and AB but not O, A, or Oh erythrocytes (B greater than A2B greater than A1B). However, no specific staining was observed in tests with monoclonal anti-B. Monoclonal antibodies G10 and anti-A and polyclonal anti-A and -B blood typing sera will be useful in the further characterization of the molecular nature of the ABH antigens.