Improving transfusion safety: implementation of a comprehensive computerized bar code-based tracking system for detecting and preventing errors.

BACKGROUND: To transfuse blood products safely, health care workers must accurately identify patients, blood samples, and the blood components. A comprehensive bar code-based computerized tracking system was developed and implemented to identify and prevent transfusion errors. STUDY DESIGN AND METHODS: A data network, wireless devices, and bar-coded labels were pilot tested before the system was introduced hospitalwide. The system provided a complete audit trail for all transactions. Data from before and after implementation were analyzed. RESULTS: Incident reports decreased from a mean of 41.5 reports per month in the 6 months before the system was implemented to a mean of 7.2 reports per month after implementation. The blood sample rejection rate decreased from 1.82 percent to a mean of 0.17 percent after implementation. Errors detected by the new system were sorted into misscans, skipped steps, wrong steps, and prevented identification errors (PIEs). Misscans and skipped steps were the most common errors in the first 10 months after implementation. During the final transfusion step, PIEs occurred at the rate of about one per month and scans were omitted approximately 1 percent of the time. Therefore, it is estimated that mistransfusions could occur about once every 100 months on average with the new system. CONCLUSIONS: The bar code-based computerized tracking system detected and prevented identification and matching errors, thereby reducing the proportion of blood samples rejected and increasing patient safety.

Identification of a signal peptide for oryzacystatin-I.

A previously unidentified extension of an open reading frame from the genomic DNA of Japonica rice (Oryza sativa L.) encoding oryzacystatin-I (OC-I; access. M29259, protein ID AAA33912.1) has been identified as a 5' gene segment coding for the OC-I signal peptide. The signal peptide appears to direct a pre-protein (SPOC-I; Accession No. AF164378) to the endoplasmic reticulum, where it is processed into the mature form of OC-I. The start codon of SPOC-I begins 114 bp upstream from that previously published for OC-I. A putative proteolytic site. which may yield a mature OC-I approximately 12 residues larger than previously described, has been identified within SPOC-I between Ala-26 and Glu-27. The signal peptide sequence was amplified by polymerase chain reaction using genomic DNA from O. sativa seedlings and ligated to the 5' end of the truncated OC-I gene at the endogenous SalI site. Partially purified protein extracts from Escherichia coli expressing SPOC-I reacted with polyclonal antibodies raised against OC-I and revealed a protein of the expected molecular weight (15,355 Da). In-vitro translation of SPOC-I in the presence of microsomal membranes yielded a processed product approximately 2.7 kDa smaller than the pre-protein. Nicotiana tabacum L. cv. Xanthi plants independently transformed with the SPOC-I gene processed SPOC-I and accumulated the mature form of OC-I (approximately 12.6 kDa), which co-migrated with natural, mature OC-I extracted from rice seed when separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Coexpression of the maize delta-zein and beta-zein genes results in stable accumulation of delta-zein in endoplasmic reticulum-derived protein bodies formed by beta-zein.

Zeins, the major seed storage proteins of maize, are of four distinct types: alpha, beta, delta, and gamma. They are synthesized on the rough endoplasmic reticulum (ER) in a sequential manner and deposited in ER-derived protein bodies. We investigated the potential for producing sulfur-rich beta-zein and delta-zein proteins in leaf and seed tissues by expressing the corresponding genes in a constitutive manner in transgenic tobacco. The delta-zein and beta-zein, when synthesized individually, were stable in the vegetative tissues and were deposited in unique, zein-specific ER-derived protein bodies. Coexpression of delta-zein and beta-zein genes, however, showed that delta-zein was colocalized in beta-zein-containing protein bodies and that the level of delta-zein was fivefold higher in delta-/beta-zein plants than in delta-zein plants. We conclude that delta-zein interacts with beta-zein and that the interaction has a stabilizing effect on delta-zein.

Iron deprivation and cancer: a view beginning with studies of monoclonal antibodies against the transferrin receptor.

This review provides a perspective on the potential utility of iron deprivation treatments as components of cancer therapy. The perspective began to develop with investigations of the selective inhibitory effects on lymphocyte activation which were produced by monoclonal antibodies against the transferrin receptor. Those investigations led to the unexpected discovery that such antibodies would produce synergistic inhibition of lymphoid tumor growth in vitro when used in combination with the iron chelator deferoxamine. The perspective was further developed when additional studies in vivo indicated that combination iron deprivation treatment could prevent initial tumor outgrowth and cause regressions of established tumors in the 38C13 murine lymphoma model. The anti-tumor effects were accompanied by significant toxicities, however, and the analysis of the causes of those toxicities is now an important issue. The opportunities and problems which these results present are interpreted in the broader context of currently available information concerning the anti-tumor effects of deferoxamine and gallium nitrate in the pre-clinical and clinical settings, and questions for future research are presented.

Direct evidence that iron deprivation induces apoptosis in murine lymphoma 38C13.

We found that the mouse B cell lymphoma 38C13 underwent apoptosis in vitro when deprived of iron by three independent methods: (1) exposure to a synergistic pair of rat IgG monoclonal antibodies against the mouse transferrin receptor; (2) exposure to the iron chelator deferoxamine (DFO), and (3) exposure to a defined culture medium without any added iron (iron-poor medium). When each antibody was present at a concentration of 5 micrograms/ml, the number of living cells declined to approximately 25% after a 24-hour incubation. After 48 h, there were no surviving cells. When DFO was present at a concentration of 10 microM, the effects were similar, but delayed by 24 h. when iron-poor medium was used, the effects and kinetics were similar to those seen with antibody treatment. For each method of iron deprivation, the reduction in cell viability correlated with the development of apoptosis, as assessed by DNA fragmentation analysis and propidium iodide staining. Electron microscopy studies provided additional confirmation of apoptotic cell death. The addition of 500 microM ferric citrate completely prevented apoptosis for each of the three methods of iron deprivation. These studies provide new and compelling evidence to support the view that iron deprivation can specifically induce apoptosis and serve to strengthen the rationale for further studies of iron deprivation as a form of cancer treatment.

A phase II trial of deferoxamine in patients with hormone-refractory metastatic prostate cancer.

The management of hormone-refractory metastatic prostate cancer remains a therapeutic dilemma. We report the results of a phase II trial with deferoxamine administrated at a dose of 50 mg/kg (maximum dose 5 g) administered intravenously over 8 hr daily, repeated for 5 days at 4-week intervals for 2 courses. Fourteen patients with advanced hormone-refractory prostate cancer were treated and 28 courses were delivered. Essentially no toxicity was observed. Using combined clinical and prostate-specific antigen (PSA) criteria. 13 of 14 patients had disease progression. However, 9 of 14 patients had stable measurable or evaluable disease and progressed solely based on PSA criteria. Deferoxamine in this dose and schedule has no activity in hormone-refractory prostate cancer. Further investigation of the effect of deferoxamine on PSA production/expression is warranted.

Cell Ablation Reveals That Expression from the Phaseolin Promoter Is Confined to Embryogenesis and Microsporogenesis.

Most previous studies of the [beta]-phaseolin (phas) gene, which encodes the major storage protein in bean (Phaseolus vulgaris L.), have shown its expression to be rigorously confined to the developing seed, both in bean and transgenic tobacco (Nicotiana tabacum L. cv Xanthi) plants. To confirm unequivocally the lack of phas expression in vegetative tissues, we placed the diphtheria toxin A-chain (DT-A) coding region under the control of [beta]-phaseolin promoter sequences. Tobacco plants transgenic for phas/DT-A were phenotypically normal until flowering, when they produced anthers that were externally normal but contained no viable pollen. Microscopic examination of immature anthers revealed a normal tapetum, but the pollen mother cells did not undergo meiosis and subsequently degenerated, resulting in male-sterile plants. This demonstration of phas expression during microsporogenesis was corroborated by the expression of [beta]-glucuronidase in pollen of plants transformed with comparable phas/uidA constructs. Although these findings suggested that similarities in phas expression may exist between seed and pollen maturation, no phas activity could be detected in bean pollen. After fertilization of the DT-A-transformed plants with pollen from wild-type tobacco, 50% of the resulting embryos aborted at the heart stage, defining this as the earliest time for phas expression during embryogenesis.

Inhibition of lymphoma growth in vivo by combined treatment with hydroxyethyl starch deferoxamine conjugate and IgG monoclonal antibodies against the transferrin receptor.

Synergistic inhibition of hematopoietic tumor growth can be observed in vitro when the iron chelator deferoxamine (DFO) is used in combination with an IgG mAb against the anti-transferrin receptor antibody (ATRA). Our goal was to ascertain whether similar findings could be seen in vivo. A high molecular weight conjugate of deferoxamine, known as hydroxyethyl starch (HES) DFO or HES-DFO, was tested in conjunction with C2, a well-defined rat antimouse transferrin receptor mAb, against the 38C13 tumor in C3H/HeN mice. It was shown that while neither HES-DFO alone nor C2 alone produced consistent, significant inhibition of tumor growth, the combination of HES-DFO and C2 produced virtually complete inhibition of initial tumor outgrowth. The latter combination failed, however, to inhibit the growth of established tumors. It was then found that when C2 was used in conjunction with RL34, another IgG ATRA, the two ATRAS were themselves capable of causing synergistic inhibition of the growth of 38C13 in vitro. When the two IgG ATRAS were used together in vivo, regressions of established tumors were observed. Moreover, the addition of HES-DFO to the IgG ATRA pair then caused more frequent regressions. Although there was never any obvious toxicity seen with a single IgG ATRA, the use of the IgG ATRA pair was associated with sporadic mortality. In addition, although HES-DFO by itself was also not associated with any obvious toxicity, combined treatment with HES-DFO and a single ATRA resulted in death due to bacterial infection in about half of the mice after 10-15 days. Combined treatment with HES-DFO and the ATRA pair resulted in death attributed to infection in nearly all of the mice after 6 days. Thus, an iron deprivation treatment protocol with HES-DFO and IgG ATRAS produced both a significant antitumor effect and an increased risk of infection in a murine model system.

Accumulation of 15-Kilodalton Zein in Novel Protein Bodies in Transgenic Tobacco.

Zeins, the seed storage proteins of maize, are a group of alcohol-soluble polypeptides of different molecular masses that share a similar amino acid composition but vary in their sulfur amino acid composition. They are synthesized on the rough endoplasmic reticulum (ER) in the endosperm and are stored in ER-derived protein bodies. Our goal is to balance the amino acid composition of the methionine-deficient forage legumes by expressing the sulfur amino acid-rich 15-kD zeins in their leaves. However, it is crucial to know whether this protein would be stable in nonseed tissues of transgenic plants. The major focus of this paper is to compare the accumulation pattern of the 15-kD zein protein with a vacuolar targeted seed protein, [beta]-phaseolin, in nonseed tissues and to determine the basis for its stability/instability. We have introduced the 15-kD zein and bean [beta]-phaseolin-coding sequences behind the 35S cauliflower mosaic virus promoter into tobacco (Nicotiana tabacum) and analyzed the protein's accumulation pattern in different tissues. Our results demonstrate that the 15-kD seed protein is stable not only in seeds but in all nonseed tissues tested, whereas the [beta]-phaseolin protein accumulated only in mid- and postmaturation seeds. Interestingly, zein accumulates in novel protein bodies both in the seeds and in nonseed tissues. We attribute the instability of the [beta]-phaseolin protein in nonseed tissues to the fact that it is targeted to protease-rich vacuoles. The stability of the 15-kD zein could be attributed to its retention in the ER or to the protease-resistant nature of the protein.

Photoaffinity Labeling of Developing Jojoba Seed Microsomal Membranes with a Photoreactive Analog of Acyl-Coenzyme A (Acyl-CoA) (Identification of a Putative Acyl-CoA:Fatty Alcohol Acyltransferase.

Jojoba (Simmondsia chinensis, Link) is the only plant known that synthesizes liquid wax. The final step in liquid wax biosynthesis is catalyzed by an integral membrane enzyme, fatty acyl-coenzyme A (CoA):fatty alcohol acyltransferase, which transfers an acyl chain from acyl-CoA to a fatty alcohol to form the wax ester. To purify the acyltransferase, we have labeled the enzyme with a radioiodinated, photoreactive analog of acyl-CoA, 12-[N-(4-azidosalicyl)amino] dodecanoyl-CoA (ASD-CoA). This molecule acts as an inhibitor of acyltransferase activity in the dark and as an irreversible inhibitor upon exposure to ultraviolet light. Oleoyl-CoA protects enzymatic activity in a concentration-dependent manner. Photolysis of microsomal membranes with labeled ASD-CoA resulted in strong labeling of two polypeptides of 57 and 52 kD. Increasing concentrations of oleoyl-CoA reduced the labeling of the 57-kD polypeptide dramatically, whereas the labeling of the 52-kD polypeptide was much less responsive to oleoyl-CoA. Also, unlike the other polypeptide, the labeling of the 57-kD polypeptide was enhanced considerably when photolyzed in the presence of dodecanol. These results suggest that a 57-kD polypeptide from jojoba microsomes may be the acyl-CoA:fatty alcohol acyltransferase.

Differing sensitivity of non-hematopoietic human tumors to synergistic anti-transferrin receptor monoclonal antibodies and deferoxamine in vitro.

We tested non-hematopoietic human tumors for in vitro sensitivity to either a pair of synergistic IgG antitransferrin (Tf) receptor monoclonal antibodies (MAbs), deferoxamine (DFO) or the combination thereof. With an equimolar mixture of the two MAbs (A27.15, E2.3), two prostate tumors showed similar degrees of maximal growth inhibition (PC-3: 35%, DU 145: 38%), two breast tumors showed more variability (MDA-MB-231: 26%, SK-BR-3: 52%) and two neuroblastomas showed the most variability (SK-N-SH: 4%, SK-N-MC: 76%). When the MAbs were applied together with DFO, the D50 for DFO was reduced for all tumors (PC-3: 2.5x, DU 145: 3.7x; MDA-MB-231: 2.9x, SK-BR-3: 1.9x, and SK-N-SH: 2.6x, SK-N-MC: 7.0x). Sensitivity to MAbs was more closely correlated with the relative decrease in Tf receptor density resulting from antibody exposure than with initial receptor density. The degree of reduction of D50 for DFO resulting from the joint application with the MAbs was, however, most closely related to the growth rate of the tumors. Since some non-hematopoietic tumors exhibit sensitivity to the effects of a synergistic pair of IgG anti-Tf receptor MAbs and DFO, it appears that further preclinical studies with such tumors, especially those with higher Tf densities, would be of interest.

Expression of CD24 (BA-1) predicts monocytic lineage in acute myeloid leukemia.

Fifty-seven cases of acute myeloid leukemia (AML), which had been subtyped by French-American-British morphologic and cytochemical criteria as myeloid (M1, M2) or monocytic (M4, M5), were retrieved from the files of the Division of Hematopathology, University of Iowa. Corresponding immunophenotyping data were reviewed with attention to the expression of CD14 (MY4) and CD24 (BA-1). Of 20 cases expressing CD24, 19 were M4 or M5, whereas all 14 cases expressing CD14 were of monocyte lineage. Therefore, CD14 was a highly specific (100%) but only moderately sensitive (58%) marker for distinguishing classes M1 or M2 from M4 or M5. By contrast and unexpectedly, CD24 was nearly as specific (97%), but more sensitive (79%) in marking M4 or M5 cells. This appears to be true even though CD24 is apparently not expressed on normal monocytes. When positive staining for either or both antibodies (CD24 or CD14) was considered indicative of a monocytic leukemia, the sensitivity of immunophenotyping in distinguishing M4/M5 from M1/M2 AML rose to 92%, while maintaining 97% specificity. The authors discuss a recent observation that may help explain the unexpected expression of CD24 in monocytic AML. They conclude that the usefulness of CD24 in identifying monocytic AML may exceed that of CD14, and that the use of CD24 and CD14 in combination improves the ability of flow cytometry to distinguish myeloid from monocytic acute leukemias.

Synthesis of photoreactive phosphatidylethanolamine and its interaction with phospholipase A2.

A photoreactive derivative of phosphatidylethanolamine, N-(4-azidobenzoyl)phosphatidylethanolamine (AB-PE), was synthesized by acylation of phosphatidylethanolamine with an N-hydroxysuccinimide ester of 4-azidobenzoic acid. The substantial photosensitivity exhibited by AB-PE correlated with a marked decrease in the absorption spectra of the compound. The compound proved sensitive to lipase and phospholipase A2 hydrolysis but resistant to phospholipase C and D activities. Photolysis of a sonicated dispersion of AB-PE containing phospholipase A2 resulted in irreversible inhibition of the enzyme. Addition of natural phosphatidylethanolamine provided protection against photoinactivation.

Photoaffinity labeling of acyl-CoA oxidase with 12-azidooleoyl-CoA and 12-[(4-azidosalicyl)amino]dodecanoyl-CoA.

Synthesis of 32P-labeled CoA of high specific activity was achieved using partially purified dephospho-CoA kinase (EC 2.7.1.24) from pig liver with [gamma-32P]ATP as donor and dephospho-CoA as acceptor. A photoaffinity dodecanoic acid analog, 12-[(4-azidosalicyl)amino]dodecanoic acid was synthesized, as were its CoA derivative (ASD-CoA) and the CoA derivative of 12-azidooleic acid. The CoA derivatives were synthesized from azido fatty acid analogs by acyl-CoA synthetase. The synthesized photolabile reagents were tested as photoaffinity labels for acyl-CoA oxidase (EC 1.3.99.3) from Arthrobacter species. When a mixture of oxidase and the acyl-CoA analogs were incubated in the absence of ultraviolet light, the analogs were recognized as substrate. Acyl-CoA oxidase was incubated in the presence of acyl-CoA analogs and immediately photolyzed, which resulted in irreversible inhibition. Oleoyl-CoA and dodecanoyl-CoA protect the enzyme from photoactivated inhibition by 12-azidooleoyl-CoA and ASD-CoA, respectively. Analysis of photolyzed enzyme preparations by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed that both analogs preferentially labeled a 54,000 molecular weight protein. These results demonstrate that the photoaffinity acyl-CoA analogs have potential application as probes to identify and characterize lipid biosynthetic enzymes and to identify the active site of these proteins.

Coexistent anti-neutrophil cytoplasmic antibody and antiglomerular basement membrane antibody associated disease = report of six cases.

From a series of 95 patients biopsied for rapidly progressive glomerulonephritis, twelve patients were identified with anti-glomerular basement membrane-mediated renal disease who were also tested for antineutrophil cytoplasmic antibody (ANCA). Six patients had both anti-glomerular basement membrane and ANCA antibodies. Three of the latter six patients had significant extrarenal disease, including severe hemoptysis, while the remaining three patients had only renal disease. The three patients with extrarenal disease had either a myeloperoxidase-positive perinuclear-ANCA (two patients) or a proteinase-3-positive cytoplasmic-ANCA (one patient). Two patients with renal disease alone had a myeloperoxidase-negative and proteinase-3-negative perinuclear-ANCA, and one patient had a proteinase-positive cytoplasmic-ANCA. Renal biopsy in all six patients showed a severe necrotizing and crescentic glomerulonephritis involving 94 to 100% of glomeruli. Renal arteritis was also noted in one perinuclear-ANCA patient. Despite aggressive therapy with steroids, cyclophosphamide, and plasma exchange, two of the six double-antibody patients died and four are on dialysis. We conclude that ANCA is commonly present in anti-glomerular basement membrane-associated disease and believe that this observation may have implications in the serologic evaluation of ANCA- and anti-glomerular basement membrane-positive patients.

The 3G11+ antigen, a marker for murine CD4+ TH1 lymphocytes, is a ganglioside.

Two monoclonal antibodies (mAbs), SM3G11 and SM6C10, can be used to discriminate between functionally distinct murine CD4+ T cell subsets. In this study we use high-performance thin-layer chromatography and immunostaining techniques to show that the 3G11 mAb reacts with two bands of a ganglioside fraction from murine spleen and thymus, and rat spleen. The 6C10 antibody shows no evidence of glycolipid reactivity. The 3G11+ bands have a mobility between those of the reference gangliosides GD1a and GD1b from human brain. The 3G11+ reactive bands were eluted in the disialyl fraction of rat spleen gangliosides using DEAE anion-exchange chromatography. Treatment of spleen gangliosides with endoglycoceramidase eliminates 3G11 antibody binding over time, indicating that the antigen contains a Glc beta 1-1'ceramide linkage, characteristic of a glycosphingolipid. Treatment of thymus or spleen gangliosides with sialidase eliminates binding of 3G11, thus indicating that the 3G11 epitope is dependent on the expression of one or more sialic acid residues. Immunostaining studies with a variety of reagents indicate that the 3G11+ gangliosides: (i) probably do not contain either the asialo-GM1 or the GM1 core structures; (ii) are not recognized by mAbs specific for the oligosaccharides of asialo-GM2, GM2, GD2 and GD3 gangliosides; and (iii) are also not recognized by antibodies or reagents that are specific for several structures representative of other major glycosphingolipid classes. Overall, these studies strongly suggest that the 3G11+ gangliosides have structures that have not been previously recognized in murine lymphoid tissue. Structures that could account for the known properties of the 3G11+ molecules are described. Finally, ways in which the selective expression of 3G11+ gangliosides might be linked to functionally distinct T-cell behaviours are discussed.

The role of iron and iron binding proteins in lymphocyte physiology and pathology.

Knowledge concerning the roles of iron and iron binding proteins in lymphocyte physiology and pathology has developed rapidly over the last few years. The genes for the major iron binding proteins have been cloned and sequenced and are now being studied with respect to transcriptional and posttranscriptional regulatory mechanisms. T cells, B cells, macrophages, and natural killer cells appear to differ from one another in the ways in which they synthesize and utilize iron binding proteins and in the amount of iron they take up and store. This suggests that differential modulation of iron-dependent metabolic functions is an intrinsic part of the distinctive physiology of each cellular component of the immune system and that the distribution of iron between those components is a carefully managed facet of the immune response. Since the immune response does not seem to be dramatically impaired by alterations in iron supplies that adversely affect other organs, it may well be that the cells of the immune system are especially adapted to have both high-priority access to iron when supply is low and high-level protection against iron-related toxicity when supply is in excess.

Identification of apparent dual ANCA specificities in a subset of patients with systemic vasculitis and crescentic glomerulonephritis.

23% of a random selection of ANCA (+) patients had dual, non-cross reactive autoantibody specificities. We found no clinical difference in such patients as compared to those having a single antibody. A second group of ANCA (+) patients had neither anti-MPO nor anti-PR3 autoantibody specificity. These findings suggest that multiple, coincident ANCA specificities occur more commonly than previously reported, and that additional, unidentified ANCA specificities are present in some patients.

Differential epitope expression of Ly-48 (mouse leukosialin).

Ly-48 is a major sialoglycoprotein expressed on the surface of a variety of mouse hematopoietic cells that exhibits many characteristic isoforms and may function in signal transduction and cell adhesion. Ly-48 is recognized by the 3E8-specific monoclonal antibody (mAb) and it has been suggested that it is the same antigen recognized by another mAb known as S7. In this report, we demonstrate definitively by transfection of a Ly-48 cDNA that S7 and two previously uncharacterized mAbs, S11 and S15, recognize the same antigen as the 3E8-specific mAb. However, 2-D gel immunoblot analyses demonstrate the complex nature of Ly-48. Although all four mAbs react similarly with lysates from the M-45 B-cell myeloma line, 2-D immunoblot analyses of the EL-4 T-cell line reveal three distinct patterns of reactivity. Further, while transfection of Ly-48 into the K562 erythroleukemic cell line conferred reactivity to all four mAbs, transfection of the Ly-48 cDNA into the nonhematopoietic cell line, Line 1, conferred reactivity only to the S11 and S15 mAbs. Thus, the Line 1 transfectants suggest the importance of posttranslational modifications in the expression of the 3E8 and S7 epitopes. Interestingly, developing fetal liver cells show the same pattern of differential Ly-48-specific mAb reactivity. The developing early fetal liver cells are reactive with S11 and S15 but are negative, to very weakly, reactive with the 3E8- and S7-specific mAbs. These results show that Ly-48 epitopes can be expressed independently on cell lines in vitro and are differentially expressed on healthy cells in vivo.