Adverse events are common on the intensive care unit: results from a structured record review.

BACKGROUND: Intensive care is advanced and highly technical, and it is essential that, despite this, patient care remains safe and of high quality. Adverse events (AEs) are supposed to be reported to internal quality control systems by health-care providers, but many are never reported. Patients on the intensive care unit (ICU) are at special risk for AEs. Our aim was to identify the incidence and characteristics of AEs in patients who died on the ICU during a 2-year period. METHODS: A structured record review according to the Global Trigger Tool (GTT) was used to review charts from patients cared for at the ICU of a middle-sized Swedish hospital during 2007 and 2008 and who died during or immediately after ICU care. All identified AEs were scored according to severity and preventability. RESULTS: We reviewed 128 records, and 41 different AEs were identified in 25 patients (19.5%). Health care-associated infections, hypoglycaemia, pressure sores and procedural complications were the most common harmful events. Twenty two (54%) of the AEs were classified as being avoidable. Two of the 41 AEs were reported as complications according to the Swedish Intensive Care Registry, and one AE had been reported in the internal AE-reporting system. CONCLUSION: Almost one fifth of the patients who died on the ICU were subjected to harmful events. GTT has the advantage of identifying more patient injuries caused by AEs than the traditional AE-reporting systems used on many ICUs.

Different metastasis patterns of a human melanoma cell line in nude mice and rats: influence of microenvironment.

The metastatic capacity of intravenously injected human FEMX-I melanoma cells in athymic nude mice and rats was compared. Young rats given 1 x 10(6) ascites tumor cells all died of lung tumors with a life span of 50 +/- 10 days (mean +/- SD). In contrast, in accordance with previous findings, only extrapulmonary metastases developed in mice. This host-dependent difference in metastasis pattern permitted studies on the role of factors that may influence the organ specificity of metastases. The tissue distribution of 125I-labeled FEMX-I cells did not differ in the two nude species during the first 12 hours after cell injection. The plating efficiency of FEMX-I cells in soft agar was increased by the addition of conditioned medium prepared from rat lungs, resulting also in a significant increase in colony size. In contrast, conditioned medium prepared from mouse lungs reduced the clonogenic capacity of the FEMX-I cells in a dose-dependent manner. Conditioned media prepared from rat and mouse liver, kidney, and spleen tissues either inhibited or had no effect on colony formation. The results suggest that the unexpected differential metastatic patterns observed in vivo may reflect differences in the presence of growth-modulating paracrine factors in the host lungs.

Nude rat model for studying metastasis of human tumor cells to bone and bone marrow.

Bone metastases reproducibly developed in nude rats after an injection of LOX human malignant melanoma cells into the left ventricle, with hind leg paralyses appearing in all animals within approximately 2 weeks. Manifest metastases were present exclusively in the skeletal system, predominantly in the lumbar portion of the spine, the long bones, and occasionally in the skull. Intracardially injected 125I-labeled tumor cells and monodisperse microspheres were distributed in parallel to the various tissues. Moreover, because the levels of radioactivity were significantly lower in bone than in lung, kidney, and liver, the pattern of metastases could not be explained solely by hemodynamic factors. In chemotherapy experiments, the survival time of rats given left ventricular injections of LOX cells increased in a dose-dependent manner after the animals were treated with dacarbazine. Researchers may find the model useful for studying the biology of bone metastases and for testing the sensitivity of these lesions to drugs.

Human melanoma cell lines showing striking inherent differences in sensitivity to immunotoxins containing holotoxins.

The in vitro sensitivity of human melanoma cell lines to conjugates of whole abrin or ricin linked through disulfide bonds to the monoclonal antimelanoma antibody 9.2.27 was studied. After passage of the conjugates through a Sepharose 4B column to remove molecular species with exposed binding sites on their B-chains, toxicity of the conjugates to different melanoma cell lines and nonmelanoma tumor lines was assessed by measuring their ability to inhibit cellular protein synthesis. The abrin conjugate was far more toxic to the target cells than the corresponding ricin conjugate. The 8 melanoma cell lines studied differed widely in their sensitivities to the abrin conjugate. The differences were associated with concomitant large differences in the sensitivities of the cells to the native toxins, and the significance of the level of the antigen expression became apparent only when the sensitivities of the different cell lines were normalized with respect to their sensitivity to native abrin. The observed relationship could not be accounted for by unspecific binding via the B-chain binding site of the immunotoxin. The differential sensitivity of the melanoma cell lines to the immunotoxin seems to be related to inherent differences between the cells in their ability to internalize and to process immunotoxins and toxins. The findings may have considerable practical implications.

Reduced antineoplastic activity in mice of cisplatin administered with high salt concentration in the vehicle.

The effect of high NaCl concentration in the vehicle on the toxic and antineoplastic activities of cisplatin [cis-dichloro-diammineplatinum(II)] (CDDP) was reinvestigated in mice. The toxicity, as measured by the survival of mice given CDDP iv, was reduced by 50-60% when the NaCl concentration in the vehicle was raised from 0.9 to 4%. In ascitic P388 leukemia the antineoplastic activity of CDDP given ip was not reduced significantly. However, in all other systems studied the antitumor activity was reduced when the CDDP was dissolved in high NaCl solution. The tumor models studied included systemic P388 and L1210 leukemias, Lewis lung carcinoma, and 5 human tumor xenografts. The human tumors were studied by the subrenal capsule assay. In the case of a malignant melanoma and a malignant fibrous histiocytoma, the effect also was demonstrated in the subcutaneous nude mouse model. In one of the malignant melanomas a 50% increase in the CDDP dose did not compensate for the reduced antitumor activity caused by the high NaCl concentration in the vehicle. These results, which stand in contrast to current views, question the experimental basis for the use of high-NaCl vehicles in the "high-dose" CDDP regimens.

New indirect approach to the therapeutic use of immunotoxins.

To improve the applicability of immunotoxins (ITs), we have developed a new two-step indirect procedure. The target cells to be killed are first incubated with cell-specific mouse monoclonal antibodies (MAbs). After removal of excess unbound antibody, the cells are incubated in the presence of lactose with an indirect IT, a conjugate of whole abrin and sheep anti-mouse immunoglobulin (SAM) that binds only to cells having primary mouse MAbs on their surfaces. The SAM-abrin IT is affinity purified before use to remove molecules with exposed B-chain-binding sites; it was nontoxic in the absence of the specific mouse MAbs, demonstrating the specificity of the two-step method. We compared the indirect approach, using four different primary MAbs, with the conventional method, in which abrin is coupled directly to the mouse MAbs. In three human cell lines--the melanoma line FEMX, the Burkitt cell line Rael, and the leukemia cell line KM3--the cell kill, measured by a clonogenic assay, was consistently greater with the indirect than with the direct method. In the melanoma and Rael cells, the indirect method gave a higher cell kill than even native abrin. With a mixture of two different antibodies an additive effect was observed with the indirect but not with the direct method. The new approach greatly simplifies the therapeutic application in vitro of ITs, because it permits the use of different primary antibodies, singly or in mixtures, in conjunction with only one or a few general indirect ITs. In efforts to further improve the usefulness of the indirect method, other indirect ITs containing different toxin moieties are being examined. The possibility of employing the indirect principle in vivo is being explored.

Successful clinical use of an anti-HLA-DR monoclonal antibody for autologous bone marrow transplantation.

It has been widely assumed that anti-HLA-DR antibodies react with pluripotent stem cells and cannot be used in bone marrow purging. We report a case of non-Hodgkin's lymphoma in which an anti-HLA-DR antibody (AB4) was used for immunomagnetic purging and the subsequent autologous bone marrow transplantation resulted in rapid marrow engraftment with no serious complications. The results indicate that the AB4 antibody, which binds to an antigen encoded by the B3 gene of the DR region, can be safely used in the clinic in the purging of bone marrow from patients with AB4-positive tumors (non-T-cell acute lymphocytic leukemia, non-Hodgkin's lymphoma, and some cases of acute myelogenous leukemia.

Studies on the mechanism of action of abrin-9.2.27 immunotoxin in human melanoma cell lines.

Previously we have shown (Godal, A. et al., J. Natl. Cancer Inst., 77: 1247-1253, 1986) that the sensitivities of different melanoma cell lines to a conjugate of abrin with the anti-melanoma antibody 9.2.27 was correlated with their sensitivities to native abrin. To elucidate the underlying mechanism we have compared the binding and toxicity of the conjugate and of native abrin to two melanoma cell lines, FEMX and LOX, which differ in sensitivity to abrin. Abrin was linked by a disulfide bond to the monoclonal antibody 9.2.27, and the conjugate was purified by affinity chromatography to remove molecules with exposed galactose-binding sites on the toxin B-chain. Lactose had no effect on the binding of the immunotoxin (IT) to the cells but nevertheless reduced strongly the toxicity to the LOX cells. The differences in sensitivity to native abrin were much larger than the concurrent differences in binding. Lactose reduced the toxicity of abrin to a far greater extent than the associated reduction in binding to the cell surface. The toxicity of the immunotoxin to the FEMX cells could be prevented by pretreatment with excess 9.2.27 antibody, whereas the more abrin-sensitive LOX cells were protected only to a limited extent. Concurrent treatment of the LOX cells with antibody and lactose acted synergistically and afforded complete protection. It is suggested that the protective effect of lactose against the IT was exerted after internalization into vesicles of IT bound unspecifically to the cell surface and that the toxic moiety of the IT, the abrin A-chain, may be translocated from endocytotic vesicles to the cytosol by two alternative mechanisms, one mediated by the antibody and a second one facilitated by the B-chain and its lectin binding site. The relative significance of these mechanisms seems to differ in different target cell lines depending on their inherent sensitivities to native abrin which in turn largely reflects the ability of the cells to internalize and process surface-bound abrin.

Activity of mitozolomide (NSC 353451), a new imidazotetrazine, against xenografts from human melanomas, sarcomas, and lung and colon carcinomas.

The chemosensitivity of human tumor xenografts to mitozolomide, 8-carbamoyl-3-(2-chloroethyl)imidazo[5-1-d]-1,2,3,5-tetrazin-4(3H) -one, was studied in 3 different assay systems. In concentrations of 1 to 500 micrograms/ml, mitozolomide completely inhibited the colony-forming ability in soft agar of cell suspensions from sarcomas, melanomas, lung and colon cancers, and a mammary carcinoma. When a panel of tumors of the different histological types was tested for its sensitivity to mitozolomide in vitro, in the 6-day subrenal capsule assay in conventional mice, and, in some cases, as s.c. growing tumors in nude mice, good agreement between the different assay systems was seen. In most cases, a very pronounced antitumor effect was observed. The efficacy of mitozolomide was as good or better than that of the drugs clinically used against the tumor types tested. Tumor size measurements and histological examinations indicated that nude mice carrying a melanoma, a small cell lung cancer, and an osteosarcoma were cured of their tumors. The approach here used for evaluating the effect of a new drug on human cancers may be useful for selecting the tumor types which primarily should be studied in clinical trials. The results indicate that clinical responses to mitozolomide may be anticipated in sarcoma, melanoma, small cell lung cancer, and possibly in colon cancer.

Expression and characteristics of a novel human osteosarcoma-associated cell surface antigen.

The characteristics of a new osteosarcoma-associated cell surface antigen were studied by means of two murine monoclonal antibodies, TP-1 and TP-3, which were found to bind to two different epitopes on the same antigen, a monomeric polypeptide with a molecular weight of approximately 80,000. Immunohistochemical studies showed that the antigen was present in all osteogenic sarcomas tested, in most cases of malignant fibrous histiocytoma, in two malignant hemangiopericytomas and in a few synovial sarcomas, but not in other main groups of sarcomas and nonsarcomatous malignancies. In normal tissues it was detected only in clusters of cells in the adrenal medulla and in proximal kidney tubules. Also endothelial cells in proliferating capillaries in placenta and in most tumors were stained. The antigen was absent in resting but present in actively proliferating osteoblastic cells. The epitopes were resistant to proteolytic and sugar-cleaving enzymes but sensitive to high temperatures and could not be detected in paraffin-embedded specimens. The tissue distribution and properties of the antigen show that it is different from the sarcoma-associated antigens previously studied. In contrast to previous findings with three other anti-sarcoma monoclonal antibodies, no correlation was found between serum alkaline phosphatase activity and the amount of TP-binding substances in the same sera. Nevertheless, an apparently complex association between alkaline phosphatase and the TP-binding antigen seems to exist. Thus, the Mr 80,000 antigen extracted from an osteosarcoma cell line showed enzyme activity, whereas TP-binding molecules precipitated from patient sera contained alkaline phosphatase activity only in a few of the cases studied. Altogether our data suggest that the antigen defined by the TP antibodies may be a marker of osteoblastic differentiation. The pattern of antigen expression in malignant tumors is unique, inasmuch as the antigen is found selectively in sarcomas and in all 31 osteosarcomas tested.

A human tumor lung metastasis model in athymic nude rats.

Experimental lung metastases regularly developed in athymic Han:rnu/rnu Rowett rats after i.v. injection of LOX human malignant melanoma cells. When 5 x 10(5) tumor cells were injected into 4-week-old rats, 89% of the animals died of lung tumors, with a mean survival time of 18 days. With 5- and 6-week-old rats, however, the fraction of animals that died decreased to 80 and 46%, with mean survival times of 35 and 38 days, respectively. The number of detectable lung colonies in each animal was about 35 in 5- and 6-week-old animals, compared to nearly 300 in 4-week-old rats. In the latter, a correlation was found between the number of tumor cells injected and the number of detectable lung colonies. The capacity of the LOX tumor to grow s.c. and to form experimental lung metastases was, by and large, similar in young nude rats and in nude mice, and no significant difference in morphology between the different tumors in the two species was seen. A high-resolution radiographic method was used to visualize lung colonies in the nude rats, and single tumors with diameters as small as 2-4 mm could be detected. By this method, for the first time, the effect of chemotherapy on a human tumor growing in a visceral organ of a rodent host could be followed by repeat X-ray examinations, mimicking a situation commonly faced in the clinic. This procedure may prove particularly useful for experimental chemotherapy studies, and may be extended to other human tumors that frequently metastasize to the lungs. Indications were obtained that some host-specific differences in tissue-preferenced growth might exist, a possibility that will be further explored.

Extrapulmonary, tissue-specific metastasis formation in nude mice injected with FEMX-I human melanoma cells.

FEMX-I human malignant melanoma cells, originating from a lymph node metastasis in a patient, uniquely and selectively produced extrapulmonary metastases after i.v. injection of cells prepared from xenografts into adult, nude mice. After a lag time of approximately 50 days, metastases were observed in s.c. sites at the back and front of the neck, and in axilla and inguinal regions. Tumor colony formation in lungs were never detected. The interscapular tumors showed a close relationship to brown fat, partly infiltrating this tissue, whereas the other s.c. tumors seemed to be localized to lymph nodes. Mesenterial and mediastinal lymph node metastases were frequently found, together with retroperitoneal tumors along the spine. The normal cells of the adrenal medulla were often replaced by melanoma cells, whereas the cortical tissue was not affected. The conclusion that FEMX-I cells possess an inherent ability for tissue-specific metastasis formation is supported by the metastatic pattern seen after i.p. and intrasplenic injection, as well as after inoculation of the cells in the footpads of the mice. The relatively slowly growing FEMX-I tumors showed the same differentiated morphology as the patient's tumor, independent of the site of growth and the number of passages in the animals. The FEMX-I tumor was easily established as a cell line in vitro. Such cells showed a strongly reduced metastatic capacity, indicating that the in vitro growth conditions had induced alterations in the FEMX-I cells influencing their ability to form site-specific metastases, changes that were shown to be reversible. It is suggested that structures on the surface of the tumor cells, as well as growth factors in the host tissues, may be of importance for the observed tissue specificity. The FEMX-I melanoma, which, as a human tumor in nude mice, has a unique metastatic pattern, offers possibilities for investigating mechanisms involved in site-specific metastasis formation, as well as for testing effects of antimetastatic, chemotherapeutic, and immunotherapeutic agents against human extrapulmonary micro- and macrometastases.

Elimination of B-lymphoma cells from human bone marrow: model experiments using monodisperse magnetic particles coated with primary monoclonal antibodies.

Conditions for removing B-lymphoma cells from human bone marrow using "immunobeads" (IBs) were investigated. The IBs were prepared by coupling monoclonal antibodies directly to a new type of monodisperse magnetic polymer particles (M 450). Two monoclonal immunoglobulin M antibodies, AB-1 (CD 19), a B-cell-specific antibody, and AB-4, an HLA-DR-specific antibody, were used. The IBs were incubated with Rael Burkitt lymphoma cells admixed to fresh, mononuclear human bone marrow cells. After incubation for 30 min at 4 degrees C, the IBs were removed using cobalt samarium magnets. The number of remaining clonogenic tumor cells was assayed by the Courtenay and Mills soft agar procedure, and the clonogenic capacity of the bone marrow progenitor cells was measured by granulocyte-monocyte and granulocyte-erythroid-monocyte-megakaryocyte assays. With a ratio of tumor cells to normal bone marrow cells of 0.1 or 0.01 and a ratio of immunobeads to tumor cells in excess of 75, a tumor cell depletion of more than 3 logs was achieved with the AB-4 IBs and slightly less with the AB-1 beads. After two consecutive cycles of purification with the AB-4 beads, no colonies were found, corresponding to more than 6 logs of purification. In the case of the AB-1 beads, 4 to 5 logs of purification were achieved. The concomitant reduction in clonogenic bone marrow progenitor cells was only 30 to 40%. Flow cytometric studies showed that the tumor cell population contained appreciable proportions of cells binding only small amounts of the antibodies used. The results indicate that the IB procedure is highly efficient and capable of removing tumor cells expressing low levels of antigen. Compared to other purging methods in use the procedure described seems to offer several advantages with respect to efficacy, speed, and simplicity. By the use of a panel of suitable antibodies the new immunobead procedure may be potentially useful in autologous bone marrow transplantation of B-lymphomas and non-T-leukemias with poor prognosis.

Phase I study of the plant protein ricin.

A Phase I study was carried out with ricin, a plant toxin acting by inhibiting protein synthesis, on 54 cancer patients with advanced disease. Ricin was given as i.v. bolus injections every two weeks at dose levels ranging from 4.5 to 23 micrograms/sq m of estimated body surface area. Ricin was well tolerated at doses up to 18 to 20 micrograms/sq m. At these levels and at higher levels, flu-like symptoms with fatigue and muscular pain appeared and, in some patients, nausea and vomiting occurred also. No myelo-suppression was seen. Antibodies to ricin were detected in serum after two to three ricin injections. Ricin was eliminated from blood according to first order kinetics. At each dose level, the plasma concentrations, as well as the side effects, showed only minor differences between patients. The highest dose given, 23 micrograms/sq m, gave plasma concentrations twice those found previously to be therapeutically effective in tumor-bearing mice. Of 38 evaluable patients, one patient with lymphoma had a partial response. Stable disease was observed in four patients with renal cancers, in two with soft tissue sarcomas, and in one patient each with mesothelioma, thyroid, and rectal cancer. A dose of 23 micrograms/sq m is recommended for Phase II trials of ricin.

Effect of protein synthesis inhibitors on the fidelity of translation in eukaryotic systems.

Factors influencing the accuracy of poly(U)-directed poly(Phe) synthesis in a wheat germ and in a reticulocyte system were studied. Addition of preformed phenylalanyl-tRNA, as well as increasing the ratio of poly(U) to ribosomes, significantly enhanced the poly(Phe) synthesis and concurrently reduced the misincorporation of leucine. The protein synthesis inhibitors cycloheximide, abrin and ricin had little or no effect on the misreading when the system was supplemented with 100 microM phenylalanyl-tRNA, but they reduced the relatively high error rate observed when the poly(U) system was not supplemented with the cognate substrate. Raising the incubation temperature enhanced the accuracy to the same extent whether or not ricin was present i.e., at widely different rates of elongation. The results show that the translational accuracy is not linked to the elongation rate as such. Translational inhibitors affect the fidelity by influencing the kinetics of the system. In systems containing limiting concentrations of cognate substrate, translational inhibitors will cause an increase in the limiting aminoacyl-tRNA species and thereby increase fidelity.

Studies on the structure and properties of the lectins from Abrus precatorius and Ricinus communis.

The amino acid composition of the isolated A- and B-chains of the toxic lectins abrin and ricin was determined and compared. Even though the two toxins originate from widely different plants, statistical analysis of the amino acid content indicates extensive homologies in the amino acid sequence of the 4 chains. The intact lectins contain no free SH-groups whereas the isolated A- and B-chains contain close to one free SH-group each. The results indicate that in both toxins the A- and B-chains are connected by a single S-S bond. The B-chains of abrin and ricin contain similar amounts of mannose and glucosamine. The A-chain of ricin also contains some carbohydrate, whereas the A-chain of abrin appears not to be a glycoprotein. The non-toxic abrus and ricinus agglutinins contain more carbohydrate than abrin and ricin. The isoelectric points of the different lectin preparations were measured by isoelectrofocusing. The intact lectins are much more resistant to heat, freezing and chemical treatments than the isolated A- and B-chains. The intact lectins are also very resistant to treatment with proteolytic enzymes, whereas the isolated chains are easily digested. Evidence indicating that the toxins and their chains undergo extensive conformational changes upon reduction of the S-S bond is discussed.

On the current management of osteosarcoma. A critical evaluation and a proposal for a modified treatment strategy.

The current management of osteosarcoma (OS) is critically reviewed and a modified treatment strategy is put forward for discussion. The overall treatment results in high-grade OS are less impressive than widely assumed. Whereas in 'classical OS' survival has indeed increased during the past decades from approximately 20% to at least 60%, in other subgroups, comprising more than 40% of the entire OS population, the prognosis has been only modestly improved. Today still more than half of an unselected OS population eventually succumbs to the disease despite the current multimodal primary treatments as well as second-line chemotherapy and surgical metastasectomy(ies). Analysis of the reported results indicates that a survival plateau of approximately 60% can be achieved by several different drug combinations. The inclusion of additional drugs and treatment with complex combinations to all patients has not yielded a convincing survival benefit. These expensive regimens overtreat a large number of patients, namely those who could have been cured by the previous less drastic regimens, and it increases the acute and delayed side-effect. Toxic deaths occur and life-threatening side-effects are not infrequent, necessitating interruption of the treatment or reduction in the dose intensity. A possible marginal early survival benefit may well be offset by late side-effects. For the above reasons, we propose an alternative, risk-adapted, treatment strategy, to retain the present results at a lower price in terms of acute toxicity and late morbidity. It is suggested that all patients with classical OS should be treated pre-operatively with optimal doses of only the two most active agents, methotrexate and doxorubicin. This presumably is sufficient in the majority of these patients. The most toxic treatment involving additional anticancer agents should be reserved for high-risk and relapsing patients, i.e. for situations where drastic measures are necessary and warranted. An important consideration is that relapsing patients are likely to benefit in particular from drugs to which they have not been previously exposed.

Immunoscintigraphy of bone sarcomas--results in 5 patients.

The feasibility of using the murine monoclonal antibody, TP-1, for clinical immunoscintigraphy was examined in a pilot study involving 5 patients with bone sarcomas. 131I-labelled F(ab')2 antibody fragments were injected in doses of 0.8-1.0 mg (90-130 MBq), and the accumulation of radioactivity was examined by scintigraphy, and assessed by direct measurements on biopsied tumour and normal tissue. One osteosarcoma patient had a primary tumour in the femur, whereas the other 4 had single lung metastases detected by other diagnostic methods. Immunoscintigraphy of the femoral primary was optimally visualised after 22 h. In 2 patients, the method failed to detect lung metastasis, in 1 of the cases possibly related to less than optimal methodological conditions. In 2 other patients, increased accumulation of radioactivity indicated one and three lung tumours, in addition to the single metastasis observed by X-ray and CT scanning, tumours that were later confirmed and removed surgically. The concentration of radioactivity in tumour and normal tissues 44-72 h after antibody injection could be measured in 4 patients. The tumour to blood ratios were in the range of 1.2-4.2, compared to 0.1-0.8 for various normal tissues. The results indicate that immunoscintigraphy with TP-1 antibody fragments have a potential for early detection of lung metastases in patients with bone sarcoma.

Immunomagnetic removal of B-lymphoma cells using a novel mono-sized magnetizable polymer bead, M-280, in conjunction with primary IgM and IgG antibodies.

The efficacy of a novel monosized and magnetizable polymer bead, denoted M-280, in immunomagnetic removal of Rael B-lymphoma cells was compared with that of M-450 beads, previously used in bone marrow purging. The M-280 beads which are smaller (diameter 2.8 microns) and contain less iron than the M-450 beads were coated with polyclonal IgG sheep antimouse (SAM) antibody. The two types of immunobeads were equally efficacious when used together with the mouse monoclonal IgG antibodies HH1 or FN1, giving tumor cell depletions of about 3 logs in one cycle of operation. However, when used together with the primary IgM monoclonal antibodies (MoAbs) AB1 or HH2, the new immunobeads were significantly more efficacious than the M-450 immunobeads. To elucidate the underlying mechanism flow cytometric studies and measurements of the binding of the labeled primary MoAbs to the cellular antigens as well as to the immunobeads were carried out. Competition experiments showed that in the case of IgG MoAbs, the SAM beads bind predominantly to the Fc portion, whereas in the case of the IgM MoAbs, the Fab part plays a relatively greater role in the binding. The results indicate that if M-280 immunobeads are used, IgM MoAbs may profitably be included in antibody cocktails together with IgG antibodies in immunomagnetic purging of B-lymphoma cells. They suggest that in the case of cell bound MoAbs, the epitopes on IgG are more accessible to SAM beads than those of surface bound IgM molecules.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunomagnetic removal of B-lymphoma cells from human bone marrow: a procedure for clinical use.

B-lymphoma cells were purged from human bone marrow by incubating the cell suspension with a cocktail of three different pan-B cell mouse IgG1 monoclonal antibodies, and then with immunobeads charged with sheep anti-mouse antibody, followed by magnetic separation. The primary antibodies used, HD37 (CD19), HD6 (CD22), and HH1 (CD37), bind to a very high percentage of the cells in non-Hodgkin's lymphomas of poor prognosis. The secondary antibody is directed against the Fc portion of the IgG antibodies. In model experiments Burkitt's lymphoma cells (Rael) were admixed to mononuclear bone marrow cells in the ratio 1/9. With a ratio of immunobeads/total antibody-binding B cells of 50/1 in a first treatment cycle and repeating the procedure with the same number of beads in a second cycle, a tumor cell depletion of more than 5 logs was achieved, as judged by a clonogenic assay. The concomitant reduction of CFU-GM and CFU-GEMM was about 20%. The purging procedure has been scaled up to clinical use. Equipment suitable for purging patients' marrow specimens, employing standard transfusion facilities, is described. With this equipment the efficacy of tumor cell removal was the same as in the model experiments, and the whole magnetic separation could be completed in 2 hours.