Malignant leukemic cell separation by iron colloid immunomagnetic adsorption.

We describe an immunomagnetic assay applicable to bone marrow purging of leukemic patients before an autologous bone marrow transplantation. An iron colloidal suspension with a CD10 monoclonal antibody (MoAb) against the common acute lymphoblastic leukemia antigen (CALLA) covalently bound to the surface of the particles has been used. NALM-6 cells, a pre-B leukemic cell line expressing the CALLA, were labeled with supravital DNA dye (Hoechst 33342), mixed with peripheral blood, and incubated with the MoAb coated to iron particles. Using this reagent, at a cell concentration of 2000/microliters, a purging effect greater than 3.5 logs is observed with 0.1 mg of coated particles. Three successive rounds of treatment with the same coated particles at the same dose showed approximately the same depletion after each treatment. The recovery of clonogenic myeloid progenitors (granulocyte-macrophage colony-forming units; CFU-GM) is around 75%. No depletion was observed when the iron particles were not attached to the CD10 MoAb, or when they were attached to a MoAb not recognizing CD10+ cells. A comparison with another commercially available magnetic support was also performed in order to evaluate the performance of our assay, which appears simple, efficient, cheap, and capable of handling large volumes of cells in sterile conditions and minimal time.

A case of gamma 3 heavy chain disease with vacuolated plasma cells: a clinical, immunological, and ultrastructural study.

A patient with lambda Bence-Jones proteinuria, Waldenström's macroglobulinaemia, and Franklin's disease (gamma HCD), but without clinical evidence of a lymphoproliferative disorder, is presented. The serum contained two distinct immunoglobulin abnormalities: a monoclonal immunoglobulin M (IgM) of lambda type, and a protein fragment which was immunologically related to immunoglobulin G (IgG) and devoid of light chain activity. This gamma HCD protein belongs to the gamma 3 subclass with a molecular weight of approximately 60,000 daltons. The urine contained a Bence-Jones lambda protein as well as the gamma HCD fragment. The two paraproteins were probably secreted by two different malignant clones. Ultrastructural study revealed pathological vacuolated plasma cells of a sort that has hitherto been principally described in association with micron HCD. The mechanism of the intracellular storage of pathological immunoglobulins is discussed in the light of the ultrastructural study.

A new cryopathic syndrome.

One case of a syndrome simulating a systemic disease is reported. An HLA-B, patient presented with apparent toxoplasmosis, retinal vein occlusion, autoantibodies against cephalin, contact factors and lipoproteins, immune complexes, rheumatoid factor, and cryoprecipitate. A representation of the immunological mechanisms involved is proposed.

Lactoferrin: its role as a regulator of human granulopoiesis?

Lactoferrin has been proposed recently as a physiological regulator of the granulocyte-monocyte progenitor (CFU-GM). This glycoprotein, when saturated with iron, has been said to limit the CFU-GM growth by decreasing production and release of colony stimulating activity by monocytes and macrophages. Human milk lactoferrin saturated with iron, at concentrations ranging from 10(-8) M, was added either to endogenously stimulated bone marrow cells or to mononucleated cells used as feeder layers for adherent cell-depleted marrow. Irrespective of the concentration of lactoferrin within the culture system used, no significant inhibition of the CFU-GM growth was observed. Moreover, the CFU-GM stimulating activity of medium conditioned by a 4 day incubation of 1 X 10(6) mononucleated blood cells in the presence or in the absence of lactoferrin was the same. Various possible explanations for not confirming the reported inhibiting activity of iron-saturated lactoferrin were explored: (a) masking inhibition of the system by prostaglandin E2 (PGE2), (b) masking inhibition of the system by bovine lactoferrin present in the fetal calf serum, (c) preinhibition of the system by leukemic-associated inhibitory activity possibly present in the culture system, (d) the iron and calcium content of the culture medium used, (e) the fixation of lactoferrin to plastic compounds, (f) the source of the human lactoferrin used, and (g) the marrow cell separation methods used. None of these factors was shown to play a role in vitro in the activity of lactoferrin and thus no evidence was found for a significant role of lactoferrin in the regulation of human granulopoiesis.

Interactions Between Antibody-Coated Iron Micro-Particles and Leukemic Cells as seen by Electron Microscopy.

An electron microscopy study was performed to visualize the interactions between monoclonal antibodies (MoAbs) coated to ferromagnetic microparticles and leukemic cells. The properties of this specific reagent used for the efficient purging of residual leukemic cells from autografts before autologuous bone marrow transplantation (ABMT) have been previously reported. Incubation of a CD10+ NALM6- cell suspension with CD10 or CD8 MoAb conjugated particles was performed following a time-course schedule ranging from 0 to 90 min. at 4°C and 37°C. Transmission electron microscopy demonstrated that, although the CD10 MoAb-particles complex linked to the cell surface only at 4°C, they readily penetrated into cells at 37°C. In contrast, after incubation with CD8 MoAb-particles, no iron particles were seen at the cell surface, or within the cells no matter what the temperature and duration of the incubation were. This observation offers the unique opportunity to deliver a pharmaceutical agent crosslinked to the same magnetic carrier inside a cell, using an interesting site-specific drug delivery concept.

[Ex-vivo treatment of a bone marrow graft]. / Traitement ex-vivo d'un greffon médullaire.

The use of bone marrow transplantation is increasing in the management of advanced cancers. In autologous bone marrow transplantation (ABMT), many investigators have attempted to purge the graft of residual tumor cells because of concern that reinfused tumor cells might contribute to relapse. The feasibility of various methods (exposure to chemical agents, monoclonal antibodies (MoAbs), toxins, dye, magnetic microparticles ... ) has been confirmed. In allogeneic bone marrow transplantation, clinical studies have related the prevention of graft-versus-host disease reaction through the partial depletion of T lymphocytes in the donor graft limited to 1 log to maintain a graft-versus-leukemia (GVL) effect. Similarly, the feasibility of different assays (soybean agglutinin, Moabs and magnetic microparticles) have been shown. However, the clinical benefit of BM purging remains to be demonstrated. For ABMT, only recent data on B-cell lymphoma and leukemia strongly support the clinical usefulness of an ex-vivo purging. For allogeneic BMT, one question remains controversial: is T lymphocytes depletion the best method for GVHD prevention?

Lactoferrin: no evidence for its role in regulation of CSA production by human lymphocytes and monocytes.

Lactoferrin (LF) has been recently proposed as a physiologic regulator of the granulocyte monocyte progenitor (CFU-GM). This glycoprotein, when saturated with iron, has been said to limit CFU-GM growth by decreasing production and release of colony stimulating activity (CSA) by monocytes and macrophages. Human milk LF saturated with iron, at concentrations ranging from 10(-18) to 10(-8) M was added either to endogenously stimulated bone marrow cells or to mononucleated cells used as feeder layers for adherent cell-depleted marrow. Irrespective of the concentration of LF within the culture system used, no significant inhibition of CFU-GM growth was observed. Moreover, the CFU-GM stimulating activity of medium conditioned by a 4-day incubation of 1 X 10(6) mononucleated blood cells in the presence or in the absence of LF was the same. Various possible explanations for not confirming the reported inhibiting activity of iron saturated LF were explored: 1) masking inhibition of the system by prostaglandin E2 (PGE2), 2) masking inhibition of the system by bovine LF still detectable in the fetal calf serum after heating, 3) preinhibition of the system by leukemic-associated inhibitory activity (LIA) possibly present in the culture system, 4) the iron and calcium content of the culture medium used, 5) the fixation of LF to plastic compounds, 6) the source of the human LF used, 7) the marrow cell separation methods used. None of these factors was shown to play a role in vitro in the activity of LF and thus no evidence was found for a significant role of LF in the regulation of CSA production by monocytes. Peripheral blood human monocytes isolated by elutriation and incubated in albumin free medium in the presence of either 125I-LF or colloidal gold-labeled LF showed no LF binding.

Comparison of the effectiveness of four clinical chemical assays in classifying patients with chest pain.

We compared the usefulness of four serum assays for classifying patients originally suspected of having an acute myocardial infarction. One of these is the long-used measurement of total creatine kinase (CK) activity. The other three are relatively new immunoassays: myoglobin by RIA, CK-BB by RIA, and CK-MB by immunoinhibition. When we evaluated test effectiveness with use of conventionally derived reference ranges, the results were misleading. However, by using receiver operating characteristic curves, we were able to effectively compare the four tests at all possible decision levels, rather than at only one. Multiple closely sequential serum specimens were obtained during the first four days after the onset of chest pain. Total CK, CK-MB, and CK-BB all behaved similarly, reaching peak diagnostic effectiveness at 18-20 h, when all three correctly classified 95% of the infarct patients, with a zero false-positive rate. However, total CK was more useful in identifying infarcts later in their courses than were the two CK isoenzyme tests. Myoglobin assay was most effective earlier in the course, at about 7 to 8 h. Our results indicate (a) that the tests for myoglobin and for CK or its isoenzymes are complementary and (b) that of the three CK tests, measurement of total CK activity provides the most information over the broadest segment of a patient's course.

Hinge-thiol coupling of monoclonal antibody to silanized iron oxide particles and evaluation of magnetic cell depletion.

Iron oxide particles of average size 0.5-1.5 microns, covered by a silane coat carrying amino groups (Bio-Mag, Advanced Magnetics, Boston), were derivatized by reaction with N-[(gamma-maleimidobutyryl)oxy]-succinimide (GMBS), N-hydroxysuccinimidyl iodoacetate (NHIA), 2-iminothiolane (2-It), or N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP). The derivatized particles were suitable for the reaction with sulfhydryl groups and subsequently coated with monoclonal antibodies (MoAbs) of different classes and isotypes (IgM, IgG1, IgG2a, IgG2b, IgG3) as well as polyclonal rabbit anti-mouse IgG (RAM). The antibodies were reduced by dithiothreitol (DTT) and covalently conjugated to the BioMag derivatives via liberated sulfhydryls of the hinge region. The observed conjugation ratios, expressed as protein/iron (micrograms/mg), could be reproducibly varied for optimization. These ratios were dependent on the type and amount of antibody offered for coupling to the derivatized particles, decreasing as follows: polyclonal = IgM greater than IgG2b greater than IgG2a = IgG3 greater IgG1. The conjugation ratios were also dependent on the type and amount of the spacer used to derivatize the BioMag particles, decreasing as follows: GMBS greater than NHIA greater than 2-It greater than SPDP. The magnetically responsive magnetite-antibody conjugates ("magneto-beads"), carrying MoAb BMA 081 (anti-CD8; IgG2a), MoAb BB10 (anti-CD10/CALLA; IgG2b), MoAb VIL-A1 (anti-CD10; IgM), and polyclonal RAM, coupled similarly via 3.6 mumol of GMBS spacer per mg of Fe, were further investigated with respect to a depletion effect on specific cell subsets. The rates of cell depletion were found to be strongly dependent on the individual characteristics of the antibody used.(ABSTRACT TRUNCATED AT 250 WORDS)