Automated database-guided expert-supervised orientation for immunophenotypic diagnosis and classification of acute leukemia.

Precise classification of acute leukemia (AL) is crucial for adequate treatment. EuroFlow has previously designed an AL orientation tube (ALOT) to guide towards the relevant classification panel (T-cell acute lymphoblastic leukemia (T-ALL), B-cell precursor (BCP)-ALL and/or acute myeloid leukemia (AML)) and final diagnosis. Now we built a reference database with 656 typical AL samples (145 T-ALL, 377 BCP-ALL, 134 AML), processed and analyzed via standardized protocols. Using principal component analysis (PCA)-based plots and automated classification algorithms for direct comparison of single-cells from individual patients against the database, another 783 cases were subsequently evaluated. Depending on the database-guided results, patients were categorized as: (i) typical T, B or Myeloid without or; (ii) with a transitional component to another lineage; (iii) atypical; or (iv) mixed-lineage. Using this automated algorithm, in 781/783 cases (99.7%) the right panel was selected, and data comparable to the final WHO-diagnosis was already provided in >93% of cases (85% T-ALL, 97% BCP-ALL, 95% AML and 87% mixed-phenotype AL patients), even without data on the full-characterization panels. Our results show that database-guided analysis facilitates standardized interpretation of ALOT results and allows accurate selection of the relevant classification panels, hence providing a solid basis for designing future WHO AL classifications.

Improved flow cytometric detection of minimal residual disease in childhood acute lymphoblastic leukemia.

Most current treatment protocols for acute lymphoblastic leukemia (ALL) include minimal residual disease (MRD) diagnostics, generally based on PCR analysis of rearranged antigen receptor genes. Although flow cytometry (FCM) can be used for MRD detection as well, discordant FCM and PCR results are obtained in 5-20% of samples. We evaluated whether 6-color FCM, including additional markers and new marker combinations, improved the results. Bone marrow samples were obtained from 363 ALL patients at day 15, 33 and 78 and MRD was analyzed using 6-color (218 patients) or 4-color (145 patients) FCM in parallel to routine PCR-based MRD diagnostics. Compared with 4-color FCM, 6-color FCM significantly improved the concordance with PCR-based MRD data (88% versus 96%); particularly the specificity of the MRD analysis improved. However, PCR remained more sensitive at levels <0.01%. MRD-based risk groups were similar between 6-color FCM and PCR in 68% of patients, most discrepancies being medium risk by PCR and standard risk by FCM. Alternative interpretation of the PCR data, aimed at prevention of false-positive MRD results, changed the risk group to standard risk in half (52%) of these discordant cases. In conclusion, 6-color FCM significantly improves MRD analysis in ALL but remains less sensitive than PCR-based MRD-diagnostics.

Three-color flowcytometric analysis of mature and immature hematological malignancies. A guideline of the Dutch Foundation for Immunophenotyping of Hematological Malignancies (SIHON).

Multiparameter flowcytometry offers an insight into differentiation pathways, maturation stages and abnormal features of cell (sub)populations thus helping to establish and classify hematological malignancies. The Dutch Foundation for Immunophenotyping of Hematological Malignancies (SIHON) has formulated a guideline for a rapid screening followed by confirmation and classification in a standardized way. For this aim seven carefully composed monoclonal antibody combinations are elucidated for screening the test sample in a first phase. In this phase a relative frequency distribution of the cells will be established and a decision will be made about abnormal cells present, as well as their mature or immature state and the cell lineage they belong to. In a second phase, panels with cell lineage dependent monoclonal antibody combinations may be used to confirm and classify the abnormal cell population indicated in phase 1, as well as to establish the presence or absence of an abberant immunophenotype.

Frequency of clonal dominance in the specific antibody response to DNP-HSA in CBA and C57BL mice reflects their susceptibility to age-associated development of monoclonal gammopathies.

The effects of age, genetic background, and neonatal thymectomy on the levels and the heterogeneity of the specific antibody response were investigated in an experimental mouse model. Both intact and neonatally thymectomized (NTx) C57BL/KaLwRij (C57BL) and CBA/BrARij (CBA) mice were immunized at the age of 3 ("young") or 22 months ("old"). Highly sensitive antigen-specific immunoblotting techniques (ABL), in combination with agar-electrophoresis and isoelectric focusing (IEF), were used to investigate total specific antibody levels, the number of responding antigen-specific clonotypes, and the dominance of responding B cell clones in the antibody response against dinitrophenylated human serum albumin. After immunization, the specific antibody levels progressively increased in all experimental groups with the exception of old C57BL mice. All mice responded with a specific polyclonal heterogeneous response. In addition, some mice showed a clonal dominance of antibody-producing cells, as is reflected in the appearance of distinct homogeneous antibody components (H-Ab) in the sera. This clonal dominance was scarce in CBA mice but frequent in C57BL mice. Age at time of immunization and NTx had little if any additive effect on the incidence of H-Ab in either mouse strain. All dominant clones showed different electrophoretic mobility, indicating the proliferation of various clonotypes and not a strain-specific dominance of one clone. In old C57BL mice the specific antibody response was more restricted in heterogeneity, as is illustrated by more visible spectrotype bands in IEF and subsequent ABL. Hence, in old C57BL mice smaller amounts of specific antibodies were produced by fewer clones. Still, the incidence of H-Ab in this group was the same as that in the group of young C57BL mice. This indicates that at old age the responding B cell clones are more prone to becoming clonally dominant in C57BL mice. This tendency correlates with the high incidence of spontaneously developing monoclonal gammopathies in aging C57BL mice.

A monoclonal antibody (ER-HR3) against murine macrophages. I. Ontogeny, distribution and enzyme histochemical characterization of ER-HR3-positive cells.

We describe ER-HR3, a monoclonal antibody directed against bone marrow-derived haemopoietic reticulum cells. ER-HR3-positive cells have the electron-microscopic and enzyme-histochemical characteristics of macrophages. Additionally, they are able to phagocytoze. The ER-HR3 antigen is expressed by a majority of blood monocytes and is present on a subpopulation of resident macrophages in multiple organs. ER-HR3-positive cells are abundant in the bone marrow, the splenic red pulp, the mesenteric lymphoid paracortex and the interfollicular areas of the Peyer's patch. Few ER-HR3-positive cells have been observed in the thymic cortex and the connective tissues of the gastro-intestinal tract, the dermis and the renal medulla. Moreover, epidermal Langerhans cells express the antigen. No cross-reactivity with other cell types has been found. It is concluded that ER-HR3 has a unique distribution pattern distinct from other macrophage-specific antibodies.

A monoclonal antibody (ER-HR3) against murine macrophages. II. Biochemical and functional aspects of the ER-HR3 antigen.

We describe the purification and intracellular distribution of an antigen present on a subpopulation of murine macrophages and recognized by monoclonal antibody ER-HR3 against bone marrow-derived haemopoietic reticulum cells. Using the ER-HR3 antibody as an immobilizing ligand, two proteins were isolated as determined by SDS polyacrylamide gel electrophoresis. Under non-reducing conditions, there was a major band with an apparent molecular mass of 69 kDa and a minor band of 55 kDa. Under reducing conditions, the apparent molecular mass of each band was estimated as 76 kDa and 67 kDa, respectively. Intracellularly, these proteins occurred in close association with membranous structures, as demonstrated with gold-labelled protein A in an electron-microscopic study of the ER-HR3-positive cell line AP284. Some of the antigen was present in vesicles. To gain further insight into the possible function of the ER-HR3 antigen, its tissue distribution was investigated under distinct experimental conditions. In mice infected with Bacillus Calmette Gurèrin, ER-HR3-positive cells were observed in many, but not all, granulomata of the spleen, the lung and the liver. The ER-HR3 reactivity in these mice clearly differed from that of other antimacrophage monoclonal antibodies, such as F4/80, M5/114 and M1/70. Furthermore, phenylhydrazine-induced extramedullary erythropoiesis in the liver was accompanied by ER-HR3 expression on a subpopulation of macrophages. Finally, the addition of ER-HR3 to an antigen-specific T cell proliferation assay did not inhibit T cell proliferation.

Selective lesions by manganese and extensive damage by iron after injection into rat striatum or hippocampus.

Regional 45Ca2+ accumulation and analysis of monoamines and metabolites in dissected tissues were used to localize, quantify, and characterize brain damage after intracerebral injections of Mn2+ into striatum and hippocampus. The specificity of Mn(2+)-induced lesions is described in relation to brain damage produced by local Fe2+ or 6-hydroxydopamine (6-OHDA) injections. In striatum, Fe2+ and Mn2+ produced dose-dependent (0.05-0.8 mumol) dopamine (DA) depletion, with Fe2+ being 3.4 times more potent than Mn2+. Studies examining the time course of changes in monoamine levels in striatum following local application of 0.4 mumol of Mn2+ revealed maximal depletion of all substances investigated (except 5-hydroxyindoleacetic acid) after 3 days. The effects on DA (87% depletion at day 3) and its major metabolites were most pronounced and lasted until at least 90 days (40% depletion), whereas serotonin and noradrenaline levels recovered within 21 and 42 days, respectively. In addition, levels of 3-methoxytyramine, which is used as an index of DA release, also recovered within 42 days, indicating a functional restoration of DA neurotransmission despite substantial loss of DA content. Intrastriatal Mn2+ (0.4 mumol) produced time-dependent 45Ca2+ accumulation in striatum, globus pallidus, entopeduncular nucleus, several thalamic nuclei, and substantia nigra pars reticulata ipsilateral to the injection site. In contrast, 6-OHDA injected at a dose equipotent in depleting DA produced significantly less 45Ca2+ accumulation in striatum and globus pallidus and no labeling of other brain areas, whereas Fe2+ (0.4 mumol) produced extensive 45Ca2+ accumulation throughout basal ganglia, accumbens, and cerebral cortex. In hippocampus, high Mn2+ (0.4 mumol) produced limited 45Ca2+ accumulation in subiculum and dentate gyrus, whereas low Fe2+ (0.1 mumol) produced widespread 45Ca2+ accumulation throughout hippocampus, thalamus, and cerebral cortex. It is concluded that (a) Mn2+ is selectively neurotoxic to pathways intrinsic to the basal ganglia, (b) intrastriatal injections can be used as a model for systemic Mn2+ intoxications, and (c) high endogenous Fe3+ and/or catecholamine levels potentiate the neurotoxicity of Mn2+.

Time- and dose-dependent 45Ca2+ accumulation in rat striatum and substantia nigra after an intrastriatal injection of quinolinic acid.

To evaluate the use of cerebral 45Ca accumulation as an index for neurotoxic brain damage, the dose and time dependency of regional 45Ca accumulation in the rat brain following an unilateral injection of quinolinic acid (QUIN) into the striatum was investigated. Accumulation of radioactivity was assessed 6 h or 1, 2, 7, 21, 42, and 84 days after QUIN and 6 h or 1 day after ip 45CaCl2 injection using liquid scintillation counting of dissected brain tissues and semiquantitative autoradiography of brain sections. Dissected striata were also used for assay of glutamic acid decarboxylase (GAD) activity and after autoradiography brain sections were stained with thionine or for cholinesterase. One week after QUIN (0-50 micrograms) a dose-dependent increase in 45Ca accumulation in the injected striatum and the ipsilateral substantia nigra, but not in frontal cortex or cerebellum, was observed. In addition, 45Ca accumulation and GAD reduction in the striatum was highly significantly correlated. In the striatum 45Ca accumulated rapidly within 48 h and after this time point--by this time GAD had reached a minimum--the 45Ca content continued to increase in the lesioned striatum until Day 42. Six hours after QUIN 45Ca accumulated in the immediate vicinity of the injection site, whereas the increase 6 weeks later is primarily due to accumulation of 45Ca in striatal tissue more remote from the injection site. In the ipsilateral substantia nigra 45Ca accumulation reached a maximum 7 days after QUIN, probably reflecting delayed transneuronal death rather than degeneration of striatonigral nerve terminals.(ABSTRACT TRUNCATED AT 250 WORDS)

Development of aging-associated monoclonal gammapathies with antibody activity to the antigen used for immunization of young mice.

The influence of immunization with dinitrophenylated human serum albumin (DNP-HSA) at a young age on the development of age-related monoclonal gammapathies (MG) was investigated in a longitudinal study in intact and neonatally thymectomized (NTx) C57BL/KaLwRij and CBA/BrARij mice. Three-month-old mice were immunized four times in monthly intervals with DNP-HSA. Control mice received saline and adjuvant only. Mice immunized with DNP-HSA responded with heterogeneous antibodies, occasionally with some clonal dominance. The antibody levels further declined and were hardly detectable when the mice were 21 months old. Eighteen of 87 experimental mice developed homogeneous antibody components (H-Ab) to DNP-HSA with aging. Their frequencies per individual groups were 5, 22, 24, and 29% for intact CBA, NTx-CBA, NTx-C57BL, and intact C57BL mice, respectively. Some H-Ab had the same mobility and similar spectrotypes as dominant clonal products at the peak of the response. However, the majority of H-Ab appearing at old age were "new" H-Ab. While most of H-Ab in the CBA mice were transient and of a low concentration, the majority of H-Ab in the C57BL mice had all characteristics of a benign monoclonal gammapathy. The results indicate that memory cells of the B cell clones involved in the original specific response may in a susceptible strain become targets for events leading to the development of benign monoclonal gammapathy.

Improved fixation of frozen lympho-haemopoietic tissue sections with hexazotized pararosaniline.

In this study, a simple single-step fixation method for frozen tissue sections is introduced using the hexazotized salt of Pararosaniline as preservative agent. Tissue preservation by this method was shown to be superior to the commonly-used fixation with acetone. Fixation with hexazotized Pararosaniline caused a minimal loss of antigenicity as demonstrated using twenty-three monoclonal antibodies directed against lympho-haemopoietic and stromal cells.

Immunoblotting techniques for the detection of low level homogeneous immunoglobulin components in serum.

Because of the increasing demand for simple and reliable techniques for the detection of low concentrations of paraproteins against a highly heterogeneous serum background, two techniques were investigated for their sensitivity: isoelectric focusing (IEF) and Wieme high resolution electrophoresis, each with subsequent blotting by diffusion. The techniques were compared using isolated mouse monoclonal antibodies (mAb) of known concentration and specificity. Wieme electrophoresis in combination with immunoblotting (IBL) or antigen-specific immunoblotting (ABL) has a detection limit of 100 ng/ml and 10 ng/ml, respectively. For IEF in combination with IBL or ABL these limits were 1000 and 30 ng/ml, respectively. For ABL, polyvinylidene difluoride (PVDF) and nylon-supported nitrocellulose (NSNC) membranes gave similar detection limits, although for IBL, PVDF is preferred to NSNC. While IEF is essential for investigating the spectrum of the antibody repertoire. Wieme electrophoresis is the most powerful technique for the detection of homogeneous immunoglobulin components (H-Ig). After separation of the proteins. IBL is fast, simple and sensitive enough for routine detection and characterization of H-Ig. However, when the antibody specificity is known, ABL should be chosen for its superior sensitivity.