Let them fly or light them up: matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry and fluorescence in situ hybridization (FISH)

This review focuses on clinical bacteriology and by and large does not cover the detection of fungi, viruses or parasites. It discusses two completely different but complementary approaches that may either supplement or replace classic culture-based bacteriology. The latter view may appear provocative in the light of the actual market penetration of molecular genetic testing in clinical bacteriology. Despite its elegance, high specificity and sensitivity, molecular genetic diagnostics has not yet reached the majority of clinical laboratories. The reasons for this are manifold: Many microbiologists and medical technologists are more familiar with classical microbiological methods than with molecular biology techniques. Culture-based methods still represent the work horse of everyday routine. The number of available FDA-approved molecular genetic tests is limited and external quality control is still under development. Finally, it appears difficult to incorporate genetic testing in the routine laboratory setting due to the limited number of samples received or the lack of appropriate resources. However, financial and time constraints, particularly in hospitals as a consequence of budget cuts and reduced length of stay, lead to a demand for significantly shorter turnaround times that cannot be met by culture-dependent diagnosis. As a consequence, smaller laboratories that do not have the technical and personal equipment required for molecular genetic amplification techniques may adopt alternative methods such as fluorescence in situ hybridization (FISH) that combines easy-to-perform molecular hybridization with microscopy, a technique familiar to every microbiologist. FISH is hence one of the technologies presented here. For large hospital or reference laboratories with a high sample volume requiring massive parallel high-throughput testing we discuss matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) of nucleic acids, a technology that has evolved from the post-genome sequencing era, for high-throughput sequence variation analysis (1, 2).     

Near-haploid clones in a malignant fibrous histiocytoma

Near-haploid solid tumors are very rare. In a storiform-pleomorphic malignant fibrous histiocytoma (MFH) of bone, we found three cell populations: one with a near-haploid, a second with a near-diploid, and a third with a near-tetraploid chromosome number. The near-haploid cells had few structural rearrangements: i(12p) and t(13q21q) in one clone, and these two and an additional t(19;?)(p11;?) in another clone. One structurally normal copy of all chromosomes was also present, except that the only chromosome 13 was involved in the t(13q21q). There were also two near-diploid clones, one without the t(19;?) and one with a single copy of this derivative chromosome. This is the first MFH reported to have a near-haploid modal chromosome number, and also the first tumor with i(12p) among bone and soft tissue tumors.     

Oxygen metabolites induced by phorbol myristate acetate increase lateral diffusion of wheat germ agglutinin-labeled glycoconjugates in human polymorphonuclear leukocytes

To assess the general effects of protein kinase C (PKC) activation on cell membrane receptor mobility in human neutrophilic polymorphonuclear leukocytes (PMNLs), the lateral diffusion of fluoresceinated succinylated wheat germ agglutinin (S-WGA-FITC)-labeled membrane glycoconjugates was measured using fluorescence recovery after photobleaching (FRAP). Activation of PKC was achieved by incubating the PMNLs with different concentrations (5–100 nM) of phorbol myristate acetate (PMA). The membrane effects of dimethyl sulfoxide (DMSO), another possible membrane perturbant, were also studied. We found that PMA treatment ( 10 nM) increased the glycoconjugate diffusion coefficient (D) 2–2.5-fold. The mobile fraction (R) remained constant, around 30%. With DMSO, no effect on the diffusion was seen. The increase in lateral mobility due to cell stimulation with PMA was totally inhibited by catalase (200 units/ml) but only partly with superoxide dismutase (2000 units/ml). Exogenous hydrogen peroxide (0.01–5 mM) had no effect on glycoconjugate mobility in unstimulated cells. We therefore propose that activation of PKC mediates augmented mobility of glycoconjugate receptors in PMNL, a reaction that seems to be critically dependent on formation of reactive oxygen metabolites. The results indicate that endogenous formation of reactive metabolites upon receptor stimulation may have a general effect on receptor mobility.     

Low-dose inhaled budesonide once or twice daily for 27 months in children with mild asthma

This study is an extended follow-up for 24 months of a 12-week trial to study the long-term clinical efficacy of low-dose inhaled budesonide (BUD) once or twice daily in children with mild asthma. A total of 122 children (mean age 9.7 years, girls/boys; 42/80) with mild asthma (FEV1 103.7% of predicted, reversibility in FEV1 3.5%, and fall in FEV1 after exercise 12.2%), not previously treated with inhaled steroids, were included in a double-blind, randomized, parallel-group study. The children were treated with inhaled BUD 100 or 200 microg administered via Turbuhaler once daily in the morning, 100 microg twice daily, or placebo for 27 months. Exercise and methacholine challenges were performed at 3-month intervals the first year and at 6-month intervals the second year, in a total of seven visits. A significant dose-response effect favoring BUD 200 microg daily (vs 100 microg daily) was found when comparing changes in FEV1, FEF25%, and FEV50%; the fall in FEV1 after an exercise test; and the effect on blood eosinophils. Bronchial hyperreactivity to methacholine decreased significantly on three visits in patients treated with BUD 200 microg daily compared to placebo. Growth rate was not significantly affected except in children aged 7-11 years at baseline after 12 months of treatment. In conclusion, 100 or 200 microg daily of inhaled BUD for 27 months is safe and effective in protecting against exercise-induced asthma and achieving nearly normal lung function. Baseline lung function was not significantly affected in this group of children with mild asthma.     

Isolation of human immunodeficiency virus (HIV) from plasma during primary HIV infection

Human immunodeficiency virus (HIV) has been isolated from plasma in 6 of 7 patients showing clinical symptoms of a primary HIV infection. Parallel cultures from peripheral blood mononuclear cells (PBMC) yielded virus in 5 patients. In one case, virus could only be isolated from the cerebrospinal fluid but not from peripheral blood. Detectable viremia was transient and preceded the appearance of HIV specific antibodies. After cessation of acute symptoms, the frequency of HIV isolations was similar to that of asymptomatic carriers (23 and 26%, respectively). The role of the immune response in terminating detectable viremia remains to be established.     

Karyotypic evolution in Ph-positive chronic myeloid leukemia in relation to management and disease progression

In a prospective study of 32 patients with chronic myeloid leukemia the frequency of chromosome abnormalities in addition to the Philadelphia chromosome (Ph) increased when the disease progressed. Before metamorphosis, 10 patients (31%) had developed additional abnormalities. Such abnormalities were present in three of them at the time of diagnosis; in the other seven, they were detected late in the chronic phase. New clonal abnormalities heralded or accompanied a more malignant phase of the disorder, usually a blastic leukemia. During metamorphosis, 78% of the patients had additional abnormalities, which in 68% of these cases comprised at least one of +8, +22q- or i(17q). Clones with additional abnormalities disappeared in eight cases, either spontaneously or in association with cytostatic therapy during the chronic or blastic phase. Involvement of chromosome #8, usually in the form of a trisomy, was found in 7 of 12 patients treated with busulfan, but was not found in any of the 10 hydroxyurea-treated patients, of whom 8 were splenectomized early during the chronic phase. Cells from the spleen, obtained by fine needle aspiration or splenectomy were cytogenetically examined in 18 cases during the chronic phase, but abnormalities in addition to the Ph were noted in only one patient, who was examined in the late chronic phase. The same abnormalities were present in bone marrow cells of this patient.     

Identification of a family with nonspecific mental retardation (MRX79) with the A140V mutation in the MECP2 gene: Is there a need for routine screening?

Mutations in the methyl-CpG-binding protein 2 (MECP2) cause Rett syndrome, a severe neurodevelopmental disorder occurring predominantly in females. Male patients with Rett syndrome are extremely rare, as the Rett-causing mutations in the MECP2 gene are usually lethal in hemizygous males. However, different mutations in the same gene were reported to cause mental retardation, both in sporadic non-syndromic males as well as in syndromic families with disease manifestation in carrier females. The majority of the reported MECP2 mutations in mentally retarded patients cause amino acid substitutions and, especially in isolated cases, discrimination between a disease-causing mutation and a rare polymorphism is not obvious and the significance of each individual variation should be verified. We mapped a new non-syndromic X-linked family (MRX79) to the chromosomal region Xq27.3-Xq28 and identified an A140V mutation in the MEPC2 gene in all patients with the disease haplotype. In addition to data published by others, this suggests that A140V is a recurrent mutation (and not a polymorphism) found in patients with X-linked mental retardation.     

Differences in adsorption of serum proteins and production of IL-1ra by human monocytes incubated in different tissue culture microtiter plates

In vitro cell culture models can be of great value in order to further analyze the regulatory mechanisms underlying the inappropriate function of the immune system in diseases such as autoimmunity and cancer. Cell culture conditions have to be well controlled in a way that they mirror the in vivo situation. The objective of this study was to compare tissue culture microtiter plates from different manufacturers with respect to their ability to support monokine production by human monocytes cultured in human serum. Tissue culture ware, made of polystyrene, undergoes treatment by the manufacturers to make the surface more suitable for culture of adherent cell populations. It is possible that quality differences in this treatment can lead to variations in protein binding properties and thereby influence the adherence and functional properties of monocytes. We measured spontaneous interleukin-1 receptor antagonist (IL-1ra) production by peripheral blood monocytes, cultured in human serum, in five different microtiter plates made for adherent cell culture. Culture in plates from two of the five manufacturers resulted in significantly lower amounts of secreted IL-1ra. IL-1ra release by human monocytes can be induced by adherent IgG cross-linking membrane receptors for the Fc part of IgG (FcgammaR). We found that reduced IL-1ra production coincided with a reduced capacity for binding of serum IgG in one case. Furthermore, this brand of microtiter plate also displayed the lowest level of adsorption of human albumin. We conclude that the protein adsorption properties of the plastic tissue culture ware have to be taken into consideration when assessing monokine production by human monocytes in vitro.     

Cytogenetic studies of bone marrow and extramedullary tissues and clinical course during metamorphosis of chronic myelocytic leukemia

Of 33 consecutive patients with chronic myelocytic leukemia, examined during metamorphosis, 82% showed chromosome abnormalities in addition to the Ph1. Aberrations most frequently encountered were +8 (39%), +22q - (30%), and i(17q) (18%). Translocations other than the Ph1 were observed in four cases and - Y clones in four cases. Discrepancies in the cytogenetic pattern between bone marrow and extramedullary tissues or blood were noted in a total of 15 patients. In six cases, transformation occurred in extramedullary organs at a time when it was not present in the marrow. In three cases the bone marrow transformation was preceded by a lymph node blastic infiltrate; in one case, by a skin infiltrate; and in one case, by a subdural blastoma. Clonal abnormalities additional to the Ph1 were identified in the tumor tissue from all these cases. Patients with primary extramedullary transformation tended to have a lower median age at onset of metamorphosis, shorter survival, and higher incidence of chromosome abnormalities than the cases without extramedullary involvement. Patients with only Ph1-positive cells and no other anomalies had a slightly longer duration of metamorphosis and longer total survival. Basophilia and thrombocytopenia were more marked in cases with i(17q) than in the rest of the series.