Diagnostic value of dominant T-cell clones in peripheral blood in 363 patients presenting consecutively with a clinical suspicion of cutaneous lymphoma.

It is now widely accepted that polymerase chain reaction (PCR) analysis of cutaneous T-cell clonality is of diagnostic value in cutaneous T-cell lymphomas (CTCLs) and most helpful in the diagnosis of mycosis fungoides (MF). However, the diagnostic and prognostic value of circulating clonal T cells remains unclear. We studied T-cell clonality in the peripheral blood (PB) and the cutaneous lesion, sampled at the same time, in 363 consecutively seen patients with a clinical suspicion of cutaneous lymphoma. Using a PCR technique providing a specific imprint of T-cell clones (PCRgamma-denaturing gradient gel electrophoresis), we found that detection of identical circulating and cutaneous T-cell clones was associated with the diagnosis of CTCL (P <.001). Detection of circulating tumor cells in patients with MF was infrequent (12.5%), except in those with erythrodermic MF (42%; P =.003). Moreover, among the 46 patients who had identical circulating and cutaneous T-cell clones, 25 (56%) had erythroderma. The finding of a dominant clone in the PB but not in the skin was frequent, regardless of the clinicohistologic classification; it occurred in 30% of patients with CTCL, 41% with non-CTCL malignant infiltrates, and 34% with benign infiltrates. This pattern was significantly more frequent in patients over 60 years of age (P <.002), even in the CTCL group (P <. 01). In conclusion, dominant T-cell clones detected in the PB of patients with MF by using a routine PCR technique are rarely tumoral and are more often related to age. A multicenter prospective study is under way to establish the prognostic value of circulating tumor cells.

Value of clonality studies of cutaneous T lymphocytes in the diagnosis and follow-up of patients with mycosis fungoides.

Histological features of early mycosis fungoides (MF) can simulate numerous inflammatory lesions and histological confirmation of MF is often delayed, compared with clinical diagnosis. Recently, using molecular techniques, the detection of a dominant T-lymphocyte clone has been reported in cutaneous lesions of MF. The aim of the present study was to determine the diagnostic value of a dominant T-lymphocyte clone as assessed by PCR-DGGE in early MF. Histopathological and molecular analyses were performed on cutaneous lesions from 104 patients clinically suspected as having MF. In this population, the positive predictive value of a PCR gamma(+) was 0.86. In addition, four of six patients whose lesions were PCR gamma(+) (detectable dominant T-cell clone) but not histologically MF progressed to MF within 2-48 months. In order to evaluate the relevance of PCR gamma-DGGE in MF follow-up, serial biopsies were performed in 24 patients. In 89 per cent of cases, the presence or absence of a PCR gamma(+) was constant during the course of the disease. When present, the DGGE imprint of PCR products was case-specific. These data demonstrate the diagnostic value in MF of T-lymphocyte clonality assessed by PCR gamma-DGGE on cutaneous lesions and show that the technique can be used in MF follow-up to evaluate residual disease with high specificity.

VJ rearrangements of the TCR gamma locus in peripheral T-cell lymphomas: analysis by polymerase chain reaction and denaturing gradient gel electrophoresis.

Using Southern blotting for the diagnosis of clonality in peripheral T-cell lymphomas (PTCLs), analysis of the T-cell receptor (TCR) gamma gene rearrangement was shown to be more informative than that of the TCR beta gene rearrangement. In order to amplify every VJ gamma rearrangement, a polymerase chain reaction (PCR) procedure using newly designed GC-clamp primers has been developed. All primers can be mixed in a single multiplex PCR. PCR products are analysed by denaturing gradient gel electrophoresis (DGGE), providing tumour-specific imprints inasmuch as the procedure characterizes N sequence polymorphism at the VJ junctions. In a series of 30 PTCL cases, the PCR procedure demonstrated 27 cases to be clonally rearranged and failed in three cases. PCR was more accurate than Southern blotting, showing 47 rearranged gamma alleles, four of which were undetectable on the Southern blot. When lymphomas were studied at different sites and at relapse, the DGGE pattern remained unchanged. In PTCL, the proposed PCR is helpful for the diagnosis and staging of the disease and should improve the follow-up monitoring. The undetectability of clonal rearrangements in a few cases is discussed in the light of concepts of lymphomagenesis and T-cell differentiation.

Cutaneous T-cell infiltrates: analysis of T-cell receptor gamma gene rearrangement by polymerase chain reaction and denaturing gradient gel electrophoresis.

In cutaneous T-cell infiltrates, the demonstration of a clonal T-cell receptor (TCR) gene rearrangement has been considered helpful to distinguish Cutaneous T-cell lymphomas from reactive lymphoproliferation. Hence, a polymerase chain reaction (PCR) method using GC-clamp primers and denaturing gradient gel electrophoresis has been developed in our laboratory to analyze the TCR gamma locus configuration. Two hundred eleven cutaneous samples from 155 patients were analyzed. A detectable clonal TCR gamma rearrangement was significantly associated with cutaneous T-cell lymphomas as defined by morphologic and immunologic criteria. A clonal TCR gamma rearrangement was also detected frequently in lymphomatoid papulosis, never in reactive lymphocytic infiltrates and B-cell lymphomas, and rarely in parapsoriasis en plaque and cutaneous lymphoid hyperplasia. Forty five patients had both a cutaneous and a peripheral blood sample. Fifteen had a detectable clonal rearrangement in the two samples and 22 were negative. Six patients had a positive skin sample and a negative blood sample, whereas two patients had a positive blood sample and a negative skin sample. Four lymph node samples were analyzed and the PCR results were the same as in the skin. Finally, 21 patients had sequential samples of recurrent skin lesions. The PCR results were concordant in all and, when detectable, the clonal TCR gamma rearrangement remained unchanged in a given patient. Because of its simplicity and accuracy, the newly designed PCR procedure improves the monitoring of diagnosis, staging, and follow-up in cutaneous T-cell infiltrates.

Serum 5'-nucleotidase and alkaline phosphatase activities after high dose chemotherapy and bone marrow transplantation in cases of malignancy in children.

After bone marrow transplantation in children, it is essential to detect secondary liver diseases and hepatotoxic effects of immunosuppressive therapy. These can be revealed by cytolytic syndromes sometimes associated with cholestasis. It is therefore important to find an early and specific cholestasis enzymatic marker. A retrospective study of the changes in levels of biological parameters has been carried out in 13 children who underwent one or more bone marrow transplantations. During the 3 months following bone marrow transplantation, all patients developed liver injury characterized by an early and very elevated 5'-nucleotidase activity (sometimes more than 40 times the upper reference limit), a moderate increase in alkaline phosphatase activity, a variable increase in alanine aminotransferase activity and inconstant changes in total bilirubin levels. These results show that cytolytic syndrome and cholestasis are often associated with increases in 5'-nucleotidase and alkaline phosphatase activities. These increases are not correlated, probably due to the influence of therapy on the synthesis and release of both enzymes in the liver. 5'-nucleotidase seems to be the best marker for the detection and follow-up of liver disease in this patient group.

Recombination pattern of the TCR gamma locus in human peripheral T-cell lymphomas.

The recombination events of the gamma and beta T-cell receptor (TCR) loci were analysed in a series of 39 peripheral T-cell lymphomas (PTCLs) in association with the expression of TCR chains. In TCR alpha beta PTCLs, 22/23 cases showed a gamma-gene rearrangement while only 18/23 showed a concomitant beta-gene rearrangement. The germline configuration of the beta locus was found in angiommunoblastic lymphadenopathy and lymphoepithelioid lymphomas. Three gamma delta PTCLs rearranged both gamma and beta genes. TCR silent PTCLs showed three different patterns of gamma- and beta-gene rearrangements. Three cases were in germline configuration for both loci; five cases had a rearranged gamma and a germline beta locus; and five cases had the two loci rearranged. Regarding the variable genes in the gamma-rearranged alleles, members of the V gamma I subgroup were the most frequently presented (39/50), followed by V gamma II, V gamma III, and V gamma IV (9/50, 1/50, and 1/50, respectively). Joining segment usage was as follows: J1 or J2 (32/50), JP1 or JP2 (17/50), and JP (1/50). Taken together, these data demonstrate that the gamma locus is more frequently rearranged whatever the TCR expression. The gamma-locus analysis provides a better diagnostic yield than the beta locus in the study of PTCL clonality.

Stability of diltiazem and its metabolites in human blood samples.

The stability of diltiazem and its metabolites in blood samples from patients under chronic diltiazem therapy was investigated. When whole blood was kept for 1 h at room temperature between sampling and centrifugation, the concentration of N-demethyldiltiazem (MA) decreased significantly, with an average loss of 24%. Under the same conditions, an average loss of 14% of diltiazem occurred, whereas the concentrations of the metabolites deacetyldiltiazem and N-demethyldeacetyldiltiazem did not change significantly. No significant decrease in MA and diltiazem concentrations was observed when whole blood was stored for 1 h in an ice bath. In spiked plasma samples kept at room temperature, only MA was unstable, with an average loss of 13% after 4 h. The present study shows the importance of observing rigorous conditions for the transport and treatment of blood samples. To achieve accurate determination of diltiazem and related compounds, the blood must be centrifuged immediately after collection or kept on ice for up to 1 h. The plasma samples must be immediately frozen at -80 degrees C and can be stored for up to 5 weeks before analysis. Using these rigorous conditions, we observed that MA is the main metabolite of diltiazem in plasma from patients under chronic oral diltiazem therapy.

[Optimization of the determination in urine of alanine aminopeptidase, gamma-glutamyltransferase and N acetyl-beta-D-glucosaminidase]. / Optimisation de la mesure dans l'urine de l'alanine aminopeptidase, de la gamma-glutamyltransférase et de la N-acétyl-bêta-D-glucosaminidase.

The authors describe the optimization of determination of alanine aminopeptidase (AAP), gamma-glutamyltransferase (GGT) and N-acetyl-beta-D-glucosaminidase (NAG) in urine by multivariate analysis. The optimal conditions found are: for AAP at 30 degrees C TRIS HCl buffer 300 mmol/l pH 7.9, L-alanine-4-nitroanilide 5.8 mmol/l, for GGT at 30 degrees C buffer glycylglycine 150 mmol/l pH 8.0, gamma-L-glutamyl-3-carboxy-4-nitroanilide 9.0 mmol/l, for NAG at 37 degrees C citrate buffer 50 mmol/l pH 5.8, m cresolsulfonphtaleinyl-N-acetyl-beta-D-glucosaminide 5.5 mmol/l. These methods are easy to perform, apply to urine without pretreatment through Sephadex: therefore complete automatization is possible. The stability of enzymatic activities in urine is of ten days at +4 degrees C in the presence of sodium azide at neutral pH. Freezing resulted in a considerable loss of activity for AAP and GGT.

Urinary alanine aminopeptidase assay improved as result of multivariate response-surface analysis.

Optimization of determination of alanine aminopeptidase in urine by univariate study led to a method involving pretreatment of urine with Sephadex G50. Re-examination of the optimization by multivariate study led us to recommend higher optimal concentrations: 5.8 mmol/L for the substrate and 300 mmol/L for the Tris buffer. Under these new conditions, pretreatment of urine was no longer necessary and the assay could be completely automated.

Creatine kinase: reassessment of optimal concentrations for adenosine-5'-diphosphate and magnesium.

Whereas univariate studies led to an European agreement for the choice of optimal reagent concentrations of 2 mmol/L for ADP and 10 mmol/L for Mg2+ for determining creatine kinase (EC 2.7.3.2) activity in serum, whatever its isoenzyme pattern, the results of our bivariate study led us to recommend higher optimal concentrations: 4.1 to 4.7 mmol/L for ADP and 22 mmol/L for Mg2+. The zone of maximal activity was in fact a broad plateau such that more than 99% of maximal enzyme activity was attained at ADP concentrations between 3 and 5 mmol/L and Mg2+ concentrations between 17 and 26 mmol/L. Under these new conditions the maximum activity measured was modestly increased (about 10%) over the previously recommended method but the assay could be expected to be more resistant to the variations of ADP and Mg2+ concentrations. It may become necessary to modify the European recommended method.

Neonatal hypercalcemia in preterm infants fed with human milk.

Hypercalcemia (serum Ca greater than or equal to 2.83 mmol/l) was detected in 10 premature infants (gestational age: 31-37 weeks and birthweight: 1100-1950 g). All were fed with pooled human breast milk. Urinary Ca excretion was high (greater than 0.200 mmol/kg/24 h) in all but one infant while serum phosphorus (P) concentration and urinary P excretion were low. Serum immunoreactive parathyroid hormone and plasma 25-hydroxyvitamin-D concentrations were normal. A significant positive correlation was found between serum Ca concentration and urinary Ca excretion, and a negative correlation between serum Ca concentration and serum P concentration or urinary P excretion. Hypercalcemia disappeared spontaneously in two patients, was corrected by a humanized milk in three patients and by P supplementation in five patients. These data suggest that neonatal hypercalcemia is related to P depletion induced by human breast milk in premature infants.

[Humoral factors of immunity in maternal milk. Comparison of their levels in the breast milk of mothers with premature and at-term delivery]. / Les facteurs humoraux de l'immunité du lait maternel. Comparaison de leur concentration dans le lait de mère accouchant prématurément ou à terme.

Eight proteins (Immunoglobulin A, G, M, C3 and C4 fractions of complement, alpha 1-glycoprotein, lactoferrin and alpha 1- antitrypsin) were measured by immuno-diffusion or laser nephelometry in 50 milk samples. Thirty nine were heated thrice at 62 degrees C for 20 minutes. Twenty six came from mothers who delivered prematurely (less than 37 weeks) and 13 from mothers who delivered at term. Eleven samples were, used to determine the effect of the heating process. There was no significative difference of the concentrations of the eight proteins between the breast milk obtained at term or prematurely, even when the comparisons were made between colostral milks or transitional milks. The heating process reduced the concentration by 47 % for IgA, more than 88 % for IgG and IgM, 41 to 74 % for the other proteins; only orosomucoid seemed little affected (-16 %). These data suggest that the heating process impairs the immunologic effect of breast milk. This effect must be particularly considered in regard to the absence of any significant difference between the milks obtained at term or prematurely.

Late evolution of serum immunoreactive parathyroid hormones, calcitonin and plasma 25-hydroxy cholecalciferol concentrations in very low birthweight infants.

The plasma concentrations of 25-hydroxycholecalciferol (25-OH-CC), immunoreactive parathyroid hormone (iPTH) and calcitonin (iCT) were measured at the age of 30 and 66 days in thirteen preterm neonates (birthweight: 970 to 1300 g). At the age of 30 days when all infants were fed only with breast milk (BM) serum iCT and iPTH levels were normal. During the second month 7 infants were fed with BM only (control group) and 6 infants were supplemented with formula (supplemented group). At the age of 66 days, mean +/- S.D. serum iPTH concentration was higher in the supplemented group than in the control group: 169 +/- 79 vs. 60 +/- 33 microliterEq/ml (p less than 0.01). Serum iCT levels remained undetectable (less than 150 pg/ml) in both groups. Plasma 25-OH-CC concentrations were normal and similar in both groups. Serum iPTH concentrations were positively correlated with phosphorus intake and negatively correlated with calcium intake from BM only. The results suggest that secondary hyperparathyroidism can be detected in very low birthweight infants supplemented with a formula, probably because of a phosphorus load or decreased intestinal absorption of calcium.

Laser nephelometry of orosomucoid in serum of newborns: reference intervals and relation to bacterial infections.

Orosomucoid was evaluated by laser-nephelometry in 1790 sera collected from 1170 newborns. Within-run precision (CV) was 2.1 to 4.2%, between-run 2.9 to 5.2%. Results correlated well with radial immunodiffusion (r = 0.989). Results can be obtained within 1 h. Orosomucoid concentrations in serum at birth range from 130 to 200 mg/L and are influenced by gestational age during the first two days of postnatal life. Thereafter, the values increase very rapidly in the first week of life, concentrations being the same as in adults by about 10 months. In 66 of 78 cases of severe bacterial infections, orosomucoid concentrations were above normal. Evidently, serum orosomucoid constitutes an useful index in diagnosis and monitoring of bacterial infections in the neonatal period.

Serum orosomucoid concentration in newborn infants.

Serum orosomucoid concentration was measured by laser nephelometry in 1970 serum samples collected from 1170 full term and preterm infants. The determinations were carried out in 1 h. Reference values are given: they show that the low levels at birth are influenced by gestational age. The concentrations increase rapidly during the first week in all infants, the adult values being reached by 10 months of age. High levels of orosomucoid concentration were detected in 85% of the infants with severe bacterial infections. Serum orosomucoid concentration proved less valuable in viral and parasitic infection. Twenty-six per cent of the sick infants without infection had a slightly elevated orosomucoid level which decreased rapidly. In the bacterial infections the evolution of serum orosomucoid concentration followed the clinical course. Thus serum orosomucoid concentration was a useful parameter for diagnosis and monitoring of bacterial infection in neonates.

Comparison of the EMIT test with high-performance liquid chromatography for the determination of phenobarbital in serum.

The new enzyme immunoassay technique (EMIT) is compared to high-performance liquid chromatography used for determining phenobarbital in serum. After resuming the methods of both techniques, we compare the results of 116 sera collected from patients receiving treatment for epilepsy. The equation of the correlation curve is: HPLC = 0.98 EMIT + 0.12. The reliability characteristics are comparable. The enzyme immunoassay technique is less specific but more practicable.