No abstract

We have found a sickling variant, Hb S Antilles, O2PI3 23 Vai-][e), that has the same electrophoretic mobility as Hb S but a distinct isoelectric focus and produces sickling in the carriers of the Hb A/S Antilles trait. The carriers' erythrocytes tend to sickle at 02 partial pressures similar to those that induce sickling in Hb S/C disease. Pure deoxy-Hb S Antilles is 50% as soluble as deoxy-Hb S (saturating concentration = 11 g dl-1 compared to 18.4 for Hb S). MATERIALS AND METHODS Routine hematological parameters were determined by using a Coulter S Counter and standard procedures were used for the preparation of erythrocyte hemolysates. Methods for electrophoresis of hemoglobin on cellulose acetate and in citrate agar and of globin chains at alkaline and acidic conditions have been described (5). IEF was performed as in refs. 6 and 7. Abnormal hemoglobin was isolated by chromatography on DEAE-cellulose (Whatman DE-52) (8). After removal of hemin by precipitation with acid/acetone the abnormal chains were prepared by CM-cellulose/8 M urea chromatography (9), aminoethylated, and hydrolyzed with trypsin treated with L-1-tosylamido-2-phenylethyl chloromethyl ketone (Worthington) (10).Tryptic digests were separated by reverse-phase HPLC using a ,uBondapak C18 column (Waters Associates) operated at room temperature. The buffers used were buffer A, 20 mM ammonium acetate, and buffer B, 50% (vol/vol) acetonitrile in 10 mM ammonium acetate, pH 5.7. All buffers were degassed and filtered before use. The column was equilibrated in 3% buffer B. Elution was performed at room temperature at a flow-rate of 1.5 ml/min and the absorbance of the fractions was monitored at 214 nm. A three-step linear gradient was applied for elution: 3-25% buffer B for 30 min, 25-50% for 10 min, and 50-60% for 15 min. Prior to elution, 1 mg of tryptic digest dissolved in 200 ,ul of 1o (vol/vol) acetic acid was centrifuged before analysis and 100 Al of the supernatant was injected into the column. Fractions were collected, and the acetonitrile was evaporated in a nitrogen stream at 60°C in a fume hood. After hydrolysis in 6 M HCl, the samples were analyzed in a Biotronik 6001E amino acid analyzer. Abnormal peptide ,-T3 was hydrolyzed by Staphyloccocus aureus V8 protease according to ref. 11 at 37°C for 15 hr. The S. aureus V8 peptides were characterized as described for the tryptic peptides. DNA of the propositus' leukocytes was digested with the restriction enzyme Mst II according to ref. 12 and the fragments were analyzed by Southern blotting.Sickling tests on fresh erythrocyte suspensions were done in vitro as previously described (13). Phthalate ester density profiles were determined with erythrocytes from several patients according to ref. 14. For preparative purposes, the propositus' erythrocytes were fractionated according to their density with a discontinuous albumin/Percoll gradient (15). Solubility of the abnormal hemoglobin was studied by measuring its Csat (saturating hemoglobin concentration) value. This was then compared...

We have developed a strategy based on polymerase chain reaction (PCR) for detecting all possible gamma T-cell receptor (gamma TCR) rearrangements and the most common delta TCR rearrangements found in B- lineage and T-acute lymphoblastic leukemia (T-ALL). The segments amplified from blasts are then directly sequenced to derive clonospecific probes. From a series of 45 patients aged 1 to 15 years (42 B-lineage ALL, 3 T-ALL), 35 (83%) could be followed for minimal residual disease with at least one clonospecific probe. Detection of clonal markers using clonospecific probes routinely allowed the detection of 1 to 10 blasts out of 10(5) cells as determined by serial dilutions of the initial samples. Residual disease was quantitated by a competitive PCR assay based on the coamplification of an internal standard. Twenty children were prospectively followed for periods varying from 7 to 30 months. In most children, a progressive decrease of the tumor load was observed, and blasts became undetectable within 6 months after the initiation of treatment. A slower kinetics of decrease in tumor cells was found in three children. These three patients relapsed with blasts that continued to display the initial clonospecific markers. Three other children had a central nervous system relapse despite the absence of detectable medullary residual disease. The use of both delta and gamma TCR genes as clonal markers, as well as simplification in the methods to detect and quantify residual blasts reported here, will allow the study of the large number of patients required to determine the role of the detection of minimal residual disease by PCR in the follow-up of childhood ALL.

A cluster sampling survey was performed in 1989 in Libreville, Gabon, to determine HTLV-I and HTLV-II prevalence and to compare the efficacy of polymerase chain reaction (PCR) and serology in detecting HTLV-I and HTLV-II infections. A total of 322 sera from adults were tested by ELISA and by Western blot (WB). The WB patterns were interpreted according to WHO criteria and those of the manufacturer. PCR analysis using primer pairs in the gag and pol region, with a specific probe for HTLV-I and HTLV-II, was performed on the lymphocytes of the 322 adults. In addition, 134/322 samples were re-tested with tax primers, in a second laboratory. Using WHO criteria, 8/322 (2.5%) samples were positive on WB and 25 were indeterminate; with the criteria of the kit, 26/322 (8.1%) were positive and 7 were indeterminate by WB. By PCR, 13 (4%) samples were positive, including 12 for HTLV-I (3.7%) and one for HTLV-II (0.3%). All 8 seropositive samples (by the WHO criteria) were positive by PCR, as were 4 out of 25 indeterminate samples. Only one out of 289 seronegative samples was positive by PCR. In contrast, only 12/26 positive samples by the kit criteria were confirmed by PCR. These results confirm the relatively high HTLV-I/II seroprevalence observed in Gabon. HTLV-I infection is preponderant, but HTLV-II is also present. The WHO criteria for WB give a better fit with PCR results than the kit criteria for WB. In the absence of a specific confirmatory test and based on the uncommon "seronegative" HTLV-I/II infection, the indication for PCR appears limited to the positive WB samples (to differentiate HTLV-I and II infection) and to the indeterminate WB samples.

We have developed a strategy based on polymerase chain reaction (PCR) for detecting all possible gamma T-cell receptor (gamma TCR) rearrangements and the most common delta TCR rearrangements found in B- lineage and T-acute lymphoblastic leukemia (T-ALL). The segments amplified from blasts are then directly sequenced to derive clonospecific probes. From a series of 45 patients aged 1 to 15 years (42 B-lineage ALL, 3 T-ALL), 35 (83%) could be followed for minimal residual disease with at least one clonospecific probe. Detection of clonal markers using clonospecific probes routinely allowed the detection of 1 to 10 blasts out of 10(5) cells as determined by serial dilutions of the initial samples. Residual disease was quantitated by a competitive PCR assay based on the coamplification of an internal standard. Twenty children were prospectively followed for periods varying from 7 to 30 months. In most children, a progressive decrease of the tumor load was observed, and blasts became undetectable within 6 months after the initiation of treatment. A slower kinetics of decrease in tumor cells was found in three children. These three patients relapsed with blasts that continued to display the initial clonospecific markers. Three other children had a central nervous system relapse despite the absence of detectable medullary residual disease. The use of both delta and gamma TCR genes as clonal markers, as well as simplification in the methods to detect and quantify residual blasts reported here, will allow the study of the large number of patients required to determine the role of the detection of minimal residual disease by PCR in the follow-up of childhood ALL.