Acquired storage-pool disorders occurring late after allogeneic bone marrow transplantation: partial activation of platelets in asymptomatic patients.

Bone marrow transplantation (BMT) may be complicated by coagulation abnormalities. The present study evaluated whether platelets might be activated in patients who had undergone BMT without significant coagulopathy. The patients selected had received allogeneic BMTs a median of 39 months before the study (range, 11-124 months) and had not received cyclosporine, FK506 (tacrolimus), or other medication affecting cyclo-oxygenase for at least 3 months prior to the collection of blood samples. Furthermore, patients had platelet counts greater than 100 x 10(9) cells/L and normal serum creatinine levels. Twenty-five healthy volunteers acted as controls. Platelet aggregation studies and a mepacrine assay of platelets showed abnormal aggregation and decreased staining in some patients. The platelet storage-pool adenosine 5'-triphosphate (ATP) level in 15 patients after BMT was 0.45+/-0.24 micromol per 10(11) platelets, whereas the level in 18 controls was 1.03+/-0.36 micromol per 10(11) platelets (P = .00078). The total ATP levels of platelets in patients and controls were 4.33+/-1.14 and 5.63+/-1.51 micromol per 10(11) platelets, respectively (P = .016). With the exception of 1 patient, plasma levels of thrombomodulin and von Willebrand factor were all within the normal range. The average plasma level of 11-dehydrothromboxane B2 was significantly increased in 15 patients after BMT compared with controls, 20.6+/-8.2 and 10.3+/-1.2 pg/mL, respectively (P = .0004). These findings suggest a long-term process of platelet activation in patients after BMT and, following the cessation of cyclosporine, development of acquired storage-pool disorder of platelets.

Adenovirus is a key pathogen in hemorrhagic cystitis associated with bone marrow transplantation.

Late-onset hemorrhagic cystitis (HC) is a well-known complication of bone marrow transplantation (BMT) that is mainly attributed to infection with BK virus (BKV) and adenovirus (AdV). From 1986 through 1998, 282 patients underwent BMT, and 45 of them developed HC. Urine samples tested positive for AdV in 26 patients, of which 22 showed virus type 11. Among patients who underwent allogeneic BMT, logistic regression analysis revealed acute graft-versus-host disease (grade, > or = 2) to be the most significant predictive factor for HC (P < .0001). In addition, a total of 193 urine samples regularly obtained from 26 consecutive patients who underwent allogeneic BMT were examined for BKV, JC virus (JCV), and AdV by means of polymerase chain reaction. Of patients without HC, approximately 30% of the specimens tested positive for BKV (58 samples) and JCV (55 samples), whereas 5 (3%) tested positive for AdV. Of the 3 samples obtained from patients with HC, the numbers of positive results for BKV, JCV, and AdV were 3, 1, and 1, respectively; the numbers of positive results increased to 14 of 17, 9 of 17, and 10 of 17, respectively, when we added another 14 samples obtained from 14 patients with HC (P < .0001, P = .026, and P < .0001, respectively). In conclusion, there was significant correlation between AdV and HC in the patients we studied.

[Allogeneic bone marrow transplantation for primary myelofibrosis after splenectomy].

A 46-year-old woman was given a diagnosis of primary myelofibrosis (PMF) in 1996. Because of the progression of anemia and splenomegaly, she was scheduled for allogeneic bone marrow transplantation (BMT) from an HLA-matched sibling donor in April 1998. Cyclophosphamide and busulfan were used as the conditioning regimen. Before BMT, the patient was treated with hydroxycarbamide, which did not resolve splenomegaly. She then underwent a splenectomy, which was followed by massive portal vein thrombosis without any significant clinical outcome. After BMT, the patient obtained rapid hematologic engraftment. Moreover, the alleviation of marrow fibrosis was confirmed 4 months after BMT. We concluded that allogenic BMT can cure patients with PMF, but that the issue of splenectomy and the indications for BMT need to be evaluated further.

Establishment of a double Philadelphia chromosome-positive acute lymphoblastic leukemia-derived cell line, TMD5: effects of cytokines and differentiation inducers on growth of the cells.

A double Philadelphia chromosome (Ph)-positive leukemia cell line with common-B cell phenotype, designated TMD5, was established from the blast cells of a patient with double Ph-positive acute lymphoblastic leukemia. TMD5 cells expressed 190 kDa BCR/ABL chimeric protein and 145 kDa ABL protein. The cells proliferated without added growth factors. Autocrine growth mechanism was not recognized. The addition of growth factors such as G-CSF, GM-CSF, IL-3, IL-6, or Stem Cell Factor did not affect the growth. Herbimycin A suppressed the growth of TMD5 cells at the low concentration that did not affect Ph-negative cells. It suppressed tyrosine phosphorylation of intracellular proteins in TMD5 cells. Dexamethasone and dibutyryl cyclic AMP also suppressed the growth. They, however, did not affect the phosphorylation significantly. Neither all-trans retinoic acid nor interferon-alpha affected the growth. TMD5 cells, characterized minutely here and rare in that they have double Ph chromosomes, will be a useful tool for the study of Ph-positive leukemia.

Two M-components in a single cell lineage in a patient with a dual isotype secretory B-cell tumour.

We describe a case of Waldenström's macroglobulinaemia with two M-components (IgM and IgG) with the same lambda light chain. Southern blot analysis of bone marrow cells showed rearrangements of immunoglobulin heavy and lambda light chain genes. Sequencing of the complementarity determining region 3 of the two lambda and mu transcripts showed 100% homology. Immunofluorescence study showed that most cells stained for both IgG and IgM. These findings indicated that a single population of cells was expressing two isotypic variants of IgG and IgM, as the genes responsible for production of both components had the same origin.

[Plasma cell leukemia with amyloid deposition and osteogenetic change at the site of an extramedullary plasmacytoma].

A 49-year-old man was admitted with swelling in the left lower extremity, and a mass in the left lower abdomen. Laboratory findings showed an increased WBC of 15,000/microliter with 41% plasma cells, and immunoglobulin (Ig) A of 2,557mg/dl with a monoclonal component. A roentgenogram and computed tomograph of the abdomen revealed that a 5 x 10 cm mass with calcification located in the iliopsoas muscle. Plasma cell leukemia with extramedullary plasmacytoma was diagnosed, and the patient was treated with high-dose dexamethasone (40 mg/day for 4 days), resulting in a good response with the disappearance of plasma cells in peripheral blood and a marked decrease in serum Ig A. However, the patient's condition deteriorated in spite of various treatments, and he died of heart failure 5 months after admission. With informed consent from relatives, a necropsy was performed and infiltration of plasma cells in the mass in the iliopsoas muscle was noted. We reported this case because plasma cell leukemia with amyloid deposition and osteogenesis at the site of extramedullary plasmacytoma is very rare.

A gas chromatographic-positive ion chemical ionization-mass spectrometric method for the determination of I-alpha-acetylmethadol (LAAM), norLAAM, and dinorLAAM in plasma, urine, and tissue.

l-alpha-Acetylmethadol (LAAM) is approved as a substitute for methadone for the treatment of opiate addiction. Analytical methods are needed to quantitate LAAM and its two psychoactive metabolites, norLAAM and dinorLAAM, to support pharmacokinetic and other studies. We developed a gas chromatographic-positive ion chemical ionization-mass spectrometric method for these analyses. The method uses 0.5 mL urine or 1.0 mL plasma or tissue homogenate, deuterated (d3) isotopomers as internal standards, methanolic denaturation of protein (for plasma and tissue), and extraction of the buffered sample with n-butyl chloride. For tissue homogenates, an acidic back extraction is included. norLAAM and dinorLAAM were derivatized with trifluoroacetic anhydride. Chromatographic separation of LAAM and derivatized norLAAM and dinorLAAM is achieved with a 5% phenyl methylsilicone capillary column. Positive ion chemical ionization detection using methane-ammonia as the reagent gas produces abundant protonated ions (MH+) for LAAM (m/z 354) and LAAM-d3 (m/z 357) and ammonia adduct ions (MNH4+) for the derivatized norLAAM (m/z 453), norLAAM-d3 (m/z 45 6), dinorLAAM (m/z 439), and dinorLAAM-d3 (m/z 442). The linear range of the calibration curves were matrix dependent: 5-300 ng/mL for plasma, 10-1000 ng/mL for urine, and 10-600 ng/g for tissue homogenates. The low calibrator was the validated limit of quantitation for that matrix. The method is precise and accurate with percent coefficients of variation and percent of targets within 13%. The method was applied to the analysis of human urine and plasma samples; rat plasma, liver, and brain samples; and human liver microsomes following incubation with LAAM.

Fentanyl receptor assay. II. Utilization of a radioreceptor assay for the analysis of fentanyl analogs in urine.

A radioreceptor assay has been developed to measure fentanyl and fentanyl-like drugs in biological specimens. The assay is based on the competition of these drugs with [3H]fentanyl for opioid receptors. Rats were injected intravenously with fentanyl (15 micrograms/kg), alpha-methylfentanyl (15 micrograms/kg), (+/-)-cis-3-methylfentanyl (15 micrograms/kg), butyrylfentanyl (0.48 mg/kg), and benzylfentanyl (3.19 mg/kg). Urine samples were analyzed by radioreceptor assay (RRA), radioimmunoassay (RIA), and gas chromatography/mass spectrometry (GC/MS). The time-course of urinary analysis of fentanyl analogs showed some discrepancies. RRA measurement of urine concentrations of (+/-)-cis-3-methylfentanyl that were undetectable by RIA gave results 5-10 times higher than values obtained by GC/MS. Concentrations of alpha-methylfentanyl obtained by RRA and GC/MS were similar; however, these samples were negative by RIA. Following the administration of benzylfentanyl, urinary concentrations were not detected by RIA and only slightly detectable with RRA; however, high concentrations of benzylfentanyl were found by GC/MS in the same samples. Urine samples from animals injected with butyrylfentanyl showed high cross-reactivity with fentanyl antibody, giving values measured by radioimmunoassay about two times higher than those obtained by the other two methods. These findings suggest that this radioreceptor assay is well-suited as an initial assay for the detection of active analogs of fentanyl in urine and correlates well with other techniques in the analysis of fentanyl; however, there is substantial disagreement between techniques in the quantitation of fentanyl analogs.

Fentanyl receptor assay. I. Development of a radioreceptor assay for analysis of fentanyl and fentanyl analogs in urine.

Radioreceptor binding assays may be useful methods for determining the plasma and urine concentration of drugs. We have evaluated the possibility of employing CNS receptors in an assay to measure fentanyl and fentanyl-like drugs. This in vitro assay is based on the competition of these drugs with [3H]fentanyl for opioid receptors in membrane preparations of rat forebrain. The binding is stereospecific, reversible, and saturable. Scatchard plots of saturation suggest the presence of high and low affinity binding sites. Naloxonazine, which selectively binds to mu 1-opioid site, competed with [3H]fentanyl for its high affinity binding site. Morphine and hydromorphone competed with [3H]fentanyl for the opioid receptor, but other morphine-like compounds were relatively weak displacers of [3H]fentanyl. Many other commonly abused drugs did not displace [3H]fentanyl from the opiod receptors. Urine samples from animals injected with fentanyl were evaluated by other analytical techniques, including radioimmunoassay (RIA) and gas chromatography/mass spectrometry (GC/MS), and results were compared to those from the radioreceptor assay. Urinary analysis of fentanyl showed a good correlation between all three methods.