W. Siegert

Microbiologic Contamination of Peripheral Blood Stem Cell Autografts

We have determined the incidence and clinical significance of positive microbiologic cultures in a series of 290 peripheral blood stem cell concentrates in 95 patients undergoing multiple apheresis procedures for autologous stem cell rescue. Specimens for bacterial cultures were obtained after processing of the autografts just prior to freezing. The incidence of microbial contamination was 4.5% (n = 13). The predominant pathogenic microorganism cultured was coagulasenegative Staphylococcus (n = 11). From 8 patients with contaminated leukapheresis products 6 underwent autologous stem cell transplantation. Five patients received 1–5 culture‐positive stem cell concentrates without serious sequelae, whereas the sixth patient was autografted with noncontaminated leukapheresis products, 1 concentrate contaminated with Aspergillus fumigatus being not reinfused. No microorganism present in the stem cell autograft was recovered in vivo in the posttransplantation period, although fever as a sign of infection occurred in all but 1 patient. Peripheral blood stem cell collection and ex vivo processing for cryopreservation may result in microbiologic contamination. However, our data show that infusion of contaminated stem cell autografts does not play a significant role as a source for infections in the clinical setting of autologous stem cell rescue.

Detection of herpesvirus type 6 by polymerase chain reaction in blood donors: random tests and prospective longitudinal studies

Summary. In order to evaluate the prevalence of HHV‐6 in blood donors, we examined 112 persons by polymerase chain reaction (PCR) and ELISA. HHV‐6 antibodies could be detected in 107/111 (96.4%) of the donors. The median ELISA antibody level was 0.451 (range 0.056–0.914). 14 individuals (12.5%) were PCR positive in either oral lavage fluid, urine or buffy coat. Six persons (5.4%) were PCR positive in buffy coat samples. The prospective longitudinal analysis of 11 donors for periods between 7 and 13 weeks revealed that 4/6 persons who were initially PCR negative had positive tests in 9/63 weeks studied. Two persons were consistently PCR positive over the whole observation period of 12 and 13 weeks. HHV‐6 variants could be determined in 14 persons as variant A in nine and variant B in five cases. These observations emphasize the high prevalance of HHV‐6 and suggest that some blood donors carry detectable concentrations of the virus and therefore may be a source for transmission of HHV‐6. The finding of positive PCR in antibody negative individuals suggests that antibody determination may not be sufficient to identify potentially infectious persons.

Treatment of relapse after allogeneic bone marrow transplantation with unmanipulated G-CSF-mobilized peripheral blood stem cell preparation

Donor lymphocyte infusions (DLI) are an effective treatment of leukemia relapse after allogeneic bone marrow transplantation. Undesired side-effects are the development of graft-versus-host disease (GVHD) and the occurrence of pancytopenia in some patients. In a pilot study, we investigated if unmanipulated G-CSF-mobilized peripheral blood stem cells which naturally contain large numbers of T lymphocytes (D-PBSC/LI) would be equally effective or even superior than DLI in generating a graft-versus-leukemia reaction (GVL) but could mitigate or prevent the development of pancytopenia. We treated 12 patients with CML chronic phase (n = 5), CML blast crisis (n = 2), AML (n = 2), ALL (n = 1), CLL (n = 1) and multiple myeloma (n = 1). In five patients with acute leukemia or CML blast crisis D-PBSC/LI followed intensive chemotherapy (group A), in seven patients D-PBSC/LI were given without any prior chemotherapy (group B). In group A two patients were evaluable for hematologic toxicity. Leukopenia <1000/ μl lasted for 10 and 19 days, and thrombocytopenia <20 000/μl for 11 and 13 days, respectively. in group b leukopenia <1000/μl and thrombocytopenia <20 000/μl was observed in only one patient. moderate cytopenia developed in four of five evaluable patients. a complete remission could be achieved in all seven patients with cml who all developed acute and/or chronic gvhd. none of the remaining five patients achieved a complete remission despite acute and/or chronic gvhd in two of them. four patients died from disease progression, one patient from a secondary lymphoma, and one patient as a result of uncontrolled gvhd. in conclusion, d-pbsc/li is effective in inducing gvl reaction but it does not prevent pancytopenia in each case. it remains unclear if it mitigates the incidence and severity of pancytopenia.

Molecular characterization of illegitimate TCRδ gene rearrangements in acute myeloid leukaemia

SUMMARY. Recently, we and others have shown the occurrence of TCRδ gene rearrangements in acute myeloid leukaemia (AML). In this study we describe the molecular characteristics of these rearrangements by the polymerase chain reaction (PCR) and the direct sequencing of PCR products. 11 rearrangements were characterized in blast cell samples from six patients. We found a heterogenous pattern of TCRδ gene rearrangements with involvement of Vδ1‐5 regions. These findings differ from observations in T‐ALL and B‐cell precursor ALL, where predominantly usage of Vδ1 and Vδ2 regions has been described. Furthermore, extensive diversity of junctional sites was observed, including addition of up to 37 N nucleotides, nucleotide deletions at junction sites of Vδ and Jδ segments and usage of up to three Dδ segments. The Dδ3 fragment was the most frequently used diversity element and was found in 10 rearrangements. Nine of the 11 rearrangements were non‐functional, either incomplete or out of the reading frame. Therefore a functional TCRδ cannot be expressed in these myeloid blast cells.

Automated Processing of Human Bone Marrow Grafts for Transplantation

Prior to purging or cryopreservation, we concentrated 21 bone marrow (BM) harvests using a modification of the ‘grancollect‐protocol’ of the Fresenius AS 104 cell separator with the Pl‐Y set. Within 40–70 min, the initial marrow volume of 1,265 ml (±537 ml) was processed two to three times. A mean of 47% (±21%) of the initial mononuclear cells was recovered in a mean volume of 128 ml (+36 ml). The recovery of clonogenic cells, measured by CFU‐GM assays, was 68% (±47%). Red blood cells in the BM concentrates were reduced to 7% (±4%) of the initial number. The procedure was efficient and yielded a BM cell fraction suitable for purging, cryopreservation and transplantation. At this time, 10 of the 21 patients whose BM was processed using this technique have been transplanted. Seven of these 10 patients have been grafted using the BM alone. Three of the 10 patients showed reduced cell viability and colony growth in the thawed BM samples, and therefore obtained BM and peripheral blood‐derived stem cells. All transplanted patients showed an evaluable engraftment, achieving 1,000 granulocytes per μl of peripheral blood in a mean of 18 days.

Selective enrichment of hematopoietic progenitor cells from peripheral blood

Abstract We tried to improve progenitor cell yields by different modifications on conventional leukocyte separation protocols to obtain a more selective stem cell enrichment from peripheral blood. Our data suggest that especially higher process volumes allow selective increases of progenitor cell recoveries. Differences in stem cell yields according to different malignancies are discussed.

High-dose chemotherapy as salvage treatment for seminoma

Between October 1989 and February 1997, 13 patients with refractory or relapsed seminomas were treated with high-dose chemotherapy (HDCT) as part of consecutive phase I/II studies. Six patients had failed prior cisplatin-based first-line treatments and seven patients had also failed cisplatin-based salvage treatments. After HDCT 4/12 (33%) patients became disease-free, 4/12 (33%) patients achieved partial remissions and 4/12 (33%) patients suffered progressive disease despite HDCT. One patient developed multiorgan failure and died. With a median follow-up of 4.5 years (range 3.4 to 8 years) five patients (38%) are alive and eight patients (62%) have died. Patients with non-pulmonary visceral metastases, with short relapse-free intervals and with cisplatin-refractory tumors were more likely to fail. HDCT can be curative in seminoma patients even if offered as second salvage treatment.

Overexpression of the Raf-1 proto-oncogene in human myeloid leukemia

The Raf-1 protein, a cytoplasmic serine/threonine kinase, plays an important role in signal transduction pathways. In order to examine the role of Raf-1 in human myeloid leukemia, we determined raf-1 mRNA expression by Northern blot analysis in blast cell samples from 27 acute myeloid leukemia (AML) cases and peripheral blood mononuclear cells from six healthy donors. A normal raf-1 transcript size was detected in all cases investigated. However, overexpression of raf-1 mRNA was found in 2 of 27 AML cases, both of which were erythroleukemias (AML, FAB M6).

TCRδ Gene Rearrangements in Acute Myeloid Leukemia with T-lymphoid Antigen Expression

In this review we present our data concerning T-cell receptor (TCR) δ gene rearrangements in acute myeloid leukemia with cuexpression of T-lymphoid features (CD2/CD4/CD7; Ly+ AML). We found a correlation between TCRδ gene rearrangements and coexpression of these T-lymphoid features. Ten of 66 Ly+ AML and only one of 44 AML cases without this coexpression exhibited TCRδ gene rearrangements (p =. 028). In contrast, no correlation was observed between terminal deoxynucleotidyl transferase (TdT) expression and the occurence of TCRδ gene rearrangements in AML. Rearrangements were found in two of 25 AML with and seven of 71 AML cases without TdT expression. Interestingly, nucleotide sequencing of junctional sites revealed up to 36 N-nucleotides in cases without or with only weak TdT expression indicating downregulation of TdT expression after the TCR rearrangement took place. Complete Vδ1Jδ1 and incomplete Dδ2Jδ1 gene rearrangements were observed most frequently in Ly+ AML. These recombination patterns were sim...

Diffuse pulmonary alveolar hemorrhage after allogeneic bone marrow transplantation.

SummaryDiffuse pulmonary alveolar hemorrhage (DAH) is a life-threatening complication following bone marrow transplantation (BMT). So far it has been seen preferentially after autologous BMT. Here, we describe a patient who presented with the picture of DAH after allogeneic BMT. We draw attention to the fact that the syndrome may occur after allogeneic BMT, too.