Cardiac amyloidosis: the need for early diagnosis.

Amyloidosis is a collection of systemic diseases characterised by misfolding of previously soluble precursor proteins that become infiltrative depositions, thereby disrupting normal organ structure and function. In the heart, accumulating amyloid fibrils lead to progressive ventricular wall thickening and stiffness, resulting in diastolic dysfunction gradually progressing to a restrictive cardiomyopathy. The main types of cardiac amyloidosis are amyloid light chain (AL) amyloidosis caused by an underlying plasma cell dyscrasia, amyloid transthyretin (TTR) amyloidosis of wild-type (normal) TTR at older age (ATTRwt) and hereditary or mutant amyloid TTR (ATTRm) in which a genetic mutation leads to an unstable TTR protein. Overall survival is poor once heart failure develops, underlining the need for early referral and diagnosis. Treatment for AL amyloidosis has improved markedly over the last decades, and TTR amyloidosis gene silencers and orally available transthyretin stabilisers are ready to enter the clinical arena after recent positive outcome trials. Novel therapies aiming at fibril degradation with monoclonal antibodies are under investigation. In this review, we focus on 'red flag' signs and symptoms, diagnosis and management of cardiac amyloidosis which differs considerably from the general management of heart failure. Only by increasing awareness, prognosis for patients with this devastating disease can be improved.

'Transformation' from amyloid light chain amyloidosis to symptomatic multiple myeloma.

Amyloid light chain (AL) amyloidosis and multiple myeloma (MM) are both clonal plasma cell disorders, and may be concurrently present in patients. However, symptomatic MM seldom develops in patients with AL amyloidosis, while the other way around is common. With this case report, we discuss the difficulties in the differential diagnosis between AL amyloidosis and MM, and extend on the possible mechanisms involved in the development of these overlapping disorders. In addition, we provide clinicians with tools that may help improve their management and monitoring of such patients.

Multiple myeloma patients receiving pre-emptive donor lymphocyte infusion after partial T-cell-depleted allogeneic stem cell transplantation show a long progression-free survival.

The purpose of this study was to determine the role of pre-emptive donor lymphocyte infusion (pDLI) after partial T-cell-depleted allogeneic SCT in patients with multiple myeloma (MM). A cohort of 24 MM patients was treated with partial T-cell-depleted myeloablative SCT between December 1997 and April 2002. These patients were intended to receive pDLI after SCT. The overall response rate after SCT was 83% (20 of 24 patients) with 10 patients (42%) in complete remission (CR). Transplant-related mortality within 1 year after SCT was 29%. Thirteen patients (54%) received pDLI and four patients in partial remission reached CR. GVHD>grade I after pDLI developed in 4 out of 13 patients (30%). Four patients received therapeutic DLI, without preceding pDLI. Eleven patients (46%) are alive, with a median follow-up of 67 months (range, 48-100 months). Seven of these patients (29%) are in continuous CR (CCR), which was confirmed by a negative patient-specific IgH PCR in four patients. All seven patients in CCR received pDLI. Although myeloablative SCT in MM induces high toxicity, we show that the concept of T-cell depletion followed by pDLI is promising and needs to be investigated in a reduced-intensity conditioning setting.

Synopsis of the Dutch multidisciplinary guideline for the diagnosis and treatment of hereditary haemochromatosis.

Hereditary haemochromatosis (HH) is a disease related to mutations in the HFE gene and can lead to progressive iron accumulation, especially in the liver, eventually resulting in organ damage. We have developed guidelines for the diagnosis and treatment of this disease according to CBO methodology (dutch institute for Healthcare Quality). The prevalence of clinical symptoms such as fatigue, arthropathies, impotence and diabetes mellitus among homozygotes was similar to that in a control population. Nevertheless, we recommend the assessment of serum iron indices when these symptoms remain unexplained. When transferrin saturation is >45% and ferritin exceeds local reference ranges, HFE mutations should be investigated. Homozygosity for the C282Y mutation or combined C282Y/H63d mutation confirms the diagnosis of HFE-related HH. Liver biopsy is recommended when ferritin exceeds 1000 microg/l to establish the presence or absence of cirrhosis, which will affect prognosis and management. iron accumulation confirmed by magnetic resonance imaging (MRI) in the absence of the homozygous C282Y mutation or the combined C282Y/H63d genotype may justify a search for rare hereditary forms of non-HFE HH in a specialised centre. The literature supports the benefits of adequate phlebotomy and the screening of first-degree relatives of index patients with clinically overt HH. overall, the guidelines presented here are to a great extent based on the expert opinion of the working party, as the quantity of evidence that met predefined criteria posed by the evidence-based approach was small. We therefore recommend world-wide efforts to collaboratively address these remaining issues.

Prognostic factors for donor lymphocyte infusions following non-myeloablative allogeneic stem cell transplantation in multiple myeloma.

In this retrospective study, we evaluated donor lymphocyte infusions given for relapsed (n=48) or persistent (n=15) myeloma following non-myeloablative allogeneic stem cell transplantation (Allo-SCT). Twenty-four of 63 patients (38.1%) responded: 12 patients (19.0%) with a partial response (PR) and 12 patients (19.0%) with a complete response (CR). Overall survival after donor lymphocyte infusions (DLI) was 23.6 months (1.0-50.7+). Median overall survival for non-responding patients was 23.6 months and has not been reached for the patients responding to DLI. In responders, progression-free survival after DLI was 27.8 months (1.2-46.2+). Patients with a PR had a median progression-free survival of 7.0 months, whereas patients with a CR to DLI had a median progression-free survival of 27.8 months. Major toxicities were acute graft-versus-host disease (GVHD) (38.1%) and chronic GVHD (42.9%). Seven patients (11.1%) died from treatment-related mortality. The only significant prognostic factors for response to DLI were the occurrence of acute and chronic GVHD. There was a trend towards significance for time between transplantation and DLI, and response. Donor lymphocyte infusion following non-myeloablative Allo-SCT is a valuable strategy for relapsed or persistent disease.

1-Beta-arabinofuranosylcytosine in therapy of leukemia: preclinical and clinical overview.

Although the mechanisms of therapeutic efficacy of cytosine arabinoside (Ara-C) are multifactorial, the pharmacodynamic basis for its cytotoxicity and therapeutic efficacy lies in its intracellular metabolism and the retention of the active metabolite, Ara-C triphosphate (Ara-CTP), which is a competitive inhibitor of DNA polymerase. Additional determinants of tumor cell sensitivity include Ara-CMP incorporation into cellular DNA, the size of the competing normal metabolite, deoxycytidine/5'-triphosphate pool, and the heterogeneity in growth kinetics of tumor cells, S-phase vs cells in other phases of the cell cycle. With high-dose Ara-C, substantial amounts of Ara-CTP are formed in phases of the cell cycle. The presence of high intracellular concentration with prolonged retention of Ara-CTP could lead to the inhibition of cell growth of the cells entering S-phase as a consequence of inhibition of DNA-polymerase and/or incorporation into cellular DNA, resulting in a chain termination. Pharmacokinetically, Ara-C is rapidly eliminated from plasma. In mice, pharmacokinetic parameters of Ara-C are not sufficient predictors for the observed differences in their in vivo antitumor activity. Although these mice were bearing different tumor types (L1210 Ara-C sensitive or P-388 relatively more resistant), the observed differences in tumor response were achieved under identical plasma Ara-C concentrations and area under the concentration time curve. The observed antitumor activity in L1210 cells is primarily associated with higher Ara-CTP pools and retention (T1/2 > 4 hr) in tumor cells as compared with normal bone marrow cells. In the least responsive tumor (P-388), although Ara-CTP pools were sufficiently high, retention of the drug in tumor cells and in normal cells is poor with a T1/2 < 2 hr. Thus, unlike mice bearing leukemia L1210 cells, alteration of the mode and dose of administration of Ara-C in mice bearing P-388 could only result in increased host toxicity with no therapeutic gain. Similarly in patients with acute nonlymphocyte leukemia (ANLL), there is no significant correlation between plasma Ara-C concentration and the intracellular concentrations or retentions of Ara-CTP. In some patients the highest Ara-CTP pools in leukemic myeloblast cells are achieved at a lower level of plasma Ara-C and decrease further with the increase of plasma Ara-C. Thus, in the in vivo model system and in ANLL patients with no prior chemotherapy, Ara-CTP retention is a critical factor associated with response to this agent, in particular its direct association with duration of complete response.(ABSTRACT TRUNCATED AT 400 WORDS)

Molecular diversity in AML1/ETO fusion transcripts in patients with t(8;21) positive acute myeloid leukaemia.

Patients with acute myeloid leukaemia with maturation (AML-M2) that carried the t(8;21) were tested for the presence of chimeric AML1/ETO mRNA. After RT-PCR, an expected band of 208 bp was observed on gel, as well as some slower migrating bands. The base composition of one of the additional products was determined and was found to contain a new 68-bp ETO sequence present at the fusion of AML1 and ETO genes. The derived protein sequence results in a truncated AML1 gene still containing the putative DNA binding domain. Molecular diversity in the AML1-ETO transcripts will have consequences for the detection of minimal residual disease and antisense studies.

Acquired skewing of Lyonization remains stable for a prolonged period in healthy blood donors.

The pattern of X-chromosome inactivation (XCIP), or Lyonization, can be used to distinguish monoclonal from polyclonal cell populations in females. However, a skewed XCIP exists in hematopoietic cells in approximately 40% of healthy elderly females, interfering with interpretation of clonality assays. In hematopoiesis, an active stem cell pool is assumed to be present within a larger population of inactive stem cells, with a continuous exchange of cells between the two compartments. The assumption that the active stem cell pool size decreases with age may explain the phenomenon of acquired skewing occurring by chance and predicts the XCIP of this population to fluctuate. This fluctuation should be reflected in the XCIP of peripheral granulocytes. We examined the XCIP for fluctuations in time in peripheral granulocytes, monocytes and T cells of young, middle-aged and elderly healthy females. We used an optimized HUMARA PCR assay that eliminates unbalanced DNA amplification. We found no fluctuations in XCIP in any age group in up to 18 months follow-up. We conclude that acquired skewing arises gradually in life without fluctuations in XCIP and that analysis at multiple time points cannot distinguish monoclonal hematopoiesis from normal, skewed hematopoiesis.

Idarubicin DNA intercalation is reduced by MRP1 and not Pgp.

Currently available data regarding the substrate specificity of the multi-drug resistance (MDR) mechanisms P-glycoprotein (Pgp) and MDR-associated protein (MRP1) for idarubicin are inconclusive. A multiparameter flow cytometry method was developed which allows simultaneous quantitative measurement of total cellular fluorescence and the amount of anthracyclines intercalated into the DNA. Anthracycline DNA intercalation was measured by fluorescence resonance energy transfer (FRET) between Hoechst 33342 and anthracyclines. Daunorubicin and idarubicin accumulation were studied and compared in established cell lines expressing Pgp and MRP1. The data demonstrate that daunorubicin DNA intercalation is affected by both Pgp and MRP1 whereas idarubicin DNA intercalation is affected only by MRP1. MRP1 and Pgp function could be blocked completely by 5 microM PAK 104P, while higher concentrations of verapamil, PSC 833 and cyclosporin A were necessary to attain complete blocking of MRP1 compared to Pgp. Daunorubicin DNA intercalation correlates better with cell survival and is more sensitive at physiological MDR expression as observed in hematopoietic progenitors than daunorubicin levels measured by total cellular fluorescence. In conclusion, idarubicin DNA intercalation is reduced by MRP1 but not by Pgp. PAK-104P is an effective modulator for both Pgp and MRP1 and may further improve idarubicin efficacy.

Apparent expansion of CD34+ cells during the evolution of myelodysplastic syndromes to acute myeloid leukemia.

Myelodysplastic syndromes (MDS) are highly proliferative bone marrow (BM) disorders where the primary lesion presumably affects a CD34+ early progenitor or stem cell. We investigated the proliferative characteristics of CD34+ cells of 33 untreated MDS patients (19 RA, 5 RARS, 7 RAEB, 2 RAEBt) and five patients with acute myeloid leukemia after MDS (sAML). All patients received a 1-h infusion of the thymidine analogue iodoor bromodeoxyuridine intravenously before a BM aspirate and biopsy was taken. A double-labeling immunohistochemistry technique by monoclonal anti-CD34 (QBend/10) and anti-IUdR/BrdU antibodies was developed and performed. By this technique we recognised CD34+ and CD34- cells actively engaged in DNA synthesis or not. As MDS evolves a significant increase occurred in the percentage of CD34+ cells of all myeloid cells (mean value: RA/RARS 1.67%; RAEB(t) 8.68%; sAML 23.83%) as well as in the percentage of proliferating CD34+ cells of all myeloid cells (RA/RARS 0.19%; RAEB(t) 0.43%; and sAML 3.30%). This was associated with a decreasing trend in the overall myeloid labeling index (LI: RA/RARS 25.8%, RAEB(t) 24.6% and sAML 21.5%). This decrease in overall myeloid LI is due to an exponential increase in the proportion of CD34+ cells of the proliferating compartment during MDS evolution (RA/RARS 0.35%, RAEB(t) 1.44% and sAML 11.98% of all S-phase cells). These CD34+ cells appeared to proliferate more slowly than their more mature CD34 negative counterparts, since we found a progressive increment in the mean total cell cycling time (Tc) of all myeloid cells during MDS progression (RA/RARS 39.8, RAEB(t) 45.2 and sAML 65.8 h). This study showed that during MDS evolution to sAML the CD34+ compartment develops a growth advantage leading to apparent expansion.

Specific detection of myeloma plasma cells using anti-idiotypic single chain antibody fragments selected from a phage display library.

Bone marrow plasma cells constitute the bulk of malignant cells in multiple myeloma patients. B-lymphocytes having immunoglobulin heavy chain gene rearrangements identical to those of the malignant clone (clonally related B-lymphocytes) may function as malignant plasma cell precursors. We and others proposed the use of anti-idiotypic antibodies to isolate and study clonally related B-lymphocytes. This strategy failed until now because anti-idiotypic antibodies raised by conventional hybridoma techniques proved to react frequently with epitopes shared by different idiotypes. Recently, we succeeded in selecting specific single chain Fv antibodies from phage libraries. To select single chain Fv bearing phages specifically directed against the immunoglobulin idiotype expressed by myeloma tumor cells we panned a semisynthetic phage library against purified myeloma paraprotein Fab fragments. The selection was performed in the presence of soluble polyclonal immunoglobulin as a competitor. Three independent selections for three myeloma patients yielded 10-26 clones. Between two and seven of the selected clones were reactive with patient Fab and not with polyclonal immunoglobulin in enzyme-linked immunosorbent assays. Five out of six anti-idiotypic single chain Fvs were able to specifically stain fixed monoclonal plasma cells in myeloma bone marrow. Idiotype specificity of these single chain Fvs was confirmed by flow cytometry since they did not react with monoclonal plasma cells of other patients, a panel of nine myeloma cell lines, isolated polyclonal bone marrow plasma cells and cultured B-lymphocytes. Using these anti-idiotypic reagents we were able to detect 25 myeloma plasma cells in a background of 50000 immunoglobulin isotype-matched cells of the myeloma cell line UM-1 or 50000 donor bone marrow cells (sensitivity 0.05%). This paper shows that highly specific anti-idiotypic single chain Fv antibody fragments selected from a phage display library can be used to detect rare idiotypic cells in patient samples.

The low cycling status of mobilized peripheral blood CD34+ cells is not restricted to the more primitive subfraction.

Mobilized peripheral blood progenitor cells (PBPC) have been shown to differ qualitatively from bone marrow (BM) progenitors. The released progenitor cells are predominantly in G0/G1 and show a relatively high percentage of rhodamine dull cells. Within the BM these last two features are characteristic of the more primitive progenitors. Although the mobilized PB cells can give rise to long-term repopulation and thus contain stem cells, the frequency of stem cells is not much higher if long-term initiating cell (LTC-IC) assays are used. To determine whether quiescent stem cells are selectively released or the low-cycle status of PB progenitors is related to the release from the BM microenvironment, the cell cycle status and rhodamine content in the PB and BM during mobilization were studied and compared with steady-state BM. More differentiated and more primitive progenitors were separated based on differentiation markers and cloned in single cell assay. In mobilized PB 54% of the CD34+ cells (n=5) were rhodamine dull compared to 22% in steady-state BM (P=0.014) [n=6]. The percentage of CD34+ cells in the S/G2M phases of the cell cycle was 2.1% in the mobilized PB (n=11), and 18% in steady-state BM (n=11) [P=0.002]. During mobilization the fraction of cells in the S/G2M phase of the cell cycle was 16% in BM (n=7), similar to steady-state BM (P=0.34). The released progenitors represented a selection of BM progenitors, with significantly more primitive progenitors (CD34+/13+/33dim) and less lymphoid precursors (CD34+/19+). Within the more differentiated CD34+113+/33bright, myelomonocytic precursors, both in PB as well as in BM, the percentage S/G2M was relatively higher than in the CD34+/13+/33dim subfraction: in normal BM: median 18% vs 8% (P=0.006) [n=8]; in mobilized PB 3% vs 2% (P=0.03) [n=10]; and in BM during mobilization 24% vs 7% (P=0.01) [n=6]. The cycle status of mobilized PB progenitors was low both in the primitive and more differentiated subfractions. During the mobilization period the BM progenitors are cycling as in steady-state BM. The low-cycle status of the mobilized PB progenitors may be related to the loss of contact with the micro-environment.

Characterization of tumor cell heterogeneity of a murine leukemia cell line (L1210) in response to arabinosylcytosine: quantitation using a computerized image analysis system.

Cytosine arabinoside (Ara-C) is one of the most effective drugs in inducing remission in acute nonlymphocytic leukemia (ANLL) patients. However, the high recurrence rate indicates that a subpopulation of leukemic cells escapes drug effect. This cellular heterogeneity in drug response may play a major role in chemotherapeutic outcome. We have recently developed the individual colony-formation assay (ICFA) to study drug effects on the kinetics of proliferation of individual cells and their progeny. Thus parameters of proliferation are calculated for individual colonies. Three categories of drug responses were defined, including immediate growth cessation, delayed growth cessation (growth stops several days after drug exposure) and growth slowdown (logarithmic growth at a reduced rate compared to control). In the experiments included in this report, murine leukemia (L1210) cells were exposed to various concentrations of Ara-C for 1, 6 or 24 hours, and their responses quantified. Regardless of the Ara-C concentration or exposure time, subpopulations of cells were observed in each of the three response categories: immediate or delayed arrest or growth slowdown. As expected, the fraction of cells exhibiting immediate growth cessation generally increased with increasing drug dose and was markedly increased with longer exposure time. Delayed arrest was most prevalent at intermediate drug concentrations at all exposure times. If exposure was limited to 1 hour, at least 30% of cells continued to grow, although at a reduced rate (71% control rate after exposure to 1 mM Ara-C). This limited effect was paralleled by saturation of Ara-C triphosphate (Ara-CTP) formation. Six-hour exposure left at least 6.4% of cells growing, with an average rate of 45% of control. Under these conditions, no saturation in Ara-CTP formation was observed. Even 24-hour exposure to 5 microM Ara-C left 4.8% of colonies growing, at 42% of control rate. Thus a subpopulation of cells continued to grow even after 24-hour exposure to a relatively high concentration of Ara-C. Surviving, but slowly growing, cells may represent a previously unrecognized population that may contribute to therapeutic failure.

Molecular analysis of the hematopoiesis supporting osteoblastic cell line U2-OS.

OBJECTIVE: Osteoblasts play an important role in regulating hematopoiesis in the bone marrow. Here we show that U2-OS, a widely used osteoblastic cell line derived from an osteosarcoma, has the capacity to support proliferation of human hematopoietic progenitor cells in vitro. In this study, U2-OS cells are characterized at the molecular level to unravel the molecular mechanisms underlying the support of hematopoiesis. MATERIALS AND METHODS: U2-OS was analyzed in great detail using RT-PCR and flow cytometry. In addition, a cDNA library was constructed and randomly sequenced to obtain insight in the repertoire of expressed molecules. RESULTS: A broad panel of growth factors and cytokines is expressed by U2-OS. TGF-beta, GM-CSF, c-kit ligand, and IL-7 are produced constitutively and IL-1beta, IL-6, IL-8, TNF-alpha, IFN-gamma, and MIP1-alpha are upregulated upon stimulation. In addition to those, mRNAs of the CC chemokine LARC and leukemia inhibitory factor were identified. U2-OS cells express high levels of beta1-integrins at the cell surface: VLA-2, VLA-3, VLA-4, VLA-5, VLA-6, and the integrin alphavbeta3. Besides integrins, ALCAM and NCAM are detected on the cell surface of U2-OS. Interestingly, we show that CD34(+) progenitor cells expressing ALCAM are highly proliferative when compared with CD34(+) ALCAM(low) cells, hinting at a role for ALCAM in anchoring progenitor cells to the bone marrow stroma. Interestingly, random sequencing of an U2-OS cDNA library yielded almost 10% of novel cDNAs with a potential role in hematopoiesis. The involvement of these novel molecules in hematopoiesis is an interesting target for future investigations. CONCLUSIONS: We conclude that U2-OS supports outgrowth of hematopoietic progenitor cells and accordingly expresses adhesion molecules and growth factors and a number of novel, as yet uncharacterized potentially interesting genes.

Medium-chain, triglyceride-containing lipid emulsions increase human neutrophil beta2 integrin expression, adhesion, and degranulation.

BACKGROUND: To test the hypothesis that lipid emulsions with different triglyceride structures have distinct immunomodulatory properties, we analyzed human neutrophil adhesion and degranulation after lipid incubation. METHODS: Neutrophils, isolated from the blood of 10 healthy volunteers, were incubated in medium or physiologic (2.5 mmol/L) emulsions containing long-chain (LCT), medium-chain (MCT), mixed LCT/MCT, or structured (SL) triglycerides. Expression of adhesion molecules and degranulation markers was evaluated by flow cytometry. Also, functional adhesion was investigated by means of a flow cytometric assay using fluorescent beads coated with the integrin ligand intercellular adhesion molecule (ICAM)-1. RESULTS: Although LCT and SL had no effect, LCT/MCT significantly increased expression of the beta2 integrins lymphocyte-function-associated antigen 1 (+18%), macrophage antigen 1 (+387%), p150,95 (+82%), and (alphaDbeta2 (+230%). Degranulation marker expression for azurophilic (CD63, +210%) and specific granules (CD66b, +370%) also significantly increased, whereas L-selectin (CD62L, -70%) decreased. The effects of LCT/MCT were mimicked by the MCT emulsion. ICAM-1 adhesion (% beads bound) was increased by LCT/MCT (34% +/- 4%), whereas LCT (19% +/-3%) and SL (20% +/- 2%) had no effect compared with medium (17% +/- 3%). CONCLUSIONS: LCT/MCT and MCT, contrary to LCT and SL emulsions, increased neutrophil beta2 integrin expression, adhesion, and degranulation. Apart from other emulsion constituents, triglyceride chain length might therefore be a key feature in the interaction of lipid emulsions and the phagocyte immune system.

Chronic active Epstein-Barr virus infection in an adult with no detectable immune deficiency.

INTRODUCTION: Epstein-Barr virus (EBV) establishes lifelong latent infection. In some patients the host-virus balance is disturbed, resulting in a chronic active EBV infection. The following case illustrates the difficulty in diagnosing and treating chronic EBV infection. CASE: A 30-year-old woman was referred because of recurrent swellings of lymphatic tissue of both eyelids, orbit and lymph nodes and general malaise since the age of 19. In the past, repeated biopsies showed MALT lymphoma and nonspecific lymphoid infiltrations. Now, a biopsy of an axillary lymph node showed paracortical hyperplasia with a polymorphous polyclonal lymphoid proliferation, and large numbers of EBV-encoded small RNA (EBER) positive cells, consistent with EBV infection. Laboratory investigation showed a high EBV viral load. No evidence of immunodeficiency was found. Chronic active EBV infection (CAEBV) was diagnosed. Treatment with high-dose acyclovir did not significantly reduce the viral load. Rituximab was given in an attempt to reduce the amount of EBV-infected B lymphocytes. However, soon after the second dose the patient died of a sub-arachnoidal haemorrhage. CONCLUSION: This case report illustrates CAEBV as a rare manifestation of EBV-induced disease, which will be detected more frequently with the use of EBV-EBER hybridisation of lymph nodes and polymerase chain reaction (PCR) for EBV DNA. The prognosis is poor with no established therapeutic strategies.

Peripheral blood cell harvests yield primitive multilineage progenitor cells in the CD34+/33- fraction.

The presence of primitive hematopoietic progenitor cells or stem cells in peripheral blood (PBSC's) harvests was investigated in a single cell culturing assay and compared with the results obtained in aspirates of normal bone marrow. Based on the presence of CD33, rather differentiated progenitor cells (CD34+/33+) were distinguished from more primitive cells (CD34+/33-). The growth potential of CD34+/33+ and CD34+/33- cells have been studied. Single cell sorting was performed from peripheral blood harvests, obtained from three patients with multiple myeloma during hematopoietic recovery after treatment with high dose cyclophosphamide and rhu-GM-CSF. To test the effect of "stem cell recruiting factors" the cells were sorted in 96-well plates, prefilled with liquid medium both in the presence of IL-3 + G-CSF+GM-CSF+Epo and the same growth factors supplemented with SCF+IL-6. Addition of SCF and IL-6 to the culturing medium enhanced the plating efficiency of CD34+/33- cells considerably more than that of CD34+/33+ cells. This was observed in harvests of peripheral blood as well as in aspirates of normal bone marrow. The differences between CD34+/33+ and CD34+/33- were even more pronounced when only the large colonies (> 500 cells/well) were taken into consideration. Assuming that IL-6 and SCF are "stem cell recruiting factors," the CD34+/33- fraction contains more clonogenic cells than the CD34+/33+ fraction. In all three patients the first CD34+ cells appearing in the peripheral blood (PB) after cytoreductive treatment were predominantly CD34+/33- (> 80%). At later stages when the leukocyte counts had reached higher values the CD34+/33+ cells predominated.(ABSTRACT TRUNCATED AT 250 WORDS)

X-linked sideroblastic anaemia due to ALAS2 mutations in the Netherlands: a disease in disguise.

BACKGROUND: X-linked sideroblastic anaemia (XLSA; OMIM#300751) is the most common inherited form of sideroblastic anaemia and is associated with several mutations in the erythroid specific 5-aminolevulinate synthase gene (ALAS2). This gene encodes for aminolevulinic acid synthase 2 (ALAS2), the catalytic enzyme involved in the first en rate-limiting step of haem biosynthesis.1-3 The disorder is characterised by mostly mild hypochromic microcytic anaemia with bone marrow ring sideroblasts. Even untransfused patients with mild or no anaemia are at risk for severe systemic iron overload due to ineffective erythropoiesis. To date, 61 different ALAS2 mutations have been reported in 120 families with XLSA. Descriptions of molecularly confirmed case series from the Netherlands, however, are lacking. METHODS: We reviewed age of presentation, clinical and biochemical features, ALAS₋2 defects and treatment characteristics of 15 Dutch patients from 11 unrelated families diagnosed with XLSA. RESULTS AND CONCLUSIONS: In one family a novel pathogenic c.1412G>A (p.Cys471Tyr) mutation was found. All other families shared the previously described c.1355G>A (p.Arg452His) mutation. Haplotype analysis in seven probands with the p.Arg452His mutation strongly suggests that six of them were ancestrally related. Nevertheless, their phenotype was very different. Our patients illustrate the phenotypical heterogeneity in the presentation of XLSA patients, the effectiveness of treatment regimens and the various pitfalls associated with the diagnosis, follow-up and treatment of the disease. A timely diagnosis avoids unnecessary investigations and allows adequate treatment that can prevent systemic iron load with subsequent severe life-threatening complications. Therefore, we suggest considering XLSA in both male and female patients with unexplained iron overload and÷or (mild) microcytic anaemia, also at older age.