The immature reticulocyte fraction: a negative predictor of the harvesting of CD34 cells for autologous peripheral blood stem cell transplantation.

The optimal time for the harvesting of peripheral blood stem cells following chemotherapy and growth factors for autologous transplantation is based on the CD34 cell count. In this study, 51 patients having 59 stem cell mobilizations were assessed for the timing of the harvest by a CD34 cell count and an immature reticulocyte fraction (IRF). Results from 272 preharvest tests showed that when the CD34 cells were not harvestable, defined as a CD34 cell count of < 15 cells/microl, the IRF was always < or = 0.2. A low IRF resulted in a negative predictive value of 1 for the harvesting of stem cells. The IRF is therefore a valuable negative predictor of the timing of autologous stem cell harvesting.

The myelodysplastic syndromes: an analysis of prognostic factors in 226 cases from a single institution.

Two hundred and twenty-six patients were diagnosed with myelodysplastic syndrome (MDS), according to the French-American-British (FAB) criteria, over a 13-year period, and studied retrospectively in a single institution in order to study indicators which were prognostically significant. Analysis of clinical and laboratory data indicated that the FAB classification, the Bournemouth, Dusseldorf, Goasguen, Sanz and FAB Scoring Systems were all good predictors of survival. We found advancing age, haemoglobin (Hb) < or = 9 g/dl, platelet count < or = 50 x 10(9)/l, increased peripheral total white cell count (WCC) and monocytosis, increased bone marrow blasts, dysgranulopoiesis, and bone marrow fibrosis were significant adverse prognostic variables. The commonest complication and cause of death was infection; however, infective episodes were not significantly associated with low neutrophil counts (either < or = 1.5 x 10(9)/l or < or = 0.8 x 10(9)/l) and there was also no significant association between neutropenia and survival. These findings indicate that neutrophil dysfunction plays an important role in the clinical progression of patients with MDS. The effect of new therapeutic modalities, such as the haemopoietic growth factors, on reducing infective episodes may be as significant as their effect on increasing neutrophil counts.

The Australian type of nondeletional G gamma-HPFH has a C-->G substitution at nucleotide -114 of the G gamma gene.

Nondeletional hereditary persistence of fetal haemoglobin (HPFH) results in the continued production of 2-25% haemoglobin F (Hb F) in the adult who is heterozygous for this mutation. This increase is associated with single-base mutations in the promoter region of either the G gamma- or A gamma-globin genes. Affected positions include -202, -175, -161, -158 and -114 of the G gamma gene, and -202, -198, -196, -195, -175, and -117 of the A gamma gene. There is now evidence that these mutations produce their effect by changing the binding of certain regulatory proteins. We describe a novel C-->G transversion at position -114 of the G gamma gene which is associated with the phenotype of G gamma-HPFH.

Comparative studies of nondeletional HPFH gamma-globin gene promoters.

The -198 T-->C and the -175 T-->C transitions involving the proximal gamma-globin gene promoter are associated with the hereditary persistence of fetal hemoglobin (HPFH) phenotype and have been demonstrated to increase promoter activity in erythroid cells using transient and stable transfection systems. The above base changes are thought to alter the binding of different transcription regulatory proteins. Another mutation of the proximal gamma-globin promoter, -158 C-->T, has been less clearly linked to the HPFH phenotype but has been associated with increased G gamma activity. In the present paper, the -198 T-->C, -175 T-->C and -158 C-->T mutations both singly and in various combinations were evaluated by an in vitro expression assay. gamma-Globin promoters were transfected by electroporation into K562 human erythroleukemia cells and their activity measured in a human growth hormone (hGH) reporter gene assay. A novel cotransfectant was used to assess transfection efficiency. Results confirmed the previously reported upregulation of gamma-globin activity with the -198 T-->C and -175 T-->C HPFH mutations and a cooperative effect on promoter activity when both these mutations are present in cis. No effect of the -158 C-->T mutation was seen either alone or in combination with the -175 T-->C and -198 T-->C mutations.

Linkage analysis of the hemoglobin F determinant(s) in an Australian hemoglobin Lepore (Boston) kindred.

Genetic determinants that influence the levels of fetal hemoglobin (Hb F) in a single Australian kindred with heterozygous Hb Lepore (Boston) were sought. There were 22 affected individuals, some of whom had high Hb F and others with Hb F levels within the normal range. Family members were typed for restriction fragment length polymorphisms (RFLPs) associated with the beta-globin gene complex and the nearby genetic markers D11S12, INS, HRAS, and PTH. Prior to linkage analysis, a cohort of 54 unrelated Hb Lepore heterozygotes was analyzed to establish the distribution of Hb F levels measured by alkaline denaturation (Hb FAD). An Hb F level of > 2.0% was used as the cutoff point for linkage analysis of the putative hereditary persistence of fetal hemoglobin (HPFH) determinant(s) in this kindred. Positive peak lod scores were obtained for the entire pedigree between the HPFH determinant and Hb Lepore (Zm+f = 2.35 at theta = 0.15) and beta-globin cluster (HBBC) (Zm+f = 2.38 at theta = 0.20) marker loci, indicating the possibility of an independent HPFH gene at some distance from the beta-globin gene cluster. However, most of these lod scores result from the non-Hb Lepore members of the family who, with one exception, do not have high Hb F, and when only those affected with Hb Lepore were analyzed the lod score values at these loci fell to small positive values (< 1.0). These data do not support an independent cosegregating HPFH determinant separate from the Hb Lepore locus in this pedigree. The results favor a pleiotropic effect of the Hb Lepore lesion itself influencing Hb F levels by genetic or environmental factors not yet elucidated.

Filipino beta zero thalassaemia: a high Hb A2 beta zero thalassaemia resulting from a large deletion of the 5' beta globin gene region.

A large novel deletional beta zero thalassaemia mutation associated with unusually high levels of haemoglobin (Hb) A2 in heterozygotes is described in two unrelated subjects of Filipino background. The deletion was characterised by DNA mapping including pulsed field gel electrophoresis. Filipino beta zero thalassaemia extends for approximately 45 kb beginning approximately 1.5 kb 3' to the delta globin gene. It is the largest deletion to date which gives rise to the beta zero thalassaemia phenotype. This mutation, similar to previously described deletional beta zero thalassaemias associated with high Hb A2, removes sequences 5' to the beta globin gene promoter and emphasises the functional importance of the 5' beta globin region in eliciting the unusually high level of Hb A2. This example also suggests that it is the 3' sequences which are transposed rather than the actual deletion size which are significant in the raised fetal haemoglobin (Hb F) found with some of the thalassaemias.

Molecular characterisation of Vietnamese HPFH.

A novel 30 kb deletion of the beta-globin gene cluster associated with the phenotype of hereditary persistence of fetal hemoglobin (HPFH) is described in two unrelated individuals of Vietnamese background. The Vietnamese G gamma A gamma HPFH deletion has a unique 5' breakpoint 3.5 kb downstream of the delta-globin gene. The 3' breakpoint lies approximately 8 kb upstream from the HPFH-3 breakpoint (Henthorn et al., 1986) and in the region of the 3' breakpoints of HPFH-4 (Saglio et al., 1986), German and Belgian G gamma+ (A gamma delta beta)zero-thalassemias (Anagnou et al., 1988; Losekoot et al., 1991). Characterisation of the 3' breakpoint in the present study has enabled more precise localisation of other deletion breakpoints at this locus. Further evidence is provided that the 3' breakpoint region contains functionally important sequences and that the juxtaposition of these sequences to the gamma-globin genes is a significant factor in the increased fetal hemoglobin levels.

Australian beta zero-thalassaemia: a high haemoglobin A2 beta zero-thalassaemia due to a 12 kb deletion commencing 5' to the beta-globin gene.

A large novel deletional beta zero-thalassaemia mutation associated with unusually high levels of haemoglobin A2 in heterozygotes is described in an Australian family. The deletion was characterized by restriction enzyme analysis followed by PCR amplification and sequencing of the breakpoint region. Australian beta zero-thalassaemia extends from 835 basepairs (bp) 5' to the cap site of the beta-globin gene downstream for 12.023 kb. This deletion, similar to previously described deletional beta zero-thalassaemias associated with high Hb A2, removes sequences 5' to the beta-globin gene promoter and emphasizes the functional importance of the 5' beta-globin region in eliciting the unusually high Hb A2 phenotype.

An A gamma globin promoter (four base-pair deletion) mutant shows linked polymorphic changes throughout the A gamma gene.

The A gamma fetal globin genes from a large Australian kindred with nondeletional A gamma hereditary persistence of fetal hemoglobin (HPFH) were cloned and sequenced. The -198 T----C mutation (British type HPFH) was demonstrated upstream of the A gamma gene on one allele. On the other allele, a 4-deletion was identified -222 to -225 bp upstream from the cap site. The 4-bp deletion allele was associated with a number of variations in the A gamma gene sequence: anA gamma T transition, in the second exon (T----C at +402 relative to the cap site); a HindIII polymorphism in the second intron; and a G gamma-like sequence in the 3' untranslated region (TCAC in place of CTCT, creating a SacI site). An association between the -222 to -225 deletion, the A gamma T polymorphism, and the second intron HindIII polymorphism has previously been reported. In addition, linkage of the HindIII polymorphism with the G gamma-like sequence in the 3' untranslated region of A gamma has also been described. The case described here is unique, with all four changes present in the one A gamma gene. It is also noteworthy because there is simultaneous occurrence of high (HPFH) and low (-222 to -225 deletion) expression mutants in the same patient. Despite the presence of the 4-bp deletion, the resulting hematological phenotype remained that of HPFH. When the 4-bp deletion promoter was studied in a K562-cell transient expression assay, there was found to be no statistically significant reduction in activity compared to the control A gamma promoter. The possible reasons for the observed differences between the in vivo and in vitro activity of this mutation are discussed.

Neonatal alloimmune thrombocytopenia caused by an antibody to the Bak(a) antigen.

A 31 year old woman was assessed following delivery of her second child affected by neonatal alloimmune thrombocytopenia (NAIT). Antiplatelet antibodies with specificity for Bak(a) were identified in the woman's serum and her platelets were typed as Bak(a) negative whilst her husband's were Bak(a) positive. Unlike the majority of reported anti-Bak(a) antibodies in the literature, this patient's serum contained no contaminating anti-HLA antibodies. This is the first report of NAIT caused by an anti-Bak(a) without co-existing anti-HLA antibodies. An anti-Bak(a) antibody has not previously been reported in Australia. The current status of this antigen system is reviewed.

Recurrent epistaxis, chronic pancytopenia and nasal mycosis.

A 52-year-old man with chronic, profound pancytopenia associated with a four-year history of Waldenstrom's macroglobulinemia refractory to chemotherapy began to experience major epistaxes requiring hospital admission for control. A diagnosis of invasive Aspergillus was made on nasal mucosal biopsy. A regimen of bi-weekly intravenous and sixth-hourly topical amphotericin B drops with adjuvant rifampicin successfully eradicated local nasal pain as well as the epistaxis. Resolution of infection was documented by gallium scan. It is suggested that, in addition to the aggressive craniofacial mycoses already described, lower grade nasal mycoses occur. The roles of biopsy and gallium scan in immunocompromised hosts with epistaxis are discussed.

Molecular mechanisms associated with increased fetal hemoglobin G gamma-type in part-aboriginal family with beta thalassemia.

A part-Aboriginal family with beta thalassemia and raised hemoglobin F (HbF) was studied at the molecular level to determine if there were identifiable gene changes associated with increased production of HbF. Two beta thalassemia heterozygotes aged eight years and 18 months had raised HbF levels of 2.9% and 22% respectively. HbF was predominantly G gamma in composition. Five family members were typed for restriction fragment length polymorphisms (RFLPs) using nine restriction enzymes and five DNA probes specific for the beta globin cluster on chromosome 11. RFLPs were combined to construct haplotypes for the beta thalassemia and the high HbF defects. A beta globin subhaplotype comprising only 5' RFLP markers (-(+)-(+) +) co-segregated with the high HbF determinant. This has previously been associated with increased G gamma expression in beta thalassemia and sickle cell anemia. An additional Xmnl RFLP 5' to the G gamma gene, which has been described in individuals with elevated G gamma expression, was also demonstrated in those family members with increased G gamma levels. In this study both the 5' beta globin subhaplotype (-(+)-(+) +) and the Xmnl/gamma RFLP are present in the one family but the relative contributions of each cannot be determined.

Fine needle aspiration sampling of bone marrow for DNA flow cytometric analysis.

Fine needle aspiration (FNA) was used to obtain bone marrow samples from 57 patients with a variety of hematologic disorders, and from three normal subjects. We confirm that the technique is well tolerated and show that reliable cytokinetic and cell differential data can be obtained provided marrow fragments are isolated for analysis. This requires aspiration of a larger volume of marrow than originally described but this can still be achieved with minimal discomfort to the patient. The technique thus provides a means by which marrow samples could be obtained at frequent intervals in the monitoring of chemotherapy.

Prodromal acquired immune deficiency syndrome in Australian homosexual men.

The acquired immune deficiency syndrome (AIDS) was first reported in 1981 in homosexual men. Since then, it has shown an alarming increase in frequency. The syndrome has now been described in other population groups as well, in particular in persons with haemophilia who are exposed to frequent parenteral injections of blood or blood products, and in sexual contacts of those in at-risk groups. This report deals with two cases of this syndrome which occurred in homosexual men residing in Sydney. These cases are among the first reported in Australia, but, undoubtedly, many more will be forthcoming. Both patients have a profound defect in cellular immunity. The cause of this condition remains a mystery, although viral infection has been suggested. Therapeutic approaches have not yet been established. Clinicians and laboratory workers should be aware that this condition is now present in Australia, and should take appropriate precautions to avoid infection and to limit its spread.