Dimitrios Loukopoulos

Elastic tissue abnormalities in inherited haemolytic syndromes

Over the last years, the significant prolongation of survival of patients with β thalassaemia, as a result of the intensification of treatment, in combination with the closer and systematic follow-up of patients, has gradually broadened the clinical spectrum of the disease with previously unknown manifestations. In this context, a set of clinical and laboratory signs similar to those of inherited pseudoxanthoma elasticum (PXE) have been noticed [1]. PXE is a rare hereditary connective tissue disorder, characterized by generalized degeneration of the elastic fibres with cutaneous, ocular and vascular manifestations [2]. This abnormality may lead to several serious and life-threatening complications, such as blindness, premature coronary artery disease, and cerebral and gastrointestinal haemorrhage [2]. The occurrence of PXE findings in β thalassaemia patients has led, and continues to lead, an increasing number of investigators to study various aspects of the phenomenon and a growing body of evidence is being accumulated [1]. Accordingly, the potential risk of PXErelated complications prompted Cianciulli et al . to perform a long-term follow-up of thalassaemia cases, while other groups expanded the investigation to other inherited haemolytic syndromes and re-evaluated some similar findings observed previously in sickle cell disease (SCD) [3]. The first manifestations of a potential elastic tissue defect associated with haemoglobinopathies were angioid streaks, which represent breaks in the elastic fibres in the membrane of Bruch and are the typical ocular findings in inherited PXE. Angioid streaks have been described in SCD since the late 1950s [4]. Subsequent studies have reported a frequency of angioid streaks in SCD ranging from 1% to 22%, depending on the patients’ age [5–7]. In β thalassaemia, an age-related occurrence of 20% was reported [1]. Angioid streaks have also been encountered in sickle thalassaemia with a frequency of 10% [8]. Sporadically, they have been seen in α thalassaemia (haemoglobin H disease) [9], haemoglobin AC disease (haemoglobin C trait carrier) [10] and β thalassaemia minor [11,12]. In a large series of angioid streaks, 50% of cases had PXE on diagnosis [13]. The possibility of a similar diffuse connective tissue disorder in SCD was raised by the report of the PXE syndrome in at least seven adult cases with SCD [14]. However, blind skin biopsies, performed in SCD patients with angioid streaks, failed to reveal any evidence of PXE [5,7]. In contrast, an autopsy series of unselected SCD patients revealed histopathological findings in the dermis and the arterial walls of multiple organs identical to those found in PXE [15]. A clearer relationship was introduced by Lippman et al . in 1985. Based on histopathology and biochemical analysis of skin biopsies, these investigators concluded that SCD appears to be associated with a wide spectrum of elastic tissue disorders resembling PXE, although less severe than PXE [16]. Subsequently, accumulated evidence from the literature has supported the concept of an underlying, generalized elastic tissue defect in the same haemoglobinopathies that were previously associated with angioid streaks. In β thalassaemia, the occurrence of PXE findings proved to be more profound and structurally identical to the inherited PXE [17]. In a 100-patient group with β thalassaemia, 16% of cases had PXE cutaneous lesions, 20% had angioid streaks and 26% had at least one of the two findings; however, none of the patients under the age of 19 years nor any of the 70 family members of cases with PXE findings showed any skin or ocular lesions [18]. The study by Cianciulli et al . confirms the high incidence of PXE-like skin, ocular and arterial manifestations in patients with β thalassaemia as well as their positive correlation with age [3]. In a previous study, the incidence of at least one of these findings reached the impressive percentage of 85% in older thalassaemic patients, aged over 30 years, with calcification of First Department of Internal Medicine, University of Athens, Medical School, ‘Laiko’ General Hospital, Athens 115 27, Greece (A. Aessopos, D. Farmakis, D. Loukopoulos).

Reticulocyte counting in thalassemic and other conditions with the R-1000 Sysmex analyzer.

SummaryPrecise reticulocyte counts are difficult to obtain by the manual method when their percentage in the blood is low or normal. In these instances, rapid reticulocyte counting by flow cytometry appears to offer more accuracy and precision. The purpose of this study was to establish reticulocyte counts in heterozygous β-thalassemia for reference purposes and to evaluate the performance of the recently introduced apparatus R-1000 (Sysmex) in the very heterogeneous thalassemic and sickle-cell syndromes. We studied a total of 364 samples; 102 heterozygous β-thalassemia carriers, 180 normal matched controls, 36 patients with thalassemia major or intermedia, and 46 patients with various sickle-cell syndromes. Reticulocyte counts (both as percentage and as total number) were higher in heterozygous β-thalassemia than in normal controls (p<0.001) and showed an inverse correlation with the respective hemoglobin values (p<0.001). These results confirm the proposed slightly increased erythropoietic activity in heterozygous β-thalassemia carriers. A drawback of the technique is that the reticulocyte-platelet discrimination error is signaled frequently in all conditions displaying a marked red cell heterogeneity, especially when these are associated with high reticulocyte numbers. This calls probably for readjustment of the corresponding algorithm. In addition, all these conditions show a significantly increased auramine-O mature red-cell nonspecific fluorescence.

Molecular analysis of transferrin receptor mRNA expression in acute myeloid leukaemia.

Transferrin receptor (TfR, CD71) is an integral membrane glycoprotein that mediates cellular uptake of iron. In most tissues, TfR expression is correlated positively with proliferation and regulated at the post‐transcriptional level. The available data regarding the pattern of TfR gene expression in haematological malignancies are very limited. In the present study, we evaluated TfR gene expression at the molecular level in bone marrow (BM) samples of 44 patients with de novo acute myeloid leukaemia (AML) at diagnosis with BM blasts > 85%. TfR mRNA levels were determined by densitometric analysis of quantitative reverse transcription polymerase chain reaction products corresponding to TfR exons 15–17. Each sample was tested in at least two independent experiments. In 13/44 patients, TfR messages were not detected (this is probably an underestimate as some positive results may be attributed to residual normal erythroid cells present in the samples). In 17/44, TfR mRNA levels were low–intermediate, and were high in the remaining patients (14/44). TfR mRNA positivity was significantly associated with older age. No statistically significant correlations were found either with specific French–American–British (FAB) subtypes or attainment of complete remission, incidence of relapse and survival (after adjusting accordingly for age and FAB subtype). The absence of TfR mRNA transcripts in a significant minority of cases suggests that alternative mechanisms of iron uptake may function in AML blast cells.

Exercise-induced myocardial perfusion abnormalities in sickle β-thalassemia: Tc-99m tetrofosmin gated SPECT imaging study ☆

PURPOSE To determine the mechanism of myocardial ischemia in patients with sickle beta-thalassemia, we performed a scintigraphic evaluation of myocardial perfusion during exercise. SUBJECTS AND METHODS We studied 30 patients with sickle beta-thalassemia, (mean [+/-SD] age, 37 +/- 10 years) who had no electrocardiographic (ECG), radiographic, or echo-Doppler signs of pulmonary hypertension, left ventricular hypertrophy, or impaired contractility. All patients had a hemoglobin level greater than 7 g/dL. Treadmill exercise test was performed according to the Bruce protocol. Myocardial perfusion was assessed by single-photon emission computed tomography, using Tetrofosmin Tc-99 m Myoview as radiotracer, at peak exercise and again 4 hours later. RESULTS Eight patients (27%) developed stress-induced scintigraphic perfusion abnormalities that were reversible in all but 1 patient. Subsequent coronary angiograms were normal in all 8 patients. ST segment depression was seen during exercise in 5 of the 7 patients who had reversible perfusion defects. Except for a significantly greater white blood cell count, these 5 patients did not differ from the rest of patients by sex, age, hemoglobin level, percentage hemoglobin F, beta-thalassemia genotype, or risk factors for coronary artery disease. Three of the 5 patients with perfusion and ECG abnormalities (and another with only perfusion defects) developed a stress-induced sickling crisis. CONCLUSION Physical stress may induce myocardial ischemia in sickle beta-thalassemia patients with normal coronary arteries and elicit painful crises. The sickling process, activated by exercise, could be the common underlying mechanism.

Trisomy 8 in a patient who responded to therapy with all‐trans‐retinoic acid and developed paroxysmal nocturnal haemoglobinuria

Trisomy 8 is the most common numerical chromosomal abnormality in myelodysplastic syndromes (MDS). Paroxysmal nocturnal haemoglobinuria (PNH) is an aquired haemolytic anaemia, clonal in nature, due to somatic mutation. PNH may evolve to aplastic anaemia, to MDS or to acute myeloid leukaemia. We present a patient who had trisomy 8 mosaicism at disease presentation who received therapy with all‐trans‐retinoic acid, responded to therapy, and developed PNH in the course of the disease. Cytogenetics at the time of PNH diagnosis showed a normal karyotype.

Lymphocyte subpopulations and interleukin levels in high-risk melanoma patients treated with high-dose interferon A-2B.

Immunologic effects of high-dose interferon are still unclear. We have evaluated changes in blood lymphocyte subpopulations, immunoglobulins, and multiple interleukin in patients with high-risk cutaneous melanoma on adjuvant treatment with high-dose interferon and compared pretreatment values with normal controls. Samples were obtained before treatment, 1 month after induction treatment and at 3, 6, and 12 months of maintenance treatment from 24 patients with high-risk melanoma. Lymphocyte subpopulations were measured by flow cytometry and interleukin and immunoglobulin levels by radioimmunoassay. A statistically significant reduction in B-lymphocytes (p < 0.001), natural killer (NK) cells (p = 0.0004), and monocytes (p = 0.04), and an elevation in CD4/CD8 ratio (p < 0.0001) was observed after 1 month of intravenous interferon. No changes were seen in CD3, CD4, and CD8 lymphocytes. No changes in interleukin (IL)-2, -4, or -5 were observed during 1 year of treatment. IL-2 pretreatment levels were significantly lower than healthy blood donors (p = 0.001), and IL-5 pretreatment levels were significantly higher (p = 0.0056). IL-10 levels significantly dropped after 6 months of treatment (p = 0.01). Immunoglobulins (IgG, IgA, IgM) remained within normal ranges. Three patients had elevated pretreatment levels of IgE. There is a time- and dose-dependent impact of interferon on numbers of circulating B lymphocytes, NK cells, monocytes, and CD4/CD8 ratio. Defects in cellular and humoral immunity are suggested by the low IL-2 and high IL-5 levels, measured in patients with melanoma as compared with healthy controls.

Development of plasma cell tumors during treatment of multiple myeloma

Plasma cell tumors (plasmacytomas-PCT) of the bone, or extramedullary PCT, may be diagnosed in patients with or without the diagnostic criteria for systemic multiple myeloma (MM). The reason for the local development of these tumors is not clear. Recent reports emphasize the contribution of CT and MRI in the detection of bone lesions and their expansion into the soft tissues. We report the development of PCT in nine patients with MM under maintenance treatment with α-IFN, of whom six had no evidence of systemic relapse and three had indications of early relapse. The PCT were located in the pelvis (4), thoracic (3), cervical (1), and lumbar (2) spine and in 8/9 cases were not demonstrable on plain X-rays. These observations suggest that frequent screening with advanced imaging techniques may detect local disease expansion in asymptomatic patients. Early application of radiochemotherapy may improve prognosis.