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Approximately 80% of α-thalassemia mutations are deletions in the α-globin cluster on chromosome 16 and about 10% of β-thalassemia mutations are deletions in the β-globin gene cluster on chromosome 11. Larger deletions involving the β-globin gene cluster lead to (δβ)-, (γδβ)-, (εγδβ)-thalassemia, or hereditary persistence of fetal hemoglobin (HPFH). Array comparative genomic hybridization (CGH) was applied to screen for deletions in the α- and β-globin gene clusters not detected by routine gap-PCR. In total, in 13 patients with hypochromia and inclusion bodies (IBs) the α-globin gene cluster was analyzed and in 13 patients with increased fetal hemoglobin levels with or without hypochromia the β-globin gene cluster was examined. All samples were subsequently investigated by multiplex ligation-dependent probe amplification (MLPA). In 9 out of 13 patients deletions of the α-globin gene cluster were identified; 5 of these deletions remove the entire α-globin cluster and extend to the telomere. Additional sequencing of the remaining 4 patients revealed polyadenylation mutation in 1 of them. 7 deletions were identified in the β-globin gene cluster in 13 patients. Additional sequencing of the remaining 6 patients revealed mutations in one of the γ-globin gene promoters in 3 of them and a KLF1-mutation in 1 of them. Array CGH is a reliable method to screen for deletions in thalassemia and hemoglobinopathy. The method offers the advantage of a high resolution with the possibility to characterize breakpoints on sequence level., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Aim: With the availability of newer hematology analysers, novel measurement techniques are being introduced into daily routine. As analyte stability may differ according to the employed measurement technique, the aim of this study was to assess the stability of hematologic analytes on different hematology analysers., Methods: We investigated the effect of storage time and storage temperature on sample stability on three newer analytical systems (Advia 120, Bayer Diagnostics; XE 2100, Sysmex and LH 750, Beckman Coulter). Samples were obtained from 64 healthy volunteers and stored at room temperature as well at 4-8 degrees C in order to conduct analysis at different timepoints up to 72 h after blood was drawn. Sample stability was assessed by the graphical truncated normal sequential test. A parameter was considered stable, when its average change was smaller than one coefficient of variation CV (%) of the assessed method, allowing a 5% risk of error., Results: Red blood cell counts, mean corpuscular hemoglobin (MCH) and hemoglobin are least affected by storage temperature and showed stability for at least 48 h, depending on analyser utilized. While reticulocytes, mean corpuscular volume (MCV), hematocrit and leukocyte counts were more stable at 4-8 degrees C, thrombocytes exhibited a better stability at RT. The white blood cell (WBC) subpopulations changed over time with a decrease in eosinophils and lymphocytes and an increase in neutrophils at 4-8 degrees C. Further, the stability pattern of WBC subpopulations was significantly different among the 3 investigated analysers with some analytes only displaying a stability of 4 h., Conclusions: Analyte stability of hematological parameters varies not only according to the investigated parameter but also according to storage temperature and the employed measurement system. Depending on the analyte sample stability differences among the different analytical systems are considerable.

Iron is an element which is essential to life but also potentially toxic. Therefore, clever mechanisms exist in the human body for uptake, transport and storage of iron. Hepcidin, which seems to be the master protein for regulation of intestinal iron absorption, is known for a short time. The expression of hepcidin is not only influenced by iron levels but also by mediators of inflammation and growth factors of erythropoiesis. Hence hepcidin plays also a crucial role in the development of anemia of chronic disease and iron overload due to ineffective erythropoiesis. Serum ferritin is a reliable parameter to estimate the storage iron. It is an acute phase protein which is elevated during infections and inflammations, though. In these situations, measurement of soluble transferrin receptors is a useful tool to differentiate between iron deficiency and anemia of chronic disease. Newer parameters as erythrocyte zink protoporphyrin or percentage of hypochromic erythrocytes (%HYPO) are suited to detect a functional iron deficiency. Early diagnosis of iron overload is essential to prevent organ damage. Serum ferritin and transferrin are useful parameters to screen for iron overload. If no clear reason for a secondary iron overload can be found, the search for a hereditary haemochromatosis is recommended. Most of these hereditary haemochromatoses are a result of mutations in the HFE gene (homozygous state for Cys282Tyr or compound heterozygosity for Cys282Tyr/ His63Asp) which can be detected by PCR technique. Liver biopsy is still the gold standard for quantification of storage iron. However, a method of increasing importance for quantification of iron overload is magnetic resonance imaging with new approaches as for example T2*.

The reference values of the most commonly used parameters in hematology were evaluated in a metaanalysis using practices of a group of major hospitals in Switzerland and in detail review of the literature. Extensive differences of the reference values have been noted being caused mainly by selection of different patient/control collectives. Whenever possible, reference values were separately evaluated for age, gender and race. The reported reference values approximated a Gauss distribution allowing for statistical evaluation accordingly. Due to recent standardization (ICSH and NCCLS), differences caused by instrumentation and preanalytics were found to be of less importance. Our presented validated reference values in hematology should allow for better discrimination of classical hematological disease entities such as an iron deficiency anemia, thalassemia and hemolysis.

Haemoglobinopathies constitute entities that are generated by either an abnormal haemoglobin or thalassaemias. While abnormal haemoglobins are caused by a qualitative structural abnormality of the haemoglobin molecule, thalassaemias result by diminished synthesis of the globin chain. Due to increased immigration from Asia, Africa and the Mediterranean to Northern Europe, haemoglobin S, haemoglobin C, haemoglobin E are also encountered commonly in Switzerland, while other abnormal haemoglobins are rare, yet can cause clinically relevant symptoms. This include haemolysis, polyglobulia, cyanosis or a combination thereof Thalassaemia-syndroms constitute with two million affected individuals to the most prelevant monogenetic diseases worldwide. Due to migration into Switzerland, they are also found quite commonly among our patients with 10-15 per cent of all hypochromic, microcytic, anemia second only to iron deficiency. Importantly, thalassaemias and haemoglobinopathies can occur concomitantly sometimes even with a normal haemoglobin variant. This results in wide-spread presentations, making diagnosis and clinical judgement difficult. We describe in this article not only physiological mechanisms and clinical presentation but also propose a step-wise diagnostic algorithm including selective use of molecular biology methods.

Objective: Reports on heparin-induced thrombocytopenia (HIT) in paediatrics are confined to isolated case reports. The objective was to systematically combine the published data., Design and Setting: Cases were identified by MEDLINE search and review of bibliographies., Patients: We included subjects reported with HIT, collecting patient demographics, clinical and laboratory characteristics, therapeutic regimens and outcomes., Measurements and Results: Reports on 70 patients were retrieved. In a majority of children, HIT occurred during hospitalisation in a paediatric ICU. In most patients, the typical onset pattern was reported, although rapid-onset-pattern HIT occurred in some. The median platelet-count nadir was 54x10(9)/l; 11% of reported patients had nadirs in the normal range. Clinical symptoms included isolated thrombocytopenia and solitary or combined venous, arterial and intracardiac thromboembolism, sometimes catheter-related. Pulmonary embolism and major bleeding were rarely described. Confirmatory functional or antigenic testing of HIT antibodies showed a similar cumulative sensitivity of about 88%. An unfavourable outcome (death/limb amputation) was reported in 42.1% of patients without therapy and in 18% of patients treated with danaparoid, lepirudin, or argatroban., Conclusions: HIT in children mainly occurs in paediatric intensive care with diagnostic features and outcomes similar to those seen in adults. HIT cannot be ruled out based on normal platelet counts or occurrence after fewer than 5 days of heparin exposure. Children should be presumed to suffer from HIT based on clinical grounds and treated accordingly (immediate heparin withdrawal and alternative anticoagulation). Alternative anticoagulation with danaparoid, lepirudin and argatroban appears to improve outcomes.

According to standard-protocols, real time quantitative RT-PCR (RQ-PCR) for quantification of BCR-ABL fusion transcripts in CML patients is performed with the construction of a standard curve for each run, each sample is analyzed at least in duplicate and 10-40 ml peripheral blood are processed. This approach is appropriate for a research laboratory, but is not suitable for a routine laboratory setting. We show that the calibration curve based on the common 5 dilution standards (between 10 and 10(6) copies) is strongly influenced by the large variability of the measurements below 100 copies of the gene. In other words, including a standard with 10 copies is a source of error, which cannot be reduced through the construction of a standard curve with each run. Adding additional dilutions between 10 and 100 copies to the standard curve, the variance of the obtained curve is much reduced. As a conclusion, it is unnecessary to construct a calibration curve with each run since only negligible inaccuracy of calibration is added to the inaccuracy of measurement. Running of the samples in duplicate seems unnecessary since the inaccuracy of the method can be correctly estimated. Finally, we propose a standardized collection and isolation of total RNA from only 2.5 ml blood using an integrated system, which allows RNA stabilization for up to 5 days and provides snapshots of BCR-ABL fusion transcripts with higher accuracy than with non-stabilized blood samples.

Anemia is often encountered in clinical practice. It has to be noted that anemia is only a symptom of an underlying disease. Early, correct and consequent diagnosis is crucial in order to prevent late detection of fatal disease or to prescribe contraindicated therapy. The combined use of a morphologic and physiologic classification allows for understanding and correct classification of anemia in a specific patient. In this respect, the determination of reticulocyte counts is of special importance. Further targeted biochemical, morphological and immunological analysis leads to the definitive diagnosis of anemia. A large proportion of the anemias can be diagnosed and treated by the general practitioner. However, involvement of a haematologist is recommended in several clinical situations, such as newly diagnosed haemolytic anemia, as well as in patients with complex, equivocal or therapy-refractory anemia or certain normocytic hyporegenerative anemias. This paper presents a differentiated stepwise approach in diagnosing anemic disorders.

Laboratory investigation plays a crucial role in the workup of hematological disease. The well established method of morphological analysis of blood components has been continuously complemented by other methods. On one hand, these consist of considerable improvements of methods employed for automated cell enumeration allowing for early and accurate detection of cell subpopulations, and quantification of valuable red cell parameters, which are of use in the differential diagnosis of anemia. On the other hand, several parameters for the differentiation of microcytic anemia have become available often allowing for the sometimes difficult diagnosis of anemia of chronic disease, iron deficiency anemia, or thalassemia (ferritin, soluble transferrin receptor, transferrin saturation, RDW, zinc protoporphyrin, as well as reticulocyte indices CHr, Ret-Y Hypo%). In macrocytic anemia, introduction of methods to measure methylmalonic acid (MMA), homocystein, holotranscobalamin (holo-TC), complement the determinations of vitamin concentrations (vitamin B12, folic acid in serum and erythrocytes). Employing these newer parameters in addition to the well established ones allows for detection of early or combined disease. The clinician has to know the diagnostic characteristics not only of the old but also of the newer parameters.