Water immersion methods do not alter muscle damage and inflammation biomarkers after high-intensity sprinting and jumping exercise.

PURPOSE: The aim of this study was to compare the efficacy of three water immersion interventions performed after active recovery compared to active recovery only on the resolution of inflammation and markers of muscle damage post-exercise. METHODS: Nine physically active men (n = 9; age 20‒35 years) performed an intensive loading protocol, including maximal jumps and sprinting on four occasions. After each trial, one of three recovery interventions (10 min duration) was used in a random order: cold-water immersion (CWI, 10 °C), thermoneutral water immersion (TWI, 24 °C), contrast water therapy (CWT, alternately 10 °C and 38 °C). All of these methods were performed after an active recovery (10 min bicycle ergometer), and were compared to active recovery only (ACT). 5 min, 1, 24, 48, and 96 h after exercise bouts, immune response and recovery were assessed through leukocyte subsets, monocyte chemoattractant protein-1, myoglobin and high-sensitivity C-reactive protein concentrations. RESULTS: Significant changes in all blood markers occurred at post-loading (p < 0.05), but there were no significant differences observed in the recovery between methods. However, retrospective analysis revealed significant trial-order effects for myoglobin and neutrophils (p < 0.01). Only lymphocytes displayed satisfactory reliability in the exercise response, with intraclass correlation coefficient > 0.5. CONCLUSIONS: The recovery methods did not affect the resolution of inflammatory and immune responses after high-intensity sprinting and jumping exercise. It is notable that the biomarker responses were variable within individuals. Thus, the lack of differences between recovery methods may have been influenced by the reliability of exercise-induced biomarker responses.

Red blood cell transfusion in southern Finland from 2011 to 2016: a quality audit.

OBJECTIVES: The current trends in RBC use and pre- and post-transfusion Hb levels were analysed to improve practice and to provide international comparison. BACKGROUND: Indications for RBC transfusion have changed with growing scientific evidence. The lowest acceptable haemoglobin (Hb) level has decreased, and transfusing single units instead of pairs has become the new standard. Evidence-based guidelines and patient blood management (PBM) programmes increase clinician awareness of rational RBC use. In Finland, however, no formal PBM programme has been established to date. METHODS: The study was registry-based, retrospective and observational. All RBC transfusions for adult patients from 2011 to 2016 in the southern region of Finland were analysed. RESULTS: RBC usage decreased from 34·9 to 27·5 units per 1000 population (P < 0·001). The percentage of single-unit transfusions increased from 57·9 to 66·7%, and the median pre- and post-transfusion Hb levels decreased from 8·4 to 8·2 g dL-1 (P < 0·001) and 9·9 to 9·6 g dL-1 (P < 0·001), respectively. The proportion of transfusions with pre-transfusion Hb ≥ 9·0 g dL-1 decreased during the study period but remained high, being 29·5% in 2011 and still 25·2% in 2016. CONCLUSIONS: Consumption of RBCs has decreased despite aging population and increasing healthcare performance demands. The results indicate more rational and evidence-based RBC use. Nevertheless, the transfusion rate and pre- and post-transfusion Hb are still sufficiently high to enable more restrictive transfusion practice.

Combined aerobic and resistance training decreases inflammation markers in healthy men.

Our primary aim was to study the effects of 24 weeks of combined aerobic and resistance training performed on the same day or on different days on inflammation markers. Physically active, healthy young men were randomly divided into three groups that performed: aerobic and resistance training consecutively in the same training session (SS) 2-3 days wk-1 or on alternating days (AD) 4-6 days wk-1 as well as control (C). The total training volume was matched in the training groups. The control group was asked to maintain their habitual physical activity and exercise level. Maximal leg press strength (1RM) and peak oxygen uptake (VO2peak ) were measured. Abdominal fat mass was estimated with dual-energy absorptiometry (DXA). High-sensitivity C-reactive protein (hs-CRP), interleukin-6 (IL-6), monocyte chemoattractant protein 1 (MCP-1), tumor necrosis factor alpha (TNF-α), and adipocytokines resistin, adiponectin, and leptin were analyzed from plasma samples. Training significantly reduced circulating hs-CRP, leptin, and resistin in both training groups (P<.05), whereas MCP-1 and TNF-α decreased only in AD (P<.05). Significant correlations were observed between changes in abdominal fat mass and corresponding changes in MCP-1, leptin, adiponectin, and resistin. Long-term combined aerobic and resistance training reduced markers of subclinical inflammation in healthy young men. The results indicate that a higher frequency of individual exercise sessions might be more beneficial with respect to the anti-inflammatory effects of physical activity. The decreases in inflammation markers seem to be related to decreases in abdominal fat mass.

Double-blinded, randomised, placebo-controlled trial of convalescent plasma for COVID-19: analyses by neutralising antibodies homologous to recipients' variants.

INTRODUCTION: Convalescent plasma (CP) emerged as potential treatment for COVID-19 early in the pandemic. While efficacy in hospitalised patients has been lacklustre, CP may be beneficial at the first stages of disease. Despite multiple new variants emerging, no trials have involved analyses on variant-specific antibody titres of CP. METHODS: We recruited hospitalised COVID-19 patients within 10 days of symptom onset and, employing a double-blinded approach, randomised them to receive 200 ml convalescent plasma with high (HCP) or low (LCP) neutralising antibody (NAb) titre against the ancestral strain (Wuhan-like variant) or placebo in 1:1:1 ratio. Primary endpoints comprised intubation, corticosteroids for symptom aggravation, and safety assessed as serious adverse events. For a preplanned ad hoc analysis, the patients were regrouped by infused CP's NAb titers to variants infecting the recipients i.e. by titres of homologous HCP (hHCP) or LCP (hLCP). RESULTS: Of the 57 patients, 18 received HCP, 19 LCP and 20 placebo, all groups smaller than planned. No significant differences were found for primary endpoints. In ad hoc analysis, hHCPrecipients needed significantly less respiratory support, and appeared to be given corticosteroids less frequently (1/14; 7.1%) than those receiving hLCP (9/23; 39.1%) or placebo (8/20; 40%), (p = 0.077). DISCUSSION: Our double-blinded, placebo-controlled CP therapy trial remained underpowered and does not allow any firm conclusions for early-stage hospitalised COVID-19 patients. Interestingly, however, regrouping by homologous - recipients' variant-specific - CP titres suggested benefits for hHCP. We encourage similar re-analysis of ongoing/previous larger CP studies. TRIAL REGISTRATION: ClinTrials.gov identifier: NCT0473040.

Strong coupling between surface plasmon polaritons and ß-carotene in nanolayered system.

In this article we experimentally demonstrate the strong coupling between surface plasmon polaritons (SPP) and the S(2) state of ß-carotene. The SPPs are excited by prism coupling technique on a thin silver film with ß-carotene embedded in a polymer layer on top of that. Rabi splittings with energies 80 and 130 meV are observed in the recorded dispersion relations. Both coupled oscillator model and transfer matrix method are used to fit the experimental results. The scattered radiation of the propagating strongly coupled SPP-S(2) hybrids is collected and an increase of the low energy splitting to 120 meV is observed compared to the reflectivity data. In addition, we performed molecule excitation by laser and analyzed the emission patterns revealing clear surface plasmon coupled fluorescence of ß-carotene. By increasing the concentration of ß-carotene we are able to collect also surface plasmon coupled Raman scattering. This study substantially extends the SPP-molecular excitation strong coupling studies to biomolecules, and energy transfer and coupling properties of excited states of carotenoids.

Average demands and most demanding passages of national-level female soccer matches: do small- and large-sided games replicate match demands?

Introduction: This study aims to (1) determine the average and most demanding passage (MDP) load of national-level female soccer matches and (2) evaluate the relationship between average and MDP load during small-sided games (SSGs), large-sided games (LSGs), and matches. Methods: A total of 37 national-level female soccer players from a single club senior team and the U18 team participated. The average and 1-, 3-, and 5-min MDP external (total, high-speed, and very-high-speed running distances, acceleration and deceleration distances, average metabolic power, and high-metabolic load distance) and internal loads (average heart rate, rate of perceived exertion) of the 29 league matches, ten 4 vs. 4 + goalkeepers SSGs, and six 8 vs. 8 + goalkeepers LSGs were analyzed by the Polar Team Pro player tracking system. Results: In matches, the external load variables during 1-, 3-, and 5-min MDPs were 167%-1,165%, 135%-504%, and 126%-359%, of match average values, respectively. In LSGs, all external load variables reached higher average values compared with those during matches, except for the very-high-speed running distance; however, no variable reached higher values of 1-min MDP compared with those during the matches. In SSGs, the average acceleration and deceleration distances were higher compared with those during the matches. Discussion: The findings from the present study suggest that LSGs and SSGs can be used to overload the average values of the selected external load variables compared with those during the matches; however, other training options must be explored to overload 1-min match MDPs.

Influence of the Menstrual Cycle Phase on Marathon Performance in Recreational Runners.

PURPOSE: To examine the potential impact of fluctuations in sex steroid hormones across the menstrual cycle (MC) on marathon running performance of recreational female athletes. METHODS: A survey questionnaire was administered to recreational, nonelite runners who had completed multiple marathons within the last 18 months. RESULTS: A total of 599 questionnaires were returned and deemed viable for review. From these, 185 survey participants were found to have complete information and eligibility to have their surveys used in the statistical analysis. A total of 106 women had their best marathon performance in the luteal phase (high sex steroid hormones) of the MC, and 79 had their best performance in the follicular phase (low sex steroid hormones) of the MC (responses were significantly different; z-score value = 1.11; P < .05). CONCLUSION: Recreational female runners have varying performances in the marathon across their MC phases, specifically performing better in the luteal phase of the cycle.

Changes in inflammation markers after a 10-week high-intensity combined strength and endurance training block in women: The effect of hormonal contraceptive use.

OBJECTIVES: The influence of hormonal contraceptives (HC) on inflammation and body composition after high-intensity combined strength and endurance training was investigated. DESIGN: Active healthy women formed two training groups: HC users (HCU, n = 9) and those who had never used HC (NHC, n = 9). Training included two strength training sessions and two high-intensity interval training sessions per week for 10 weeks. METHODS: Before (PRE) and after (POST) the training intervention, high-sensitivity C-reactive protein (hs-CRP), tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and interleukin-1beta (IL-1ß) concentrations were measured. Dual-energy X-ray absorptiometry was used to estimate fat mass (FM), abdominal fat mass (aFM), and lean mass (LM). RESULTS: Circulating concentrations of hs-CRP decreased significantly in the NHC from pre to post with -0.46 mg l-1 (95% CI: -0.78, -0.14, p = 0.009, ES = 0.434), whereas a significant increase was observed in HCU from pre to post with 0.89 mg l-1 (95% CI: 1.66, 0.12, p = 0.048, ES = 1.988) with a significant between-group difference (p = 0.015). In addition, hs-CRP concentration was significantly higher in HCU than in NHC after training (p = 0.036) at post. Lean mass increased significantly more in NHC than in HCU (p = 0.049). CONCLUSIONS: High-intensity combined strength and endurance training can modify inflammation and body composition of women. The present study showed that inflammation, in terms of hs-CRP was higher post training in HCU than NHC, which may be associated with smaller gains in lean mass in response to training.

Age-related decrease in stimulated glutamate release and vesicular glutamate transporters in APP/PS1 transgenic and wild-type mice.

We assessed baseline and KCl-stimulated glutamate release by using microdialysis in freely moving young adult (7 months) and middle-aged (17 months) transgenic mice carrying mutated human amyloid precursor protein and presenilin genes (APdE9 mice) and their wild-type littermates. In addition, we assessed the age-related development of amyloid pathology and spatial memory impaired in the water maze and changes in glutamate transporters. APdE9 mice showed gradual spatial memory impairment between 6 and 15 months of age. The stimulated glutamate release declined very robustly in 17-month-old APdE9 mice as compared to 7-month-old APdE9 mice. This age-dependent decrease in stimulated glutamate release was also evident in wild-type mice, although it was not as robust as in APdE9 mice. When compared to individual baselines, all aged wild-type mice showed 25% or greater increase in glutamate release upon KCl stimulation, but none of the aged APdE9 mice. There was an age-dependent decline in VGLUT1 levels, but not in the levels of VGLUT2, GLT-1 or synaptophysin. Astrocyte activation as measured by glial acidic fibrillary protein was increased in middle-aged APdE9 mice. Blunted pre-synaptic glutamate response may contribute to memory deficit in middle-aged APdE9 mice.

Calcitonin gene methylation in chronic myeloproliferative disorders.

Alterations in DNA methylation appear to be an integral part of the malignant transformation. For example, the p15 region of chromosome 11 with multiple genes related to cell growth regulation exhibits different methylation patterns in the 5' area of the calcitonin A gene in healthy bone marrow cells, and in leukemic cell populations. In this work the methylation status of the 5' area of the calcitonin gene in myeloproliferative disorders (MPD) other than chronic myeloid leukemia (CML) is studied. A total number of 37 patients with polycythemia vera, essential thrombocythemia, or myelofibrosis were studied. A control group of 18 healthy persons and patients with reactive hematologic changes was included. The DNA isolated from peripheral blood or bone marrow cells was digested with the methylation-sensitive HpaII restriction enzyme. A Southern blot was hybridized with a 1.7 kb probe specific to the 5' area of the calcitonin gene. The result was visualized autoradiographically and analyzed with a densitometer. The results have been expressed as ratios between the abnormal and normal autoradiography band intensities, referred to as the calc-value or CALC. An increase in the calc-value signifies increasing methylation. In the control group the calc-value had a mean of 0.274. The myelofibrosis patients exhibited very strong hypermethylation in the calcitonin gene 5' area, with a mean calc-value of 11.1 (median 2.6). The polycythemia vera patients showed considerable variation in their methylation status, with a mean value of 1.52. The essential thrombocythemia patients exhibited weak hypermethylation, with a mean calc-value of 0.58. A correlation between karyotypic abnormalities and hypermethylation was observed. Complicated forms of MPD exhibited higher levels of methylation than the uncomplicated disease forms.

Hypermethylation of the calcitonin gene in the myelodysplastic syndromes.

It is well documented that the calcitonin gene area in the short arm of chromosome 11 is hypermethylated in most acute leukemias as well as in chronic lymphatic leukemia. In contrast, the gene is normally methylated during the chronic phase of the chronic myeloid leukemia but turns hypermethylated as the disease escalates. As the methylation of the calcitonin gene correlates with the disease activity in chronic myeloid leukemia, it seemed worthwhile to study the gene methylation in other premalignant hematologic conditions with a potential to terminate in fulminant acute leukemia. We report here on the calcitonin gene methylation in patients with myelodysplastic syndromes (MDS) using a methylation sensitive restriction enzyme HpaII and standard Southern blotting techniques. Bone marrow aspirates from a total of 26 MDS patients were studied. In 24 of these patients, the calcitonin gene was hypermethylated. There was no correlation between the methylation status and the morphological stage of the disease. All six patients with a blast count < 5% had a hypermethylated gene. Of the 19 patients with a blast count > 5%, 17 were hypermethylated only two having normal methylation status of the gene. It appears that the hypermethylation of the calcitonin gene area in the short arm of chromosome 11 may be an early event in the pathogenesis of the myelodysplastic syndromes. The methylation analysis may thus be of value as a diagnostic tool in MDS but an abnormal methylation pattern does not seem to have a direct relation with the degree of blast infiltration.

Towards automatic detection of point mutations: use of scintillating microplates in solid-phase minisequencing.

Simplification of molecular genetic techniques is one of the main features of large-scale clinical applications of mutation analysis. The solid-phase minisequencing method, which is based on single-nucleotide primer extension by a DNA polymerase on a solid support, is an easy way of detecting point mutations of previously known locations. Here the procedure was further simplified by the use of microplates made of scintillating plastics, a microplate format scintillation counter and an automatic microplate washer. DNA samples from patients with either a hereditary aspartylglucosaminidase (AGA) gene point mutation or an acquired N-ras gene mutation were analyzed by three different minisequencing detection procedures utilizing tritiated nucleotides. The new counting method with scintillating plates was compared to traditional liquid scintillation counting in scintillation vials or to another microplate format procedure, which requires addition of scintillation liquid. In all three methods, normal individuals, heterozygous carriers of the AGA mutation and homozygous patients could be unequivocally discriminated. The N-ras mutation in leukemic blasts could also be detected with high resolution. The coefficients of variation and reproducibility of the scintillating microplate method were almost identical to those of the traditional liquid scintillation assay, which was used as a reference method. The technical innovations adopted here for performing minisequencing assays reduce significantly the labor required without affecting the quality of the results.

Diagnosis of pancreatic lesions using fine needle aspiration cytology: detection of K-ras point mutations using solid phase minisequencing.

AIMS: To improve the diagnostic value of fine needle aspiration biopsy of pancreatic lesions using a simple mutation detection method based on the polymerase chain reaction (PCR). METHODS: Fine needle aspirates from 21 suspected pancreatic lesions were analysed for K-ras codon 12 point mutations using solid phase minisequencing. RESULTS: A point mutation in codon 12 of the K-ras gene was detected in 14 of 17 cases of pancreatic carcinoma. No false positive results were recorded. The concordance of the result with routine cytology was 78%. All patients diagnosed as having malignant disease on cytology also had a K-ras point mutation. Additional information on the presence of malignancy was obtained using molecular genetic analysis in two cases. CONCLUSIONS: PCR based minisequencing is a promising method for the analysis of cytological material. K-ras point mutation analysis was modified to enable it to be carried out in a clinical laboratory. Advantages of the method include its simplicity and speed. Adequate sampling guidance is important but analysis can be performed even with small amounts of cellular material.

Comparison of dopamine and noradrenaline release in mouse prefrontal cortex, striatum and hippocampus using microdialysis.

In vivo release of dopamine (DA) and noradrenaline (NA) in mouse medial prefrontal cortex, medial striatum and hippocampus was characterized using in vivo microdialysis. Basal release of NA was similar in these areas, but DA in striatum was 13-30 times higher than in other areas. Unconditioned stimuli (handling, novelty) induced strong increases, except for striatal DA. Striatal NA was more sensitive to handling than NA in other areas.

alpha(2C)-Adrenoceptors modulate the effect of methylphenidate on response rate and discrimination accuracy in an operant test.

The present study investigated the role of alpha(2C)-adrenoceptors in the regulation of activity and discrimination accuracy in an operant chamber test. We trained food deprived control and alpha(2C)-adrenoceptor knockout mice to collect liquid food rewards in an operant chamber during the light (20 s) period. No food reward was delivered during the dark period (40 s). The alpha(2C)-adrenoceptor knockout mice tended to make fewer total responses and collect less rewards than their controls after saline treatment. However, only response accuracy of alpha(2C)-adrenoceptor knockout mice was significantly lower than that of the control mice. Methylphenidate, a drug blocking dopamine re-uptake and increasing dopamine release, dose-dependently decreased the number of total responses and collected food rewards in control mice but increased those measures in alpha(2C)-adrenoceptor knockout mice. In addition, the effect of methylphenidate on discrimination accuracy differed between knockout and control mice. Our results indicate that alpha(2C)-adrenoceptors may regulate dopamine-mediated functions.

Thoracic lesions: diagnosis by ultrasound-guided biopsy.

We reviewed the results of US-guided fine-needle biopsies of peripheral pulmonary, pleural, mediastinal and chest wall lesions in 200 patients. Sufficient material for cytological analysis was obtained in 95%, 92%, 96% and 100%, respectively. Sensitivity was 88%, 94%, 96%, 100% and specificity 89%, 100% and 100%, respectively. The ratio of false-negative results was 7%. A cutting needle biopsy was additionally performed in 24 patients. All but two of the histological samples (92%) were adequate for diagnostic purposes and a correct diagnosis was established in 86% (19/22) of these. 8 patients (4%) with pleural or pulmonary targets had minor complications (5 pneumothorax, 3 haemoptysis), which did not require treatment. Cutting needle biopsies and biopsy of mediastinal lesions proved safe. Due to the many advantages US may be considered for guidance in peripheral larger-sized pulmonary lesions, particularly those abutting the pleura, and also in pleural, thoracic wall and mediastinal masses.

Ultrasonic depolymerization of aqueous polyvinyl alcohol.

Ultrasonication has proved to be a highly advantageous method for depolymerizing macromolecules because it reduces their molecular weight simply by splitting the most susceptible chemical bond without causing any changes in the chemical nature of the polymer. Most of the effects involved in controlling molecular weight can be attributed to the large shear gradients and shock waves generated around collapsing cavitation bubbles. In general, for any polymer degradation process to become acceptable to industry, it is necessary to be able to specify the sonication conditions which lead to a particular relative molar mass distribution. This necessitates the identification of the appropriate irradiation power, temperature, concentration and irradiation time. According to the results of this study the reactors constructed worked well in depolymerization and it was possible to degrade aqueous polyvinyl alcohol (PVA) polymer with ultrasound. The most extensive degradation took place at the lowest frequency used in this study, i.e. 23 kHz, when the input power was above the cavitation threshold and at the lowest test concentration of PVA, i.e. 1% (w/w). Thus this study confirms the general assumption that the shear forces generated by the rapid motion of the solvent following cavitational collapse are responsible for the breakage of the chemical bonds within the polymer. The effect of polymer concentration can be interpreted in terms of the increase in viscosity with concentration, causing the molecules to become less mobile in solution and the velocity gradients around the collapsing bubbles to therefore become smaller.

Excitation energy transfer in native and unstacked thylakoid membranes studied by low temperature and ultrafast fluorescence spectroscopy.

In this work, the transfer of excitation energy was studied in native and cation-depletion induced, unstacked thylakoid membranes of spinach by steady-state and time-resolved fluorescence spectroscopy. Fluorescence emission spectra at 5 K show an increase in photosystem I (PSI) emission upon unstacking, which suggests an increase of its antenna size. Fluorescence excitation measurements at 77 K indicate that the increase of PSI emission upon unstacking is caused both by a direct spillover from the photosystem II (PSII) core antenna and by a functional association of light-harvesting complex II (LHCII) to PSI, which is most likely caused by the formation of LHCII-LHCI-PSI supercomplexes. Time-resolved fluorescence measurements, both at room temperature and at 77 K, reveal differences in the fluorescence decay kinetics of stacked and unstacked membranes. Energy transfer between LHCII and PSI is observed to take place within 25 ps at room temperature and within 38 ps at 77 K, consistent with the formation of LHCII-LHCI-PSI supercomplexes. At the 150-160 ps timescale, both energy transfer from LHCII to PSI as well as spillover from the core antenna of PSII to PSI is shown to occur at 77 K. At room temperature the spillover and energy transfer to PSI is less clear at the 150 ps timescale, because these processes compete with charge separation in the PSII reaction center, which also takes place at a timescale of about 150 ps.

Excitation energy transfer pathways in Lhca4.

EET in reconstituted Lhca4, a peripheral light-harvesting complex from Photosystem I of Arabidopsis thaliana, containing 10 chlorophylls and 2 carotenoids, was studied at room temperature by femtosecond transient absorption spectroscopy. Two spectral forms of Lut were observed in the sites L1 and L2, characterized by significantly different interactions with nearby chlorophyll a molecules. A favorable interpretation of these differences is that the efficiency of EET to Chls is about two times lower from the "blue" Lut in the site L1 than from the "red" Lut in the site L2 due to fast IC in the former case. A major part of the energy absorbed by the "red" Lut, approximately 60%-70%, is transferred to Chls on a sub-100-fs timescale from the state S(2) but, in addition, minor EET from the hot S(1) state within 400-500 fs is also observed. EET from the S(1) state to chlorophylls occurs also within 2-3 ps and is ascribed to Vio and/or "blue" Lut. EET from Chl b to Chl a is biphasic and characterized by time constants of approximately 300 fs and 3.0 ps. These rates are ascribed to EET from Chl b spectral forms absorbing at approximately 644 nm and approximately 650 nm, respectively. About 25% of the excited Chls a decays very fast-within approximately 15 ps. This decay is proposed to be related to the presence of the interacting Chls A5 and B5 located next to the carotenoid in the site L2 and may imply some photoprotective role for Lhca4 in the photosystem I super-complex.

Idiopathic macrocytic anaemia in the aged: molecular and cytogenetic findings.

Macrocytosis in the elderly is often caused by abnormalities of haematological stem cell differentiation. In this study, a group of elderly patients was analysed for four molecular and cell biological parameters. The aim of the study was to screen elderly patients with idiopathic macrocytic anaemia or MDS for a set of alterations which are related to haematological dysplasia. The analyses used were: DNA-methylation at the calcitonin A gene 5'-area, NRAS point mutations at codons 12 and 13, in vitro colony formation of peripheral blood progenitor cells and cytogenetics of bone marrow cells. The results show that a significant portion of elderly patients with idiopathic macrocytosis have one or more of the abnormalities analysed. Hypermethylation of the calcitonin A gene 5'-area at the chromosome 11 band p15 is relatively common (7/15). Chromosomal aberrations (3/12) and NRAS oncogene point mutations (0/15) were rare findings. In vitro culture of erythroid progenitor cells was relatively frequently abnormal (7/15). Eight of our nine macrocytic patients who did not fulfill the FAB criteria for MDS had at least one of the alterations studied; this suggests that these patients might represent early phases of a stem cell disorder.