Differential fluorescence nanoparticle tracking analysis for enumeration of the extracellular vesicle content in mixed particulate solutions.

BACKGROUND: A major concern for the extracellular vesicle (EV) field is the current lack of accurate methods for EV quantification. Total protein measurement fails to reliably quantify EVs from serum-containing conditioned media and classical nanoparticle tracking analysis (NTA) allows quantification and size determination of particles, but fails to discriminate between membrane-bounded EVs, lipids and protein aggregates. However, EVs can be fluorescently labelled with non-specific membrane markers or with antibodies specifically recognizing EV surface marker proteins. Fluorescence-based NTA (F-NTA) is thus emerging as a method for counting and phenotyping of EVs. We have validated a differential NTA/F-NTA method using specific antibodies against surface markers in analogy to flow cytometric analyses. METHODS: EVs from umbilical cord mesenchymal stromal cells (UC-MSCs) were isolated by a combined tangential flow filtration and ultracentrifugation protocol. EV preparations from 2 × 107 cells were stained with AlexaFluor 488-conjugated specific antibodies or corresponding isotype controls. Amount and size of particles in normal scattering light mode (N mode) versus fluorescence mode (F mode, laser wavelength 488 nm) was measured using ZetaView Nanoparticle Tracking Analyzer (Particle Metrix). Cryo electron microscopy (EM) was used to verify the presence of membrane bilayer surrounded nanoparticles. RESULTS: All UC-MSC-EV preparations were found positive for typical EV marker proteins and negative for MHC class I. Novel and improved devices that include more sensitive cameras for detection in the fluorescent mode further increase the detection limit. CONCLUSION: Differential NTA/F-NTA facilitates determination of the percentage of EV marker protein-positive nanoparticles within a mixed particulate solution. The set of markers can be extended to other MSC-EV positive and negative surface proteins in order to establish F-NTA-based profiling as a supporting method for the quantification of EVs.

T-cell death, phosphatidylserine exposure and reduced proliferation rate to validate extracorporeal photochemotherapy.

BACKGROUND AND OBJECTIVES: Extracorporeal photochemotherapy (ECP) is an established therapy in various diseases, such as cutaneous T-cell lymphoma and graft-versus-host disease. This study was performed to investigate the practicability of a flow cytometric T-cell evaluation after ECP as a tool to validate the quality of ECP procedures and to enable the comparability of treatments with different ECP devices. MATERIALS AND METHODS: Peripheral blood mononuclear cells (PBMNCs) of healthy volunteer blood donors were treated by offline ECP. To quantify the effect of ECP on T cells in vitro, phosphatidylserine exposure and 7-aminoactinomycin D (7-AAD) reactivity as well as the proliferative activity of phytohaemagglutinin-induced, viable CD3(+) lymphocytes were analysed by flow cytometry. RESULTS: The expected T-cell death after ECP was confirmed by 7-AAD measurements. Phosphatidylserine exposure gradually increased between 20 and 70 h after ECP. Treatment-related inhibition of T-cell proliferation was 92.6 ± 1.4%. CONCLUSION: The combination of viability, phosphatidylserine exposure and T-cell division analyses by flow cytometry in a single-platform system provides a valuable tool to validate ECP procedures.

Function and activation state of platelets in vitro depend on apheresis modality.

BACKGROUND AND OBJECTIVES: In multicomponent collection, various blood components are prepared during one apheresis process. The aim of this prospective crossover study was to compare the function, metabolic parameters and activation state of fresh and stored platelets (PLTs) collected by two different cell separators. MATERIALS AND METHODS: Twenty-four donors underwent apheresis on each of two cell separators (Fenwal Amicus(®) and CaridianBCT Trima Accel(®)) with an interval of at least 2 months between donations. Per donation, one double dose of PLT concentrate (PC) and one unit of packed red-blood-cells were collected. In total, 48 single unit PCs were tested for pH, glucose, bicarbonate, lactate, potassium and LDH concentration during 7 days of storage. PLT function was analysed by aggregometry, rotation thrombelastometry and hypotonic shock response. The PLT surface expression of P-selectin (CD62P) and LAMP-3 (CD63) was estimated by flow cytometry. RESULTS: During storage, metabolic parameters were well maintained in both groups, but levels of glucose and pH were significantly lower, while lactate and LDH were significantly higher in Amicus(®)-PCs. Amicus(®)-derived PLTs were significantly more activated as evidenced by higher CD62P and CD63 expression. In parallel, the in vitro function of Amicus(®)-PLTs was significantly reduced compared to Trima(®)-PLTs. CONCLUSION: In multicomponent apheresis, standardized PLT collection is effective and well tolerated. The higher activation of Amicus(®)-derived PLTs may be because of the divergent centrifugation modalities during collection. Possible consequences for the clinical outcome of thrombocytopenic patients will be evaluated in further trials.

Red blood cell alloimmunization in 184 patients with myeloid neoplasms treated with azacitidine - A retrospective single center experience.

Alloimmunization to Red Blood Cell (RBC) antigens frequently occurs in patients with myeloid neoplasms (AML, MDS and CMML) and potentially poses the patient at risk for delayed hemolytic transfusion reactions and limited supply of compatible RBC-units. However, there is comparatively little data on transfusion associated characteristics in this patient cohort. We therefore retrospectively analyzed transfusion requirements and clinical outcomes of 184 patients with myloid neoplasms treated with azacitidine at the Paracelsus Medical University Salzburg, which were included in the Austrian Registry of Hypomethylating Agents. The mean blood component requirements for AML, MDS and CMML were 39.8, 67.4 and 31.4 RBC units and 31.7, 27.6 and 19.1 platelet (PLT) units respectively. In spite of an extended and stringent RBC unit matching policy (ABO, RhD, RhCcEe and K antigens), 20 (11%) patients formed at least one alloantibody ("allo-group"), whereas 164 patients (89%) did not ("non-allo-group"). The most frequent antibody specificity was anti-E, followed by anti-Wra -Lua, -D, -C and -Jka. Alloimmunization was associated with higher numbers of transfused RBC units (68 vs. 38; p=0.001), as well as with longer time under transfusion (16.7 vs. 9.4 months; p=0.014). Median overall survival (OS) did not differ significantly between the "allo"- and "non-allo-group".

Biological and karyotypic characterization of a new cell line derived from human gliosarcoma.

A cell line (NCE-G28) was established from the biopsy material of a human gliosarcoma of low histological differentiation. The initial cultures showed a mixed population of cells which in later stages became more uniform due to loss of slower growing constituents. The cells have been growing steadily for 20 months. A suspension of NCE-G28 cells injected s.c. as well as i.p. into nude mice produced solid tumors in all cases. Histologically these tumors closely resembled the original tumor. The original tumor, the nude mouse tumor, and NCE-G28 cells were immunochemically positive for glial fibrillary acidic protein as well as for neural plasma membrane antigen A2B5 expression. Two cell strains, 9B2C and 9B2E, were obtained by cloning of the initial cultures and another strain, NCE-G28T, was derived after explantation of a mouse heterotransplant. The two subclones were negative for glial fibrillary acidic protein expression but stained for cell surface fibronectin. NCE-G28T cells initially were positive for glial fibrillary acidic protein but lost this property within 8 months of cultivation. Karyotype analysis of NCE-G28 and the three strains revealed hyperdiploidy and six structurally altered marker chromosomes five of which were shared by nearly all cells. Receptors for epidermal growth factor were detected in all cell lines with the highest levels (about 300,000 receptors/cell) in the parental cell line. The epidermal growth factor receptors had an affinity of 2.5 nM (Kd) and by affinity cross-linking analysis a molecular weight of 170,000 was found. Initially, NCE-G28 cells responded to epidermal growth factor as well as fibroblast growth factor with increased rates of proliferation, while platelet derived growth factor had no effect. In higher passages the growth factor sensitivity was reduced. Using antibodies directed against synthetic protooncogene peptides the production of c-sis immunoreactive material was detected. NCE-G28 cells produce an autocrine factor which stimulated proliferation. This factor is present in conditioned medium and is active on cultured meningiomas and other glioma cell lines. NCE-G28 cells can be maintained in serum-free defined medium on plastic coated with fibronectin or an extracellular matrix from bovine corneal endothelial cells. The NCE-G28 cell line with its strains provide an in vitro model system in which the complexity of gliosarcoma cell populations and the interaction of the cloned cellular constituents can be studied.

Cytotoxicity of spin trapping compounds.

Spin trapping compounds are used frequently to detect free radicals released by cells. Their cytotoxicity has to be considered in order to prevent perturbations of normal cell growth and viability. Eleven spin traps (eight nitrones and three nitroso traps) have been tested for their effects on bovine aortic endothelial cells (toxicity range, 50% survival rate). The lowest cytotoxicity was found for 5,5-dimethylpyrroline-1-oxide and 2,2,4-trimethyl-2H-imidazole-1-oxide whereas nitrosobenzene and 2-methyl-2-nitrosopropane exerted the strongest cytotoxic effects. In addition, three nitronyl nitroxides were tested. Their cytotoxicity was found to be dependent on substitution, and the toxic concentration of a lipophilic derivative was found to be more than two orders lower as compared to a hydrophilic derivative. The results of this study indicate that most spin traps can be used in cell cultures at customary (i.e. millimolar) concentrations; caution is recommended when nitroso spin traps are applied to cells.

The sera of spontaneously hypertensive rats contain agonistic auto-antibodies against the beta 1-adrenoceptor.

OBJECTIVE: To examine auto-antibodies in the sera of spontaneously hypertensive rats (SHR) and investigate the possibility of abnormalities of the immune system in those rats. DESIGN: Blood samples were taken from 18-month-old SHR and age-matched Wistar-Kyoto (WKY) rats. The immunoglobulin fraction was prepared from the rat sera. METHODS: Immunoglobulins were investigated in a sensitive biological test system using spontaneously beating neonatal rat heart myocytes. RESULTS: Immunoglobulins prepared from the sera of the 18-month-old SHR increased the beating frequency of cultured rat heart myocytes. This positive chronotropic effect induced by the auto-antibody-containing immunoglobulin fraction was realized via the beta 1-adrenergic receptor. CONCLUSIONS: The sera of the investigated ageing SHR contain agonistic auto-antibodies directed against the beta 1-adrenoceptor. These anti-beta-adrenoceptor antibodies in the SHR, like those in the sera of cardiomyopathic patients, recognize in most cases the second extracellular loop of the beta 1-adrenoceptor as the site of the antigenic determinant, and act in a similar way to the antibodies observed in the sera of cardiomyopathic patients. The present findings provide the opportunity of using ageing SHR as a model to investigate the development and possible pathogenic role of the agonistic auto-antibodies that recognize the beta 1-adrenoceptor.

Growth factors in cultured human glioma cells: differential effects of FGF, EGF and PDGF.

Primary cell cultures were initiated from surgically obtained specimens from human gliomas. After 4-10 passages in vitro the cells from 18 individual cases were seeded onto cluster dishes and left to attach for 24 h in medium containing fetal calf serum. Thereafter the medium was changed to serumfree, defined conditions and the following growth factors were added: EGF 1.5 x 10-8 M, PDGF 35 ng/ml, FGF 80 pg/ml. The growth factors were added fresh every 2 days. After 9 or 11 days in culture, the cells on one multiwell-plate were counted. In 8 cases another set which was cultured also in a multiwell-plate but on glass cover slides was immunostained for glial fibrillary acidic protein (GFAP), fibronectin (FN), A2B5 and galactocerebroside (galC). The proliferative response pattern in consequence to the growth factor addition varied greatly between different cases. In all cases a pronounced proliferative response was accompanied by marked changes on the culture morphology. Usually the best proliferative response was obtained with PDGF (10 cases) or FGF (8 cases) whereas EGF was most effective only in one case. Two cases showed only minimal response, one of which was an oligodendroglioma D according to Ludwig and another a gliosarcoma which was re-evaluated after 1 year in culture. It could be noted, however, that FGF had a tendency to be less effective in the group of malignant astrocytomas. The staining pattern of the cultures with the afore mentioned markers was not affected by the growth factor treatment. The proliferative response usually resulted in increased staining for fibronectin and never an induction of GFAP. A2B5 staining was positive only in one case of gliosarcoma and galC staining was regularly negative.

Corticotropin-releasing hormone (CRH) receptors in the mesenteric small arteries of rats resemble the (2)-subtype.

The potencies of the corticotropin-releasing hormone (CRH) agonistic peptides oCRH, h/rCRH, frog sauvagine, and carp urotensin I and of the antagonistic peptide alpha-helical CRH9-41 were compared in 3 different in vitro assays: (a) receptor binding to rat brain membranes; (b) release of ACTH/beta-endorphin from rat pituitary cells; and (c) relaxation of rat mesenteric small arteries. From their potency profiles, especially from the high potency of sauvagine relative to CRH in the relaxation assay, it is concluded that the receptors mediating the hypotensive action of systemic CRH in vascular smooth muscle are different from those in the pituitary and brain, and may be identical or very similar to the recently cloned new CRH receptor type 2.

Culture of human brain tumors on an extracellular matrix derived from bovine corneal endothelial cells and cultured human glioma cells.

Cell culture has become an integral part of the daily routine of most oncology laboratories. It has enabled researchers to investigate a wide range of cellular parameters in a defined system in which the experimental conditions can be controlled and repeated. Although the manufacturers of tissue culture materials are continually improving their products, cell attachment and initial survival of primary cultures from tumor cells are still problems in many laboratories. Many different approaches have been taken to circumvent this problem, and coating of tissue culture dishes with attachment enhancers, such as polyamino acids (1), fibronectin (2), laminin (3), and collagen (4), has been found helpful. For a long time, it was known that endothelial cells produce a basement membrane, and this led to the use of bovine corneal basement membrane by Gospodarowicz et al. (5,6), in their research into the phenomenon of regeneration and nonregeneration of corneal endothelium in different species. The application of this bovine corneal extracellular matrix (bECM) has since been greatly expanded (5,6). bECM has found broad approval, and has been used for mammary carcinoma (7), urological tumors (8), and different kinds of pituitary adenomas (9,10) as well as CNS tumors (11).

Inhibitory effects of sulfonated shale oils (ammonium bituminosulphonates, Ichthyols) on enzymes of polyenoic fatty acid metabolism.

The two commercial pharmaceutical preparations of ammonium bituminosulphonates, Leukichthol and Dark Ichthyol, were shown to inhibit the formation of 5S-hydroxy-6E,8Z,11Z,14Z-eicosatetraenoic acid (5-HETE) from external arachidonic acid by human polymorphonuclear leukocytes stimulated by ionophore A-23187 in a dose-dependent manner. Pure arachidonate 15-lipoxygenases from rabbit reticulocytes and soya beans, and the particulate prostaglandin endoperoxide synthase from sheep vesicular glands, were also inhibited. With the reticulocyte lipoxygenase, the Ichthyols suppressed the enzyme activity by two different mechanisms: (1) a prolongation of the lag period typical of lipoxygenase catalysis, and (2) by a lowering of the maximal enzymatic activity after the end of lag period. As expected, the first effect was reversed by the addition of the lipoxygenase product 13S-hydroperoxy-9Z,11E-octadecadienoic acid (13-HpODE). Ammonium bituminosulphonates are thus universal inhibitors of lipoxygenase activities, and the latter are of potential importance in inflammatory dermatoses.

Effects of endotoxin-induced mastitis on the pharmacokinetic properties of aditoprim in dairy cows.

Plasma disposition of aditoprim, a new dihydrofolate reductase inhibitor, was studied in healthy cows and cows with endotoxin-induced mastitis. A single dose of 5 mg of aditoprim/kg of body weight was administered IV to 5 healthy cows and to the same cows 3 weeks later at 2 hours after intramammary infusion of 0.1 mg of endotoxin into the rear quarters. Mastitis developed in all endotoxin-infused quarters and cows had systemic signs of disease (fever, tachycardia, depression) from 2 to 10 hours after infusion of endotoxin. Pharmacokinetic characteristics of aditoprim in healthy cows were a large volume of distribution (6.28 L/kg), a systemic clearance of 0.82 L/h/kg, and an elimination half-life of 7.26 hours. In cows with mastitis, plasma concentrations of aditoprim were lower between 5 and 26 hours after injection. The systemic clearance (1.00 L/h/kg) and the volume of distribution (12.25 L/kg) were significantly higher in cows with mastitis, but elimination half-life was not significantly different. The lower plasma concentrations of aditoprim between 5 and 26 hours after injection in cows with mastitis are explained by fluid compartment shifts and/or blood flow changes induced by mastitis, although increased elimination of aditoprim in cows with mastitis cannot completely be ruled out. The antibacterial activity of aditoprim is nearly the same as that of trimethoprim. The longer elimination half-life time of aditoprim, however, indicates that it may have a practical pharmacotherapeutic advantage over trimethoprim.

Hemodynamic response and effects on myocardial energetics of 3-cyano-2-morpholino-5-(pyrid-4-yl)pyridine (AWD 122-14) in anesthetized minipigs.

AWD 122-14, a new positive inotropic and vasodilating agent, was investigated in comparison to amrinone, milrinone and dopamine in anesthetized minipigs. AWD 122-14 (1.17.10(-7)-37.5.10(-7) mol/kg) increased dose-dependent left ventricular contractility (LV dp/dtmax) (122x5 +/- 11x3%; ED50 = 8.1x10(-7) to mol/kg). Dopamine (2.64x10(-8)-21x12 x 10(-8) mol/kg) in comparison increased contractility up to 153.1 +/- 44.9% of control value and is about 20 times more potent than AWD 122-14 at the ED50 value and about 10 times more potent at the ED30 value. Amrinone (1.60x10(-6)-16.90x10(-6) mol/kg) and milrinone (1.48x10(-7)-23.70x10(-7) mol/kg) only slightly increased contractility in anesthetized minipigs, but they appear to posses a similar pharmacological profile like AWD 122-14. The hemodynamic effects were associated with an increase in myocardial oxygen consumption (E1: 18.8 +/- 10.0%) due to the marked increases in LV dp/dtmax and heart rate. LVMW was unchanged and LVSW decreased (-29.0 +/- 10.2%) after application of AWD 122-14. The reduction in left ventricular work (LVMW, LVSW) and the increase in myocardial oxygen consumption led to a decrease of left ventricular external mechanical efficiency of the non-failing minipig heart (Etam: -21.1 +/- 9.4%). Additional hemodynamic effects of AWD 122-14 were studied under calcium channel blockade (verapamil, nifedipine). After pretreatment with verapamil the agent (1.17.10(-7)-18.75.10(-7) mol/kg i.v.) increased left ventricular contractility between 42.9 +/- 41.6% and 58.5 +/- 33.3%. After pretreatment with nifedipine the agent induced a dose-dependent increase in LV dp/dtmax between 11.1 +/- 7.7% and 47.8 +/- 23.7%.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of 3-cyano-2-morpholino-5-(päyrid-4-yl)pyridine (AWD 122-14) and dopamine on left ventricular function during acute volume load in 18-month-old spontaneously hypertensive rats and Wistar-Kyoto rats.

Effects of AWD 122-14, a new cardiotonic agent, and dopamine were studied in an experimental model of congestive heart failure in 12- and 18-month-old spontaneously hypertensive rats (SHR rats) in comparison to normotensive Wistar-Kyoto rats (WKY rats) as control group. This model combines an acute volume overloading with an already existing chronic pressure overload. Heart rate (HR), peak left ventricular pressure (PLVP), left ventricular enddiastolic pressure (LVEDP), and left ventricular contractility index (LV dp/dtmax) were significantly elevated in SHR rats versus WKY rats. Left ventricular mass (LVM) to body mass (bw) ratio was increased in SHR rats and there was a parallel, rightward shift of the left ventricular diastolic pressure-volume-relationship in the 18-month-old SHR rats. Thus for a given LVEDP, there is an increased left ventricular enddiastolic volume (LVEDV) in SHR rats, indicating a true structural outgrowth of the left ventricular lumen. During acute volume overloading LVEDP increased in the 12- and 18-month-old WKY rats and in the 12-month-old SHR rats. In contrast, the 18-month-old SHR rats showed no increase of the already very high baseline level of LVEDP. Dopamine and AWD 122-14 increased LV dp/dtmax in all groups. AWD 122-14 was able to reduce left ventricular filling pressure of the 18-month-old SHR rats. A further interesting finding was that AWD 122-14 reduced the content of thiobarbituric acid material in the left ventricle in the 18-month-old SHR rats (reduced lipid peroxidation), suggesting a possible cardioprotective action of this substance.

[The cardiovascular effect of Cordemcura on various animal models in vivo]. / Zur Herz-Kreislaufwirkung von Cordemcura an verschiedenen Tiermodellen in vivo.

The cardiotonic activity of Cordemcura was investigated in anesthetized pigs, cats, and rats. Hemodynamic measurements were made under normal conditions and under drug-induced heart failure (hexobarbital, isoproterenol). Cordemcura caused significant positive inotropic effects with rapid onset and relatively short duration of action (0.2-3.2 mg/kg i. v.). In the pig dp/dtmax was increased by 31.4% (p less than 0.02) at 1.6 mg/kg, in the cat by 34.2% (p less than 0.05) at 0.5 mg/kg, and in the rat by 14.4% (ns) at 0.5 mg/kg. The inotropic response was associated with a significant reduction of total peripheral resistance (TPR) (pig: -21.4%, p less than 0.02; cat: -13.7), and relatively small changes in heart rate, cardiac output, and blood pressure. Under drug-induced heart failure there was no further increase in the cardiotonic activity of Cordemcura. The hemodynamic effects of Cordemcura were compared with the effects of dopamine (5-100 micrograms/kg i. v.) and ouabain (10-40 micrograms/kg i. v.).

[The action of Cordemcura on the energy balance of the isolated rabbit heart]. / Zur Wirkung von Cordemcura auf die Energiebilanz am isolierten Kaninchenherzen.

The effect of Cordemcura, a new cardiotonic substance, was tested on retrograd perfused isolated rabbit hearts in comparison with equieffective doses of dopamine. The substances were applied continously via the perfusion medium in the following dosages: Cordemcura 3.34 X 10(-4) mol/l, 2.67 X 10(-3) mol/l; dopamine 3.9 X 10(-6) mol/l, 1.6 X 10(-5) mol/l). The effects of the substances were studied during normoxic and hypoxic perfusion. The heart rate was increased under normoxic perfusion of Cordemcura about 37.1% (3.34 X 10(-4) mol/l) and 17.9 (2.67 X 10(-3) mol/l). The oxygen consumption in relation to the performance of the hearts (contractility. heart rate) was increased by Cordemcura on 1.45 and 1.93 (3.34 X 10(-4) mol/l and 2.67 X 10(-3) mol/l) in the same way like by dopamine (1.30 and 2.66; 3.9 X 10(-6) mol/l and 1.6 X 10(-5) mol/l). The content of adenine nucleotides decreased in the hypoxic perfused hearts on nearly 20% of the control values. There was no difference between control, Cordemcura and dopamine treated hearts. Cordemcura increased the level of cAMP in the myocardium after 90 min of hypoxic perfusion in comparison with control hearts (control hearts 2.8 +/- 0.7 nmol/g protein; Cordemcura 4.7 +/- 0.8 nmol/g protein). In comparison to control hearts and dopamine treated hearts the hypoxic perfusion with Cordemcura resulted in a significant lower release of lactate dehydrogenase from the heart.

["Inflammatory aneurysm" of the aorta. Diagnosis, therapy, results]. / Das "inflammatorische Aneurysma" der Aorta. Diagnose, Therapie, Ergebnisse.

From 1970 to 1987 among 964 patients with aortic aneurysms 52 (5.4%) underwent aortic graft replacement for inflammatory aortic aneurysm. 79.2% were symptomatic, 18.9% ruptured at the time of admission. CT-scan is of main diagnostic value. The perioperative mortality rate was 15.1%. At follow-up (28 months mean) 35 of 38 living patients (92.7%) were examined clinically, by sonography and in most cases by CT-scan. The late complication rate was 20% (n = 7, atrophic kidney 3, anastomotic aneurysms 4). In contrast to abdominal aortic aneurysms inflammatory aneurysms present an elevated morbidity and mortality rate which has to be reduced by exact preoperative diagnosis and modified surgical technique.

Reticulocyte hemoglobin content allows early and reliable detection of functional iron deficiency in blood donors.

BACKGROUND: Frequent blood donations may lead to a depletion of body iron stores resulting in manifest anemia. Reticulocyte hemoglobin content (CHr) - a marker for impaired hemoglobinisation (IH) caused by functional iron deficiency (FID) - was investigated regarding its value as a routine screening parameter in frequent whole blood donors. METHODS: In a prospective study, 917 frequent blood donors and 688 first time or reactivated donors were tested for iron status and red blood cell count, including CHr. The ferritin index as a marker to indicate absent iron stores (AIS) was calculated. RESULTS: Depending on the number of donations during the preceding 12 months, AIS were detected in up to 21.4% of male and 27.8% of female donors, respectively. IH was present in up to 6.4% male and 16.7% female donors with 2 and 4 preceding donations, respectively. The defined CHr cut-off value was 28.0 pg to detect IH in frequent whole blood donors with AIS, leading to a test specificity of 98.2% (positive predictive value, PPV: 57.7%) in male and of 97.8% (PPV: 82.9%) in female donors. CONCLUSION: Determination of CHr is feasible to detect FID resulting in IH in frequent blood donors. It may help to prevent the development of anemia in frequent blood donors and also can help to decide whether donor deferral or even iron substitution need to be recommended.