Assessing warm ischemic injury of pig livers at hypothermic machine perfusion.

BACKGROUND: Livers originating from donation after circulatory death (DCD) donors are exposed to warm ischemia (WI) before liver transplantation (LTx). Currently, there are no objective tests to evaluate the damage sustained before LTx. This study aims to identify surrogate markers for liver injury that can be assessed during hypothermic machine perfusion (HMP) preservation. In addition, we want to use mathematical equation modeling combining these markers to improve our assessment of DCD livers for transplantation. MATERIALS AND METHODS: Porcine livers were exposed to incremental periods of WI (0-120 min) and subsequently HMP preserved for 4 h. Biochemical and hemodynamic parameters were repeatedly measured in the perfusate during HMP. Subsequently, to mimic LTx, normothermic isolated-liver perfusion was applied for 2 h and the injury assessed using a morphological score. RESULTS: With increasing WI periods, the perfusate became more acidotic, and levels of aspartate aminotransferase (AST), liver fatty acid binding protein, redox-active iron, and arterial vascular resistance increased. A damage index, combining AST and pH (damage index = 2 - 37 × ß(AST) - 257 × ß(pH)) based on multifactorial analysis of the changing pattern of these markers, had increased sensitivity and specificity to reflect WI and reperfusion injury. CONCLUSIONS: This proof of concept study demonstrated the potential role for objective evaluation of DCD porcine livers during HMP and the advantage to use multifactorial analysis on the markers' changing pattern.

Pulmonary administration of a water-soluble curcumin complex reduces severity of acute lung injury.

Local or systemic inflammation can result in acute lung injury (ALI), and is associated with capillary leakage, reduced lung compliance, and hypoxemia. Curcumin, a plant-derived polyphenolic compound, exhibits potent anti-inflammatory properties, but its poor solubility and limited oral bioavailability reduce its therapeutic potential. A novel curcumin formulation (CDC) was developed by complexing the compound with hydroxypropyl-γ-cyclodextrin (CD). This results in greatly enhanced water solubility and stability that facilitate direct pulmonary delivery. In vitro studies demonstrated that CDC increased curcumin's association with and transport across Calu-3 human airway epithelial cell monolayers, compared with uncomplexed curcumin solubilized using DMSO or ethanol. Importantly, Calu-3 cell monolayer integrity was preserved after CDC exposure, whereas it was disrupted by equivalent uncomplexed curcumin solutions. We then tested whether direct delivery of CDC to the lung would reduce severity of ALI in a murine model. Fluorescence microscopic examination revealed an association of curcumin with cells throughout the lung. The administration of CDC after LPS attenuated multiple markers of inflammation and injury, including pulmonary edema and neutrophils in bronchoalveolar lavage fluid and lung tissue. CDC also reduced oxidant stress in the lungs and activation of the proinflammatory transcription factor NF-κB. These results demonstrate the efficacy of CDC in a murine model of lung inflammation and injury, and support the feasibility of developing a lung-targeted, curcumin-based therapy for the treatment of patients with ALI.

Cyclodextrin-complexed curcumin exhibits anti-inflammatory and antiproliferative activities superior to those of curcumin through higher cellular uptake.

Curcumin, a yellow pigment present in the spice turmeric (Curcuma longa), has been linked with multiple beneficial activities, but its optimum potential is limited by poor bioavailability, in part due to the lack of solubility in aqueous solvents. To overcome the solubility problem, we have recently developed a novel cyclodextrin complex of curcumin (CDC) and examined here this compound for anti-inflammatory and antiproliferative effects. Using the electrophoretic mobility shift assay, we found that CDC was more active than free curcumin in inhibiting TNF-induced activation of the inflammatory transcription factor NF-kappaB and in suppressing gene products regulated by NF-kappaB, including those involved in cell proliferation (cyclin D1), invasion (MMP-9), and angiogenesis (VEGF). CDC was also more active than free curcumin in inducing the death receptors DR4 and DR5. Annexin V staining, cleavage of caspase-3 and PARP, and DNA fragmentation showed that CDC was more potent than free curcumin in inducing apoptosis of leukemic cells. Antiproliferative assays also demonstrated that CDC was more active than free curcumin in suppressing proliferation of various cancer cell lines. The cyclodextrin vehicle had no effect in these assays. Compared with free curcumin, CDC had a greater cellular uptake and longer half-life in the cells. Overall we demonstrated that CDC had superior attributes compared with free curcumin for cellular uptake and for antiproliferative and anti-inflammatory activities.

Multifactorial biological modulation of warm ischemia reperfusion injury in liver transplantation from non-heart-beating donors eliminates primary nonfunction and reduces bile salt toxicity.

OBJECTIVE: To design a multifactorial biological modulation approach targeting ischemia reperfusion injury to augment viability of porcine liver grafts from non-heart-beating donors (NHBD). BACKGROUND DATA: Liver Transplantation (LTx) from NHBD is associated with an increased risk of primary nonfunction (PNF) and biliary complications. In porcine NHBD-LTx, we previously reported a 50% risk of PNF and toxic bile formation in grafts exposed to > or =30' warm ischemia (WI). METHODS: Porcine livers exposed to 45' WI were cold stored, transplanted and either modulated (n = 6) or not (controls, n = 9). In the modulation group, donor livers were flushed with warm Ringers (avoiding cold-induced vasoconstriction), streptokinase (eliminating stagnating thrombi), and epoprostenol (vasodilator, platelet aggregation inhibitor) prior to cold storage. In recipients, glycine (Kupffer cell stabilizer), alpha1-acid-glycoprotein (anti-inflammatory protein), MAPKinase-inhibitor (pro-inflammatory cytokine generation inhibitor), alpha-tocopherol and glutathione (anti-oxidants), and apotransferrin (iron chelator) were administrated intravenously. PNF, survival, lactate, transaminase, TNF-alpha, redox-active iron, and biliary bile salt-to-phospholipid ratio were monitored. RESULTS: No PNF was observed in modulated versus 55% in control pigs (P = 0.025). Survival was 83% in modulated versus 22% in control pigs (P = 0.02). At 180' postreperfusion, lactate was lower in modulated (5.4 +/- 1.9 mmol/L) versus control pigs (9.4 +/- 2.2 mmol/L; P = 0.011). At 60' postreperfusion, there was a trend for lower AST in modulated versus control pigs at 60' (939 +/- 578 vs. 1683 +/- 873 IU/L; P = 0.089). Postreperfusion, TNF-alpha remained stable in modulated pigs (49 +/- 27 pg/mL at 15' and 85 +/- 26 pg/mL at 180'; P = 0.399) but increased in control pigs (107 +/- 36 pg/mL at 15' and 499 +/- 216 pg/mL at 180'; P = 0.023). At 180' postreperfusion, redox-active iron was higher in control pigs versus modulated pigs (0.21+/-0.18 vs. 0.042+/-0.062 mum; P = 0.038). Biliary bile salt-to-phospholipid ratio post-LTx was lower in modulated versus control pigs (1128 +/- 447 vs. 4836 +/- 4619; P = 0.05). CONCLUSIONS: A multifactorial biological modulation eliminates PNF, improves liver function and increases survival. Biochemically, TNF-alpha and redox-active iron are suppressed and biliary bile salt toxicity is reduced. Translating this strategy clinically may lead to wider and safer use of NHBD.

Non-transferrin-bound iron in haematological patients during chemotherapy and conditioning for autologous stem cell transplantation.

Free iron induced hydroxyl radical formation is one possible mechanism for tissue injury during cytotoxic therapy. We studied the appearance of free, non-transferrin-bound iron (NTBI) at baseline and during the 20-d period after the onset of cytotoxic chemotherapy in patients with haematological malignancy undergoing intensive chemotherapy or conditioning for autologous stem cell transplantation (aSCT). NTBI was detected on average for 15.6 d in patients treated with chemotherapy only, and for 6.1 d in patients undergoing aSCT. The recovery of the bone marrow function coincided with the disappearance of NTBI. The type of the conditioning regimen was also associated with the appearance of NTBI. The timing of the presence of NTBI accords with the presence of the most important non-infectious complication of intensive chemotherapy and autologous transplantation, mucosal injury, and free iron is likely to contribute to this and probably other complications of the intensive treatments.

Transplantation of allogeneic T cells alters iron homeostasis in NOD/SCID mice.

Iron overload is common in patients undergoing allogeneic hematopoietic cell transplantation (HCT), but the mechanisms leading to overload are unknown. Here, we determined iron levels and the expression of iron regulatory proteins in the liver and gut of nonobese diabetic-severe combined immunodeficient (NOD/SCID) mice that underwent transplantation with syngeneic (histocompatible) or allogeneic (histoincompatible) T lymphocytes. Infusion of histoincompatible T cells resulted in a significant rise in serum iron levels and liver iron content. Iron deposition was accompanied by hepatocyte injury and intestinal villous damage. Feeding of low- or high-iron diet was associated with appropriate ferroportin 1 and hepcidin responses in mice given histocompatible T cells, whereas mice given histoincompatible T cells showed inappropriate up-regulation of duodenal ferroportin 1 and a loss of expression of hepatic hepcidin. These findings suggest that alloreactive T cell-dependent signals induced dysregulation of intestinal iron absorption, which contributed to liver iron overload after HCT.

Inhibition of erythroid and granulocyte-macrophage colony formation by non-transferrin-bound iron in vitro: protective effect of apotransferrin.

OBJECTIVES: The aim of the study was to investigate in vitro the effect of free iron on erythroid and granulocyte-macrophage colony formation and the effect of binding free iron with apotransferrin. METHODS: Normal haematopoietic progenitors were cultured in vitro with different concentrations of free iron in the form of ferric nitrilotriacetic acid (FeNTA). Parallel cultures were performed after the preincubation of FeNTA with apotransferrin. RESULTS: Free iron inhibited colony formation by erythroid and granulocyte-macrophage progenitors and reduced the size of the colonies in a dose-dependent manner. Preincubation of FeNTA with apotransferrin diminished the inhibitory effect of FeNTA on colony formation increasing both the number and the size of colonies. CONCLUSIONS: Free iron was toxic to haematopoietic progenitors in in vitro cultures; the toxic effect could be reduced with apotransferrin.

Inhibition of ERK1/2 activation by phenolic antioxidants protects kidney tubular cells during cold storage.

BACKGROUND: Cold storage of tissues induces reactive oxygen species (ROS), which contribute to cell injury. We have compared different antioxidants in protection of renal tubular cells against hypothermia injury and studied their effect on cold-induced mitogen-activated protein (MAP) kinase activation. METHODS: Cultured renal tubular epithelial cells (LLC-PK1) were stored in University of Wisconsin solution supplemented with compounds tested for 16 hr at 4 degrees C. Release of lactate dehydrogenase and cellular adenosine triphosphate were measured. Activation of MAP kinases was determined by Western blotting. Intracellular ROS were monitored with a fluorescent probe. RESULTS: Cold storage resulted in a substantial loss of cell viability. The simple phenol butylated hydroxyanisol (BHA) most effectively prevented hypothermia-induced cell injury, whereas about 100-fold higher concentration of the polyphenol epigallocatechin gallate (EGCG) was needed, although EGCG most effectively scavenged intracellular ROS elicited by serum withdrawal. The MEK inhibitor U0126 and reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor diphenyleneiodonium effectively protected the cells against hypothermia injury. ERK1/2 was rapidly activated during chilling of the cells and this was inhibited by BHA but not by EGCG. CONCLUSION: The results suggest that chilling of renal epithelial cells induces ROS generation by NADPH oxidase, which leads to rapid activation of the MEK-ERK1/2 cascade and initiation of cell injury. This can be prevented by antioxidants.

Primary graft nonfunction and Kupffer cell activation after liver transplantation from non-heart-beating donors in pigs.

More extensive use of non-heart-beating donors (NHBD) could reduce mortality on liver transplantation waiting lists, but this is associated with more primary nonfunction (PNF). We assessed which parameters are involved in the development of PNF in livers from NHBD in a previously validated pig liver transplantation model, in which livers were transplanted after exposure to incremental periods of warm ischemia. The risk of PNF was unacceptably high (>50%) when livers were exposed to >30 minutes' warm ischemia before a short cold ischemic period. This study examined how PNF is affected by Kupffer cell activation (beta-galactosidase), the generation of cytokines tumor necrosis factor alpha and interleukin 6, antioxidant mechanisms (ascorbic acid, alpha-tocopherol, reduced glutathione), circulating redox-active iron, and sinusoidal endothelial cell function (hyaluronic acid clearance). Kupffer cells were more activated in PNF recipients, as suggested by higher beta-galactosidase levels (15 minutes after reperfusion), and secondarily, by higher production of tumor necrosis factor alpha and interleukin 6 (180 minutes after reperfusion). In addition, alpha-tocopherol and reduced glutathione were lower, and ascorbic acid and redox-active iron higher in PNF recipients. Finally, PNF grafts displayed progressively decreasing hyaluronic acid clearance (suggesting sinusoidal endothelial cell dysfunction) and parenchymal edema. Consequently, a reduced-flow phenomenon was documented. In grafts from NHBD that are destined to fail, beta-galactosidase activity (a surrogate of Kupffer cell activation) is higher, proinflammatory cytokines are overproduced, some antioxidant mechanisms fail, and circulating redox-active iron is more rapidly released. A no-flow phenomenon is eventually observed in these failing grafts.

Effect of repeated apotransferrin administrations on serum iron parameters in patients undergoing myeloablative conditioning and allogeneic stem cell transplantation.

Myeloablative conditioning prior to allogeneic stem cell transplantation causes a rapid increase in transferrin saturation and potentially toxic non-transferrin-bound iron (NTBI) in plasma. We have studied the ability of repeatedly administered apotransferrin to maintain this iron in a transferrin-bound form. Twenty adult patients undergoing myeloablative conditioning and allogeneic stem cell transplantation were enrolled to receive apotransferrin with one of three dosage regimens. Ten consecutive patients with the same preconditioning were studied as controls. At the highest dose level, full transferrin saturation and appearance of NTBI were prevented in five of the eight patients. Serum iron increased significantly more in the patients receiving apotransferrin than in the controls and remained elevated until erythropoietic recovery. From the increment of iron saturation and the amount of endogenous and administered apotransferrin, an average 180 mumol of iron per day was bound to transferrin during the first 4 d after the start of the conditioning therapy. Thereafter, iron accumulation levelled off in most patients. The results suggested that about half of the amount of iron normally transported to erythropoiesis was initially released to plasma after induction of the erythroid arrest. Complete iron binding with apotransferrin would apparently require very high apotransferrin doses.

Bicarbonate inhibits the growth of Staphylococcus epidermidis in platelet concentrates by lowering the level of non-transferrin-bound iron.

BACKGROUND: Platelet concentrates (PCs) contain non-transferrin-bound iron (NTBI) owing to the displacement of iron from plasma-derived transferrin by citrate. NTBI in the PC medium supports the growth of Staphylococcus epidermidis. The possibilities of lowering the level of NTBI have been studied with the aim to inhibit the growth of S. epidermidis in the PC medium. STUDY DESIGN AND METHODS: NTBI in PC supernatants was determined by a chelation method and by the bleomycin-detectable iron assay. Iron binding by transferrin was determined by spectrophotometry. The growth of inoculated S. epidermidis in PC supernatants was monitored by optical density and determination of viable counts. RESULTS: Bicarbonate enhanced in a dose-dependent manner transferrin iron binding in citrate-containing solutions, including citrated plasma and PAS-II. The use of a modified anticoagulant supplemented with bicarbonate effectively lowered the level of NTBI and inhibited bacterial growth in citrated plasma. Supplementation of bicarbonate to the additive solution to increase the ratio of bicarbonate to citrate in a reconstituted PC medium further inhibited bacterial growth. Maintenance of stable pH and bicarbonate level in the reconstituted medium necessitated storage under 5 percent CO(2). CONCLUSIONS: The relatively low bicarbonate level in PC medium promotes iron displacement by citrate from plasma-derived transferrin. The appearance of NTBI can be decreased and iron-dependent bacterial growth can be inhibited by increasing bicarbonate level in citrated plasma and PC medium. To achieve the same beneficial effect in blood banking, other more practical ways to bind NTBI in a harmless form should be developed.

Non-transferrin-bound iron in platelet concentrates promotes the growth of Staphylococcus epidermidis.

BACKGROUND: Staphylococcus epidermidis, the most common organism implicated in bacterial contamination of platelet (PLT) concentrates (PCs), does not grow in serum unless transferrin is fully saturated and there is non-transferrin-bound iron (NTBI) available. Here, the occurrence and origin of NTBI in PCs has been studied. STUDY DESIGN AND METHODS: NTBI in PC supernatants was determined by a chelation method and by the bleomycin-detectable iron assay. Iron binding by transferrin was determined by spectrophotometry, and transferrin iron forms, by urea gel electrophoresis. The growth of inoculated S. epidermidis in PC supernatants was monitored by optical density and determination of viable counts. RESULTS: PCs contained approximately 0.14 micromol per L redox-active iron measured by the bleomycin assay and approximately 0.7 micromol per L NTBI by the chelation method. As a further indication of the presence of NTBI, the growth of S. epidermidis in the PC supernatants was inhibited by iron chelation with deferoxamine. Transferrin in the PC medium was only partially saturated with iron, and the reason for the presence of NTBI was found to be impaired iron binding by transferrin. Iron was displaced from transferrin by citrate at molar ratios to transferrin that occur in citrated plasma and in PLT additive solution (AS). Citrated plasma supported the growth of S. epidermidis whereas serum did not. CONCLUSIONS: PCs stored in plasma or AS contain a low level of NTBI because of the displacement of iron from plasma-derived transferrin by citrate. NTBI in the PC medium supports the growth of S. epidermidis.

Regulation of monocyte migration by amphoterin (HMGB1).

Amphoterin (HMGB1) is a 30-kD heparin-binding protein involved in process extension and migration of cells by a mechanism involving the receptor for advanced glycation end products (RAGE). High levels of amphoterin are released to serum during septic shock. We have studied the expression of amphoterin in monocytes and the role of amphoterin and RAGE in monocyte transendothelial migration. Un-activated monocytes in suspension did not reveal amphoterin on their surface, but adherent monocytes exported amphoterin to the cell surface. Immunohistochemical staining of arterial thrombi in vivo revealed amphoterin in mononuclear cells and in surrounding extracellular matrix. Amphoterin was secreted from phorbol ester and interferon-gamma (IFN-gamma)-activated macrophages, and the secretion was inhibited by blocking the adenosine 5'-triphosphate (ATP)-binding cassette transporter-1, a member of the multidrug resistance protein family. Amphoterin was specifically adhesive for monocytes in peripheral blood leukocyte adhesion assay. Adhesion caused an extensive spreading of cells, which was inhibited by the dominant-negative RAGE receptor (soluble ectodomain of RAGE), and adhesion up-regulated chromogranin expression in monocytes, also suggesting a RAGE-dependent interaction. Monocyte transendothelial migration was efficiently inhibited by anti-amphoterin and anti-RAGE antibodies and by the soluble RAGE. We suggest that amphoterin is an autocrine/paracrine regulator of monocyte invasion through the endothelium.

Erratum to "Apotransferrin administration prevents growth of Staphylococcus epidermidis in serum of stem cell transplant patients by binding of free iron". [FEMS Immunol. Med Microbiol. 37 (2003) 45-51].

We investigated the effect of free, non-transferrin-bound iron occurring in haematological stem cell transplant patients on growth of Staphylococcus epidermidis in serum in vitro, and prevention of bacterial growth by exogenous apotransferrin. S. epidermidis did not grow in normal serum at inoculated bacterial densities up to 10(3) cfu ml(-1) but slow growth could be detected at higher initial inocula. Addition of free iron abolished the growth-inhibitory effect of serum, whereas addition of apotransferrin again restored it. Appearance of free iron and loss of growth inhibition coincided in patient serum samples taken daily during myeloablative therapy. Intravenously administered apotransferrin effectively bound free iron and restored the growth inhibition in patient sera. The results suggest that exogenous apotransferrin might protect stem cell transplant patients against infections by S. epidermidis and possibly other opportunistic pathogens.

Reduction of circulating redox-active iron by apotransferrin protects against renal ischemia-reperfusion injury.

BACKGROUND: Warm ischemia-reperfusion (I/R) injury plays an important role in posttransplant organ failure. In particular, organs from marginal donors suffer I/R injury. Although iron has been implicated in the pathophysiology of renal I/R injury, the mechanism of iron-mediated injury remains to be established. The authors therefore investigated the role of circulating redox-active iron in an experimental model for renal I/R injury. METHODS: Male Swiss mice were subjected to unilateral renal ischemia for 45 min, followed by contralateral nephrectomy and reperfusion. To investigate the role of circulating iron, mice were treated with apotransferrin, an endogenous iron-binding protein, or iron-saturated apotransferrin (holotransferrin). RESULTS: Renal ischemia induced a significant increase in circulating redox-active iron levels during reperfusion. Apotransferrin, in contrast to holotransferrin, reduced the amount of circulating redox-active iron and abrogated renal superoxide formation. Apotransferrin treatment did not affect I/R-induced renal apoptosis, whereas holotransferrin aggravated apoptotic cell death. Apotransferrin, in contrast to holotransferrin, inhibited the influx of neutrophils. Both apo- and holotransferrin reduced I/R-induced complement deposition, indicating that the effects of transferrin are differentially mediated by its iron and protein moiety. Finally, apotransferrin, in contrast to holotransferrin, dose-dependently inhibited the loss of renal function induced by ischemia. CONCLUSIONS: Redox-active iron is released into the circulation in the course of renal I/R. Reducing the amount of circulating redox-active iron by treatment with apotransferrin protects against renal I/R injury, inhibiting oxidative stress, inflammation, and loss of function. Apotransferrin could be used in the treatment of acute renal failure, as seen after transplantation of ischemically damaged organs.

Apotransferrin administration prevents growth of Staphylococcus epidermidis in serum of stem cell transplant patients by binding of free iron.

We investigated the effect of free, non-transferrin-bound iron occurring in haematological stem cell transplant patients on growth of Staphylococcus epidermidis in serum in vitro, and prevention of bacterial growth by exogenous apotransferrin. S. epidermidis did not grow in normal serum at inoculated bacterial densities up to 10(3) cfu ml(-1) but slow growth could be detected at higher initial inocula. Addition of free iron abolished the growth-inhibitory effect of serum, whereas addition of apotransferrin again restored it. Appearance of free iron and loss of growth inhibition coincided in patient serum samples taken daily during myeloablative therapy. Intravenously administered apotransferrin effectively bound free iron and restored the growth inhibition in patient sera. The results suggest that exogenous apotransferrin might protect stem cell transplant patients against infections by S. epidermidis and possibly other opportunistic pathogens.

Effective binding of free iron by a single intravenous dose of human apotransferrin in haematological stem cell transplant patients.

Myeloablative treatment results in iron accumulation and the appearance of non-transferrin-bound iron (NTBI) in the circulation, which may contribute to treatment-related organ damage and susceptibility to infections. The aim of this study was to investigate the efficacy of human apotransferrin in the binding of NTBI in patients receiving an allogeneic stem cell transplant after myeloablative conditioning. A single intravenous 100 mg/kg dose of apotransferrin was given to six adult patients on d 3 after the transplantation. Initially, all patients had serum transferrin saturation above 80% and NTBI in their serum. After the apotransferrin injection, serum NTBI became undetectable in all patients and transferrin saturation decreased to 30-50%. Serum transferrin increased by an average of 1.95 g/l. The administered apotransferrin was subsequently converted into monoferric and diferric transferrin forms. NTBI reappeared and transferrin saturation again exceeded 80% 12-48 h after the injection in four patients and after 6 d in one patient. NTBI remained non-detectable for the whole 12 d follow-up period in one patient. The apotransferrin injection was well tolerated and no adverse events with probable association with the apotransferrin were observed. Repeated apotransferrin infusions might completely eliminate NTBI and iron-induced toxicity during myeloablative therapy.

Bleomycin-detectable iron assay for non-transferrin-bound iron in hematologic malignancies.

BACKGROUND: A microwell modification of the bleomycin assay for determining non-transferrin-bound iron (NTBI) was evaluated and compared with a chelation method. METHODS: The bleomycin assay reagent and sample volumes were halved, and measurements were done in microwell plates. Samples from patients treated for hematologic malignancies were studied. The chelation method was based on mobilization of NTBI with a chelator and measurement of the ultrafiltered iron-chelator complex. NTBI results were also compared with transferrin saturation and the distribution of transferrin iron forms by urea-polyacrylamide gel electrophoresis. RESULTS: The bleomycin assay intraassay imprecision (CV) was 7.7% and 8.2% and the interassay imprecision was 18% and 9.8% for a low (0.2 micromol/L) and a high (1.5 micromol/L) contrtrol, respectively. Hemolysis increased measured NTBI. A detection limit of 0.1 micromol/L was established based on the interference of nonvisible hemolysis and on accuracy studies. In patient samples, NTBI exceeded the detection limits only when transferrin saturation was >80%. Compared with the chelation method, the bleomycin assay gave clearly lower NTBI concentrations. The chelation method also gave positive results at <80% transferrin saturation. The recovery of iron added as ferric nitrilotriacetate to serum was 33% by the bleomycin assay and 64% by the chelation assay. CONCLUSIONS: The microwell version of the bleomycin assay is reproducible. When hemolyzed samples were excluded, bleomycin-detectable iron was found only when the transferrin saturation was >80%, suggesting high specificity. Bleomycin-detectable iron constitutes only a portion of the NTBI measured by the chelation method.