A comparison of liver fat fraction measurement on MRI at 3T and 1.5T.

PURPOSE: Volumetric liver fat fraction (VLFF) measurements were made using the HepaFat-Scan® technique at 1.5T and 3T to determine the agreement between the measurements obtained at the two fields. METHODS: Sixty patients with type 2 diabetes (67% male, mean age 50.92 ± 6.56yrs) and thirty healthy volunteers (50% male, mean age 48.63 ± 6.32yrs) were scanned on 1.5T Aera and 3T Skyra (Siemens, Erlangen, Germany) MRI scanners on the same day using the HepaFat-Scan® gradient echo protocol with modification of echo times for 3T (TEs 2.38, 4.76, 7.14 ms at 1.5T and 1.2, 2.4, 3.6 ms at 3T). The 3T analyses were performed independently of the 1.5T analyses by a different analyst, blinded from the 1.5T results. Data were analysed for agreement and bias using Bland-Altman methods and intraclass correlation coefficients (ICC). A second cohort of 17 participants underwent interstudy repeatability assessment of VLFF measured by HepaFat-Scan® at 3T. RESULTS: A small, but statistically significant mean bias of 0.48% was observed between 3T and 1.5T with 95% limits of agreement -2.2% to 3.2% VLFF. The ICC for agreement between field strengths was 0.983 (95% CI 0.972-0.989). In the repeatability cohort studied at 3T the repeatability coefficient was 4.2%. The ICC for agreement was 0.971 (95% CI 0.921-0.989). CONCLUSION: There is minimal bias and excellent agreement between the measures of VLFF using the HepaFat-Scan® at 1.5 and 3T. The test retest repeatability coefficient at 3T is comparable to the 95% limits of agreement between 1.5T and 3T suggesting that measurements can be made interchangeably between field strengths.

Hepatic iron concentration correlates with insulin sensitivity in nonalcoholic fatty liver disease.

Rodent and cell-culture models support a role for iron-related adipokine dysregulation and insulin resistance in the pathogenesis of nonalcoholic fatty liver disease (NAFLD); however, substantial human data are lacking. We examined the relationship between measures of iron status, adipokines, and insulin resistance in patients with NAFLD in the presence and absence of venesection. This study forms part of the Impact of Iron on Insulin Resistance and Liver Histology in Nonalcoholic Steatohepatitis (IIRON2) study, a prospective randomized controlled trial of venesection for adults with NAFLD. Paired serum samples at baseline and 6 months (end of treatment) in controls (n = 28) and patients who had venesection (n = 23) were assayed for adiponectin, leptin, resistin, retinol binding protein-4, tumor necrosis factor α, and interleukin-6, using a Quantibody, customized, multiplexed enzyme-linked immunosorbent assay array. Hepatic iron concentration (HIC) was determined using MR FerriScan. Unexpectedly, analysis revealed a significant positive correlation between baseline serum adiponectin concentration and HIC, which strengthened after correction for age, sex, and body mass index (rho = 0.36; P = 0.007). In addition, there were significant inverse correlations between HIC and measures of insulin resistance (adipose tissue insulin resistance (Adipo-IR), serum insulin, serum glucose, homeostasis model assessment of insulin resistance, hemoglobin A1c, and hepatic steatosis), whereas a positive correlation was noted with the insulin sensitivity index. Changes in serum adipokines over 6 months did not differ between the control and venesection groups. Conclusion: HIC positively correlates with serum adiponectin and insulin sensitivity in patients with NAFLD. Further study is required to establish causality and mechanistic explanations for these associations and their relevance in the pathogenesis of insulin resistance and NAFLD. (Hepatology Communications 2018;2:644-653).

Post-mortem study of the association between cardiac iron and fibrosis in transfusion dependent anaemia.

BACKGROUND: Heart failure related to cardiac siderosis remains a major cause of death in transfusion dependent anaemias. Replacement fibrosis has been reported as causative of heart failure in siderotic cardiomyopathy in historical reports, but these findings do not accord with the reversible nature of siderotic heart failure achievable with intensive iron chelation. METHODS: Ten whole human hearts (9 beta-thalassemia major, 1 sideroblastic anaemia) were examined for iron loading and fibrosis (replacement and interstitial). Five had died from heart failure, 4 had cardiac transplantation for heart failure, and 1 had no heart failure (death from a stroke). Heart samples iron content was measured using atomic emission spectroscopy. Interstitial fibrosis was quantified by computer using picrosirius red (PSR) staining and expressed as collagen volume fraction (CVF) with normal value for left ventricle <3%. RESULTS: The 9 hearts affected by heart failure had severe iron loading with very low T2* of 5.0 ± 2.0 ms (iron concentration 8.5 ± 7.0 mg/g dw) and diffuse granular myocardial iron deposition. In none of the 10 hearts was significant macroscopic replacement fibrosis present. In only 2 hearts was interstitial fibrosis present, but with low CVF: in one patient with no cardiac siderosis (death by stroke, CVF 5.9%) and in a heart failure patient (CVF 2%). In the remaining 8 patients, no interstitial fibrosis was seen despite all having severe cardiac siderosis and heart failure (CVF 1.86% ±0.87%). CONCLUSION: Replacement cardiac fibrosis was not seen in the 9 post-mortem hearts from patients with severe cardiac siderosis and heart failure leading to death or transplantation, which contrasts markedly to historical reports. Minor interstitial fibrosis was also unusual and very limited in extent. These findings accord with the potential for reversibility of heart failure seen in iron overload cardiomyopathy. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT00520559.

Cardiac iron load and function in transfused patients treated with deferasirox (the MILE study).

OBJECTIVES: To assess the effect of iron chelation therapy with deferasirox on cardiac iron and function in patients with transfusion-dependent thalassemia major, sickle cell disease (SCD), and myelodysplastic syndromes (MDS). METHODS: This phase IV, single-arm, open-label study over 53 wk evaluated the change in cardiac and liver iron load with deferasirox (up to 40 mg/kg/d), measured by magnetic resonance imaging (MRI). RESULTS: Cardiac iron load (myocardial T2*) significantly improved (P = 0.002) overall (n = 46; n = 36 thalassemia major, n = 4 SCD, n = 6 MDS). Results were significant for patients with normal and moderate baseline cardiac iron (P = 0.017 and P = 0.015, respectively), but not in the five patients with severe cardiac iron load. Liver iron concentration (LIC) significantly decreased overall [mean LIC 10.4 to 8.2 mg Fe/g dry tissue (dw); P = 0.024], particularly in those with baseline LIC >7 mg Fe/g dw (19.9 to 15.6 mg Fe/g dw; P = 0.002). Furthermore, myocardial T2* significantly increased in patients with LIC <7 mg Fe/g dw, but not in those with a higher LIC. Safety was consistent with previous reports. CONCLUSIONS: Once-daily deferasirox over 1 yr significantly increased myocardial T2* and reduced LIC. This confirms that single-agent deferasirox is effective in the management of cardiac iron, especially for patients with myocardial T2* >10 ms (Clinicaltrials.gov identifier: NCT00673608).

Stereological Analysis of Liver Biopsy Histology Sections as a Reference Standard for Validating Non-Invasive Liver Fat Fraction Measurements by MRI.

BACKGROUND AND AIMS: Validation of non-invasive methods of liver fat quantification requires a reference standard. However, using standard histopathology assessment of liver biopsies is problematical because of poor repeatability. We aimed to assess a stereological method of measuring volumetric liver fat fraction (VLFF) in liver biopsies and to use the method to validate a magnetic resonance imaging method for measurement of VLFF. METHODS: VLFFs were measured in 59 subjects (1) by three independent analysts using a stereological point counting technique combined with the Delesse principle on liver biopsy histological sections and (2) by three independent analysts using the HepaFat-Scan® technique on magnetic resonance images of the liver. Bland Altman statistics and intraclass correlation (IC) were used to assess the repeatability of each method and the bias between the methods of liver fat fraction measurement. RESULTS: Inter-analyst repeatability coefficients for the stereology and HepaFat-Scan® methods were 8.2 (95% CI 7.7-8.8)% and 2.4 (95% CI 2.2-2.5)% VLFF respectively. IC coefficients were 0.86 (95% CI 0.69-0.93) and 0.990 (95% CI 0.985-0.994) respectively. Small biases (≤3.4%) were observable between two pairs of analysts using stereology while no significant biases were observable between any of the three pairs of analysts using HepaFat-Scan®. A bias of 1.4±0.5% VLFF was observed between the HepaFat-Scan® method and the stereological method. CONCLUSIONS: Repeatability of the stereological method is superior to the previously reported performance of assessment of hepatic steatosis by histopathologists and is a suitable reference standard for validating non-invasive methods of measurement of VLFF.

MRI Measurements of Iron Load in Transfusion-Dependent Patients: Implementation, Challenges, and Pitfalls.

Magnetic resonance imaging (MRI) has played a key role in studies of iron overload in transfusion-dependent patients, providing insights into the relations among liver and cardiac iron loading, iron chelator dose, and morbidity. Currently, there is rapid uptake of these methods into routine clinical practice as part of the management strategy for iron overload in regularly transfused patients. Given the manifold methods of data acquisition and analysis, there are several potential pitfalls that may result in inappropriate decision making. Herein, we review the challenges of establishing suitable MRI techniques for tissue iron measurement in regularly transfused patients.

Tissue Iron Distribution Assessed by MRI in Patients with Iron Loading Anemias.

Bone marrow, spleen, liver and kidney proton transverse relaxation rates (R2), together with cardiac R2* from patients with sickle cell disease (SCD), paroxysmal nocturnal hemoglobinuria (PNH) and non-transfusion dependent thalassemia (NTDT) have been compared with a control group. Increased liver and bone marrow R2 values for the three groups of patients in comparison with the controls have been found. SCD and PNH patients also present an increased spleen R2 in comparison with the controls. The simultaneous measurement of R2 values for several tissue types by magnetic resonance imaging (MRI) has allowed the identification of iron distribution patterns in diseases associated with iron imbalance. Preferential liver iron loading is found in the highly transfused SCD patients, while the low transfused ones present a preferential iron loading of the spleen. Similar to the highly transfused SCD group, PNH patients preferentially accumulate iron in the liver. A reduced spleen iron accumulation in comparison with the liver and bone marrow loading has been found in NTDT patients, presumably related to the differential increased intestinal iron absorption. The correlation between serum ferritin and tissue R2 is moderate to good for the liver, spleen and bone marrow in SCD and PNH patients. However, serum ferritin does not correlate with NTDT liver R2, spleen R2 or heart R2*. As opposed to serum ferritin measurements, tissue R2 values are a more direct measurement of each tissue's iron loading. This kind of determination will allow a better understanding of the different patterns of tissue iron biodistribution in diseases predisposed to tissue iron accumulation.

Texture-based classification of liver fibrosis using MRI.

PURPOSE: To investigate the ability of texture analysis of MRI images to stage liver fibrosis. Current noninvasive approaches for detecting liver fibrosis have limitations and cannot yet routinely replace biopsy for diagnosing significant fibrosis. MATERIALS AND METHODS: Forty-nine patients with a range of liver diseases and biopsy-confirmed fibrosis were enrolled in the study. For texture analysis all patients were scanned with a T2 -weighted, high-resolution, spin echo sequence and Haralick texture features applied. The area under the receiver operating characteristics curve (AUROC) was used to assess the diagnostic performance of the texture analysis. RESULTS: The best mean AUROC achieved for separating mild from severe fibrosis was 0.81. The inclusion of age, liver fat and liver R2 variables into the generalized linear model improved AUROC values for all comparisons, with the F0 versus F1-4 comparison the highest (0.91). CONCLUSION: Our results suggest that a combination of MRI measures, that include selected texture features from T2 -weighted images, may be a useful tool for excluding fibrosis in patients with liver disease. However, texture analysis of MRI performs only modestly when applied to the classification of patients in the mild and intermediate fibrosis stages.

Mapping iron in human heart tissue with synchrotron x-ray fluorescence microscopy and cardiovascular magnetic resonance.

BACKGROUND: MRI assessment of cardiac iron is particularly important for assessing transfusion-dependent anaemia patients. However, comparing the iron distribution from histology or bulk samples to MRI is not ideal. Non-destructive, high-resolution imaging of post-mortem samples offers the ability to examine iron distributions across large samples at resolutions closer to those used in MRI. The aim of this ex vivo case study was to compare synchrotron X-ray fluorescence microscopy (XFM) elemental iron maps with magnetic resonance transverse relaxation rate maps of cardiac tissue samples from an iron-loaded patient. METHODS: Two 5 mm thick slices of formalin fixed cardiac tissue from a Diamond Blackfan anaemia patient were imaged in a 1.5 T MR scanner. R2 and R2* transverse relaxation rate maps were generated for both slices using RF pulse recalled spin echo and gradient echo acquisition sequences. The tissue samples were then imaged at the Australian Synchrotron on the X-ray Fluorescence Microscopy beamline using a focussed incident X-ray beam of 18.74 keV and the Maia 384 detector. The event data were analyzed to produce elemental iron maps (uncalibrated) at 25 to 60 microns image resolution. RESULTS: The R2 and R2* maps and profiles for both samples showed very similar macro-scale spatial patterns compared to the XFM iron distribution. Iron appeared to preferentially load into the lateral epicardium wall and there was a strong gradient of decreasing iron, R2 and R2* from the epicardium to the endocardium in the lateral wall of the left ventricle and to a lesser extent in the septum. On co-registered images XFM iron was more strongly correlated to R2* (r = 0.86) than R2 (r = 0.79). There was a strong linear relationship between R2* and R2 (r = 0.87). CONCLUSIONS: The close qualitative and quantitative agreement between the synchrotron XFM iron maps and MR relaxometry maps indicates that iron is a significant determinant of R2 and R2* in these ex vivo samples. The R2 and R2* maps of human heart tissue give information on the spatial distribution of tissue iron deposits.

Multicenter validation of spin-density projection-assisted R2-MRI for the noninvasive measurement of liver iron concentration.

PURPOSE: Magnetic resonance imaging (MRI)-based techniques for assessing liver iron concentration (LIC) have been limited by single scanner calibration against biopsy. Here, the calibration of spin-density projection-assisted (SDPA) R2-MRI (FerriScan®) in iron-overloaded ß-thalassemia patients treated with the iron chelator, deferasirox, for 12 months is validated. METHODS: SDPA R2-MRI measurements and percutaneous needle liver biopsy samples were obtained from a subgroup of patients (n = 233) from the ESCALATOR trial. Five different makes and models of scanner were used in the study. RESULTS: LIC, derived from mean of MRI- and biopsy-derived values, ranged from 0.7 to 50.1 mg Fe/g dry weight. Mean fractional differences between SDPA R2-MRI- and biopsy-measured LIC were not significantly different from zero. They were also not significantly different from zero when categorized for each of the Ishak stages of fibrosis and grades of necroinflammation, for subjects aged 3 to <8 versus ≥8 years, or for each scanner model. Upper and lower 95% limits of agreement between SDPA R2-MRI and biopsy LIC measurements were 74 and -71%. CONCLUSION: The calibration curve appears independent of scanner type, patient age, stage of liver fibrosis, grade of necroinflammation, and use of deferasirox chelation therapy, confirming the clinical usefulness of SDPA R2-MRI for monitoring iron overload.

Diagnostic performance of a rapid magnetic resonance imaging method of measuring hepatic steatosis.

OBJECTIVES: Hepatic steatosis is associated with an increased risk of developing serious liver disease and other clinical sequelae of the metabolic syndrome. However, visual estimates of steatosis from histological sections of biopsy samples are subjective and reliant on an invasive procedure with associated risks. The aim of this study was to test the ability of a rapid, routinely available, magnetic resonance imaging (MRI) method to diagnose clinically relevant grades of hepatic steatosis in a cohort of patients with diverse liver diseases. MATERIALS AND METHODS: Fifty-nine patients with a range of liver diseases underwent liver biopsy and MRI. Hepatic steatosis was quantified firstly using an opposed-phase, in-phase gradient echo, single breath-hold MRI methodology and secondly, using liver biopsy with visual estimation by a histopathologist and by computer-assisted morphometric image analysis. The area under the receiver operating characteristic (ROC) curve was used to assess the diagnostic performance of the MRI method against the biopsy observations. RESULTS: The MRI approach had high sensitivity and specificity at all hepatic steatosis thresholds. Areas under ROC curves were 0.962, 0.993, and 0.972 at thresholds of 5%, 33%, and 66% liver fat, respectively. MRI measurements were strongly associated with visual (r(2) = 0.83) and computer-assisted morphometric (r(2) = 0.84) estimates of hepatic steatosis from histological specimens. CONCLUSIONS: This MRI approach, using a conventional, rapid, gradient echo method, has high sensitivity and specificity for diagnosing liver fat at all grades of steatosis in a cohort with a range of liver diseases.

The effect of magnetically induced linear aggregates on proton transverse relaxation rates of aqueous suspensions of polymer coated magnetic nanoparticles.

It has been recently reported that for some suspensions of magnetic nanoparticles the transverse proton relaxation rate, R(2), is dependent on the time that the sample is exposed to an applied magnetic field. This time dependence has been linked to the formation of linear aggregates or chains in an applied magnetic field via numerical modeling. It is widely known that chain formation occurs in more concentrated ferrofluids systems and that this has an affect on the ferrofluid properties. In this work we examine the relationships between colloidal stability, the formation of these linear structures, and changes observed in the proton transverse relaxation rate of aqueous suspensions of magnetic particles. A series of iron oxide nanoparticles with varying stabilizing ligand brush lengths were synthesized. These systems were characterized with dynamic light scattering, transmission electron microscopy, dark-field optical microscopy, and proton transverse relaxation rate measurements. The dark field optical microscopy and R(2) measurements were made in similar magnetic fields over the same time scale so as to correlate the reduction of the transverse relaxivity with the formation of linear aggregates. Our results indicate that varying the ligand length has a direct effect on the colloidal arrangement of the system in a magnetic field, producing differences in the rate and size of chain formation, and hence systematic changes in transverse relaxation rates over time. With increasing ligand brush length, attractive inter-particle interactions are reduced, which results in slower aggregate formation and shorter linear aggregate length. These results have implications for the stabilization, characterization and potentially the toxicity of magnetic nanoparticle systems used in biomedical applications.

Identification of nonferritin mitochondrial iron deposits in a mouse model of Friedreich ataxia.

There is no effective treatment for the cardiomyopathy of the most common autosomal recessive ataxia, Friedreich ataxia (FA). This disease is due to decreased expression of the mitochondrial protein, frataxin, which leads to alterations in mitochondrial iron (Fe) metabolism. The identification of potentially toxic mitochondrial Fe deposits in FA suggests Fe plays a role in its pathogenesis. Studies using the muscle creatine kinase (MCK) conditional frataxin knockout mouse that mirrors the disease have demonstrated frataxin deletion alters cardiac Fe metabolism. Indeed, there are pronounced changes in Fe trafficking away from the cytosol to the mitochondrion, leading to a cytosolic Fe deficiency. Considering Fe deficiency can induce apoptosis and cell death, we examined the effect of dietary Fe supplementation, which led to body Fe loading and limited the cardiac hypertrophy in MCK mutants. Furthermore, this study indicates a unique effect of heart and skeletal muscle-specific frataxin deletion on systemic Fe metabolism. Namely, frataxin deletion induces a signaling mechanism to increase systemic Fe levels and Fe loading in tissues where frataxin expression is intact (i.e., liver, kidney, and spleen). Examining the mutant heart, native size-exclusion chromatography, transmission electron microscopy, Mössbauer spectroscopy, and magnetic susceptibility measurements demonstrated that in the absence of frataxin, mitochondria contained biomineral Fe aggregates, which were distinctly different from isolated mammalian ferritin molecules. These mitochondrial aggregates of Fe, phosphorus, and sulfur, probably contribute to the oxidative stress and pathology observed in the absence of frataxin.

Renal iron load in sickle cell disease is influenced by severity of haemolysis.

Spin density projection-assisted R2-magnetic resonance imaging (R2-MRI; FerriScan(®)) scans from 40 chelation-naïve sickle cell patients were used to assess renal iron load by measuring renal R2 (R-R2). Clinical data were collected retrospectively for the 2-year period preceding the scan. R-R2 showed no significant correlation with transfusional iron load (assessed by liver iron concentration), but correlated significantly with serum bilirubin (R = 0·61, P < 0·0001) and lactate dehydrogenase (R = 0·58, P < 0·0001). Mean (±standard deviation) R-R2 was higher (P = 0·02) in patients with renal hyperfiltration (29·8 ± 10·3/s) than those without (23·11 ± 6·6/s). Five patients had significantly lower signal intensity in the renal cortex, as compared to the medulla. These patients had a significantly higher (P < 0·0001) mean R-R2 than those showing no cortico-medullary difference. We postulate that the increased R-R2 is associated with haemolysis rather than transfusional iron load in sickle cell disease.

Nuclear magnetic resonance: a tool for malaria diagnosis?

Malaria control can be improved by rapid, sensitive, low-cost detection of infection. Several such strategies are being pursued. Rapid diagnostic tests can detect infections at parasite densities above 200 µL(-1). Polymerase chain reaction methods can detect low parasite densities, but are slow and prone to contamination under field conditions. Methods that detect hemozoin presence in blood have been proposed as alternatives for rapid detection of infection. In this study, we used a benchtop nuclear magnetic resonance (NMR) device to detect hemozoin. This device could be deployed in malaria-endemic settings. We measured synthetic hemozoin in phosphate-buffered saline and malaria parasites in human blood. The NMR detected hemozoin in suspensions of 4 ng µL(-1) and parasites at densities of 8,000-10,000 µL(-1) (0.2% parasitemia). Thus, our preliminary NMR approach, although providing very rapid measurements, is unlikely to achieve the required sensitivity and specificity for malaria diagnosis, unless a preliminary concentration step is performed.

A sub-microscopic gametocyte reservoir can sustain malaria transmission.

BACKGROUND: Novel diagnostic tools, including PCR and high field gradient magnetic fractionation (HFGMF), have improved detection of asexual Plasmodium falciparum parasites and especially infectious gametocytes in human blood. These techniques indicate a significant number of people carry gametocyte densities that fall below the conventional threshold of detection achieved by standard light microscopy (LM). METHODOLOGY/PRINCIPAL FINDINGS: To determine how low-level gametocytemia may affect transmission in present large-scale efforts for P. falciparum control in endemic areas, we developed a refinement of the classical Ross-Macdonald model of malaria transmission by introducing multiple infective compartments to model the potential impact of highly prevalent, low gametocytaemic reservoirs in the population. Models were calibrated using field-based data and several numerical experiments were conducted to assess the effect of high and low gametocytemia on P. falciparum transmission and control. Special consideration was given to the impact of long-lasting insecticide-treated bed nets (LLIN), presently considered the most efficient way to prevent transmission, and particularly LLIN coverage similar to goals targeted by the Roll Back Malaria and Global Fund malaria control campaigns. Our analyses indicate that models which include only moderate-to-high gametocytemia (detectable by LM) predict finite eradication times after LLIN introduction. Models that include a low gametocytemia reservoir (requiring PCR or HFGMF detection) predict much more stable, persistent transmission. Our modeled outcomes result in significantly different estimates for the level and duration of control needed to achieve malaria elimination if submicroscopic gametocytes are included. CONCLUSIONS/SIGNIFICANCE: It will be very important to complement current methods of surveillance with enhanced diagnostic techniques to detect asexual parasites and gametocytes to more accurately plan, monitor and guide malaria control programs aimed at eliminating malaria.

Short report: Quantification of Plasmodium falciparum gametocytes by magnetic fractionation.

A method of gametocyte quantitation in human blood was developed based on magnetic fractionation using commercially available magnetic fractionation columns (MFCs) and exploiting the magnetic susceptibility of mature Plasmodium falciparum gametocytes. The technique uses magnetic microspheres as a calibration standard. Microspheres are added to each blood sample to a known concentration. When exposed to a magnetic field, gametocytes and magnetic microspheres are preferentially captured inside MFCs. After removal of the magnetizing field, the magnetically captured material can be eluted, placed on a microscope slide that is stained, and counted by using conventional methods. The limits of quantitation for P. falciparum gametocytes were determined from serial dilutions of blood samples with known gametocyte density. The upper limit was 1,000 gametocytes/µL. Quantitative analysis above this threshold is difficult because of an over-abundance of gametocytes. The lower limit was 0.1 gametocytes/µL, and there is a significant probability of a false-negative result below this level.

Parameterization of high magnetic field gradient fractionation columns for applications with Plasmodium falciparum infected human erythrocytes.

BACKGROUND: Magnetic fractionation of erythrocytes infected with Plasmodium falciparum has several research uses including enrichment of infected cells from parasite cultures or enhanced detection of P. falciparum gametocytes. The aim of the present study was to quantitatively characterize the magnetic fractionation process and thus enable optimization of protocols developed for specific uses. METHODS: Synchronized cultures of P. falciparum parasites incubated with human erythrocytes were magnetically fractionated with commercially available columns. The timing of the fractionation experiments was such that the parasites were in second half of their erythrocytic life cycle with parasite densities ranging from 1 to 9%. Fractionations were carried out in a single pass through the columns. Cells were enumerated and differentiated in the initial samples as well as in the positive and negative fractions. The capture of cells by the fractionation column was described by a saturation binding model. RESULTS: The magnetic binding affinity to the column matrix was approximately 350 times greater for infected cells compared with uninfected cells. The purity of infected cells in the captured fraction was generally >80% but decreased rapidly (to less than 50%) when the number of infected cells that passed through the column was substantially decreased (to less than 9 +/- 5 x 105 cells). The distribution of captured parasite developmental stages shifted to mature stages as the number of infected cells in the initial samples and flow rate increased. The relationship between the yield of infected cells in the captured fraction and flow rate of cells conformed to a complementary cumulative log-normal equation with flow rates >1.6 x 105 cells per second resulting in yields <50%. CONCLUSIONS: A detailed quantitative analysis of a batchwise magnetic fractionation process for malaria infected erythrocytes using high gradient magnetic fractionation columns was performed. The models applied in this study allow the prediction of capture efficiency if the initial infected cell concentration and the flow rate are known.

A comparative study of a flow-cytometry-based assessment of in vitro Plasmodium falciparum drug sensitivity.

BACKGROUND: Recently developed Sybr Green-based in vitro Plasmodium falciparum drug sensitivity assays provide an attractive alternative to current manual and automated methods. The present study evaluated flow cytometry measurement of DNA staining with Sybr Green in comparison with the P. falciparum lactate dehydrogenase assay, the tritiated hypoxanthine incorporation assay, a previously described Sybr Green based plate reader assay and light microscopy. METHODS: All assays were set up in standardized format in 96-well plates. The 50% inhibitory concentrations (IC50) of chloroquine, mefloquine and dihydroartemisinin against the laboratory adapted P. falciparum strains 3D7, E8B, W2mef and Dd2 were determined using each method. RESULTS: The resolution achieved by flow cytometry allowed quantification of the increase in individual cell DNA content after an incubation period of only 24 h. Regression, and Bland and Altman analyses showed that the IC50 values determined using the flow cytometry assay after 24 h agreed well with those obtained using the hypoxanthine incorporation assay, the P. falciparum lactate dehydrogenase assay, the Sybr Green plate reader assay and light microscopy. However the values obtained with the flow cytometry assay after 48 h of incubation differed significantly from those obtained with the hypoxanthine incorporation assay, and the P. falciparum lactate dehydrogenase assay at low IC50 values, but agreed well with the Sybr Green plate reader assay and light microscopy. CONCLUSIONS: Although flow cytometric equipment is expensive, the necessary reagents are inexpensive, the procedure is simple and rapid, and the cell volume required is minimal. This should allow field studies using fingerprick sample volumes.