The Vitamin D Metabolite Ratio (VMR) is a Biomarker of Vitamin D Status That is Not Affected by Acute Changes in Vitamin D Binding Protein.

BACKGROUND: 25-hydroxyvitamin D[25(OH)D] may be a poor marker of vitamin D status due to variability in levels of vitamin D binding protein (VDBP). The vitamin D metabolite ratio (VMR) is the ratio of 24,25-dihydroxyvitamin D[24,25(OH)2D3] to 25(OH)D3 and has been postulated to reflect vitamin D sufficiency independent of variability in VDBP. Therapeutic plasma exchange (TPE) is a procedure that removes plasma, including VDBP, and may lower bound vitamin D metabolite concentrations. Effects of TPE on the VMR are unknown. METHODS: We measured 25(OH)D, free 25(OH)D, 1,25-dihydroxyvitamin D[1,25(OH)2D], 24,25(OH)2D3, and VDBP in persons undergoing TPE, before and after treatment. We used paired t-tests to assess changes in these biomarkers during a TPE procedure. RESULTS: Study participants (n = 45) had a mean age of 55 ± 16 years; 67% were female; and 76% were white. Compared to pretreatment concentrations, TPE caused a significant decrease in total VDBP by 65% (95%CI 60,70%), as well as all the vitamin D metabolites-25(OH)D by 66% (60%,74%), free 25(OH)D by 31% (24%,39%), 24,25(OH)2D3 by 66% (55%,78%) and 1,25(OH)2D by 68% (60%,76%). In contrast, there was no significant change in the VMR before and after a single TPE treatment, with an observed mean 7% (-3%, 17%) change in VMR. CONCLUSIONS: Changes in VDBP concentration across TPE parallel changes in 25(OH)D, 1,25(OH)2D, and 24,25(OH)2D3, suggesting that concentrations of these metabolites reflect underlying VDBP concentrations. The VMR is stable across a TPE session despite a 65% reduction in VDBP. These findings suggest that the VMR is a marker of vitamin D status independent of VDBP levels.

Identification of a Functionally Efficient and Thermally Stable Outward Sodium-Pumping Rhodopsin (BeNaR) from a Thermophilic Bacterium.

Rhodopsins are transmembrane proteins with retinal chromophores that are involved in photo-energy conversion and photo-signal transduction in diverse organisms. In this study, we newly identified and characterized a rhodopsin from a thermophilic bacterium, Bellilinea sp. Recombinant Escherichia coli cells expressing the rhodopsin showed light-induced alkalization of the medium only in the presence of sodium ions (Na+), and the alkalization signal was enhanced by addition of a protonophore, indicating an outward Na+ pump function across the cellular membrane. Thus, we named the protein Bellilinea Na+-pumping rhodopsin, BeNaR. Of note, its Na+-pumping activity is significantly greater than that of the known Na+-pumping rhodopsin, KR2. We further characterized its photochemical properties as follows: (i) Visible spectroscopy and HPLC revealed that BeNaR has an absorption maximum at 524 nm with predominantly (>96%) the all-trans retinal conformer. (ii) Time-dependent thermal denaturation experiments revealed that BeNaR showed high thermal stability. (iii) The time-resolved flash-photolysis in the nanosecond to millisecond time domains revealed the presence of four kinetically distinctive photointermediates, K, L, M and O. (iv) Mutational analysis revealed that Asp101, which acts as a counterion, and Asp230 around the retinal were essential for the Na+-pumping activity. From the results, we propose a model for the outward Na+-pumping mechanism of BeNaR. The efficient Na+-pumping activity of BeNaR and its high stability make it a useful model both for ion transporters and optogenetics tools.

Allochromatium tepidum, sp. nov., a hot spring species of purple sulfur bacteria.

We describe a new species of purple sulfur bacteria (Chromatiaceae, anoxygenic phototrophic bacteria) isolated from a microbial mat in the sulfidic geothermal outflow of a hot spring in Rotorua, New Zealand. This phototroph, designated as strain NZ, grew optimally near 45 °C but did not show an absorption maximum at 915 nm for the light-harvesting-reaction center core complex (LH1-RC) characteristic of other thermophilic purple sulfur bacteria. Strain NZ had a similar carotenoid composition as Thermochromatium tepidum, but unlike Tch. tepidum, grew photoheterotrophically on acetate in the absence of sulfide and metabolized thiosulfate. The genome of strain NZ was significantly larger than that of Tch. tepidum but slightly smaller than that of Allochromatium vinosum. Strain NZ was phylogenetically more closely related to mesophilic purple sulfur bacteria of the genus Allochromatium than to Tch. tepidum. This conclusion was reached from phylogenetic analyses of strain NZ genes encoding 16S rRNA and the photosynthetic functional gene pufM, from phylogenetic analyses of entire genomes, and from a phylogenetic tree constructed from the concatenated sequence of 1090 orthologous proteins. Moreover, average nucleotide identities and digital DNA:DNA hybridizations of the strain NZ genome against those of related species of Chromatiaceae supported the phylogenetic analyses. From this collection of properties, we describe strain NZ here as the first thermophilic species of the genus Allochromatium, Allochromatium tepidum NZT, sp. nov.

Relationship between Microorganisms Inhabiting Alkaline Siliceous Hot Spring Mat Communities and Overflowing Water.

The compositions of Octopus Spring and Mushroom Spring (Yellowstone National Park, Wyoming, USA) microbial mats have been thoroughly studied, but the compositions of the effluent waters that flow above the mats have not. In this study, cells in the mats and overflowing waters of both springs were investigated at multiple sites where Synechococcus spp. are the dominant cyanobacteria (ca. 72°C to ca. 50°C), and on several dates. In addition to microscopic analyses of stained and autofluorescent cells, 16S rRNA gene sequencing was used to characterize the major taxa present and a protein-encoding gene (psaA) was sequenced and analyzed by ecotype simulation to predict species of Synechococcus The mats of both springs were similar in terms of the downstream distribution of predominant taxa detected previously. However, waters above these mats were predominated by taxa that reside in upstream mats or communities above the upper-temperature limit of the mat. A disturbance/recolonization study was performed at a site normally predominated by Synechococcus species adapted to low temperatures. After removing indigenous Synechococcus cells, Synechococcus species adapted to higher temperatures, which were predominant in the water overflowing this site, colonized the newly forming mat. Differences in recolonization under reduced and UV-screened irradiance suggested that, in addition to physical transport, environmental conditions likely select for species that are better adapted to these different conditions and can influence mat recovery. A transport model was developed and used to predict that, in Mushroom Spring, erosion predominates in the narrower and deeper upstream effluents and deposition predominates over erosion in wider and shallower downstream effluents.IMPORTANCE In flowing aquatic systems, cell erosion and deposition are important to the dispersal of cells from one location to another. Very little is known about microbial dispersal and the physical processes that underlie it. This study demonstrates its importance to colonization of downstream surfaces and especially to the recolonization and functioning of disturbed sites. Ecological systems in flowing environments are often, roughly speaking, pseudosteady, in that nutrients enter the system and by-products leave at relatively steady rates. Over time, material inputs and outputs must balance. Measurements of input fluxes (e.g., growth rates and proxies, such as photosynthesis rates) are frequent. However, erosion and deposition of cells are seldom measured and ecological significance is sometimes neglected. The importance of these parameters is immediately evident in any attempt to construct a model of long-time community behavior, as spatial ecological structure is significantly impacted and can be dominated by migration of organisms, even in small numbers.

Short-Term Stable Isotope Probing of Proteins Reveals Taxa Incorporating Inorganic Carbon in a Hot Spring Microbial Mat.

The upper green layer of the chlorophototrophic microbial mats associated with the alkaline siliceous hot springs of Yellowstone National Park consists of oxygenic cyanobacteria (Synechococcus spp.), anoxygenic Roseiflexus spp., and several other anoxygenic chlorophototrophs. Synechococcus spp. are believed to be the main fixers of inorganic carbon (Ci), but some evidence suggests that Roseiflexus spp. also contribute to inorganic carbon fixation during low-light, anoxic morning periods. Contributions of other phototrophic taxa have not been investigated. In order to follow the pathway of Ci incorporation into different taxa, mat samples were incubated with [13C]bicarbonate for 3 h during the early-morning, low-light anoxic period. Extracted proteins were treated with trypsin and analyzed by mass spectrometry, leading to peptide identifications and peptide isotopic profile signatures containing evidence of 13C label incorporation. A total of 25,483 peptides, corresponding to 7,221 proteins, were identified from spectral features and associated with mat taxa by comparison to metagenomic assembly sequences. A total of 1,417 peptides, derived from 720 proteins, were detectably labeled with 13C. Most 13C-labeled peptides were derived from proteins of Synechococcus spp. and Roseiflexus spp. Chaperones and proteins of carbohydrate metabolism were most abundantly labeled. Proteins involved in photosynthesis, Ci fixation, and N2 fixation were also labeled in Synechococcus spp. Importantly, most proteins of the 3-hydroxypropionate bi-cycle for Ci fixation in Roseiflexus spp. were labeled, establishing that members of this taxocene contribute to Ci fixation. Other taxa showed much lower [13C]bicarbonate incorporation.IMPORTANCE Yellowstone hot spring mats have been studied as natural models for understanding microbial community ecology and as modern analogs of stromatolites, the earliest community fossils on Earth. Stable-isotope probing of proteins (Pro-SIP) permitted short-term interrogation of the taxa that are involved in the important process of light-driven Ci fixation in this highly active community and will be useful in linking other metabolic processes to mat taxa. Here, evidence is presented that Roseiflexus spp., which use the 3-hydroxypropionate bi-cycle, are active in Ci fixation. Because this pathway imparts a lower degree of selection of isotopically heavy Ci than does the Calvin-Benson-Bassham cycle, the results suggest a mechanism to explain why the natural abundance of 13C in mat biomass is greater than expected if only the latter pathway were involved. Understanding how mat community members influence the 13C/12C ratios of mat biomass will help geochemists interpret the 13C/12C ratios of organic carbon in the fossil record.

In Situ Hydrogen Dynamics in a Hot Spring Microbial Mat during a Diel Cycle.

UNLABELLED: Microbes can produce molecular hydrogen (H2) via fermentation, dinitrogen fixation, or direct photolysis, yet the H2 dynamics in cyanobacterial communities has only been explored in a few natural systems and mostly in the laboratory. In this study, we investigated the diel in situ H2 dynamics in a hot spring microbial mat, where various ecotypes of unicellular cyanobacteria (Synechococcus sp.) are the only oxygenic phototrophs. In the evening, H2 accumulated rapidly after the onset of darkness, reaching peak values of up to 30 µmol H2 liter(-1) at about 1-mm depth below the mat surface, slowly decreasing to about 11 µmol H2 liter(-1) just before sunrise. Another pulse of H2 production, reaching a peak concentration of 46 µmol H2 liter(-1), was found in the early morning under dim light conditions too low to induce accumulation of O2 in the mat. The light stimulation of H2 accumulation indicated that nitrogenase activity was an important source of H2 during the morning. This is in accordance with earlier findings of a distinct early morning peak in N2 fixation and expression of Synechococcus nitrogenase genes in mat samples from the same location. Fermentation might have contributed to the formation of H2 during the night, where accumulation of other fermentation products lowered the pH in the mat to less than pH 6 compared to a spring source pH of 8.3. IMPORTANCE: Hydrogen is a key intermediate in anaerobic metabolism, and with the development of a sulfide-insensitive microsensor for H2, it is now possible to study the microdistribution of H2 in stratified microbial communities such as the photosynthetic microbial mat investigated here. The ability to measure H2 profiles within the mat compared to previous measurements of H2 emission gives much more detailed information about the sources and sinks of H2 in such communities, and it was demonstrated that the high rates of H2 formation in the early morning when the mat was exposed to low light intensities might be explained by nitrogen fixation, where H2 is formed as a by-product.

Automated data extraction from in situ protein-stable isotope probing studies.

Protein-stable isotope probing (protein-SIP) has strong potential for revealing key metabolizing taxa in complex microbial communities. While most protein-SIP work to date has been performed under controlled laboratory conditions to allow extensive isotope labeling of the target organism(s), a key application will be in situ studies of microbial communities for short periods of time under natural conditions that result in small degrees of partial labeling. One hurdle restricting large-scale in situ protein-SIP studies is the lack of algorithms and software for automated data processing of the massive data sets resulting from such studies. In response, we developed Stable Isotope Probing Protein Extraction Resources software (SIPPER) and applied it for large-scale extraction and visualization of data from short-term (3 h) protein-SIP experiments performed in situ on phototrophic bacterial mats isolated from Yellowstone National Park. Several metrics incorporated into the software allow it to support exhaustive analysis of the complex composite isotopic envelope observed as a result of low amounts of partial label incorporation. SIPPER also enables the detection of labeled molecular species without the need for any prior identification.

Prophylactic red blood cell exchange for ABO-mismatched hematopoietic progenitor cell transplants.

BACKGROUND: To enhance donor availability, almost half of hematopoietic progenitor cell transplants (HPCTs) cross ABO blood type boundaries. ABO-incompatible HPCTs are well tolerated; however, there is an increased risk of delayed hemolysis in patients with minor and bidirectional ABO mismatches. Delayed hemolysis generally occurs 1 to 2 weeks after HPCT and is related to production of alloantibodies directed against recipient ABO red blood cell (RBC) antigens by passenger donor lymphocytes. One previous study has suggested that prophylactic RBC exchange in patients with minor and bidirectional ABO-mismatched HPCT reduces the risks of severe immune hemolysis, but this recommendation is controversial. STUDY DESIGN AND METHODS: Herein we describe our experience using prophylactic RBC exchange in patients with minor and bidirectional ABO-mismatched HPCTs who were deemed to be at high risk for immune hemolysis. We compare the group of patients that received prophylactic RBC exchange with a historical cohort of ABO-mismatched patients who underwent HPCT without prophylactic RBC exchange. RESULTS: Our study suggests that prophylactic RBC exchange in minor and bidirectional ABO-mismatched HPCT does not reduce severe immune hemolysis, nor does it improve 1-year survival, the number of RBC units transfused after transplant, or length of hospitalization after HPCT. CONCLUSION: This study failed to identify a clear role for selected prophylactic RBC exchange in patients who were deemed at risk for severe post-HPCT immune hemolysis.

The selective therapeutic apheresis procedures.

Selective apheresis procedures have been developed to target specific molecules, antibodies, or cellular elements in a variety of diseases. The advantage of the selective apheresis procedures over conventional therapeutic plasmapheresis is preservation of other essential plasma components such as albumin, immunoglobulins, and clotting factors. These procedures are more commonly employed in Europe and Japan, and few are available in the USA. Apheresis procedures discussed in this review include the various technologies available for low-density lipoprotein (LDL) apheresis, double filtration plasmapheresis (DFPP), cryofiltration, immunoadsorption procedures, adsorption resins that process plasma, extracorporeal photopheresis, and leukocyte apheresis.

Geographic and Ecological Diversity of Green Sulfur Bacteria in Hot Spring Mat Communities.

Three strains of thermophilic green sulfur bacteria (GSB) are known; all are from microbial mats in hot springs in Rotorua, New Zealand (NZ) and belong to the species Chlorobaculum tepidum. Here, we describe diverse populations of GSB inhabiting Travel Lodge Spring (TLS) (NZ) and hot springs ranging from 36.1 °C to 51.1 °C in the Republic of the Philippines (PHL) and Yellowstone National Park (YNP), Wyoming, USA. Using targeted amplification and restriction fragment length polymorphism analysis, GSB 16S rRNA sequences were detected in mats in TLS, one PHL site, and three regions of YNP. GSB enrichments from YNP and PHL mats contained small, green, nonmotile rods possessing chlorosomes, chlorobactene, and bacteriochlorophyll c. Partial 16S rRNA gene sequences from YNP, NZ, and PHL mats and enrichments from YNP and PHL samples formed distinct phylogenetic clades, suggesting geographic isolation, and were associated with samples differing in temperature and pH, suggesting adaptations to these parameters. Sequences from enrichments and corresponding mats formed clades that were sometimes distinct, increasing the diversity detected. Sequence differences, monophyly, distribution patterns, and evolutionary simulation modeling support our discovery of at least four new putative moderately thermophilic Chlorobaculum species that grew rapidly at 40 °C to 44 °C.

Complete genome of Candidatus Chloracidobacterium thermophilum, a chlorophyll-based photoheterotroph belonging to the phylum Acidobacteria.

Candidatus Chloracidobacterium thermophilum, which naturally inhabits microbial mats of alkaline siliceous hot springs in Yellowstone National Park, is the only known chlorophototroph in the phylum Acidobacteria. The Ca. C. thermophilum genome was composed of two chromosomes (2,683,362 bp and 1,012,010 bp), and both encoded essential genes. The genome included genes to produce chlorosomes, the Fenna-Matthews-Olson protein, bacteriochlorophylls a and c as principal pigments, and type-1, homodimeric reaction centres. Ca. C. thermophilum is an aerobic photoheterotroph that lacks the ability to synthesize several essential nutrients. Key genes of all known carbon fixation pathways were absent, as were genes for assimilatory nitrate and sulfate reduction and vitamin B(12) synthesis. Genes for the synthesis of branched-chain amino acids (valine, isoleucine and leucine) were also absent, but genes for catabolism of these compounds were present. This observation suggested that Ca. C. thermophilum may synthesize branched-chain amino acids from an intermediate(s) of the catabolic pathway by reversing these reactions. The genome encoded an aerobic respiratory electron transport chain that included NADH dehydrogenase, alternative complex III and cytochrome oxidase. The chromosomes of the laboratory isolate were compared with assembled, metagenomic scaffolds from the major Ca. C. thermophilum population in hot-spring mats. The larger chromosomes of the two populations were highly syntenous but significantly divergent (~13%) in sequence. In contrast, the smaller chromosomes have undergone numerous rearrangements, contained many transposases, and might be less constrained by purifying selection than the large chromosomes. Some transposases were homologous to those of mat community members from other phyla.

Identification and distribution of high-abundance proteins in the octopus spring microbial mat community.

A shotgun metaproteomics approach was employed to identify proteins in a hot spring microbial mat community. We identified 202 proteins encompassing 19 known functions from 12 known phyla. Importantly, we identified two key enzymes involved in the 3-hydroxypropionate CO(2) fixation pathway in uncultivated Roseiflexus spp., which are known photoheterotrophs.

Therapeutic apheresis for renal disorders.

This review summarizes the clinical evidence and practical details for the use of plasmapheresis and other apheresis modalities for each indication in nephrology. Updated information on the molecular biology and immunology of each renal disease is discussed in relation to the rationale for apheresis therapy and its place amid other available treatments. Autoantibody-mediated diseases, such as anti-GBM (anti-glomerular basement membrane) glomerulonephritis (GN), ANCA (antineutrophil cytoplasmic antibody)-related GN and the antibody-mediated type of TTP (thrombotic thrombocytopenic purpura), and alloantibody-mediated diseases such as kidney transplant sensitization and humoral rejection, can be treated by various plasmapheresis methods. These include standard plasmapheresis with a replacement volume, or plasmapheresis with online plasma purification using adsorption columns or secondary filtration. However, it should be noted that the pathogenic molecules implicated in FSGS (focal segmental glomerulosclerosis), myeloma cast nephropathy, and perhaps other diseases are too small to be removed by most online purification methods. A great majority of controlled trials and series on which evidence-based treatment recommendations are made were performed using centrifugal plasmapheresis; it is presumed that membrane-separation plasmapheresis is equally efficacious. For some rarer diseases, such as MPGN (membranoproliferative GN) type 2 with factor H abnormalities or C3Nef (C3 nephritic factor) autoantibodies, there are only a few case reports, but enough scientific understanding to warrant a trial of plasmapheresis in severe cases. Photopheresis, which is effective for cell-mediated rejection in heart and lung transplantation, has not yet found a place in the routine treatment of kidney transplant rejection.

Therapeutic plasma exchange in the intensive care unit: Rationale, special considerations, and techniques for combined circuits.

Therapeutic plasma exchange (TPE) is an extracorporeal blood purification technique with proven efficacy in a variety of conditions, including in the intensive care setting. It is not uncommon for a critically ill patient to require more than one extracorporeal procedure in addition to TPE. This review focuses on the combination of TPE with other extracorporeal circuits in a critical care setting via a single vascular access (either in-series, parallel, or a hybrid mode) which is often referred to as performing procedures "in tandem." Authors performed literature review via pubmed.gov using search terms: plasma exchange, plasmapheresis, apheresis, tandem circuits, combined circuits, critical care, ICU, CRRT, hemodialysis, and ECMO. Thirty-eight English-language, peer-reviewed papers were appraised that satisfied the content of this review on techniques for combining circuits with plasma exchange, as well as describing the advantages of tandem procedures and potential complications that can arise. Performing these procedures simultaneously can be advantageous in reducing total procedure and staffing time, avoiding placement of additional central lines, reducing overall need for anticoagulation, and limiting multiple blood primes in certain populations. However, the described combined circuits are complex, associated with higher complications, and require a skilled team to understand and mitigate the potential complications associated with these combined procedures.

Fine-scale distribution patterns of Synechococcus ecological diversity in microbial mats of Mushroom Spring, Yellowstone National Park.

Past analyses of sequence diversity in high-resolution protein-encoding genes have identified putative ecological species of unicellular cyanobacteria in the genus Synechococcus, which are specialized to 60°C but not 65°C in Mushroom Spring microbial mats. Because these studies were limited to only two habitats, we studied the distribution of Synechococcus sequence variants at 1°C intervals along the effluent flow channel and at 80-µm vertical-depth intervals throughout the upper photic layer of the microbial mat. Diversity at the psaA locus, which encodes a photosynthetic reaction center protein (PsaA), was sampled by PCR amplification, cloning, and sequencing methods at 60, 63, and 65°C sites. The evolutionary simulation programs Ecotype Simulation and AdaptML were used to identify putative ecologically distinct populations (ecotypes). Ecotype Simulation predicted a higher number of putative ecotypes in cases where habitat variation was limited, while AdaptML predicted a higher number of ecologically distinct phylogenetic clades in cases where habitat variation was high. Denaturing gradient gel electrophoresis was used to track the distribution of dominant sequence variants of ecotype populations relative to temperature variation and to O2, pH, and spectral irradiance variation, as measured using microsensors. Different distributions along effluent channel flow and vertical gradients, where temperature, light, and O2 concentrations are known to vary, confirmed the ecological distinctness of putative ecotypes.

Influence of molecular resolution on sequence-based discovery of ecological diversity among Synechococcus populations in an alkaline siliceous hot spring microbial mat.

Previous research has shown that sequences of 16S rRNA genes and 16S-23S rRNA internal transcribed spacer regions may not have enough genetic resolution to define all ecologically distinct Synechococcus populations (ecotypes) inhabiting alkaline, siliceous hot spring microbial mats. To achieve higher molecular resolution, we studied sequence variation in three protein-encoding loci sampled by PCR from 60°C and 65°C sites in the Mushroom Spring mat (Yellowstone National Park, WY). Sequences were analyzed using the ecotype simulation (ES) and AdaptML algorithms to identify putative ecotypes. Between 4 and 14 times more putative ecotypes were predicted from variation in protein-encoding locus sequences than from variation in 16S rRNA and 16S-23S rRNA internal transcribed spacer sequences. The number of putative ecotypes predicted depended on the number of sequences sampled and the molecular resolution of the locus. Chao estimates of diversity indicated that few rare ecotypes were missed. Many ecotypes hypothesized by sequence analyses were different in their habitat specificities, suggesting different adaptations to temperature or other parameters that vary along the flow channel.

Extracorporeal photopheresis: how, when, and why.

Extracorporeal photopheresis (ECP) is a well-tolerated procedure that suppresses T-lymphocyte activity in a clonally-specific way. It is an effective therapy that has established indications in the management of cutaneous T-cell lymphoma, graft-versus-host disease and some scenarios of solid-organ transplant rejection. It is being used increasingly around the world. Its applications are evolving, including exploration of its potential for treating autoimmune diseases where cytotoxic T-cell-mediated mechanisms appear to be involved, such as Crohn's disease. This article reviews scientific insights into its mechanism of action on the immune system, details of the clinical procedure, its clinical applications in various diseases, and the current evidence for its efficacy and place in medical therapeutics.

Conventional apheresis therapies: a review.

This article reviews advances in the scientific basis and medical practice of plasmapheresis and cytapheresis therapies. Newly-characterized autoantibodies in neuromyelitis optica, Guillain-Barre variants, anti-neutrophil cytoplasmic antibody (ANCA) vasculitides, etc., exemplify the modern molecular biology which now provides a rigorous framework of understanding for the clinical practice of plasmapheresis. Clinical trials continue to clarify the appropriate use of therapeutic plasmapheresis (TPE) in these and other diseases. Centrifugal (cTPE) and membrane filtration (mTPE) types of plasmapheresis are compared, with details of the plasmapheresis prescription, anticoagulation choices, replacement fluids and other practical considerations. Plasma removal is more efficient with cTPE; mTPE systems have a lower plasma extraction ratio, and therefore require higher blood flow rates or longer procedure times. Autoantibodies and other pathogenic macromolecules targeted for removal by plasmapheresis can be depleted predictably when the plasma is discarded, as in conventional TPE. On-line plasma processing to regenerate the patient's own plasma avoids the need for replacement albumin solutions or plasma transfusion, but is inherently less efficient at removing the target molecule, so usually requires a longer procedure. Therapeutic white cell reduction (leukapheresis), platelet reduction (thrombocytapheresis) and red cell exchange (erythrocytapheresis) require centrifugal apheresis systems.