HLAMatchmaker is effective for selecting appropriate platelet units for alloimmunised thrombocytopaenic patients who are refractory to random donor platelets.

BACKGROUND: HLAMatchmaker has been used in the Irish Blood Transfusion Service (IBTS) to select platelets for HLA-alloimmunised, platelet refractory thrombocytopaenic patients since 2006. Although available since 2002, only three studies have been published supporting the programme's effectiveness for this indication. OBJECTIVES: We sought to examine increments to HLA-matched platelets (HMPs) at various matchmaker scores and to examine the impact of transfusing older platelets and ABO-mismatched platelets to this patient group. METHODS/MATERIALS: A total of 20 consecutive HLA-alloimmunised thrombocytopaenic patients were retrospectively studied. Data collected included: pre- and post-transfusion platelet count, indication for transfusion, HLAMatchmaker score, age of unit and degree of ABO mismatch. Data were also collected on the last 3 U of (RDPs). The Mann-Whitney U-test was used to compare increments between transfusion episodes of random donor platelets (RDPs) vs HMPs with matchmaker scores <3, 4-7 or >8. RESULTS: Increments at <2 h were available for 63 transfusion episodes. Increments at 2-24 h were available for 93 transfusion episodes. Increments were higher when transfusing HMPs than when transfusing RDPs. Increments for HMPs that were ABO mismatched were no different than for ABO-identical units, with the exception of late increments post-transfusing HMPs with a major ABO mismatch. Age of platelets did not influence increments. CONCLUSION: The use of HLAMatchmaker to select platelet units for thrombocytopaenic HLA-alloimmunised patients produced satisfactory platelet increments. When providing HLAMatchmaker-selected HMPs, ABO mismatch and the use of platelet units that are up to 7 days old should be permissible.

Red cell alloimmunisation following intrauterine transfusion and the feasibility of providing extended phenotype-matched red cell units.

OBJECTIVES: To analyse the incidence of additional alloantibody formation following intrauterine red cell transfusion and to evaluate the feasibility of providing extended phenotype-matched red cells in future intrauterine transfusion (IUT). BACKGROUND: IUT is performed in severe, life-threatening fetal anaemia, usually in alloimmunised pregnancies. Its complications include the formation of additional alloantibodies to other red cell antigens. MATERIALS AND METHODS: This was an 11-year retrospective, observational study of additional alloantibody formation in patients receiving IUT in the National Maternity Hospital, Dublin. The study included evaluation of the donor population in the Republic of Ireland (RoI) with regards to the feasibility of providing extended phenotype-matched units in future IUT. RESULTS: Following IUT, 22% of mothers formed additional red cell alloantibodies. In 67% of cases, the transfused donor red cells expressed the cognate antigen. Suitable donors are available for most combinations of Fy, Jk and Ss antigens. CONCLUSIONS: In our population, it is feasible to provide more extensively phenotype-matched red cells for future IUT. These can be supplied from the current donor pool with no significant extra phenotyping required. We consider their provision to be a reasonable proactive step in a known at-risk group.

ACORN2: new developments of the ACORN concept.

The density-modification procedures incorporated in ACORN, available in the CCP4 package, have proved to be very successful in solving and refining high-resolution crystal structures from very poor starting sets. These can be calculated from a correctly positioned initial fragment containing between 1 and 8% of the scattering power of the total structure. Improvements of ACORN, reported here and incorporated in the program ACORN2, have lowered the size of the fragment required and examples are given of structures solved with only 0.25% of the scattering power in the fragment, which may be a single atom. Applications of ACORN2 to structures with space group P1 have shown the remarkable property that when the starting point is a pair of equal atoms, or even a single atom placed at the origin, the refinement process breaks the centric nature of the initial phases and converges to phases corresponding to one of the two possible enantiomorphs. Examples are given of the application of ACORN2 to the solution and/or refinement of a number of known trial structures and to the refinement of structures when phases are available either from MAD or from a molecular-replacement model.

ACORN: a review.

The ACORN system was originally developed as a means of ab initio solution of protein structures when atomic resolution data were available. The first step is to obtain a starting set of phases, which must be at least slightly better than random. These may be calculated from a fragment of the structure, which can be anything from a single metal atom to a complete molecular-replacement model. A number of standard procedures are available in ACORN to orientate and position such a fragment. The fragment provides initial phases that give the first of a series of maps that are iteratively refined by a dynamic density-modification (DDM) process. Another FFT-based procedure is Sayre-equation refinement (SER), which modifies phases better to satisfy the Sayre equation. With good-quality atomic resolution data, the final outcome of applying DDM and SER is a map similar in appearance to that found from a refined structure, which is readily interpreted by automated procedures. Further development of ACORN now enables structures to be solved with less than atomic resolution data. A critical part of this development is the artificial extension of the data from the observed limit to 1 A resolution. These extended reflections are allocated unit normalized structure amplitudes and then treated in a similar way to observed reflections except that they are down-weighted in the calculation of maps. ACORN maps, especially at low resolution, tend to show C atoms less well, in particular C(alpha) atoms which fall within the first diffraction minimum of their three neighbours. Two new density-modification procedures (DDM1 and DDM2) and a density-enhancement procedure (ENH) have been devised to counter this problem. It is demonstrated that high-quality maps showing individual atoms can be produced with the new ACORN. ACORN has also been demonstrated to be very effective in refining phase sets derived from physical processes such as those using anomalous scattering or isomorphous derivative data. Future work will be directed towards applying ACORN to resolutions down to 2 A.

Simulation of the generation and processing of Doppler ultrasound fetal heart signals to obtain directional motion information.

In fetal heart monitoring using Doppler ultrasound signals the cardiac information is commonly extracted from non-directional signals. As a consequence often some of the cardiac events cannot be observed clearly which may lead to the incorrect detection of the valve and wall motions. Here, directional signals were simulated to investigate their enhancement of cardiac events, and hence provide clearer information regarding the cardiac activities. First, fetal Doppler ultrasound signals were simulated with signals encoding forward and reverse motion then obtained using a pilot frequency. The simulation results demonstrate that the model has the ability to produce realistic Doppler ultrasound signals and a pilot frequency can be used in the mixing process to produce directional signals that allow the simulated cardiac events to be distinguished clearly and correctly.

A modified ACORN to solve protein structures at resolutions of 1.7 A or better.

ACORN has previously been shown to provide an efficient density-modification procedure for the solution of protein structures using diffraction data to better than 1.3 A. The initial phase set could be obtained from a variety of sources such as the position of a heavy atom, a set of scatterers such as S that had been positioned from anomalous dispersion measurements, a fragment or a very low homology model placed from a molecular-replacement search. Several structures solved using the early version of ACORN have been reported in the literature. Here, the effect of applying the original ACORN procedures at lower resolution is reported and new procedures that yield good-quality maps with data sets of resolution down to 1.7 A are described. These new procedures involve the artificial extension of data to atomic resolution and new density-modification processes that develop density at atomic positions that was previously suppressed. The test calculations were aimed firstly towards a proof of principle using a small fragment of a known structure to demonstrate that the procedure could generate correct density and a derived model in initially empty regions of the cell. Further tests addressed the use of more realistic starting models.

Detrended fluctuation analysis: a suitable method for studying fetal heart rate variability?

We evaluate the suitability of an enhanced detrended fluctuation analysis for studying fetal heart rate series involving imperfect quality of information. Our results indicate that to explore persistent long-range correlations, or fractality, the collection requirements of the data can be relaxed by allowing the possibility of using averaged fetal heart rate series. In addition, it also appears feasible to employ, without producing major alterations in the long-range scaling behaviour, fragmented fetal heart rate series involving up to 50% of random missing values, or up to 50 min of consecutive missing samples in recordings of approximately equal to 8 h length. These are crucial advantages to overcome the often variable quality of fetal data. Consequently, these findings may open the possibility of obtaining information concerning the development of neural processes from fetal heart rate series, despite their non-stationary and fragmented nature.

Application of region-based segmentation and neural network edge detection to skin lesions.

This paper proposes two approaches to the skin lesion image segmentation problem. The first is a mainly region-based segmentation method where an optimal threshold is determined iteratively by an isodata algorithm. The second method proposed is based on neural network edge detection and a rational Gaussian curve that fits an approximate closed elastic curve between the recognized neural network edge patterns. A quantitative comparison of the techniques is enabled by the use of synthetic lesions to which Gaussian noise is added. The proposed techniques are also compared with an established automatic skin segmentation method. It is demonstrated that for lesions with a range of different border irregularity properties the iterative thresholding method provides the best performance over a range of signal to noise ratios. Iterative thresholding technique is also demonstrated to have similar performance when tested on real skin lesions.

Independent component extraction methods in biosignal processing.

The paper discusses methods for independent source identification within multiple channels electroencephalographical (EEG) signals recordings. The focus is to compare the independent component analysis (ICA) technique to a novel proposed method for individual components separation - the phase space method (PSM). Methods are suitable to be used for any multi-lead signal especially within biomedical signals processing area where independence is a key issue.

Interpretation of heart rate variability via detrended fluctuation analysis and alphabeta filter.

Detrended fluctuation analysis (DFA), suitable for the analysis of nonstationary time series, has confirmed the existence of persistent long-range correlations in healthy heart rate variability data. In this paper, we present the incorporation of the alphabeta filter to DFA to determine patterns in the power-law behavior that can be found in these correlations. Well-known simulated scenarios and real data involving normal and pathological circumstances were used to evaluate this process. The results presented here suggest the existence of evolving patterns, not always following a uniform power-law behavior, that cannot be described by scaling exponents estimated using a linear procedure over two predefined ranges. Instead, the power law is observed to have a continuous variation with segment length. We also show that the study of these patterns, avoiding initial assumptions about the nature of the data, may confer advantages to DFA by revealing more clearly abnormal physiological conditions detected in congestive heart failure patients related to the existence of dominant characteristic scales.

Application of empirical mode decomposition to heart rate variability analysis.

The analysis of heart rate variability, involving changes in the autonomic modulation conditions, demands specific capabilities not provided by either parametric or non-parametric spectral estimation methods. Moreover, these methods produce time-averaged power estimates over the entire length of the record. Recently, empirical mode decomposition and the associated Hilbert spectra have been proposed for non-linear and non-stationary time series. The application of these techniques to real and simulated short-term heart rate variability data under stationary and non-stationary conditions is presented. The results demonstrate the ability of empirical mode decomposition to isolate the two main components of one chirp series and three signals simulated by the integral pulse frequency modulation model, and consistently to isolate at least four main components localised in the autonomic bands of 14 real signals under controlled breathing manoeuvres. In addition, within the short time-frequency range that is recognised for heart rate variability phenomena, the Hilbert amplitude component ratio and the instantaneous frequency representation are assessed for their suitability and accuracy in time-tracking changes in amplitude and frequency in the presence of non-stationary and non-linear conditions. The frequency tracking error is found to be less than 0.22% for two simulated signals and one chirp series.

A direct-method ab initio phasing of a protein, cupredoxin amicyanin, at 1.31 A resolution.

The direct-methods program MULTAN88 has been applied successfully to redetermine the structure of a protein, cupredoxin amicyanin, containing 808 non-H atom sites, one Cu atom and 132 ordered water molecules in the asymmetric unit using data at 1.31 A resolution. Starting with initially random phases, useful phase sets selected by figures of merit could be obtained from multiple trials. The E maps corresponding to the best eight phase sets in order of combined figures of merit (CFOM2) revealed a distorted tetrahedral geometry around the Cu site. The phase estimates from the metal and a few neighbouring atoms in the initial E map corresponding to the set with the highest CFOM2 could be improved by the density-modification procedure PERP and led to an interpretable electron-density map.

A direct-method ab initio phasing of a protein, pseudoazurin, at 1.55 A resolution.

The direct-method program MULTAN88 has been applied to solve a known protein, pseudoazurin, space group P6(5), unit-cell parameters a = b = 50.0 (1), c = 98.5 (3) A, with 917 protein atoms, a Cu atom and 93 solvent water molecules in the asymmetric unit (>6,000 non-H atoms in the unit cell) and data at 1.55 A resolution. One of several trials with sets of initially random phases yielded phase estimates for 1,000 reflections with a mean phase error of 68.3 degrees that was recognized as the best available solution by the figure of merit being used. Phase extension to 2,000 largest Es showed a distorted tetrahedral geometry around the Cu site. Density modification was applied to improve the phases and the quality of the maps, starting with phases calculated from the atomic positions indicated by the first map.

A flexible and efficient procedure for the solution and phase refinement of protein structures.

An ab initio method is described for solving protein structures for which atomic resolution (better than 1.2 A) data are available. The problem is divided into two stages. Firstly, a substructure composed of a small percentage ( approximately 5%) of the scattering matter of the unit cell is positioned. This is used to generate a starting set of phases that are slightly better than random. Secondly, the full structure is developed from this phase set. The substructure can be a constellation of atoms that scatter anomalously, such as metal or S atoms. Alternatively, a structural fragment such as an idealized alpha-helix or a motif from some distantly related protein can be orientated and sometimes positioned by an extensive molecular-replacement search, checking the correlation coefficient between observed and calculated structure factors for the highest normalized structure-factor amplitudes |E|. The top solutions are further ranked on the correlation coefficient for all E values. The phases generated from such fragments are improved using Patterson superposition maps and Sayre-equation refinement carried out with fast Fourier transforms. Phase refinement is completed using a novel density-modification process referred to as dynamic density modification (DDM). The method is illustrated by the solution of a number of known proteins. It has proved fast and very effective, able in these tests to solve proteins of up to 5000 atoms. The resulting electron-density maps show the major part of the structures at atomic resolution and can readily be interpreted by automated procedures.

On the application of phase relationships to complex structures. XXXVI: some experiments with a small protein without heavy atoms.

The direct-methods program MULTAN88 has been applied to a known protein, ribonuclease (RNAP1), containing 808 non-H atoms, including five S atoms, plus 83 ordered solvent water molecules. Phase sets with mean phase errors between 69 and 75 degrees were selected by modified figures of merit for trials with the full data at 1.17 A resolution and also with restricted data at 1.25 and 1.5 A resolution. These figures of merit had previously only been applied to protein structures containing heavy atoms, and this is the first demonstration of their usefulness with no heavy atom present. An initial set of 1091 phases from a 1.17 A trial was developed by an objective procedure to give the full structure with a residual of 0. 21, which agrees well with the published structure.

Detection of fetal electrocardiogram signals using matched filters with adaptive normalisation.

The authors discuss the application of matched litters to the detection of R-waves in fetal electrocardiogram (FECG) data, recorded during labour using a scalp electrode. When using the basic matched filter, one correlates a template representing the clean signal with the noisy signal. This method is optimal when the underlying noise is white in nature. However, it is known that false detection of R-waves can occur in the presence of extraneous peaks which have a similar shape to the fetal R-wave. It is proposed to switch between two different normalisations of the impulse response of the matched filter to alleviate this problem. When the signal-to-noise ratio is lower than a predetermined threshold, then normalisation to the geometric mean of the template and noisy data energies is carried out, otherwise only normalisation to the template energy is made. In the former case, the background noise and spikes that are larger than the underlying FECG are attenuated, hence increasing the probability of detection of the R-waves. In the latter case, noise, which has a lower amplitude than the underlying R-wave, is reduced. The effectiveness of this method is demonstrated by application to scalp electrode data.

Channels in the gramicidin S-with-urea structure and their possible relation to transmembrane ion transport.

The structure of membrane-active antibiotic cyclodecapeptide gramicidin S in the crystals of its complex with urea, C(60)H(92)N(12)0(10).0.(5)[(NH(2))(2)CO].7.94H(2)0, has been investigated with three-dimensional X-ray data by the automatic sequential approximation method. The crystals are trigonal, space group P3(1)21, a = 25.80(3), c= 21.49 (2) A, M(r) = 7968, calculated density = 1.088 mg m(-3), Z = 1. Conventional R factor: R1 = 0.0943, wR2 = 0.2478 [I> 2sigma(I)]. The molecule possesses an antiparallel twisted beta-structure, with turns involving the Phe-Pro peptides. The Orn side chains extend on one side of the sheet, while the non-polar Val and Leu side chains are located on the other face. One of the Orn residues (namely Orn2) is linked by an intermolecular hydrogen bond to the O atom of Phe4 residue, the other is free. The side chains of the Phe residues have trans orientation (chi(1) approximately 180 degrees ) and those of the Val, Orn, Leu residues, except those of Orn2, have the preferential gauche orientation with the chi(1) angle close to 60. Two side chains show statistical disorder and conformation of the Pro residues is C(s)-C(beta)-exo. There is half a urea molecule and also 7.94 water molecules distributed on 13 positions for each antibiotic molecule. A partially occupied and poorly ordered alcohol molecule had been identified. The gramicidin S molecules are arranged around the 3(1) axis in the form of a left-handed double spiral forming suggestive channels. The outer hydrophobic surface of the spiral is made of uncharged side radicals while the inside surface consists of the main-chain atoms, mainly O and N, and of ornithine side chains with N atoms at the ends. By changing the Orn side-chain conformation, the inner diameter of the channels may change from 3.4 to 6.3 A. Thus, ions and particles of rather large size may pass through the channel. The possibility of the creation of the gramicidin S channels in mitochondrial membranes has been noted by some biochemists. The channel complexes are close-packed in a hexagonal arrangement in the crystal. The CI(-) ions, present in abundance in the mother solution, are not found ordered in the crystals, which may indicate the absence of the charges in the terminal N atoms of the Orn residues.

New techniques for applying anomalous-scattering and isomorphous-replacement data incorporated in ANOMIR - a general application package.

A computer package ANOMIR is described which can derive phases from anomalous scattering and/or isomorphous-replacement data in any combination. For anomalous scattering it incorporates five methods of applying one-wavelength data and three methods for multiple-wavelength data including SPIN, reported here for the first time. In addition there are three procedures for multiple-wavelength data - the first modifying data for different wavelengths to make them mutually consistent, the second estimating the contributions of the anomalous scatterers alone and the third which finds anomalous differences. For single isomorphous replacement or one-wavelength anomalous scattering the phase ambiguity can be resolved by the direct method [Fan, Han, Qian & Yao (1984). Acta Cryst. A40, 489- 495] but for multiple isomorphous replacement the main method is an adaptation of the probability-curve method [Blow & Crick (1959). Acta Cryst. 12, 794-802]. A new statistical method is described for estimating the standard error in measuring magnitudes which is independent of having subsets of centric reflections. A method is described whereby the weights associated with phase estimates are used to generate probability curves, through which it is possible to combine estimates from different methods and to produce a 'best phase' and figure-of-merit for every reflection. ANOMIR procedures are also available for handling combinations of one-wavelength anomalous scattering with single- or multiple-isomorphous replacement. A final process, which is always beneficial, is a single parallel application of the tangent formula. The ANOMIR package has been designed for easy use and is controlled throughout by KEYWORDS. Results for several structures are given and compared with those found from the MLPHARE program in the CCP4 package.

Direct-space methods in phase extension and phase refinement. V. The histogram moments method.

Any distribution is completely defined by its moments. It is shown that a process of phase refinement can be carried out, based on Fourier transforms, which modifies the moments of electron density, separately in the protein and solvent regions, towards target values. Tests have been carried out on two moderate-sized proteins with 800-900 atoms in the asymmetric unit, one containing heavy atoms and the other not. It has been found that refinement using the third moment about zero in the protein region is most effective and that refinement with higher moments, or in the solvent region, adds nothing useful. Two kinds of weights are necessary in the method. One is for giving a weighted mixture of new phase indications with original phase estimates from, say, multiple isomorphous replacement. The other weights are applied to the Fourier coefficients of density maps to give the best possible signal:noise ratio. These weights have been explored empirically and the best ones found are described. It is concluded that since the moments method, which changes phases in reciprocal space, is independent of other histogram-matching procedures, which change density in real space, it has something to offer in a refinement package containing several procedures.