Comparison of methods for assessing abdominal adipose tissue from magnetic resonance images

Objective: To compare the inter‐rater and intra‐rater reliability and analysis time of two methods for quantifying visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) volumes from magnetic resonance (MR) images. Research Methods and Procedures: Ten subjects (BMI, 27.0 ± 2.1 kg/m²; 56 years of age ± 4 years) underwent MR imaging of the abdomen. Ten transverse T1‐weighted images were selected from each scan and analyzed using two software packages that differ in principle. The first method, ANALYZE version 5.0, represents the manual threshold method, and the second, HIPPO version 1.3, is based on the fuzzy clustering approach. Inter‐rater reliability for each method was assessed by comparing the intra‐class correlation coefficients (ICCs) for VAT and SAT results from two evaluators, and intra‐rater reliability for each method was assessed by comparing ICCs for VAT and SAT analyses performed 1 week apart by the same evaluator. The total time for analysis also was compared between methods. Results: The inter‐rater reliability for VAT was greater with HIPPO than with ANALYZE (ICC = 0.996 vs. 0.828), whereas inter‐rater reliability for SAT did not differ between methods (ICC = 0.975 and 0.987). The intra‐rater reliability was equally high with HIPPO and ANALYZE for both VAT (ICC = 0.998 vs. 0.992) and SAT (ICC = 0.996 vs. 0.992). HIPPO required less than one‐half as much analysis time as ANALYZE (15.9 ± 4.4 vs. 36.5 ± 8.2 minutes, p < 0.0001). Discussion: HIPPO software appears advantageous for the quantification of VAT from multislice MR images because inter‐rater results are more reliable, and it is more time‐efficient than less automated methods.     

Efficiency considerations in the analysis of inter-observer agreement

The reliability of binary assessments is often measured by the proportion of agreement above chance, as estimated by the kappa statistic. In this paper, we develop a model to estimate inter-rater and intra-rater reliability when each of the two observers has the opportunity to obtain a pair of replicate measurements on each subject. The model is analogous to the nested beta-binomial model proposed by Rosner (1989, 1992). We show that the gain in precision obtained from increasing the number of measurements per rater from one to two may allow fewer subjects to be included in the study with no net loss in efficiency for estimating the inter-rater reliability.     

Intra-Rater and Inter-Rater Reliability of a Medical Record Abstraction Study on Transition of Care after Childhood Cancer

BackgroundThe abstraction of data from medical records is a widespread practice in epidemiological research. However, studies using this means of data collection rarely report reliability. Within the Transition after Childhood Cancer Study (TaCC) which is based on a medical record abstraction, we conducted a second independent abstraction of data with the aim to assess a) intra-rater reliability of one rater at two time points; b) the possible learning effects between these two time points compared to a gold-standard; and c) inter-rater reliability.MethodWithin the TaCC study we conducted a systematic medical record abstraction in the 9 Swiss clinics with pediatric oncology wards. In a second phase we selected a subsample of medical records in 3 clinics to conduct a second independent abstraction. We then assessed intra-rater reliability at two time points, the learning effect over time (comparing each rater at two time-points with a gold-standard) and the inter-rater reliability of a selected number of variables. We calculated percentage agreement and Cohen’s kappa.FindingsFor the assessment of the intra-rater reliability we included 154 records (80 for rater 1; 74 for rater 2). For the inter-rater reliability we could include 70 records. Intra-rater reliability was substantial to excellent (Cohen’s kappa 0-6-0.8) with an observed percentage agreement of 75%-95%. In all variables learning effects were observed. Inter-rater reliability was substantial to excellent (Cohen’s kappa 0.70-0.83) with high agreement ranging from 86% to 100%.ConclusionsOur study showed that data abstracted from medical records are reliable. Investigating intra-rater and inter-rater reliability can give confidence to draw conclusions from the abstracted data and increase data quality by minimizing systematic errors.     

Reliability and intermethod agreement for body fat assessment among two field and two laboratory methods in adolescents

To increase knowledge about reliability and intermethods agreement for body fat (BF) is of interest for assessment, interpretation, and comparison purposes. It was aimed to examine intra- and inter-rater reliability, interday variability, and degree of agreement for BF using air-displacement plethysmography (Bod-Pod), dual-energy X-ray absorptiometry (DXA), bioelectrical impedance analysis (BIA), and skinfold measurements in European adolescents. Fifty-four adolescents (25 females) from Zaragoza and 30 (14 females) from Stockholm, aged 13-17 years participated in this study. Two trained raters in each center assessed BF with Bod-Pod, DXA, BIA, and anthropometry (DXA only in Zaragoza). Intermethod agreement and reliability were studied using a 4-way ANOVA for the same rater on the first day and two additional measurements on a second day, one each rater. Technical error of measurement (TEM) and percentage coefficient of reliability (%R) were also reported. No significant intrarater, inter-rater, or interday effect was observed for %BF for any method in either of the cities. In Zaragoza, %BF was significantly different when measured by Bod-Pod and BIA in comparison with anthropometry and DXA (all P < 0.001). The same result was observed in Stockholm (P < 0.001), except that DXA was not measured. Bod-Pod, DXA, BIA, and anthropometry are reliable for %BF repeated assessment within the same day by the same or different raters or in consecutive days by the same rater. Bod-Pod showed close agreement with BIA as did DXA with anthropometry; however, Bod-Pod and BIA presented higher values of %BF than anthropometry and DXA.     

Multiple regression for molecular-marker,quantitative trait data from large F2 populations

Molecular marker-quantitative trait associations are important for breeders to recognize and understand to allow application in selection. This work was done to provide simple, intuitive explanations of trait-marker regression for large samples from an F₂ and to examine the properties of the regression estimators. Beginning with a(- 1,0,1) coding of marker classes and expected frequencies in the F₂, expected values, variances, and covariances of marker variables were calculated. Simple linear regression and regression of trait values on two markers were computed. The sum of coefficient estimates for the flanking-marker regression is asymptotically unbiased for an included additive effect with complete interference, and is only slightly biased with no interference and moderately close (15 cM) marker spacing. The variance of the sum of regression coefficients is much more stable for small recombination distances than variances of individual coefficients. Multiple regression of trait variables on coded marker variables can be interpreted as the product of the inverse of the marker correlation matrix R and the vector a of simple linear regression estimators for each marker. For no interference, elements of the correlation matrix R can be written as products of correlations between adjacent markers. The inverse of R is displayed and used to illustrate the solution vector. Only markers immediately flanking trait loci are expected to have non-zero values and, with at least two marker loci between each trait locus, the solution vector is expected to be the sum of solutions for each trait locus. Results of this work should allow breeders to test for intervals in which trait loci are located and to better interpret results of the trait-marker regression.     

Validity and reliability of a visual scoring method for masticatory ability using test gummy jelly

Background: For quantitative evaluation of masticatory ability of the elderly patients, there should be a simple and reliable method without special techniques and instruments. Objective: The purpose of this study was to examine the validity and reliability of a visual scoring method for assessing masticatory performance. Materials and Methods: A 10‐stage scale for visually scoring was rated based on the range of the glucose concentration dissolved from comminuted jelly. Photographic images of comminuted jellies were produced as a standard material for each score. Fifty subjects were recruited as raters who graded the visual score for 50 photographic images of comminuted jellies on the screen of a lap‐top three times in random order. Results: There were strong correlations (rs = 0.911– 0.981, Spearman’s rank coefficient) between the actual scores determined from the glucose concentration and the visual scores graded by subjects in all three measurements. The intraclass correlation coefficients (ICCs) of the inter‐rater reliability and the ICCs of the intra‐rater reliability of the visual scoring ranged from 0.946 to 0.947 and from 0.860 to 0.987 in three measurements, respectively. Conclusions: These results indicated that the visual scoring method was valid and reliable for evaluation of masticatory performance.     

Step matrices and the interpretation of homoplasy

Assumptions about the costs of character change, coded in the form of a step matrix, determine most-parsimonious inferences of character evolution on phylogenies. We present a graphical approach to exploring the relationship between cost assumptions and evolutionary inferences from character data. The number of gains and losses of a binary trait on a phylogeny can be plotted over a range of cost assumptions, to reveal the inflection point at which there is a switch from more gains to more losses and the point at which all changes are inferred to be in one direction or the other. Phylogenetic structure in the data, the tree shape, and the relative frequency of states among the taxa influence the shape of such graphs and complicate the interpretation of possible permutation-based tests for directionality of change. The costs at which the most-parsimonious state of each internal node switches from one state to another can also be quantified by iterative ancestral-state reconstruction over a range of costs. This procedure helps identify the most robust inferences of change in each direction, which should be of use in designing comparative studies.     

Exotic Dreams: A Cross-Cultural Study

The purpose of this study was to identify exotic (i.e., puzzling, unusual, extraordinary, anomalous) dreams in a sample of 1,666 dream reports from six countries, and to make gender comparisons as well. Research participants were members of dream seminars that one of us conducted between 1990 and 1998 in Argentina, Brazil, Japan, Russia, Ukraine, and the United States. Only one dream report per participant was utilized, 910 dream reports from women and 756 from men. Scoring criteria were determined in advance for creative, lucid, healing, dreams within dreams, out-of-body, telepathic, mutual (and shared), clairvoyant, precognitive, past-life, initiation, and visitation dreams. When a dream fell into two categories, it received a score of 0.5 for each of the categories, rather than a score of 1.0, awarded when a dream represented a single category. In the sample of 1,666 dreams, there were 135 (8.1%) exotic dreams. Female dreamers reported 77 (8.5% of all female reports) exotic dreams, while male dreamers reported 58 (7.7% of all male reports), the difference was not statistically significant. The country with the highest percentage of exotic dream reports was Russia (12.7% of the total number reported by Russian seminar participants), followed by Brazil (10.9%), Argentina (9.0%), Japan (8.1%), Ukraine (5.9%), and the United States (5.7%). When chi square statistics were applied, it was found that Russian dreamers reported significantly more exotic dreams than dreamers in Ukraine or the United States.     

Personality in nonhuman primates: a review and evaluation of past research

Scientific reports of personality in nonhuman primates are now appearing with increasing frequency across a wide range of disciplines, including psychology, anthropology, endocrinology, and zoo management. To identify general patterns of research and summarize the major findings to date, we present a comprehensive review of the literature, allowing us to pinpoint the major gaps in knowledge and determine what research challenges lay ahead. An exhaustive search of five scientific databases identified 210 relevant research reports. These articles began to appear in the 1930s, but it was not until the 1980s that research on primate personality began to gather pace, with more than 100 articles published in the last decade. Our analyses of the literature indicate that some domains (e.g., sex, age, rearing conditions) are more evenly represented in the literature than are others (e.g., species, research location). Studies examining personality structure (e.g., with factor analysis) have identified personality dimensions that can be divided into 14 broad categories, with Sociability, Confidence/Aggression, and Fearfulness receiving the most research attention. Analyses of the findings pertaining to inter‐rater agreement, internal consistency, test–retest reliability, generally support not only the reliability of primate personality ratings scales but also point to the need for more psychometric studies and greater consistency in how the analyses are reported. When measured at the level of broad dimensions, Extraversion and Dominance generally demonstrated the highest levels of inter‐rater reliability, with weaker findings for the dimensions of Agreeableness, Emotionality, and Conscientiousness. Few studies provided data with regard to convergent and discriminant validity; Excitability and Dominance demonstrated the strongest validity coefficients when validated against relevant behavioral criterion measures. Overall, the validity data present a somewhat mixed picture, suggesting that high levels of validity are attainable, but by no means guaranteed. Discussion focuses on delineating major theoretical and empirical questions facing research and practice in primate personality. Am. J. Primatol. 72:653–671, 2010. © 2010 Wiley‐Liss, Inc.     

Reducing the number of microlocations in oligonucleotide microchip matrices by the application of degenerate oligonucleotides

The application of degenerate oligonucleotides to DNA Sequencing by Hybridisation with Oligonucleotide Matrix (SHOM) is proposed. The use of degenerate oligonucleotides is regarded as an example of pooling methods that are suitable for various laboratory procedures requiring numerous samples to be assayed. As each DNA sequence coded by four letters (A, G, C, T) may be defined by two sequences: a sequence coded by W and S (W-weak-A or T, S-strong-G or C) and a sequence coded by R and Y (R-purine-A or G, Y-pirymidine-T or C), n4n -nucleotide sequences may be defined with the help of 2xn2sequences. In the place of the originally described microchip matrix composed of all possible unambiguous octanucleotides (4(8)=65 536) attached to the equal number of 65 536 microlocations a matrix composed of 512 microlocations containing 256 2(8)-degenerate octanucleotides is proposed. The matrix contains all 256 possible octanucleotides coded by W and S variations and all 256 possible octanucleotides coded by R and Y variations. The 512 256-degenerate octanucleotides allows to retrieve the same information as 65 536 unambiguous octanucleotides. A variant of the DNA sequence reconstruction method applicable to this system is presented. The use of degenerate oligonucleotides also gives the possibility to apply matrices composed of longer oligonucleotides without increasing the number of microlocations in matrices, which would enable increasing the length of unambiguously reconstructed sequence, e.g. a matrix comprising 131 072 16-mer oligonucleotides i.e. 65 536 65 536-fold degenerate oligonucleotide coded by W and S variations and 65 536 65 536-fold degenerate oligonucleotide coded by R and Y variations could replace one matrix comprising all possible unambiguous 16-mer oligonucleotides (ca. 4.3x10(9)).     

The ZZP Questionnaire. Reliability of a new resource utilization measure

Data to determine the resource utilization of care recipients need to be reliable and the items that are measured need to be useful. In 2006, the Dutch Ministry of Health and Welfare has mandated all nursing homes and homes for the elderly to measure the Resource Utilization of all residents with the ZZP Questionnaire. Are the data resulting from this measurement reliable and is each of the 54 items of the ZZP Questionnaire useful? To answer this we tested the reliability of the data in a nursing home and a home for the elderly in two wards each. For 122 residents questionnaires were completed such that the inter- and intra-rater reliability of the answers could be assessed. Ten of the 54 items in the questionnaire showed insufficient inter rater reliability (<0.40) on the weighted Cohen kappa and another sixteen moderate (0.40 - 0.60). On the intra rater reliability test seven items had an insufficient kappa and another fifteen moderate. Besides, ten clusters of items could be formed with in-cluster Spearman correlation rates of .75 or higher. From the results of the reliability tests and the item intercorrelation rates we concluded that a substantial number of items needs to be improved and that in the ZZP Questionnaire 15 of the 54 items appear to be redundant on statistical grounds.     

Use of Multiple Genetic Markers in Prediction of Breeding Values

Genotypes at a marker locus give information on transmission of genes from parents to offspring and that information can be used in predicting the individuals' additive genetic value at a linked quantitative trait locus (MQTL). In this paper a recursive method is presented to build the gametic relationship matrix for an autosomal MQTL which requires knowledge on recombination rate between the marker locus and the MQTL linked to it. A method is also presented to obtain the inverse of the gametic relationship matrix. This information can be used in a mixed linear model for simultaneous evaluation of fixed effects, gametic effects at the MQTL and additive genetic effects due to quantitative trait loci unlinked to the marker locus (polygenes). An equivalent model can be written at the animal level using the numerator relationship matrix for the MQTL and a method for obtaining the inverse of this matrix is presented. Information on several unlinked marker loci, each of them linked to a different locus affecting the trait of interest, can be used by including an effect for each MQTL. The number of equations per animal in this case is 2m + 1 where m is the number of MQTL. A method is presented to reduce the number of equations per animal to one by combining information on all MQTL and polygenes into one numerator relationship matrix. It is illustrated how the method can accommodate individuals with partial or no marker information. Numerical examples are given to illustrate the methods presented. Opportunities to use the presented model in constructing genetic maps are discussed.     

Care quality: reliability and usefulness of observation data in bench marking nursing homes and homes for the aged in the Netherlands

Before including quality of care indicators in the Benchmark of Nursing Homes and Homes for the Aged in the Netherlands the reliability of the patient data collection, and usefulness had to be established. The patient data items were derived from the Resident Assessment Instruments (RAI) and a questionnaire on social interaction in elderly people. Three nursing homes and three homes for the aged participated in the test with 550 patients. 279 x 2 assessments were collected by independent raters for an inter rater reliability test; 259 x 2 by the same rater for a reliability test-retest; and 24 by a single rater. The scores on paired assessment forms were compared with the weighted Kappa agreement test. The test results allowed 10 of the 13 quality indicators from RAI to be retained. In addition new quality indicators could be defined on 'giving attention' and 'unrespectful addressing'. We estimate on the basis of a questionnaire for the raters that on average 9 to 12 minutes per patient are needed to collect and enter data for the resulting 12 quality indicators.     

The Use of Global Rating Scales for OSCEs in Veterinary Medicine

OSCEs (Objective Structured Clinical Examinations) are widely used in health professions to assess clinical skills competence. Raters use standardized binary checklists (CL) or multi-dimensional global rating scales (GRS) to score candidates performing specific tasks. This study assessed the reliability of CL and GRS scores in the assessment of veterinary students, and is the first study to demonstrate the reliability of GRS within veterinary medical education. Twelve raters from two different schools (6 from University of Calgary [UCVM] and 6 from Royal (Dick) School of Veterinary Studies [R(D)SVS] were asked to score 12 students (6 from each school). All raters assessed all students (video recordings) during 4 OSCE stations (bovine haltering, gowning and gloving, equine bandaging and skin suturing). Raters scored students using a CL, followed by the GRS. Novice raters (6 R(D)SVS) were assessed independently of expert raters (6 UCVM). Generalizability theory (G theory), analysis of variance (ANOVA) and t-tests were used to determine the reliability of rater scores, assess any between school differences (by student, by rater), and determine if there were differences between CL and GRS scores. There was no significant difference in rater performance with use of the CL or the GRS. Scores from the CL were significantly higher than scores from the GRS. The reliability of checklist scores were .42 and .76 for novice and expert raters respectively. The reliability of the global rating scale scores were .7 and .86 for novice and expert raters respectively. A decision study (D-study) showed that once trained using CL, GRS could be utilized to reliably score clinical skills in veterinary medicine with both novice and experienced raters.     

Norovirus infections in preterm infants: wide variety of clinical courses

Background

The Clubfoot Assessment Protocol (CAP) was developed for follow-up of children treated for clubfoot. The objective of this study was to analyze reliability and validity of the six items used in the domain CAPMotion Quality using inexperienced assessors.

Findings

Four raters (two paediatric orthopaedic surgeons, two senior physiotherapists) used the CAP scores to analyze, on two different occasions, 11 videotapes containing standardized recordings of motion activity according to the domain CAPMotion Quality These results were compared to a criterion (two raters, well experienced CAP assessors) for validity and for checking for learning effect. Weighted kappa statistics, exact percentage observer agreement (Po), percentage observer agreement including one level difference (Po-1) and amount of scoring scales defined how reliability was to be interpreted. Inter- and intra rater differences were calculated using median and inter quartile ranges (IQR) on item level and mean and limits of agreement on domain level. Inter-rater reliability varied between fair and moderate (kappa) and had a mean agreement of 48/88% (Po/Po-1). Intra -rater reliability varied between moderate to good with a mean agreement of 63/96%. The intra- and inter-rater differences in the present study were generally small both on item (0.00) and domain level (-1.10). There was exact agreement of 51% and Po-1 of 91% of the six items with the criterion. No learning effect was found.

Conclusion

The CAPMotion quality can be used by inexperienced assessors with sufficient reliability in daily clinical practice and showed acceptable accuracy compared to the criterion.     

Betrouwbaarheid van de Zorgzwaartepakket (ZZP) scorelijst

The ZZP Questionnaire. Reliability of a new resource utilization measure. Data to determine the resource utilization of care recipients need to be reliable and the items that are measured need to be useful. In 2006, the Dutch Ministry of Health and Welfare has mandated all nursing homes and homes for the elderly to measure the Resource Utilization of all residents with the ZZP Questionnaire. Are the data resulting from this measurement reliable and is each of the 54 items of the ZZP Questionnaire useful? To answer this we tested the reliability of the data in a nursing home and a home for the elderly in two wards each. For 122 residents questionnaires were completed such that the inter- and intra-rater reliability of the answers could be assessed. Ten of the 54 items in the questionnaire showed insufficient inter rater reliability (<0.40) on the weighted Cohen kappa and another sixteen moderate (0.40 – 0.60). On the intra rater reliability test seven items had an insufficient kappa and another fifteen moderate. Besides, ten clusters of items could be formed with in-cluster Spearman correlation rates of .75 or higher. From the results of the reliability tests and the item intercorrelation rates we concluded that a substantial number of items needs to be improved and that in the ZZP Questionnaire 15 of the 54 items appear to be redundant on statistical grounds.Tijdschr Gerontol Geriatr 2007; 38: 166-173     

Psychometric Properties of the Problem Areas in Diabetes (PAID) Instrument in Singapore

Background

Emotional distress is an important dimension in diabetes, and several instruments have been developed to measure this aspect. The Problem Areas in Diabetes (PAID) scale is one such instrument which has demonstrated validity and reliability in Western populations, but its psychometric properties in Asian populations have not been examined.

Methods

This was a secondary analysis of data from patients with Type 2 diabetes mellitus recruited through convenience sampling from a diabetes specialist outpatient clinic in Singapore. The following psychometric properties were assessed: Construct validity through confirmatory factor analysis (CFA) and Rasch analysis, concurrent validity through correlation with related scales (Kessler Psychological Distress Scale, Diabetes Health Profile—psychological distress, Audit of Diabetes Dependent Quality of Life), reliability through assessment of internal consistency and floor and ceiling effects, and sensitivity by estimating effect sizes for known clinical and social functioning groups.

Results

203 patients with mean age of 45±12 years were analysed. None of the previously published model structures achieved a good fit on CFA. On Rasch analysis, four items showed poor fit and were removed. The abridged 16-item PAID mapped to a single latent trait, with a high degree of internal consistency (Cronbach ɑ 0.95), but significant floor effect (24.6% scoring at floor). Both 20-item and 16-item PAID scores were moderately correlated with scores of related scales, and sensitive to differences in clinical and social functioning groups, with large effect sizes for glycemic control and diabetes related complications, nephropathy and neuropathy.

Conclusion

The abridged 16-item PAID measures a single latent trait of emotional distress due to diabetes whereas the 20-item PAID appears to measures more than one latent trait. However, both the 16-item and 20-item PAID versions are valid, reliable and sensitive for use among Singaporean patients with diabetes.     

Updating the reference population to achieve constant genomic prediction reliability across generations

The reliability of genomic breeding values (DGV) decays over generations. To keep the DGV reliability at a constant level, the reference population (RP) has to be continuously updated with animals from new generations. Updating RP may be challenging due to economic reasons, especially for novel traits involving expensive phenotyping. Therefore, the goal of this study was to investigate a minimal RP update size to keep the reliability at a constant level across generations. We used a simulated dataset resembling a dairy cattle population. The trait of interest was not included itself in the selection index, but it was affected by selection pressure by being correlated with an index trait that represented the overall breeding goal. The heritability of the index trait was assumed to be 0.25 and for the novel trait the heritability equalled 0.2. The genetic correlation between the two traits was 0.25. The initial RP (n=2000) was composed of cows only with a single observation per animal. Reliability of DGV using the initial RP was computed by evaluating contemporary animals. Thereafter, the RP was used to evaluate animals which were one generation younger from the reference individuals. The drop in the reliability when evaluating younger animals was then assessed and the RP was updated to re-gain the initial reliability. The update animals were contemporaries of evaluated animals (EVA). The RP was updated in batches of 100 animals/update. First, the animals most closely related to the EVA were chosen to update RP. The results showed that, approximately, 600 animals were needed every generation to maintain the DGV reliability at a constant level across generations. The sum of squared relationships between RP and EVA and the sum of off-diagonal coefficients of the inverse of the genomic relationship matrix for RP, separately explained 31% and 34%, respectively, of the variation in the reliability across generations. Combined, these parameters explained 53% of the variation in the reliability across generations. Thus, for an optimal RP update an algorithm considering both relationships between reference and evaluated animals, as well as relationships among reference animals, is required.     

Integrating ambiguously aligned regions of DNA sequences in phylogenetic analyses without violating positional homology

Phylogenetic analyses of non-protein-coding nucleotide sequences such as ribosomal RNA genes, internal transcribed spacers, and introns are often impeded by regions of the alignments that are ambiguously aligned. These regions are characterized by the presence of gaps and their uncertain positions, no matter which optimization criteria are used. This problem is particularly acute in large-scale phylogenetic studies and when aligning highly diverged sequences. Accommodating these regions, where positional homology is likely to be violated, in phylogenetic analyses has been dealt with very differently by molecular systematists and evolutionists, ranging from the total exclusion of these regions to the inclusion of every position regardless of ambiguity in the alignment. We present a new method that allows the inclusion of ambiguously aligned regions without violating homology. In this three-step procedure, first homologous regions of the alignment containing ambiguously aligned sequences are delimited. Second, each ambiguously aligned region is unequivocally coded as a new character, replacing its respective ambiguous region. Third, each of the coded characters is subjected to a specific step matrix to account for the differential number of changes (summing substitutions and indels) needed to transform one sequence to another. The optimal number of steps included in the step matrix is the one derived from the pairwise alignment with the greatest similarity and the least number of steps. In addition to potentially enhancing phylogenetic resolution and support, by integrating previously nonaccessible characters without violating positional homology, this new approach can improve branch length estimations when using parsimony.