Rare (uro-)genital pathologies in young girls mimicking sexual abuse.

Examinations of young children for suspicions of sexual abuse are challenging for the involved medical specialists because the consequences of the interpretation of the findings can be severe and dramatic. A broad knowledge of differential diagnoses including rare pathologies like urethral prolapse and failure of the midline fusion of the perineum, known as perineal groove, is essential in order to avoid unnecessary diagnostics and treatment, prejudgment, and to reduce patient family's anxiety. We report two independent cases of girls aged 7 months and 5 years suffering from these rare pathologies, one presenting with painless lower genital tract bleeding, the other showing a lesion of the perineum as random finding during a neuropediatrician's consultation. In both cases, the pathologies were initially misdiagnosed as injuries due to sexual assault, and judicial investigation procedures were initiated. In this paper, the characteristic symptoms and morphology of urethral prolapse and perineal groove are presented to enhance the awareness of these pathologies among forensic experts and help to establish the correct diagnosis.

Two Cases of Fatal Inhalation of Easily Available "Recreational" Substances.

ABSTRACT: We present 2 cases of fatal inhalation of easily available highly volatile substances that occurred in a recreational context. Case 1 concerns an 18-year-old man who was found dead with a 25-L plastic bag pulled over his head and a whipped cream steel siphon connected to the bag. The deceased was known to previously have inhaled nitrous oxide. Autopsy results were unremarkable, toxicological analysis using static headspace gas chromatography coupled to mass spectrometry analysis proved the presence of nitrous oxide in lung tissue and blood. Asphyxiation was ascertained as the cause of death. Case 2 describes the death of a 54-year-old man found dead on his bed wearing a rubber gas mask. A bottle with ethyl chloride-containing cold spray was found beside him. Autopsy did not reveal relevant pathological findings; a subsequent toxicological analysis proved the presence of ethyl chloride. Respiratory arrest because of ethyl chloride inhalation was established as the cause of death. The 2 cases presented here demonstrate the danger of easily available, volatile substances with a high potential for abuse. A careful investigation of the death scene, proper specimen collection during the autopsy, and extensive toxicological tests, including headspace gas chromatography coupled to mass spectrometry analysis, are necessary to prove inhalation of these substances.

T2 and T2∗ mapping in ex situ porcine myocardium: myocardial intravariability, temporal stability and the effects of complete coronary occlusion.

Diagnosis of ischaemia-related sudden cardiac death in the absence of microscopic and macroscopic ischaemic lesions remains a challenge for medical examiners. Medical imaging techniques increasingly provide support in post-mortem examinations by detecting and documenting internal findings prior to autopsy. Previous studies have characterised MR relaxation times to investigate post-mortem signs of myocardial infarction in forensic cohorts. In this prospective study based on an ex situ porcine heart model, we report fundamental findings related to intramyocardial variability and temporal stability of T2 as well as the effects of permanent coronary occlusion on T2 and T2∗ relaxation in post-mortem myocardium. The ex situ porcine hearts included in this study (n= 19) were examined in two groups (Ss, n= 11 and Si, n= 8). All magnetic resonance imaging (MRI) examinations were performed ex situ, at room temperature and at 3 T. In the Ss group, T2 mapping was performed on slaughterhouse porcine hearts at different post-mortem intervals (PMI) between 7 and 26 h. Regarding the intramyocardial variability, no statistically significant differences in T2 were observed between myocardial segments (p= 0.167). Assessment of temporal stability indicated a weak negative correlation (r=- 0.21) between myocardial T2 and PMI. In the Si group, animals underwent ethanol-induced complete occlusion of the left anterior descending artery. T2 and T2∗ mapping were performed within 3 h of death. Differences between the expected ischaemic and remote regions were statistically significant for T2 (p= 0.007), however not for T2∗ (p= 0.062). Our results provide important information for future assessment of the diagnostic potential of quantitative MRI in the post-mortem detection of early acute myocardial infarction.

Post-mortem MR angiography: quantitative investigation and intravascular retention of perfusates in ex situ porcine hearts.

As the implementation of minimally invasive imaging techniques in both forensic and pathological practice increases, research in this area focuses on addressing recognised diagnostic weaknesses of current approaches. Assessment of sudden cardiac death (SCD) can be considered one such area in which post-mortem imaging still shows diagnostic weaknesses. We hypothesise that magnetic resonance imaging (MRI) with an angiographic adjunct may improve the visualisation and interpretation of cardiac pathologies in a post-mortem setting. To systematically investigate this hypothesis, selected perfusates (paraffin oil, Gadovist®;-doped physiological solution and polyethylene glycol (PEG)) were injected into the left anterior descending (LAD) artery of ex situ porcine hearts to assess the visualisation of perfusates in MRI as well as their intravascular retention over 12 h. Morphological images were acquired and quantitative T1 maps were generated from inversion recovery data. Visualisation of vascular structure and image quality were assessed using signal-to-noise and contrast-to-noise ratios. Intravascular retention was assessed both visually and statistically using a volume of interest (VOI) approach to analyse significant changes in signal intensity in and around the filled LAD artery, as well as changes in the longitudinal relaxation time (T1) in adjacent myocardium. In addition to presenting possible mechanisms explaining perfusate extravasation given the increased permeability of post-mortem vessels, the potential diagnostic consequences of this phenomenon and the importance of contrast stability and extended intravascular retention are discussed. In light of our findings and these considerations, paraffin oil emerged as the preferred perfusate for use in post-mortem MR angiography.

Temperature dependence of viscosity, relaxation times (T1, T2) and simulated contrast for potential perfusates in post-mortem MR angiography (PMMRA).

Developments in post-mortem imaging increasingly focus on addressing recognised diagnostic weaknesses, especially with regard to suspected natural deaths. Post-mortem MR angiography (PMMRA) may offer additional diagnostic information to help address such weaknesses, specifically in the context of sudden cardiac death. Complete filling of the coronary arteries and acceptable contrast with surrounding tissue are essential for a successful approach to PMMRA. In this work, the suitability of different liquids for inclusion in a targeted PMMRA protocol was evaluated. Factors influencing cooling of paraffinum liquidum + Angiofil® (6 %) in cadavers during routine multiphase post-mortem CT angiography were investigated. The temperature dependence of dynamic viscosity (8-20 °C), longitudinal (T1) and transverse (T2) relaxation (1-23 °C) of the proposed liquids was quadratically modelled. The relaxation behaviour of these liquids and MR scan parameters were further investigated by simulation of a radiofrequency (RF)-spoiled gradient echo (GRE) sequence to estimate potentially achievable contrast between liquids and post-mortem tissue at different temperatures across a forensically relevant temperature range. Analysis of the established models and simulations indicated that based on dynamic viscosity (27-33 mPa · s), short T1 relaxation times (155-207 ms) and a minimal temperature dependence over the investigated range of these parameters, paraffin oil and a solution of paraffin oil + Angiofil® (6 %) would be most suitable for post-mortem reperfusion and examination in MRI.

About the power of biostatistics in sibling analysis-comparison of empirical and simulated data.

The determination of potential sibship is a common task in routine kinship analysis, but often the putative parents are not available for analysis anymore. Then, a sibling analysis has to be conducted investigating only the potential siblings, thus reducing the power of the conclusion. In an attempt to determine meaningfulness of biostatistical calculations, 346 dizygotic twin pairs, 30 confirmed half siblings, and 112 unrelated people (to generate 6216 pair comparisons) were studied, all genetically typed using at least the Powerplex® 16 STRs. From every pair, the probabilities for a full sibship (identical parents) and half sibship (different fathers) were calculated using a commercially available computer program. Additionally, we simulated marker data for one million pairs of full sibs, half sibs, and unrelated persons each. Ninety-five percent of full sibling pairs demonstrated a likelihood ratio (LR) > 9 (W-value > 90 %) and less than 4% of these showed a LR < 3 (W-value < 75%) for full sibship after analysis of 15 STRs. The results for half siblings are less unambiguous. Here, only 57% achieved a LR > 9 and 23% a LR < 3. Regarding the unrelated pairs, more than 90% had a LR < 1/9 and only 2% reached a LR > 9. All in all, our results show that 15 to 20 STRs have sufficient power for analyses in kinship. Moreover, our data provide a statistical basis for the determination of the information content of a LR/W-value in a sibship case. Investigating an identical number of full siblings and unrelated pairs, it could be shown that 92% of pairs with a LR > 9 for full sibship probability really are full siblings. So, setting a cutoff level for full sibship at LR > 9, less than 10% of pairs will be wrongly assumed as full siblings even though they are unrelated.

The auditory ossicles as a DNA source for genetic identification of highly putrefied cadavers.

Genetic identification of putrefied bodies is a common task in forensic medicine. With advancing putrefaction, however, DNA integrity is rapidly decreasing and genetic typing of tissue might be impaired or impossible. Since DNA stability is generally higher in hard tissues, bones or teeth are frequently used as DNA source in such cases. However, isolation of DNA from hard tissues is usually very time-consuming and labor-intensive. This can be especially important in (forensic) cases where time is short and identification has to be carried out as fast as possible. Here, we present the identification of dead bodies by analyzing DNA from the auditory ossicles. These minuscule bones provided DNA of sufficient quality and quantity for identification purposes in all 40 investigated cases. Additionally, processing of the bones proved to be amazingly easy and fast, and a successful extraction is possible using a variety of different methods. We present a detailed protocol, results, and cases in which this new method has been successfully applied.

The new guidelines for paternity analysis in Germany-how many STR loci are necessary when investigating duo cases?

The requirements in the new German guidelines for paternity analysis have not only changed according to the so-called Gendiagnostikgesetz, the new German law regulating human genetic as well as paternity analyses, but also regarding the minimal number of short tandem repeats (STRs) which should be investigated (15 STRs) and the minimal required average exclusion chance (99.999 %). Even in paternity analyses involving only two people (e.g., father and child or mother and child), this exclusion chance is mandatory. A retrospective analysis of 330 father-child cases from our routine investigations showed in 142 cases (43 %) an individual exclusion chance below 99.999 % when using 15 STRs as required, in our routine work provided by the Powerplex® 16 kit which is reported to have an average exclusion chance of 99.988 %. Therefore, these same 330 father-child pairs were additionally analysed using the Powerplex® 21 kit and 120 of these duos were additionally analysed using the Powerplex® ESX17 kit enabling the analysis of 20 or 16 loci respectively. Now, an individual exclusion chance of more than 99.999 % could be achieved in 95.5 % (Powerplex® 21; calculation without the results of D6S1043), 98.8 % (Powerplex® 21; calculation with the results of D6S1043, using allele frequencies established in this study for a German and a West African population) and 98.3 % (Powerplex® ESX17). These data clearly demonstrate that in duo cases, more than the required 15 STR loci have to be investigated to obtain sufficient results.

The SNPforID Assay as a Supplementary Method in Kinship and Trace Analysis.

OBJECTIVE: Short tandem repeat (STR) analysis using commercial multiplex PCR kits is the method of choice for kinship testing and trace analysis. However, under certain circumstances (deficiency testing, mutations, minute DNA amounts), STRs alone may not suffice. METHODS: We present a 50-plex single nucleotide polymorphism (SNP) assay based on the SNPs chosen by the SNPforID consortium as an additional method for paternity and for trace analysis. The new assay was applied to selected routine paternity and trace cases from our laboratory. RESULTS AND CONCLUSIONS: Our investigation shows that the new SNP multiplex assay is a valuable method to supplement STR analysis, and is a powerful means to solve complicated genetic analyses.

Genetic identification of highly putrefied bodies using DNA from soft tissues.

The identification of putrefied bodies is a common task in forensic routine work. The deceased are usually identified by dental records, fingerprinting, or--in cases where no such data are available--DNA analysis. However, with progressive putrefaction, DNA integrity is rapidly decreasing. Genetic analysis may then be greatly impaired, if not impossible. The aim of our study was to establish an efficient procedure to successfully extract and amplify DNA from soft tissues of bodies in different stages of putrefaction. Soft tissues-unlike teeth or bones-usually allow the application of fast and easy-to-use extraction protocols. DNA was extracted from different tissues (aorta, kidney, liver, and skeletal muscle) taken at autopsy using a commercially available DNA extraction kit, and DNA quality and quantity were controlled by agarose gel electrophoresis and real-time polymerase chain reaction (PCR). Presence of mitochondrial DNA was tested using a highly sensitive duplex PCR. Short tandem repeat analysis was done using the AmpFlSTR Identifiler kit. Additionally, mitochondrial DNA sequencing was performed. After DNA extraction from at least two different tissues-preferably the kidney and the aorta-with the extraction kit based on the Nucleobond method, a successful amplification of at least eight loci was possible in 17 out of 18 cases, and 12 or more loci could be amplified in 15 cases.

First experiences using the new Powerplex® ESX17 and ESI17 kits in casework analysis and allele frequencies for two different regions in Germany.

DNA databases are the most efficient tools in criminal investigations with unknown perpetrators. Due to a significant number of random matches in cross-border DNA profile exchanges, the European Network of Forensic Science Institutes (ENFSI) proposed the addition of further short tandem repeats (STRs) to European DNA databases. Therefore, the new Powerplex® ESX17 and Powerplex® ESI17 kits from Promega comprised the 11 established DNA database STRs and additionally the well-known loci D1S1656 and D12S391, as well as D2S441, D10S1248, and D22S1045. The latter three STRs are thereby established as so-called mini-STRs to fulfill the increasing requirements regarding sensitivity and reproducibility for analysis of minute amounts of DNA. Here, we provide allele frequencies for the five additional STRs from two populations from Germany. A test regarding suitability and robustness of the new kits for routine trace analysis showed that it is more likely to obtain a meaningful profile using Powerplex® ESX17 and Powerplex® ESI17 kits compared to the Powerplex® ES kit. However, for both new kits the range of template DNA amount is rather small, e.g., slightly more DNA than recommended resulted in DNA profiles which could not be reliably evaluated due to allelic drop-in or imbalances and overshoots. In our opinion, the new kits are very promising new tools in forensic trace analysis even though handling and evaluation should yet be carried out with great caution.

The new Powerplex® ESX17 and ESI17 kits in paternity and maternity analyses involving people from Africa--including allele frequencies for three African populations.

Paternity and maternity investigations in immigration procedures are frequently done in Germany. Since mostly only one parent and one or more children are investigated, the occurrence of possible mutational events has to be interpreted with great care and the analysis of as many STRs as possible is recommended. The new Powerplex® ESX17 and Powerplex® ESI17 kits from Promega comprising both eleven established STRs and additionally the loci D1S1656, D2S441, D10S1248, D12S391, and D22S1045 (in different order) are potential tools in such paternity or maternity analyses, but only few allele frequency data for the five new loci exist. Here, we provide allele frequencies for the five additional STRs from three different populations from Africa. In addition, we present two maternity cases and one paternity case in which a clear inclusion or exclusion of the alleged parent could only be achieved by the additional application of the new Powerplex® ESX17 kit.

Bone age estimation with the Greulich-Pyle atlas using 3T MR images of hand and wrist.

The age estimation of the hand bones by means of X-ray examination is a pillar of the forensic age estimation. Since the associated radiation exposure is controversial, the search for ionizing radiation-free alternatives such as MRI is part of forensic research. The aim of the current study was to use the Greulich-Pyle (GP) atlas on MR images of the hand and wrist to provide reference values for assessing the age of the hand bones. 3T hand MR images of 238 male participants between the ages of 13 and 21 were acquired using 3D gradient echo sequences (VIBE, DESS). Two readers rated the images using the X-ray-based GP atlas method. A descriptive analysis and a transitional analysis were used for the statistical processing of the data. The agreement between and within the raters was assessed. In addition, a comparison was made with the chronological age and with X-ray studies. The descriptive analysis and the transition analysis showed similar results. Both evaluations showed good agreement with X-ray studies. The comparison with the chronological age showed a difference of 0.37 and 0.54 years for the two readers. The age estimate based on the cross-validated transition analysis showed a mean error of -0.28 years. Inter- and intra-rater agreement were good. In summary, it can be concluded that age estimation of hand bones with MR images is routinely applicable with the GP atlas as an alternative without ionizing radiation. However, in order to reduce the estimation error, a multi-factorial assessment based on examinations of several body regions is still recommended.

Regulation of activin A synthesis in microglial cells: pathophysiological implications for bacterial meningitis.

Previous studies have shown that activin A, a neuroprotective cytokine and dimeric polypeptide composed of two betaA subunits, is elevated in the cerebrospinal fluid of patients suffering from bacterial meningitis. In this study, to elucidate further the functional significance and pathophysiological implications of these findings, we demonstrated that microglial cells are not only the source but also the target cells of activin A in the central nervous system: immunohistochemistry and RT-PCR revealed expression of activin subunit betaA mRNA as well as activin receptor type I and type II mRNA in rat microglia in vitro. Further studies showed that activin enhances microglial proliferation and decreases the gamma-interferon-induced synthesis of nitric oxide, one of several microglial mediators involved in the inflammatory response in microglia activation. Furthermore, quantitative RT-PCR, Western blotting, and ELISA showed an inhibitory effect of activin on inducible nitric oxide synthase, tumor necrosis factor-alpha, interleukin-6, and interleukin-1beta gene and protein levels after lipopolysaccharide treatment. We suggest that the increased synthesis of activin A is directly involved, via influence on microglia cell functions, in the modulation of the inflammatory response in bacterial meningitis.

Overview of clinical forensic services in various countries of the European Union.

Examination of a person who has been a victim of a physical or sexual assault may be very important for upcoming legal proceedings. In the context of a clinical forensic examination, physical findings are recorded and biological trace material is gathered and secured. Ideally, all forensic findings are documented in a detailed report combined with photographic documentation, which employs a forensic scale to depict the size of the injuries. However, the integrity of such forensic findings depends particularly on two factors. First, the examination needs to be conducted professionally to ensure that the findings are properly admissible as court evidence. Second, the examination should take place as soon as possible because the opportunity to successfully secure biological samples declines rapidly with time. Access to low-threshold clinical forensic examinations is not evenly provided in all member states of the European Union (EU); in some states, they are not available at all. As part of the JUSTeU! (Juridical standards for clinical forensic examinations of victims of violence in Europe) project, the Ludwig Boltzmann Institute for Clinical Forensic Imaging in Graz, Austria created (in cooperation with its international partner consortium) a questionnaire: the purpose was to collect information about support for victims of physical and/or sexual assault in obtaining a low-threshold clinical forensic examination in various countries of the EU. Our paper provides a summary of the responses and an overview of the current situation concerning provided clinical forensic services.

The impact of DNA contamination of bone samples in forensic case analysis and anthropological research.

Contamination precautions and quality control are great issues when human bones are investigated genetically. This is especially true for historical samples with only minute amounts of usually highly degraded DNA. But also in forensic routine analysis, sometimes DNA has to be isolated from bones in equally bad conditions, e.g. from burned victims. In such cases, there are several eventualities to contaminate the sample with foreign DNA, for example caused by the recovery of the bones, by trace investigation on a crime scene, or - of course - during handling in the lab. We present the investigation of artificially contaminated historical bone samples which contained no original DNA. Three different kind of contamination were studied: (1) touching of the samples, (2) application of saliva, and (3) application of pure DNA. The samples were genetically investigated without and with the employment of a defined cleaning protocol of the bones. The results show that pure DNA can usually not be removed from the bones and that saliva is a similar thread for subsequent DNA analysis. After the cleaning procedure about 70% of saliva contaminated samples still yielded reproducible STR profiles implicating severe problems for the investigation of highly degraded bone fragments. Simple touching of the specimens seems not to be a real problem for genetic investigations since the obtained signals were not reproducible.

[Excessive use of fentanyl patches as the only means of suicide]. / Exzessiver Fentanylpflastergebrauch als alleiniges Suizidmittel.

Due to the increasing prescription there are more and more cases of abuse and accidents related to fentanyl patches. The use of fentanyl patches (e.g. Durogesic) for committing suicide is rare, however. In our case, we describe a suicide with an amount of fentanyl patches never mentioned in the literature before. A depressive 63-year-old man was found dead in his apartment. On the body of the decedent 20 nearly symmetrically arranged fentanyl patches (Durogesic) of different strength with a total dose of 1350 microg/h were found. According to the results of the police the fentanyl patches were part of the pain therapy of his late wife, who had died one year before. Neither the autopsy nor the histological examinations revealed a cause of death. The chemical-toxicological investigation showed the following fentanyl concentrations: 94.9 ng/g (femoral vein blood), 45.9 ng/g (blood of the left heart), 74.8 ng/g (blood of the right heart), 101 ng/mL (urine), 468 ng/mL (bile), 745 ng/mL (stomach contents), 78.4 ng/mL (cerebrospinal fluid), 133 ng/mL (vitreous humor). The blood concentrations were in the upper range of the concentrations found in similar cases published. Hints for a postmortem increase of the fentanyl concentration because of the long postmortem interval of nearly 8 days were found.

[Verification of private paternity analysis at the Institutes of Legal Medicine in Greifswald, Jena, and Kiel]. / Richtlinienkonforme Uberprüfung von privaten Abstammungsgutachten an den rechtsmedizinischen Instituten in Greifswald, Jena und Kiel.

This investigation presents the retrospective evaluation of paternity testing done as a "second opinion" in the last four years at the Institutes of Legal Medicine in Jena, Greifswald, and Kiel (Germany). All analyses were court-ordered and were preceded by paternity tests of "private" labs. The cases were selected in chronological order without any further exclusion criteria. A total of 59 cases, in which "private" laboratories from all regions of Germany had already performed paternity tests, were evaluated. In all cases, analyses were mainly done by PCR-based STR-typing (8 - 20 STRs). 18 % of the investigated "private" expert opinions showed a false determination of alleles. In two cases, paternity was wrongly confirmed or excluded. The reasons for the mistakes of private laboratories were hard to analyse, since most labs did not provide sufficient information (e.g. alleles, kits and chemicals used) in the written test results. In several cases, not even the typing results were revealed. Furthermore, in paternity testing of "private" labs the identity of the persons examined was usually not assured (e. g. by photo documentation or fingerprints) adding to the problem of insufficient test result reliability.

Integrated computer-aided forensic case analysis, presentation, and documentation based on multimodal 3D data.

Three-dimensional (3D) crime scene documentation using 3D scanners and medical imaging modalities like computed tomography (CT) and magnetic resonance imaging (MRI) are increasingly applied in forensic casework. Together with digital photography, these modalities enable comprehensive and non-invasive recording of forensically relevant information regarding injuries/pathologies inside the body and on its surface. Furthermore, it is possible to capture traces and items at crime scenes. Such digitally secured evidence has the potential to similarly increase case understanding by forensic experts and non-experts in court. Unlike photographs and 3D surface models, images from CT and MRI are not self-explanatory. Their interpretation and understanding requires radiological knowledge. Findings in tomography data must not only be revealed, but should also be jointly studied with all the 2D and 3D data available in order to clarify spatial interrelations and to optimally exploit the data at hand. This is technically challenging due to the heterogeneous data representations including volumetric data, polygonal 3D models, and images. This paper presents a novel computer-aided forensic toolbox providing tools to support the analysis, documentation, annotation, and illustration of forensic cases using heterogeneous digital data. Conjoint visualization of data from different modalities in their native form and efficient tools to visually extract and emphasize findings help experts to reveal unrecognized correlations and thereby enhance their case understanding. Moreover, the 3D case illustrations created for case analysis represent an efficient means to convey the insights gained from case analysis to forensic non-experts involved in court proceedings like jurists and laymen. The capability of the presented approach in the context of case analysis, its potential to speed up legal procedures and to ultimately enhance legal certainty is demonstrated by introducing a number of representative forensic cases.

[Significance of additional RFLP single locus probes analysis after STR based determination of sibship]. / Die Aussagekraft zusäitzlicher RFLP-analysen bei Abstammungsbegutachtung mittels STR-Analyse.

Autosomal STR typing alone seems to be no sufficient tool for resolving deficiency cases (e.g. cases of questioned paternity or half-sibships). Therefore, we investigated whether the additional analysis of RFLP single locus probes can improve the solution of such complicated kinship cases. We analyzed 207 children and men from 101 families using the AmpFlSTRIdentifiler multiplex PCR kit and three RFLP single locus probes. A comparison between each child and all unrelated men resulted in 11,023 man / child pairs. Less than three excluding STRs were found in 125 child / unrelated man pairs (1.13%). Additional analysis of RFLP results reduced the number of ambiguous cases to 35. Half-sibling pairs were simulated using STR results from 20 cases with high paternity probabilities (group 1) and relatively low paternity probabilities (group 2). Using a commercially available computer program we calculated probabilities for 778 half-sibling pairs. In 35 pairs (4.49%) half-sibling probabilities over 90.0% could be calculated. Additional investigation of RFLP single locus probes did not lead to a more reliable evaluation of these results. The combined investigation of autosomal STRs and RFLP single locus probes can satisfactorily solve deficient paternities but does not contribute to the solution of questioned half-sibships.