pediatrics
January 2016, VOLUME137 /ISSUE 1

Translational Research in Pediatrics IV: Solid Tissue Collection and Processing

  1. Carolina Gillio-Meina, PhDa,b,
  2. H. Ronald Zielke, PhDc, and
  3. Douglas D. Fraser, MD, PhDa,b,d,e,f,g
  1. aTranslational Research Centre, London, Ontario, Canada;
  2. bChildren’s Health Research Institute, London, Ontario, Canada;
  3. cPediatrics, University of Maryland, Baltimore, Maryland;
  4. dCentre for Critical Illness Research,
  5. eCritical Care Medicine and Pediatrics,
  6. fClinical Neurologic Sciences, and
  7. gPhysiology and Pharmacology, Western University, London, Ontario, Canada
  1. Drs Gillio-Meina and Zielke contributed to drafting the review and revised the manuscript; and Dr Fraser conceptualized and initiated the review, contributed to drafting the review, and revised the manuscript. All authors approved the final manuscript as submitted.

Abstract

Solid tissues are critical for child-health research. Specimens are commonly obtained at the time of biopsy/surgery or postmortem. Research tissues can also be obtained at the time of organ retrieval for donation or from tissue that would otherwise have been discarded. Navigating the ethics of solid tissue collection from children is challenging, and optimal handling practices are imperative to maximize tissue quality. Fresh biopsy/surgical specimens can be affected by a variety of factors, including age, gender, BMI, relative humidity, freeze/thaw steps, and tissue fixation solutions. Postmortem tissues are also vulnerable to agonal factors, body storage temperature, and postmortem intervals. Nonoptimal tissue handling practices result in nucleotide degradation, decreased protein stability, artificial posttranslational protein modifications, and altered lipid concentrations. Tissue pH and tryptophan levels are 2 methods to judge the quality of solid tissue collected for research purposes; however, the RNA integrity number, together with analyses of housekeeping genes, is the new standard. A comprehensive clinical data set accompanying all tissue samples is imperative. In this review, we examined: the ethical standards relating to solid tissue procurement from children; potential sources of solid tissues; optimal practices for solid tissue processing, handling, and storage; and reliable markers of solid tissue quality.

  • Abbreviations:
    AFS
    agonal factor score
    PCR
    polymerase chain reaction
    PMI
    postmortem interval
    RIN
    RNA integrity number
  • Human tissue experiments are imperative for health research. Solid tissue is an invaluable substrate to understand the molecular and cellular mechanisms underlying childhood diseases. Tissue samples can be collected from subjects across the developmental spectrum, from fetal to late adolescent stages. Children with diseases can also live into adulthood, which may necessitate collection of tissue in later life. In the present review, which is the fourth in a series on tissue sampling and biobanking for child-health studies,13 we describe issues that arise from the collection, processing, and storage of solid tissues. Our goals were to highlight the ethical standards for solid tissue collection from children and the various factors affecting tissue integrity. In addition, we reviewed newer methods for determining tissue quality.

    Ethics Standards for Solid Tissue Research

    Tissue retrieval for research purposes can be complicated by concerns for the individual’s legitimate ethical rights, religious beliefs, and emotional well-being. Biobanks typically have a well-organized informed consent system, whereas the consent standards for archival tissues can be variable. Published ethical guidelines for biobanks suggest the following: (1) that children, as soon as mentally capable, should be asked to assent to research; (2) that biobanks collecting non-postmortem tissue should make an effort to recontact young adults for re-consent once they are capable of providing it; and (3) that minors, when permitted by law, withdraw their samples and clinical data regardless of their parents’ consent.46 Broad consent to cover unexpected studies is ideal, but if the research changes significantly from the original protocol, ethics committees should decide whether re-consent is necessary.4 Archived tissues should be anonymized and consented for by the patients and/or their legal guardian. Internal resource committees should approve access to specimens.7

    Acquiring parental/guardian consent for tissues is often difficult due to fear and/or grieving, but many still consent because they believe that tissue research may be useful for the development of new drugs and treatments8 and/or because of a wish to prevent others from similar painful situations.9 Obtaining consent from relatives after a child dies requires biobank staff to have sensitivity and training to avoid coercion and exacerbating distress. Tissue procurement via percutaneous needle biopsy, as opposed to full open autopsy, may be of greater acceptability to parents/guardians, but this approach can introduce sampling errors.10 In addition, obtaining consent for tissues to be used in research must be coordinated with organ procurement organizations.

    Although predeath autopsy permission for research tissues may be obtained during the informed consent process,11,12 the institutional review board in accordance with law requires postmortem confirmation of the permission from the research participant’s legally authorized representative.1315

    Additional issues to be considered by biobanks include the following: recruiting properly qualified professionals with religious and cultural knowledge to participate on research ethics committees (ie, pediatricians, lawyers, community members); access to larger numbers of shared control cases; standardized protocols between laboratories; and procedures for handling genetic information, incidental findings in DNA studies, and ownership rights.

    Recent advances in human tissue engineering and organoids require unique guidelines and procedures.16 The moral status of interfering with the beginning of life when using human embryonic stem and fetal cells is always questioned. Acquired tissues should only be used for the informed research purpose, and parents often consent only to autologous use with respect to a child’s tissues. Researchers are encouraged to educate the public about their work, although it is challenging to provide details about the tissue engineering process to a lay audience.

    Internationally, human tissue research is controlled by federal regulations, and studies require approval by research ethics committees or institutions’ review boards.1214,1723 Different approaches for informed consent and/or assent should be implemented in child research according to the targeted age (from fetal to adolescent stages).6 Several guidelines are available for access, use, legal rights, handling genetic information, and the sharing of biological samples for research purposes.2428 In addition, the Genetic Privacy Act and Commentary,29,30 the Genetic Information Nondiscrimination Act,31 and the Health Insurance Portability and Accountability Act (termed the Privacy Rule)32 were created to regulate genetic information and individual identifiable health information release. Commercial biobanks have raised significant ethical and legal issues, prompting several countries to create comprehensive regulations through mechanisms such as licensure.33

    Sources and Types of Solid Tissues Collected

    Sources of solid tissue for child-health research include local collections, biobanks, and archived tissues collected as normal clinical practice by pathology laboratories. With the development of newer technologies to identify and quantify molecular biomarkers, including DNA microarrays, quantitative polymerase chain reaction (PCR), and mass spectrometry, biobanking is critical for many disease investigations, whereas archived tissue has been mainly used to detect rare diseases or to investigate specific populations.34

    A variety of solid tissues have been collected for health research (Table 1). Biopsy/surgical specimens are collected to test histopathologic features3538; to determine pathogenic mediators36,3941; to obtain a definitive diagnosis of different syndromes, disorders, or diseases37,42,43; to test the safety of different techniques44,45; to study the role of toxic elements46; and to diagnosis new or rare diseases.47 Biopsy/surgical specimens reflect the tissue in vivo state, but collection is hampered by accessibility, limited volume, or because the sampled area may not fully represent the disease.10,48 Pediatric tissue is also required in studies on the normal development of the human.4951

    TABLE 1

    Examples of Solid Tissues Used in Health Research

    Fresh tissues can be used for engineering and regenerative medicine.165 Donor somatic cells (fibroblasts from skin, adipose cells, and bone marrow; blood mononuclear cells from peripheral blood), term placenta cells, fetal and neonatal cells, and umbilical cord cells are all excellent sources of embryonic and adult stem cells. These cells can potentially be used in tissue replacement therapies, human in vitro models of disease, screening of therapeutic and toxic effects of chemicals, and personalized medicine.166

    Lack of accessibility of fresh tissue makes postmortem tissue an important alternative. Postmortem tissue has been used for studies on morphology167171; to study the mechanical properties of different organs167,168; to validate new techniques172174; to correlate morphology with microbiology findings103; to report new or rare diseases175; to study the molecular mechanisms95,123 to determine toxic element exposure87; to determine a definitive disease diagnosis88,96,104,176; and to detect infectious agents.10,68 The use of postmortem tissue is often hampered, however, by agonal factors, postmortem intervals (PMIs), and storage conditions that contribute to tissue degradation.177,178

    Solid tissues can be derived from living patient organ donors, as well as from deceased patients, resulting from neurologic or cardiac death.179183 Recovery of donor medical information before the donor tissue procurement is ideal,184 particularly in preregistered cases, and it can be supplemented with archived medical records. Importantly, the case must meet the collection criteria of the tissue bank. Exclusion criteria will vary according to project and may include drug usage, cancer, psychiatric illnesses, and extended PMI. Obtaining consent for tissue procurement is often the initial focus (if not acquired in advance), followed by tissue retrieval.

    Tissue quality, physiology, and function are critical for tissue donation, as is the patient’s medical condition at the time of death and the patient’s location (termed geographical limitation).179 The viability of the tissues is measured in hours from the moment of retrieval, which is a time critical for successful transplantation or research.20 For example, ocular tissue must be recovered within 12 hours of cardiocirculatory death for transplantation, but it can be used for research within 15 to 24 hours of cardiocirculatory death.179 Tissue banks often interact permanently with universities, hospitals, medical centers, and support foundations, and they offer a variety of organs/tissues for child-health research.180183

    Tissue that would otherwise have been discarded, including placentas and umbilical cords, can be obtained for research purposes because their collection poses little risk to the patients.185187 The placenta has been used in child-health research to investigate preeclampsia,188 maternal obesity,63 endocrine disruptors,189 attention-deficit/hyperactivity disorder,64 Smith-Lemli-Opitz syndrome,190 and vertical virus transmissions.65,66 Blood drawn from umbilical cord veins has been used for regenerative medicine72 and stem cell research.191,192 Other discarded tissues include the aborted fetus,193,194 bone,117 spine,121 ovary,140,141 and skin,195 and extra tissue fragments from surgical removal/biopsies of neoplasms that are not appropriate for diagnostic procedures.8,196

    Future tissue sources may include donor tissue not suitable for transplantation due to an extended postrecovery interval, age, abnormal macroscopic results, long ICU stay, or a positive result on the donor’s serologic test (eg, HIV, hepatitis B or C). These latter tissues include cornea,126 kidney,130 and pancreas.132 Table 2 summarizes examples of pediatric tissues/organs and the analytical techniques used in research studies.

    TABLE 2

    Examples of Pediatric Tissues/Organs Used in Specific Disease Research Studies

    Fresh, Fresh Frozen, and Fixed Tissues

    Fresh, fresh frozen, and fixed tissues are amenable to several investigative methods, including tissue engineering, microarray analysis, PCR, receptor autoradiography, proteomics, chromatography, immunochemistry, and protein sequencing (Table 3).207209

    TABLE 3

    Types of Analyses Conducted With Biopsy/Surgical and Postmortem Tissues

    Fresh tissue is used for tissue engineering research and organoid formation, following digestion of extracellular matrix and cell isolation in a sterile environment.166 When isolated cells are reprogrammed to stem cells and the end point is differentiation to specific cell lineages, specific media conditions must be used. Because tissue function often relies on several cell types in a specific 3-dimensional order, the development of novel culture environments using biomaterials and bioreactors is imperative to maintain selected aspects of native tissue.166 Mechanical stimuli, extracellular matrix components, or even long-term cell culture conditions are extremely important for the development of an organoid.166

    Fresh frozen tissue preserves biomolecules in an intact state and provides a high yield of quality DNA and RNA.225,226 Due to the small sample size of fresh frozen tissue, however, it is often difficult to correlate biochemical experiments with histologic/pathologic examinations.225 Regardless of the tissue origin (either biopsy/surgical or postmortem), snap-freezing with storage at –80°C or below are ideal to maintain the integrity of biochemical molecules.207,210,227

    Tissue samples are often fixed in formaldehyde or formalin before being embedded in paraffin wax for sectioning and subsequent histologic examination. Fixed tissue is most commonly used for protein localization and morphology studies. Paraffin blocks have been successfully used to isolate both DNA and RNA when optimal fixation and extraction methods were used.228 However, fixation of solid tissue can degrade and damage DNA and RNA,225,229 thus interfering with gene expression, mutation, and polymorphism studies. For better quality tissue, fixation should be conducted as soon as possible after collection,209 and the samples should be immediately embedded in paraffin blocks.229 DNA can be isolated from fixed tissue that has not yet been embedded,225 but the DNA is of less quality compared with DNA isolated from frozen tissue.

    Factors Influencing Solid Tissue Quality

    Many factors affect the quality of tissue directly or indirectly by interfering with protein stability and nucleotide yield or quality.67,177,230234 The parameters affecting solid tissue qualities are summarized in Table 4.

    TABLE 4

    Parameters Affecting Tissue Quality of Biopsy/Surgical and Postmortem Tissues

    Gender and Age

    Female gender and increased age at death are 2 factors that influence total tissue messenger RNA levels256 and alter specific gene expression profiles.258 Although both factors are reportedly important determinants of tissue quality,232,258 other research has shown that brain RNA integrity is not influenced by gender or age <50 years (H.R.Z., unpublished observations).

    Agonal Factors

    Agonal factors include coma, pyrexia, hypoxia, seizures, dehydration, hypoglycemia, multiple organ failure, prolonged death, head injury, respiratory arrest, and neurotoxic substance ingestion; these factors can change global gene expression via signaling pathways (ie, the stress response and apoptosis) with activation of acid ribonucleases.232,258 Consequently, there is variability of RNA integrity and divergence in gene expression profiles.232,258 Individuals who suffered prolonged agonal states (ie, respiratory arrest, multiorgan failure, coma) have decreased gene expression for energy metabolism proteins and proteolytic activities, and increased expression of genes encoding stress response proteins and transcription factors. In contrast, those who experienced rapid deaths (ie, trauma, cardiac events, asphyxia) have limited alterations in gene expression.236 Agonal factors, such as coma and hypoxia, can markedly affect the RNA quality and have a major impact on gene expression profiles in microarray analyses.232 The activation of ribonucleases during the terminal phase may introduce large divergences of global gene expression profiles in microarrays compared with gender, age, postmortem delays, or diagnoses of psychiatric disorders.232

    A sudden or prolonged death can affect tissue quality, resulting in the creation of the agonal factor score (AFS).232 Each agonal condition is scored 1 if present or 0 if absent. Agonal duration is also rated.261 Individual scores of the agonal conditions and agonal duration are summed to provide the final AFS for each sample. A lower AFS is associated with high-quality tissue.233 The AFS has the practical limitation of its inability to assess each anatomic region separately, resulting in the development of an agonal stress rating system that evaluates the degree of stress based on gene expression data.237 The agonal stress rating can reduce the number of false-positive findings by allowing a quantitative assessment of tissue quality and is useful for identifying anatomic regions that exhibit different stress outcomes.237

    Postmortem Intervals

    A PMI is defined as the difference between the time of death and the time of tissue preservation (eg, frozen, fixed).184 After death, bodies are placed at 4°C for variable times before sample collection. Traditionally, PMIs can range from 6 to 72 hours,262 depending on the period of time that parents wish to remain with their deceased child, with 1 to 6 hours as the minimum time to access the body for tissue collection.184 High-quality tissue is generally associated with low PMIs.233,263,264 Longer PMIs alter the tissue quality at the molecular level. Postmortem tissue can maintain gene expression profiles in heart tissue for at least 24 hours,78 whereas longer PMIs result in greater epithelial cell loss in cornea tissue.245 Delays in PMI can also affect protein stability, particularly receptors and cytosolic proteins.251,253,265 PMI delays >40 hours result in posttranslational modifications and decrease protein immunoreactivity in human degenerative diseases.241,254,266,267 Longer PMIs are associated with oxidation/nitration events.240

    Other Factors Affecting Solid Tissue Quality

    After death, the time to tissue collection, extraction, and processing, as well as tissue storage protocols and temperatures, are critical factors affecting tissue quality (Table 5). For example, brain RNA and protein degradation associated with postmortem delay is dependent on the storage temperature.210,234,268

    TABLE 5

    Solid Tissue Storage and Processing

    Indicators of Tissue Quality

    Solid tissue quality control is imperative for child-health research studies. Several methods have been used to ensure solid tissue quality. Some factors (ie, gender) that lead to bias can be statistically controlled by using matched designs, match-pairing of patients with disease and control subjects with the same values of factors, and/or by adding the factors as covariates in the analysis.256

    RNA Integrity

    RNA integrity is an important indicator of tissue quality. The most common and sensitive method used for determination of total RNA quality, with high reproducibility and with impartial measurements, is microfluidic chip-based capillary electrophoresis270,271 using a Bioanalyzer (Agilent Technologies, Santa Clara, CA) or Experion (Bio-Rad Laboratories, Munich, Germany) instrument. Using electrophoretic data, an RNA integrity number (RIN) or RNA quality index is generated, taking into account the 18S/28S ribosomal RNA peaks, as well as background and degradation products. The RIN can range from undetectable to 10, with undetectable being completely degraded and 10 being mostly intact RNA233; RIN values ≤6 are usually considered insufficient for RNA studies.210

    RNA integrity can be influenced by degradation, tissue type, BMI, and by other tissue quality markers, including pH, PMI, agonal factors, and relative humidity of the processing laboratory.230233 In some tissues, RNA degradation increases with longer PMIs.177,230 Prolonged agonal factors, such as coma and hypoxia, together with a pH ≤5.9, can also affect the RNA integrity and have a major impact on gene expression.232 RNA quality is reduced in a time-dependent manner at a relative humidity ≥31%231 and with freeze/thaw cycles.233

    RNA quality control is imperative when gene expression analysis is performed by using postmortem tissues, with quantitative data checked for the influence of antemortem and postmortem parameters.230 The RIN values are used to identify samples that should be excluded from analyses and to distinguish similar biological replicates.230 RNA preservation may be useful in RNA expression studies of individual subjects due to possible divergence of RIN and PMI values, as well as regional tissue differences in RIN.210 Housekeeping genes must be carefully chosen and monitored to obtain normalization.

    Importantly, RIN values should serve only as a guide to determine if the tissue appears of sufficient quality to commence actual studies of gene expression. In addition to the RIN, RNA integrity has been previously tested by using northern blot hybridization,272275 quantitative in situ hybridization,274276 and quantitative reverse transcription PCR.273 For microarrays, several RNA integrity indicators, as well as the percent present call (percentage of the total number of probe sets detected as present on the array), have been used.232

    Protein Integrity

    Tryptophan, an essential amino acid in protein biosynthesis and a biochemical precursor for the biogenic amines, increases with longer PMI and is considered a good indicator of tissue quality.55 Elevated tryptophan levels indicate protein degradation and alterations in enzyme activity. Tissue tryptophan levels also reflect freezing/packaging methods, in which lower levels are associated with dry ice storage temperatures and aluminum packing methods.55 Although the reason for selective preservation is unknown, protein integrity may vary depending on the specific protein, cellular localization, protein posttranslational modification stage, and protein function.210

    Tissue pH

    Tissue pH values ≥6.4 are associated with higher quality samples.178,233,236,277 The pH of the tissue can be directly correlated with agonal states (prolonged versus brief death) and with differential gene expression patterns. A prolonged terminal phase is associated with a significantly lower brain pH compared with that of rapid death.178 Subjects with pH ≤5.9 exhibit specific changes in gene expression.236 Tissue pH does not correlate with PMI, time in storage, freezing method, or tissue packing, but it can be influenced by gender.178 Brain tissue pH has been shown to be remarkably consistent across different anatomic regions.178,233,235 In animals, there is a rapid drop of brain tissue pH within 10 minutes after death,261 followed by a period of pH stability for 24 to 36 hours.235 Because pH is not a reliable indicator of storage delays and temperature fluctuations that directly affect tissue quality, pH is a better indicator of premortem events than postmortem events.

    Nucleotide Isolation

    Fresh or frozen tissues are an ideal source for DNA and RNA (Table 6). When RNA isolation is the end point, working in a ribonuclease-free environment should be done to avoid introducing ribonucleases into the sample.18 Fresh solid tissues are best placed in liquid nitrogen or dry ice. Tissue samples can also be stored at 21°C in an RNA stabilization solution (ie, RNAlater solution, Thermo Fisher Scientific, Waltham, MA).278

    TABLE 6

    Sample Processing and Storage Tips for DNA/RNA

    DNA is relatively resistant to PMIs, but it is vulnerable to degradation in solutions, particularly fixatives used for tissue preservation.229 Thus, better DNA yield is obtained from solid tissue stored at –80°C. Isolation of high-quality DNA can be obtained by using QIAamp Micro Qiagen (Qiagen Inc, Valencia, CA).229

    Two methods to isolate tissue are laser cutting and laser capture microdissection. Laser cutting is used to isolate large areas, particularly from hard tissues. In contrast, laser capture microdissection is gentle, and it can isolate small areas or even single cells. The latter method does not alter or damage cellular morphology and preserves biomolecule integrity, making the technique useful for collecting RNA/DNA from small numbers of cells in tissue regions of interest.225 When laser capture microdissection is combined with quantitative PCR, changes in region-specific gene expression can be detected.231 The effect of environmental humidity on RNA quality requires that a relative humidity be maintained at or below 23% for high efficiency of capture and the collection of high-quality RNA.

    Microarray analysis of postmortem tissue is a vital tool to investigate gene expression patterns. Microarrays such as GeneChip Human Exon 1.0 ST Array (Affymetrix Ltd, High Wycombe, UK) provide reliable results over a wide range of RINs (from 1–8.5) to help determine RNA quality.56 A reliable and specific parameter used with microarrays is the present call or the percentage of probe sets with signal detection above background probe levels.232 The average correlation index is another tool developed to evaluate agonal factors and RNA integrity on the basis of the similarity of gene expression profiles for each microarray among a total set of microarray data.232

    Protein Isolation

    Protein levels derived from solid tissues (Table 7) can be affected by storage temperature >4°C and PMI delays >40 hours. Bidimensional gel electrophoresis, western blot analysis, and mass spectrometry are useful tools for identifying protein degradation in postmortem tissues.234 Immunohistochemistry studies on postmortem tissue have shown stable signal intensity for several proteins during a PMI of 24 hours.250

    TABLE 7

    Sample Processing and Storage Tips for Protein and Lipids

    Tissue microarray can be a low-cost, high-throughput technique to investigate protein expression.43,243,289,290 In addition, proteomic methods can be used to investigate tissue quality, and they include 2-dimensional polyacrylamide gel electrophoresis matrix-assisted laser desorption/ionization time-of-light291,292 and surface-enhanced laser desorption ionization time-of-flight mass spectrometry. These latter methods can be used to detect different patterns of protein susceptibility to PMI and storage temperature.293

    Lipid Isolation

    Lipids can be isolated from fresh or frozen tissue (Table 7) by sample homogenization in an organic solvent at 4°C followed by an extraction protocol.288,294 Extracts can be stored frozen at –80°C until analysis.287 Different lipid subtypes can be successfully quantified by using mass spectrometric detection coupled with either gas, liquid, or thin-layer chromatography.108,222,223,295 Tandem mass spectrometry with a lipid database296 can be used to discriminate among chemical lipid variants with identical masses due to the identical number of acylic carbons and double bonds.287 Matrix-assisted laser desorption/ionization mass spectrometry permits the direct scanning of tissue slides, and it thus identifies the precise localization of different lipids in the tissue.297

    Storage Procedures

    Tissue samples can be cryopreserved, in either a –80°C freezer or in liquid nitrogen (–170°C), or fixed in formalin and embedded in paraffin (Tables 6 and 7). Choosing the optimal storage protocol depends on the study end point. Embedding in paraffin is a relatively inexpensive technique with histologic morphology of high quality, but genetic material may be damaged.18 Cryopreservation is ideal for extraction of high-quality DNA, RNA, and protein, and it is a good storage method for morphologic studies, but it is expensive.18 Specimens should be quick-frozen either in a dry ice/isobutane mix, between chilled aluminum plates or in liquid nitrogen vapor to avoid ice crystal formation. The samples should be on a flat surface to avoid distortion. Frozen specimens are best stored in heat-sealed plastic. It is desirable to use bar coding to identify the case number and tissue sample, and tissue from multiple donors with a specific disorder should be stored in different freezers if >1 freezer is available. Freezers should be equipped with an alarm system to avoid catastrophic failure and nucleotide degradation. The inner-chamber temperature of the freezer should be monitored at least weekly and recorded in a log.18 The benefits of the freezer are the ability to store many samples in a relatively small storage space, with ease of access, and fewer infrastructure requirements. The disadvantages of freezers include high cost, fragility of the equipment, and the dependence on energy.18 Liquid nitrogen storage provides better preservation of samples, independent of energy, but requires constant maintenance of the nitrogen level and is associated with a greater difficultly of accessing the samples.18

    Tissue Distribution

    A trained pathologist or technician may be necessary to identify and section tissue. For some specific conditions/diseases and tissues/organs, a systemic evaluation of the tissue by a pathologist might be necessary to confirm diagnosis before distribution for research. Efforts need to be taken to prevent thawing of the tissue during the sampling procedure. Different approaches will be necessary to section the frozen tissue, including chipping, sawing, or use of a dental drill to excise tissues from large sections (ie, coronal brain section). The sample should be heat-sealed under vacuum in plastic and immediately placed in a –80°C freezer until it is packaged for shipping. Tissues are packed in inner and outer pouches consisting of an amber translucent Teflon-Kapton (Dupont, Circleville, OH)material and then heat-sealed after packaging. The pack is vacuum-sealed within a plastic bag and placed in a disposable transport box containing dry ice.298 Commercial companies now offer a large variety of containers for temperature-sensitive shipments.299 β-ray sterilized plastic bags and vacuum sealing of fresh excised specimens at surgical theaters, followed by time-controlled transferring at 4°C to the pathology laboratory, do not affect morphology, nucleic acids, proteins, or cell viability.300303 Antifreeze solution E/P20 (containing 20% ethylene glycol and propylene glycol) preserves cells and DNA integrity derived from human blood tissue and bone marrow.304 Generic insulated shipping containers with appropriate grams of wet ice for the given time interval are used for cornea shipping (≤8°C).305

    After death, RNA integrity is influenced by the PMI, which is enhanced by higher storage temperatures.268 The levels of proteins and posttranslational modifications are also dramatically affected at higher temperatures such as 4°C to 21°C.234 Preservation of protein samples at 21°C for >12 hours decreases biochemical reliability. In addition, thawed tissue stored at 21°C and then refrozen is no longer usable for biochemical studies.234 Overall, optimal storage conditions should be obtained for solid tissues by 30 minutes (for DNA or RNA) or 2 hours (for protein) after death to prevent degradation.18,234 PMIs are often longer for medical examiner cases or for those who die at home or in hospice care. Best practices for collection, transport, and processing of solid tissues for nucleotide and protein isolation have been provided by the International Society for Biological and Environmental Repositories306 and the National Cancer Institute.24,184

    Tissue Linkages to Clinical Data

    Research using solid tissues must be linked back to the patient and his or her disease. Thus, accurate recording of clinical data and the specifics of tissue collection are essential. Table 8 outlines the recommended minimal clinical data set.

    TABLE 8

    Recommended Clinical Data Set to Accompany Solid Tissue

    Transmission of Genetic Testing Results to Families

    Thresholds have been established for reporting genetic testing results to study participants, with 3 key criteria: (1) the risk for the disease should be significant (variants with greater penetrance or associated with younger age of onset should receive priority); (2) the disease should have important health implications (ie, fatal or substantial morbidity); and (3) proven therapeutic or preventive interventions should be available. In addition, the genetic results should come only from certified laboratories.307,308

    Conclusions

    This review summarizes pertinent issues regarding the procurement and storage of solid tissues from children. Due to the variability in collection method, site, time, and storage conditions of tissues, it is imperative to understand the factors affecting tissue quality. Standardization of methods of collection and linked clinical information will aid reliable research results. Minimal quality control measures for solid tissue are required for each study,184 which may include a search of donor information, record of agonal states, standardization of PMIs, standardization of sample collection and storage processes, histologic reviews of samples to assess proper biospecimen identification, and the use of best practices for molecule isolation. Translational research on solid tissues will contribute to new diagnostics and therapeutics in child diseases.

    Footnotes

      • Accepted October 5, 2015.
    • Address correspondence to Douglas D. Fraser, MD, PhD, Paediatric Critical Care Medicine, Room C2-843, Children’s Hospital, London Health Sciences Centre, 800 Commissioners Rd East, London, ON, Canada, N6A 5W9. E-mail: douglas.fraser{at}lhsc.on.ca
    • FINANCIAL DISCLOSURE: The authors have indicated they have no financial relationships relevant to this article to disclose.

    • FUNDING: Drs Gillio-Meina and Fraser are supported by the Children’s Health Foundation (http://www.childhealth.ca) and the Lawson Health Research Institute (http://www.lawsonresearch.com).

    • POTENTIAL CONFLICT OF INTEREST: The authors have indicated they have no potential conflicts of interest to disclose.

    References