2.2 Validation of Autopsy and Specimen Collection Techniques and RNA Quality Assessment 2.2.1 BackgroundAs mentioned in the Chapter 1 that this D.Phil. project focuses on the development of molecular-based techniques to complement the understanding of pathogenesis of severe malaria gained by conventional histopathological and immunopathological approaches, this necessitates the access to human tissue with severe malaria infections and tissue of normal controls. In order to gain this access, our group has started an ongoing autopsy-based study in fatal malaria in Mozambique (details of this study will be discussed in the next section).
Before the autopsy in Mozambique commenced, a study to validate the autopsy and specimen collection techniques, which would be used in Mozambique, had been done in Bangkok, Thailand. This study allowed us to perform a feasibility study of mRNA extraction and gene expression analysis on samples of post mortem human organs, optimising retrieval for molecular pathology studies by examining effects of pre- and post- mortem factors, tissue collection, storage and RNA isolation techniques. Moreover, this study form a part of tissue bank of control patients for the use in histological, immunohistochemical and the comparison of gene expression profile between malaria and non-malaria individuals together with the baseline difference of gene expression in different genetic backgrounds.
A key concern in conducting molecular-based research from human postmortem tissue is the quality of the genetic material preserved in the tissue. Unlike animal models, where conditions prior to and at death can be strictly controlled and manipulated, human tissues can only be collected at autopsy after all the necessary treatments have been given and patients have reached the end of life naturalistically. This introduces a lot of uncontrollable confounding factors that affect the quality of the tissue. Some of these traditionally recognised confounds are related to pre-mortem events, including agonal factors (e.g. hypoxia, pyrexia, coma, seizures, acidosis, dehydration, head injury at time of death) and duration of agonal state. Others are related to post-mortem events such as postmortem interval between death and tissue fixation, temperature of the corpse and the environment, tissue collection, tissue fixation, tissue processing and storage condition. In addition, some confounds are still incompletely understood and unpredictable. There are unexplainable variations in the quality of the tissue from different areas of the same brain or from different cases with the same pre- and post- mortem condition.
Recent studies have focused on identifying measures of tissue quality for human postmortem tissue. Several studies have proved that 28S/18S ratio, the historical and commonly used measure of RNA quality, is not suitable as a marker of quality for mRNA1. The brain PH has been proposed as it correlates with some of quality markers and it affects the gene expression profile2; however, PH alone lack the ability to apply as a screening tool. Most studies agree that RNA Integrity Number (RIN), which is generated from Agilent 2100 Bioanalyzer, is by far the most accurate screening measure to evaluate the quality of RNA samples prior to their use in ultra-sensitive platform e.g. microarray analysis1 3 4. Bioanalyzer generates digital electropherograms and calculates RIN using manufacturers advanced algorithm, which takes more components of total RNA profiles into account than previously possible with standard agarose gel. Samples with RIN values higher than 7 are generally accepted as high quality and suitable for mRNA expression studies. However, not all mRNA transcripts have the same vulnerability to degradation. Thus the RIN value of 7 is not a magic number to reject or accept the use of that sample for molecular studies. The quality control measures of microarray such as percent call rate, scaling factor, spikes and GAPDH ratio are still crucial for the evaluation of hybridization and chip image, and the sample should be excluded from the analysis if these parameters are lower than the acceptable thresholds after an actual microarray is performed.
The most common mean of preserving tissues intended for RNA analysis is snap freezing, either in liquid phase or vapour phase of liquid nitrogen. This has been proved to be the gold standard of tissue preservation methods for sensitive molecular-based studies. However, conducting such method is not easy or even not possible in some particular circumstances because it requires trained technicians with necessary specialised equipments such as tanks, flasks, cryotubes and access to liquid nitrogen. Given Beira, the second largest city of Mozambique, for an example, there is no liquid nitrogen supply in Beira. Liquid nitrogen tank must be transported for days to Maputo to be refilled. The electricity network in Beira is not very stable. Power outages often occur almost everyday, which make the -70C freezer unreliable for tissue storage after snap freezing. In order to circumvent these limitations, an alternative way to preserve tissue on site has to be found.
There has been increasing number of reports that use commercially available RNA stabilizing reagents, as an alternative to snap freezing, for collection of human tissue. RNAlater (Ambion), which is compatible with most RNA isolation protocols and downstream application including RT-PCR and microarray5, is most widely tested and used. This solution eliminates the need to immediately process or snap freeze the tissue because tissue stored in this solution at room temperature for 1 week gives comparable RNA yield and quality to snap freezing tissue6. Tissue in RNAlater can also be stored at -20C or -80C for long-term preservation after allowing thorough penetration of the solution into the tissue at room temperature or 4C. Therefore, submerging tissue in RNAlater is a promising solution to the tissue collection in the autopsy study in Beira.
2.2.2 Materials and MethodsCollecting CasesThis autopsy study was embedded in non-randomized routine autopsies at the Pathology and Forensic Departments of King Chulalongkorn Memorial Hospital, Bangkok, Thailand. The autopsy was only performed on the case that written permission to conduct an autopsy and to retain tissue samples for research has been given by the next-of-kin of the deceased. Detailed medical record of the case dying after the admission to hospital was collected (n=1); however, most cases were forensic cases who died outside the hospital (n=9). Time of death, time of body put into the refrigeration, time body taken out of the refrigeration and time of performing an autopsy were recorded. In cases that the time of death was not certain, the time of death was estimated from postmortem changes and circumstances of the death. All candidates to the study were tested for, hepatitis B, hepatitis C and HIV infections using postmortem serum on commercially available rapid tests (SD Bioline Rapid Test for HBsAg, anti-HCV and anti-HIV 1&2, Standard Diagnostic Inc, Korea). Only cases with negative results of all the 3 markers were recruited into the study. The ethical permission for autopsy and the use of tissue collection was approved by the Institutional Review Board of the Faculty of Medicine, Chulalongkorn University.
The body was kept refrigerated (4oC) for variable periods of time prior to the autopsy. The autopsy was performed using standard techniques. All internal organs were removed and dissected in usual fashions and completely examined. All macroscopic findings at autopsy were recorded in a standardized form. The brain was also removed and dissected. A gross neuropathological examination was completed on each brain. Four specific areas of the brain were collected from each case, including primary motor cortex (dorsal part of precentral gyrus), anterior nucleus of thalamus, medulla oblongata and posterior lobe of cerebellar hemisphere. Samples from the lungs, liver, spleen and kidneys were also collected.
Collecting SpecimensFor each fresh postmortem specimen of the brain, kidney, lung, liver and spleen, a single tissue fragment was subdivided into 4 groups according to storage conditions; 1) snap freezing in liquid nitrogen vapour (LNV) then stored at -70oC 2) immersion in RNAlater at 4oC for 24 hours then stored at -70oC (RNAlater A) 3) immersion in RNAlater at room temperature (around 27-35 oC in Bangkok) for 24 hours then stored at -20oC (RNAlater B) 4) 10% formalin at room temperature for several days then embedded in paraffin block. Tissues immersed in RNAlater or snap freez in LNV were cut into a small piece, approximately 5x5x5 mm, in order to ensure the quick and complete immersion of RNAlater solution or immediate freezing process of LNV to avoid ice crystal artifacts. Tissues immersed in 10% formalin were cut into 20x20x5 mm dimension for later paraffin embedding and histopathological examination. This experimental design allowed the evaluation of the quality of RNA from the same tissue with 3 different tissue preservation techniques.
It is noteworthy that only brain tissues were studied in the comparison of tissue preservation techniques. In total, there were 90 samples from 3 areas of the brains of 10 cases with 3 different preservation techniques to be analysed. The other organs collected from the autopsy have been stored untouched.
RNA AnalysisThe tissue samples of the brains were homogenized by a handheld rotor-stator homogenizer (PRO200, PRO Scientific, USA) in RLT buffer provided by RNeasy Lipid Tissue Mini Kit (Qiagen, UK). Total RNA was isolated according to manufacturers instructions. RNA purity was assessed by Nanodrop-1000 (Thermo Scientific, USA) using the A260/A280 ratio and A260/A230 ratio (A260 = absorbance at 260 nm, A280 = absorbance at 280 nm,