Lec1-Specimens for Molecular Testing-67-FINAL PDF

Specimens for molecular testing Asst. Prof. Dr. Somphot Saoin Department of Medical Technology School of Allied Health Sciences University of Phayao 22 October 2024  Learning Outcome อธิบายชนิดของสิ่งส่งตรวจทีเ่ หมาะสมกับชนิ...

Specimens for molecular testing Asst. Prof. Dr. Somphot Saoin Department of Medical Technology School of Allied Health Sciences University of Phayao 22 October 2024  Learning Outcome อธิบายชนิดของสิ่งส่งตรวจทีเ่ หมาะสมกับชนิดการตรวจทีพ่ บประจา ในงานอณูชวี วิทยาได้ อธิบายกระบวนการ collection transportation processing และ storage สิง่ ส่งตรวจทีเ่ หมาะสมกับงานอณูชวี วิทยาได้ Specimens for molecular testing 2  Quality assurance Pre-analytical Process Specimen collection, transport and processing Analytical Process Laboratory testing Analysis of a result and Post-analytical Process Production of a lab report or re-testing  Pre-analytical process Pre-analytical errors make up to 60-70% of all laboratory errors occurring inside and outside the laboratories Source: Grzych G, Sivadas A. The Rising Importance of Pre-Analytical Phase in Medical and Research Laboratory, A New Challenge in the Omics Era. Biomed J Sci & Tech Res 27(4)-2020. BJSTR. MS.ID.004523.  Consideration for sample handling Nucleic integrity Stability The effects of some interfering agents Processes: Collection Transportation Storage Specimens for molecular testing 5  Types of Specimen Whole blood Nasopharyngeal swabs Serum Cervical swab Plasma Bronchoalveolar lavage Buffy coat Buccal Cells and Mouthwash Dried blood spot Stool Urine Cervical and Urethral Swabs Specimens for molecular testing 6  Blood and its components Whole blood, Plasma, Serum, Buffy coat and Bone marrow Awareness of anticoagulant Effect of heparin on the molecular assays-enzyme based molecular tests Recommend K2/K3-EDTA or acid citrate dextrose (ACD) RNA-commercial blood collection tubes containing RNA stabilizer PAXgene (QIAgen) BD PAXgene Blood RNA Tube (BD) Tempus Blood RNA Tube (Thermo) Specimens for molecular testing 7 Specimens for molecular testing 8  Whole blood Before extraction: RT for up to 24 h → genomic DNA degradation Refrigerator (2-8 °C) for 72 h (optimum time), 5- 6 days (maximum) Frozen whole blood : heme inhibit PCR reaction Extraction of RNA within 4 hours. Specimens for molecular testing 9  Plasma Plasma DNA levels gradually decrease overtime DNA levels gradually decrease in a time frame of 9 to 41 months during storage at -80°C RNA: stable 4°C up to 24 hours DNA : stable 4°C and RT stable during 24 hours It is recommended to ship the plasma at 2°C to 8°C and can be kept for up to 5 days. For longer durations, it should be stored at -20°C or lower Specimens for molecular testing 10  Serum One of the most commonly used samples in clinical laboratories DNA yield of serum may be low, it is reported to be suitable for evaluating genomic DNA Serum should be shipped frozen on dry ice and stored at −20°C prior to DNA or RNA studies Serum should be used in less than one day at RT or in 2-7 days at 4 °C. Specimens for molecular testing 11  Buffy coat Enriched white blood cell (WBC) layer provides a good source of nucleic acids for molecular assays DNA extraction within a few days, the buffy coat can be isolated and stored at -70°C RNA should be isolated from buffy coat within 1 to 4 hours of specimen collection→ Kept in RNA stabilizing solutions Specimens for molecular testing 12  Dried blood spot Dried blood spots (DBSs) can be prepared on filter paper with optimal blood volumes as low as 50 μL One of the great advantages of DBS is easy handling and shipping even in rural areas with unavailable cold chain Drying procedure takes 3 hours at RT in a safety cabinet or 1 hour at 37°C in an oven RNA in DBS samples is stable at least for 3 months at RT Specimens for molecular testing 13  Bone marrow aspiration BM aspirations can be stored at 2°C to 8°C for up to 72 hours upon reception For longer storage, specimens can be stored at -20°C for several months after the elimination of RBCs. RNA stabilizer solution→ RNA extraction should be done up to four hours after collection Specimens for molecular testing 14  Bronchoalveolar lavage Bronchoalveolar lavage sampling (BAL) is used to sample lung lesions in a less invasive manner than traditional percutaneous needle biopsy method Detect microbial agents including mycobacteria, presence of cancerous cells or some genetic diseases such as cystic fibrosis BAL cells such as neutrophils for respiratory syncytial virus (RSV) or cell free BAL fluid for herpes simplex virus (HSV) or cytomegalovirus (CMV) 15  Bronchoalveolar lavage Specimens should be transported and tested within 24 hours of collection Specimens should be stored for up to 72 hours at 4°C or longer durations at less than -70°C. For detecting mycobacteria, they should be decontaminated and digested before freezing or long-term storage of the specimens Specimens for molecular testing 16  Buccal Cells and Mouthwash Specimens Easily obtainable samples especially for large- scale population-based and epidemiologic studies Mouthwash gives a greater yield and higher quality of human DNA: high-molecular weight DNA With long-term storage in RT, the amount of non- human DNA increases due to bacterial overgrowth These specimens are stable at RT for 1 to 2 weeks or for at least 6 months if stored at -20°C Specimens for molecular testing 17 Urine Urine samples can be used for molecular evaluation of kidney, bladder and prostate neoplasia, genetic anomalies, and a number of infections Immediate analysis is ideal Adding EDTA with 40 mM final concentration has been recommended to preserve human DNA in urine for up to 28 days at RT, 4°C, and -80°C. Addition of PenStrep with no negative effect on DNA preservation has to be considered to prevent bacterial overgrowth Specimens for molecular testing 19 Nasopharyngeal Swab Respiratory virus diagnosis Influenza Respiratory syncytial virus (RSV) Parainfluenza virus Human Metapneumovirus Rhinovirus Enterovirus Adenovirus Coronavirus Specimens for molecular testing 20 Specimens for molecular testing 21 Nasopharyngeal Swab The most common acceptable specimen for detecting Influenza Viruses Throat swabs and/or nasal swabs The swab should be transported into sterile viral transport medium (VTM) at 4°C. If the shipment is delayed by 3 to 4 days, the samples should be kept at -70°C Specimens for molecular testing 22  Cervical and Urethral Swabs Detection of sexually transmitted diseases (STDs) Screening Human papillomavirus (HPV) DNA is a useful cancer screening method to prevent cervix cancer Cervical specimens for HPV collected by swabs can be stored up to 1 month at ambient temperature The results of clinical tests are highly dependent on appropriate shipping on wet ice, and storage of specimens at 2 °C to 8 °C in the laboratory within 7 days of collection Specimens for molecular testing 23  Cervical and Urethral Swabs Swabs can be transported dry inside a sealed tube or placed in transport mediums. DNA stability can be maintained at 2°C to 8°C for up to 10 days. C. trachomatis, Hemophilus ducreyi, Klebsiella granulomatis, N. gonorrhea, Mycoplasma genitalium, Treponema pallidum, T. vaginalis Specimens for molecular testing 24 Storage Specimens for molecular testing 25 Thank you for your attention Specimens for molecular testing 26