Factors to consider in molecular diagnostics testing during the preanalytical phase

Molecular diagnostics applies techniques of molecular biology to diagnose disease, as well as predict its course; select appropriate treatments; and monitor treatments’ effectiveness.¹ Testing within this medical field often involves a complex workflow of many steps that are prone to variability. Different factors can influence this variability, and if they are not controlled, they could alter specimen integrity and thus lead to patient misdiagnosis.

The preanalytical phase plays an important role in the entire workflow followed in molecular diagnostics, with steps that include ordering of the test, as well as the collection, transport, storage, and processing of the sample. This last step, which comprises the extraction of the molecular target, is a fundamental method in molecular biology, as it is the beginning of downstream processes and product development, including diagnostic kits. Errors made in each of these steps can have a significant impact on the correct diagnosis of a patient. This review analyzes important variables in these preanalytical phase steps that have an impact on molecular diagnostics testing.

Test ordering and patient health status

Medical specialists request molecular tests to enable them to reach a diagnosis and to choose a treatment strategy. In this regard, physicians requesting a molecular test should be aware of the limitations of such tests in management and decision making, as well as the cost–benefit ratio.

Errors in test ordering are due to a variety of reasons and are commonly the result of simple mistakes, such as unnecessarily repeating a test, forgetting to order a test, or simply ordering the wrong test. The implementation of electronic medical records systems has contributed to reducing these errors, resulting in a decrease in duplicate orders, as well as reduced costs and fewer issues with preauthorization.² Additionally, diagnostic stewardship programs in areas like infectious diseases can assist not only in guiding test ordering, but also in speeding up test results and their interpretation.^3,4,5

A patient’s health status also plays a role in the results of molecular diagnostic tests. Patients receiving antiviral treatment are monitored for treatment efficacy by nucleic acid amplification tests (NAAT), where viral loads decrease to low or undetectable levels with treatment. In the case of a CMV (cytomegalovirus) infection, for example, viral load has been also used not only for its prognostication, but also as a guidance for preemptive therapy and an indication of the risk of clinical relapse or drug resistance.⁶ A similar effect on disease management is applied to HIV patients undergoing preexposure prophylaxis, who in addition to showing a reduced viral load may present a delayed seroconversion, which hinders the detection of HIV RNA and antibodies.^7,8 In cases of an acute viral infection like COVID-19, patients experience evident symptoms, and the viral load is at detectable levels. However, testing during the early phases of a latent period can lead to false negative results if there are low levels of viral particles; it is therefore important to consider replication dynamics when performing molecular testing.⁹

Sample adequacy, collection, transport, and storage

When a patient is suspected of having an infection, various factors can influence a test’s efficacy in diagnosing them. False positives may be caused by lingering traces of viral DNA or RNA, specimen contamination, or other complications. False negatives are also possible, largely caused by improper sample collection and handling.³

Hemolysis of blood samples is the most frequent pre-analytical interference and a major source of error leading to unreliable test results. The effect of in vitro hemolysis should be considered in the fields of cancer diagnostics and non-invasive prenatal testing. In this sense, the release of genomic DNA from non-tumor and maternal white blood cells can lead to underestimation of the tumor DNA and fetal DNA fractions, respectively, in hemolyzed samples.¹⁰

The adequacy of the collection method used is of paramount importance when assessing the quality of molecular amplification methods in clinical diagnostics. In blood and bone marrow specimens, clotting must be inhibited, with EDTA and citrate being commonly used for this purpose. Specimen collection systems containing citrate dilute the specimen by 10%, while heparin (routinely 14.3 IU/ml of whole blood) inhibits amplification in concentrations as low as 0.05 IU per reaction volume.¹¹ Components of whole human blood (WHB), like immunoglobulin G, hemoglobin and lactoferrin can also act as PCR inhibitors. Additionally, it may be possible to completely inhibit Taq DNA and AmpliTaq Gold, common polymerases used for PCR, in the presence of less than 0.2% WHB, when direct PCR is performed.¹² PCR inhibitors generally act through the inactivation of DNA polymerases, binding of DNA polymerase co-factors, or degradation of target nucleic acids and/or primers.¹³

Collected blood can be used directly (whole blood) or be fractionated into serum, plasma, or buffy coat. If the fractions are not intended for short term usage, they should be divided into multiple aliquots in small vials and stored in freezers to avoid multiple freeze-thaw cycles. Whole blood can be temporarily stored at room temperature for up to 24 hours, or in the refrigerator (2°C – 8°C) for a maximum of 72 hours. After this time, genomic DNA (gDNA) will degrade.¹⁴ If this time is exceeded, the erythrocytes should be removed, since the heme group can inhibit the PCR.

As for RNA, whole blood should be collected in tubes containing an RNA stabilizer. If this is not possible, the specimen should be placed on ice immediately after collection and transported to the laboratory for RNA extraction. The quick addition of RNase inhibitors is also an established step in the preanalytical phase when working with RNA assays.

 When compared to whole blood plasma, serum is less favorable in molecular diagnostics due to its lower DNA yield. However, it may be suitable for evaluating gDNA. For DNA or RNA studies, serum should be shipped frozen on dry ice and stored at -20°C.^15,16

Plasma DNA concentrations gradually decrease over time and can be delayed by keeping the samples stored at a temperature ranging between 2°C to 8°C. Evaluation of the effects of different storage temperatures on RNA and DNA levels in unfiltered plasma showed that storage of samples at 4°C yielded stable RNA levels for up to 24 hours, while DNA levels were stable at both 4°C and room temperature for 24 hours.¹⁷

Buffy coats, as a layer enriched in white blood cells, are commonly used as a source of nucleic acids for molecular assays. If DNA extraction from buffy coat is performed within days, it is recommended to isolate it and store at -70°C or lower.¹⁸ If, however, DNA isolation occurs immediately after collection, it can be stored long term (for up to 9 years) in a deep-frozen state (-80°C).¹⁹ Additionally, resuspending the separated buffy coat in TRIzol and immediately cryopreserving it at -80°C is very effective for extracting RNA of high purity and quality that is suitable for sequencing.²⁰

Sample processing

Preparing a sample for molecular testing requires different processes in which the sample is mixed with reagents for extraction, stability, and amplification. Some samples, such as sputum and saliva, are difficult to process due to their high viscosity, making them difficult to handle in laboratories with automated platforms. These samples can cause pipetting errors and contamination. To prevent this from happening, homogenization procedures are implemented to liquify the sample prior to nucleic acid extraction, which can include proteinase K or dithiothreitol,²¹ as well as a mechanical disruption by glass bead beating.²²

Although working with viscous samples can cause issues in automated platforms, there are many benefits to working in an automated fashion as it can improve throughput, reproducibility, and maximize the laboratory technician's time for data analysis. Additionally, the risk of sample contamination can be reduced, as well as environmental contamination of the laboratory, with potentially infectious material.

Additional pretreatments that can have a positive impact on sample processing and downstream molecular testing involve work with pathogen inactivating agents. A chemical commonly used for this purpose is guanidinium thiocyanate, which is the main component of nucleic acid extraction kits. Buffers containing it destabilize the viral envelope and eliminate cellular nucleases, while maintaining the structure of DNA and RNA for subsequent molecular biology analysis in lower biosafety level facilities.²³ Other methods include thermal inactivation, UV radiation, and the use of formaldehyde.^24,25,26

Conclusion

The preanalytical phase of the molecular diagnostics workflow is complex and requires meticulous attention to best practice, as sample quality forms the basis for accurate patient diagnosis and treatment. While the field of molecular diagnostics constantly evolves, the preanalytical phase will continue to influence its effectiveness and trajectory.

References

1. Koellner CM, Mensink KA, Highsmith WE. Molecular Pathology. The Molecular Basis of Human Disease. In: Coleman WB, Tsongalis GJ, eds. Molecular Pathology. The Molecular Basis of Human Disease. Second Edition. Academic Press; 2018.
2. Conrad S, Gant Kanegusuku A, Conklin SE. Taking a step back from testing: Preanalytical considerations in molecular infectious disease diagnostics. Clin Biochem. 2023;115:22-32. doi:10.1016/j.clinbiochem.2022.12.003. 
3. Diagnostic stewardship interventions that make a difference. Asm.org. Published August 3, 2021. Accessed November 17, 2023. https://asm.org/Articles/2021/August/Diagnostic-Stewardship-Interventions-That-Make-a-D.
4. Zacharioudakis IM, Zervou FN. Diagnostic stewardship in infectious diseases: steps towards intentional diagnostic testing. Future Microbiol. 2022;17:813-817. doi:10.2217/fmb-2022-0070. 
5. Ku TSN, Al Mohajer M, Newton JA, et al. Improving antimicrobial use through better diagnosis: The relationship between diagnostic stewardship and antimicrobial stewardship. Infect Control Hosp Epidemiol. 2023;4:1-8. doi:10.1017/ice.2023.156. 
6. Tan SC, Yiap BC. DNA, RNA, and protein extraction: the past and the present. J Biomed Biotechnol. 2009;2009:574398. doi:10.1155/2009/574398.
7. Custer B, Quiner C, Haaland R, et al. HIV antiretroviral therapy and prevention use in US blood donors: a new blood safety concern. Blood. 2020;10;136(11):1351-1358. doi:10.1182/blood.2020006890. 
8. Grebe E, Busch MP, Notari EP, et al. HIV incidence in US first-time blood donors and transfusion risk with a 12-month deferral for men who have sex with men. Blood. 2020;10;136(11):1359-1367. doi:10.1182/blood.2020007003. 
9. Xin H, Li Y, Wu P, et al. Estimating the Latent Period of Coronavirus Disease 2019 (COVID-19). Clin Infect Dis. 2022;3;74(9):1678-1681. doi:10.1093/cid/ciab746.
10. Nishimura F, Uno N, Chiang PC, et al. The Effect of In Vitro Hemolysis on Measurement of Cell-Free DNA. J Appl Lab Med. 2019;4(2):235-240. doi:10.1373/jalm.2018.027953. 
11. Neumaier M, Braun A, Wagener C. Fundamentals of quality assessment of molecular amplification methods in clinical diagnostics. International Federation of Clinical Chemistry Scientific Division Committee on Molecular Biology Techniques. Clin Chem. 1998;44(1):12-26.
12. Cai D, Behrmann O, Hufert F, Dame G, Urban G. Direct DNA and RNA detection from large volumes of whole human blood. Sci Rep. 2018;21;8(1):3410. doi:10.1038/s41598-018-21224-0. 
13. Schrader C, Schielke A, Ellerbroek L, Johne R. PCR inhibitors - occurrence, properties and removal. J Appl Microbiol. 2012;113(5):1014-26. doi:10.1111/j.1365-2672.2012.05384.x. 
14. Malentacchi F, Ciniselli CM, Pazzagli M, et al. Influence of pre-analytical procedures on genomic DNA integrity in blood samples: the SPIDIA experience. Clin Chim Acta. 2015;2;440:205-10. doi:10.1016/j.cca.2014.12.004.
15. Shepard E, Madej RM, Alfaro MP, et al. Collection, Transport, Preparation, and Storage of Specimens for Molecular Methods. 2nd ed. CLSI guideline MM13. Clinical and Laboratory Standards Institute; 2020. 
16. ​Springer J, Morton CO, Perry M, et al. Multicenter comparison of serum and whole-blood specimens for detection of Aspergillus DNA in high-risk hematological patients. J Clin Microbiol. 2013;51(5):1445-50. doi:10.1128/JCM.03322-12.  
17.  Tsui NB, Ng EK, Lo YM. Stability of endogenous and added RNA in blood specimens, serum, and plasma. Clin Chem. 2002;48(10):1647-53.  
18. ​Austin MA, Ordovas JM, Eckfeldt JH, et al. Guidelines of the National Heart, Lung, and Blood Institute Working Group on Blood Drawing, Processing, and Storage for Genetic Studies. Am J Epidemiol. 1996;1;144(5):437-41. doi:10.1093/oxfordjournals.aje.a008948. 
19. Mychaleckyj JC, Farber EA, Chmielewski J, et al. Buffy coat specimens remain viable as a DNA source for highly multiplexed genome-wide genetic tests after long term storage. J Transl Med. 2011;10;9:91. doi:10.1186/1479-5876-9-91.  
20. Wilson C, Dias NW, Pancini S, Mercadante V, Biase FH. Delayed processing of blood samples impairs the accuracy of mRNA-based biomarkers. Sci Rep. 2022;17;12(1):8196. doi:10.1038/s41598-022-12178-5.
21. Peng J, Lu Y, Song J, et al. Direct Clinical Evidence Recommending the Use of Proteinase K or Dithiothreitol to Pretreat Sputum for Detection of SARS-CoV-2. Front Med (Lausanne). 2020;18;7:549860. doi:10.3389/fmed.2020.549860.
22. ​Lu J, Carmody LA, Opron K, et al. Parallel Analysis of Cystic Fibrosis Sputum and Saliva Reveals Overlapping Communities and an Opportunity for Sample Decontamination. mSystems. 2020;7;5(4):e00296-20. doi:10.1128/mSystems.00296-20.  
23. ​Elveborg S, Monteil VM, Mirazimi A. Methods of Inactivation of Highly Pathogenic Viruses for Molecular, Serology or Vaccine Development Purposes. Pathogens. 2022;19;11(2):271. doi:10.3390/pathogens11020271.  
24. Espinosa MF, Sancho AN, Mendoza LM, Mota CR, Verbyla ME. Systematic review and meta-analysis of time-temperature pathogen inactivation. Int J Hyg Environ Health. 2020;230:113595. doi:10.1016/j.ijheh.2020.113595.  
25. ​Ohashi H, Koi T, Igarashi T. State-of-the-art technology: Inactivation of pathogens using a 222-nm ultraviolet light source with an optical filter. J Sci Technol Light. 2021;44(0):9-11. doi:10.2150/jstl.ieij20a000006. 
26. Möller L, Schünadel L, Nitsche A, et al. Evaluation of virus inactivation by formaldehyde to enhance biosafety of diagnostic electron microscopy. Viruses. 2015;10;7(2):666-79. doi:10.3390/v7020666.