How to Cite:

Alnihier, M. M. H., Alotaibi, A. T., & Alanzi, A. S. B. (2022). Storage conditions and collection
containers for various specimen types to preserve analyte stability: Review. International Journal of
Health Sciences, 6(S9), 5121–5131. [Link]

Storage conditions and collection containers

for various specimen types to preserve analyte
stability: Review

Mohammed Mishal Hamoud Alnihier

KSA, National Guard Health Affairs
Email: AlniherMo@[Link]

Ahmed Turki Alotaibi

KSA, National Guard Health Affairs
Email: A7mdoh85@[Link]

Awad Shehab B Alanzi

KSA, National Guard Health Affairs

Abstract---Background: Molecular tests for detecting nucleic acids in

biological materials are crucial in clinical diagnosis and therapy
decision-making. However, errors in molecular labs often stem from
pre-analytical mistakes during specimen handling. The integrity of
nucleic acids can be compromised by inappropriate collection, transit,
and storage procedures. Despite the importance of pre-analytic
factors, there is a lack of standardized recommendations for molecular
assays. Aim of Work: This article aims to provide a comprehensive
overview of key considerations in sample handling before molecular
testing, covering various types of biological samples. Methods: We
conducted a review of the literature to identify key pre-analytical
factors that can impact molecular testing. We focused on specimen
collection, transportation, and storage protocols for solid tissues,
whole blood, serum, plasma, and other bodily fluids. We also
discussed pre-analytical factors relevant to prenatal diagnostic
samples. Additionally, we highlighted the importance of adhering to
established procedures to minimize errors in molecular testing.
Results: Our review identified several critical pre-analytical factors
that can affect the quality of nucleic acids in biological samples.
Factors such as inadequate sample collection, improper
transportation conditions, and suboptimal storage can lead to
degraded nucleic acids, affecting the accuracy of molecular tests.
Following standardized protocols for sample handling is essential to
ensure reliable results in molecular analysis. Conclusion: In
conclusion, pre-analytical factors play a significant role in the success
of molecular testing for nucleic acids. By understanding and

International Journal of Health Sciences E-ISSN 2550-696X © 2022.

Manuscript submitted: 27 June 2022, Manuscript revised: 18 August 2022, Accepted for publication: 6 Sept 2022
5121
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addressing key considerations in sample handling, healthcare

professionals can improve diagnostic accuracy and treatment
decisions. Standardized protocols for specimen collection,
transportation, and storage are crucial in minimizing errors and
ensuring the reliability of molecular assays. Further research and
consensus guidelines are needed to establish best practices for pre-
analytic factors in molecular testing.

Keywords---Biological fluids, Formaldehyde, Genetic material, Pre-

analytical stage.

Introduction

Nucleic acid-based diagnostic assays are seeing fast expansion as they serve to
supplement or even replace conventional diagnostic techniques in clinical labs.
Additionally, they are used for prognostication and determining the optimal
approach for treating several ailments. These tests need little quantities of DNA or
RNA samples to be conducted and are very precise. The findings can be
replicated, but like other diagnostic tests, they may have limitations due to
analytic and pre-analytic variables. Several studies indicate that pre-analytical
mistakes account for around 60-70% of all laboratory errors, both inside and
outside of laboratory settings [1,2]. There are valuable suggestions for
standardizing specimen processing in molecular labs, but there is a significant
amount of variation in how specimens are handled before they are sent to these
laboratories. This crucial stage of test performance has the potential to provide
inaccurate outcomes, either in the form of false negatives or false positives.
Implementing dependable protocols in sample processing will enhance the
precision and consistency of these diagnostic tests. The primary concerns about
sample handling are to the preservation of nucleic acid integrity and stability, as
well as the potential impact of interfering agents during sample transit,
extraction, and storage.

Recently, Compton et al. put out a set of guidelines specifically designed for tissue
and blood specimens from cancer patients (Compton et al.) [3]. This article is a
study from the Precision Medicine Project Team of the College of American
Pathologists (CAP). The team has developed a set of evidence-based
recommendations for pre-analysis procedures in tissue and blood specimens.
Their review was restricted to primary data obtained from published
recommendations by ASCO (American Society of Clinical Oncology),
Biorepositories and Biospecimen Research Branch, CAP, European Committee for
Standardization/Technical Committee, CLSI (Clinical & Laboratory Standards
Institute), and other relevant guidelines. Their emphasis was only on pre-
analytical parameters that affected the analysis data of nucleic acids utilized for
cancer patients, namely in formalin-fixed, paraffin-embedded (FFPE) tissues or
blood/plasma biospecimens.
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Aim of Work

This article examines published papers and recommendations that discuss proper
procedures for collecting, transporting, preserving, and storing various biological
samples, such as tissue and blood. These procedures are commonly followed in
pathology and translational research laboratories. Aside from the examination of
cancer biospecimens, this study also included a comprehensive analysis of
prevalent molecular assays used for the detection of various viral and bacterial
pathogens. The majority of literature regarding pre-analytical research in human
samples was gathered from the previous 20-year period. In order to choose papers
for citation, we took into account both the citation rate of the article and the
ranking of the journal in which it was published.

Form for Selecting and Requesting Tests

Multiple medical professionals often request molecular testing to accurately

diagnose, choose an appropriate therapeutic approach, or determine the
prognosis classification of patients. Clinicians who request a molecular test
should be cognizant of the limits of the test's efficacy in guiding management and
decision-making, as well as its cost-effectiveness. There are still several disorders
for which some regular non-molecular diagnostics are more reliable than the
currently available molecular testing. The doctors must ensure that the request
has been determined adequately with a rational and cost-efficient step-by-step
approach in patient care. Conducting essential but unsolicited molecular studies
after to initial diagnosis by pathologists is a recognized approach in molecular
labs [4]. Characterizing gene expression assays accurately and performing these
tests by an in-house pathologist or laboratory medicine experts (reflex testing) is
crucial for saving time in targeted treatment [5-12]. This guideline is particularly
advisable when the proportion of samples with a specific diagnosis requiring
further testing exceeds 10%. Some frequent examples of genetic abnormalities in
different types of cancer are HER2 amplification in breast cancer, EGFR
mutations and ALK rearrangements in lung cancer, BRAF mutations in malignant
melanoma, RAS mutations in colorectal cancer, and BCR/ABL1 in chronic
myelogenous leukemia [4].

Best Practices for Pre-analytical Phase in Various Biological Samples

Tissue specimens

Fixation is an essential process for preserving tissues in the long term, preventing
self-destruction and the deterioration of molecular components. Fixed tissue
specimens kept in paraffin blocks in pathology departments are often used as
useful reservoirs of nucleic acids for molecular tests. Hence, it is crucial to
examine pre-analytic factors in order to prevent any potential misunderstanding
of the findings.

Categories of Fixatives

Commonly used fixatives include neutral buffered formalin (NBF), ethanol,

methanol, Bouin, Carnoy, Zenker, and glutaraldehyde [13-15]. We provide a
5124

concise overview of the impacts of various fixatives on nucleic acids. Formalin

fixation causes the formation of cross-links between DNA and proteins, as well as
between RNA and proteins. These cross-links might hinder the effective extraction
of nucleic acids and can interfere with in situ hybridization. Nucleic acid
fragmentation and loss might result from extended fixation of the material or
changes in the fixative's pH. Fixation in un-buffered formalin causes a substantial
reduction in the amount of extracted DNA compared to buffered formalin,
resulting in less efficient DNA detection [16].

Formalin fixation may cause unpredictable damage to nucleic acids and result in
erroneous mutations [17]. The postmortem interval, which is the time elapsed
since death, and the cold ischemia time, which is the time between tissue removal
from the body and preservation, should be restricted to 48 hours and 1 hour
respectively when analyzing DNA using Fluorescence In Situ Hybridization (FISH).
Similarly, for PCR analysis, these thresholds can be defined as less than 4 days
and 24 hours. For maximum DNA integrity, it is recommended to fix the sample
in formalin for fewer than 72 hours. However, PCR may still be performed
successfully even if the fixation time is greater. This applies to both formalin
fixation and the creation of formalin-fixed paraffin-embedded (FFPE) blocks [18].
Several authors have suggested that for optimal preservation of nucleic acids, it is
advisable to begin formalin fixation within 2 hours of tissue removal, use cold
fixation at 4°C with 10% neutral formalin, fix the tissue for a duration of 3 to 6
hours, add ethylene-diamine-tetra-acetic acid (EDTA) at a concentration of 20-50
mmol/L, and avoid exposure to low pH environments [12].

Formalin may chemically modify RNA, unlike fresh material. In an experimental

investigation, the extracted RNA from tissues showed reverse transcription
inhibition in quantitative RT-PCR experiments, particularly in samples with long
length amplicons, while having satisfactory RNA integrity scores in FFPE samples.
The PAXgene fixed samples did not exhibit this inhibition (19). For RNA analysis,
the duration of fixation in NBF should not exceed 48 hours at 4°C or 8 hours at
room temperature (uncontrolled RT). Additionally, the blocks should be examined
within a maximum of 1 year. The research has generally neglected to consider the
potential effects of many parameters, such as dehydration, clearing, paraffin
reagents, and other circumstances, on DNA endpoints. To get more details, we
suggest consulting a highly commendable study authored by Bass et al. [18].
Although there are several hurdles, genomic and gene expression data obtained
from FFPE specimens often provide satisfactory outcomes when compared to the
original fresh samples.

Ethanol and methanol are very effective fixatives for maintaining both high
molecular weight DNA and RNA, while causing little chemical alterations to the
nucleic acids [12]. Aside from its ability to preserve solid tissue, ethanol may also
serve as a means of transporting and preserving tissue at room temperature (RT)
[19,20]. Kilpatrick et al. proposed that tissues preserved in ethanol for a duration
ranging from one day to two years may yield high molecular weight DNA with
great efficiency. After a period of 6 months, significant DNA destruction was seen
in tissues that were kept in 20% ethanol.
 5125

Carnoy's solution, consisting of 60% ethanol, 30% chloroform, and 10% glacial
acetic acid, is considered one of the most effective fixatives for preserving nucleic
acids, particularly high-molecular-weight RNA [12, 21]. Research has shown that
RNA obtained from epithelial cells and preserved in Carnoy's solution retains the
integrity of nucleic acids for the beta-actin gene, allowing amplification of up to
977 base pairs (bps) [22]. Carnoy's fixative demonstrates superior tissue
preservation compared to methanol-acetone when evaluated after a post-fixation
time of more than 3 months [22]. Li et al. performed DNA amplification on target
DNA sequences extracted from cells that had been fixed in Carnoy's preservative
and kept for varying periods of time, ranging from a few weeks to 6 years, at a
temperature of -20°C. The researchers found no discernible variation in the
amplification products' quality when comparing three different template sizes (400
bps for the retinoblastoma gene, 251 bps for the Duchenne muscular dystrophy
gene, and 609 bps for the sex-determining region of the Y gene). This held true for
both fresh tissue samples and Carnoy's-fixed cells [23].

The efficacy of extracting and retaining total RNA from tissue has been shown
using a fixative solution called Methacarn. Methacarn is composed of 60%
methanol, 30% chloroform, and 10% glacial acetic acid. RNA fragments ranging
from 300 to 700 base pairs in length may be amplified from methacarn-fixed
tissues [24].

Severe DNA degradation is found in Bouin's, B5, and Zenker's solutions due to
the presence of heavy metals in these fixatives. Using a fixative containing
mercury for nucleic acid separation is not recommended. Mercuric acid and
chromium not only coagulate proteins, but also induce the development of large
metal-nucleoprotein complexes, resulting in a decrease in the amount of DNA
extracted [25-27]. Furthermore, Bouin's solution, which consists of acetic acid
and formalin with a pH of 2, induces DNA de-purination and causes damage to
both DNA and RNA [25, 28]. These ribonucleic acids develop resistance to
ribonuclease enzymes, which may lead to their presence in DNA extraction
products [29].

Glutaraldehyde is a commonly used fixative in the field of electron microscopy.

Studies have shown that DNA with a high molecular weight (>50 kb) is better
retained in tissues that have been fixed in a 0.2 M phosphate-buffered solution
containing 1% glutaraldehyde (at pH 7.0) compared to tissues fixed in a 10%
buffered formalin solution [13]. There is a lack of data on RNAs that have been
preserved with glutaraldehyde.

Consequences of Decalcification

Decalcification agents often utilized, such as nitric acid and hydrochloric acid,
include powerful acids that destroy nucleic acids and reduce their recovery rate
from samples. The amount and purity of the extracted nucleic acids are
satisfactory when decalcification is performed using either 14% EDTA or formic
acid, as opposed to the previously described strong acids [30-32].
5126

Paraffin

Paraffin waxes infiltrate the tissue in a liquid state, quickly solidify upon cooling,
and maintain the structure of the tissue during sectioning [27]. There are many
types of paraffin waxes with varying quality and melting temperatures, ranging
from 47°C to 64°C. These variations may have a significant impact on the process
of nucleic acid extraction, leading to potential confusing consequences. Paraffin,
when exposed to a temperature of 60°C, has the potential to induce DNA damage
and protein cross-linking [33-35]. Paraffin removal is essential for obtaining pure
DNA since any remaining paraffin might hinder PCR, as shown in [36, 37]. Goelz
et al. first reported the inhibitory impact of remaining paraffin on the amount of
DNA obtained [38]. Chung et al. showed that thorough removal of paraffin is an
essential need for extracting nucleic acids from specimens that are embedded in
paraffin. The formalin fixation and paraffin embedding resulted in a 70% decrease
in RNA production and a reduction in amplicon length to fewer than 300 bps, as
shown in their demonstration [33]. However, a separate research demonstrated
that reverse transcription-polymerase chain reaction (RT-PCR) successfully
amplified all (100%) RNA fragments up to 151 base pairs in length for the
housekeeping gene (glucose-6-phosphate dehydrogenase) in breast cancer
samples that were preserved in paraffin. Nevertheless, it is important to
acknowledge that advancements in nucleic acid extraction techniques have led to
a reduction in the disruptive impact of paraffin [40].

While evaluating single nucleotide polymorphisms (SNPs) using real-time

techniques, Huijsmans et al. suggested the use of extraction by silica membrane-
based column and improved magnetic silica, despite the potential presence of
amplification inhibitors in these approaches. The researchers discovered that the
use of a silica membrane-based extraction technique, together with the addition
of magnetic silica, could effectively amplify DNA fragments ranging from 400 to
600 base pairs [41]. RNA fragmentation may occur in paraffin-embedded tissue
samples [42]. Páska et al. suggested that amplicons of up to 225 base pairs (bps)
may provide satisfactory outcomes in gene expression investigations [42], but
Godfrey et al. demonstrated that only RNA amplicons shorter than 130 bps could
produce acceptable findings in reverse transcription polymerase chain reaction
(RT-PCR) [43]. To enhance PCR efficiency, modifying some thermocycler settings,
such as increasing the number of cycles and the length of each cycle, was
beneficial [44, 45].

Microtomy

The ideal thickness of sections in FFPE tissue for nucleic acid assessment is
contingent upon the size and cellularity of the tissues. Typically, it is advised to
use 20-µm slices of big tissues or 40 to 80 µm thickness of smaller tissue for PCR
analysis [46]. The recommended protocol suggests using three sections that are
10 micrometers thick for each response in kidney, prostate, stomach, colon,
brain, bone marrow (BM), and lung samples. However, for breast tissue, it is
necessary to use 10 sections with a similar thickness in order to carry out DNA
extraction [47]. Based on our empirical observations, using five sections that are
10 micrometers thick may provide dependable outcomes in terms of both the
quality and amount of nucleic acid (data that has not been published). Prior to
 5127

each block sectioning, it is advisable to clean the blade of microtomes by using

100% ethanol [46] and xylene or by employing a disposable blade [48]. To ensure
the thorough cleaning of blades and prevent any contamination from previous
blocks, it is recommended to cut at least two sections of a blank paraffin block
without any tissue between each set of target blocks. The sections of these blanks
should undergo DNA extraction procedures and be evaluated simultaneously with
the samples of interest to guarantee the absence of any cross-contamination [46].

Tissue Conditions

While the composition of the tissue has a role, typically 1 to 2 grams of tissue
would provide sufficient material for molecular analysis. A minimum of 2 grams of
tissue is required for the analysis of low cellular specimens such as muscle,
fibrous, and adipose tissue [46]. During surgical operations, extended use of
anesthetic and ischemia techniques such artery ligation may result in tissue
damage due to lack of oxygen and lowered pH levels. This can subsequently
impact the expression of mRNA, leading to inaccurate quantification of different
mRNAs [12, 49]. The age of a sample is a crucial component that must be taken
into account when dealing with archival samples. According to Goelz et al., DNA
fragment sizes obtained from samples maintained for four to six years were often
lower compared to the DNA sizes of samples held for less than two years [38].
However, some labs successfully used 20-year-old FFPP blocks without
encountering any issues [50, 51].

Watanabe et al. assessed the quantity, quality, and intactness of DNA in FFPE
tissue samples. A research was done on twenty-five lung cancer FFPE samples
that had been maintained at room temperature for 0.5, 3, 6, 9, and 12 years. The
researchers used two DNA extraction techniques and observed that the duration
of storage impacts the DNA's integrity, but not its overall yield and purity in FFPE
samples, irrespective of the extraction method [52]. In a separate work, Wang et
al. demonstrated effective amplification of DNA from 40-year-old FFPE tissues.
However, the amount of DNA obtained was lower compared to fresh cells [53]. The
target tissues for molecular analysis often consist of a heterogeneous combination
of cells, not all of which are the primary focus of molecular tests. In order to
prevent the weakening of nucleic acids from unwanted cells, it is essential to
identify and designate the specific region that contains the desired cells on both
the hematoxylin and eosin (H&E) slide and the associated paraffin block. One
possible subsequent procedure may include performing shallow punching on the
paraffin block or using laser micro-dissection on the slides. To summarize, while
long-term storage of blocks has proven effective for DNA analysis, it is crucial to
acknowledge that the length of amplifiable gene fragments may diminish with
time. The literature has not thoroughly addressed the potential effects of many
parameters, such as dehydration, clearing, paraffin reagents, and other
circumstances, on DNA endpoints. These effects should be confirmed in future
investigations.

Conclusion

It is recognized that putting into practice the suggestions provided here may need
changes in processes, timetables, or supplies. However, it is vital to note that a
5128

trustworthy documenting of the pre-analytical history of patients' specimens is a

necessary need for its application. its documentation provides objective hints to
identify false positive or negative results. Specimen pre-analytical variables in
translational research facilities are neither regulated nor recorded. Furthermore,
many labs do not regularly conduct essential quality control measures that are
required for clinical purposes, which might potentially identify subpar specimen
quality prior to testing. As a result, the results produced by these laboratories
may be entirely incomprehensible or inconclusive. Hence, reaching a consensus
on the precise factors related to patient specimen collection, handling, processing,
storage, and transportation that lead to quality issues and molecular changes
would guarantee that the research data is sufficiently reliable and reproducible.
This, in turn, would facilitate the translation of findings from laboratory
experiments to clinical applications.

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