JCCLS MM5-A1(2011.12.1)

An Approved Guideline for the Quality Management ofSpecimens for Molecular Methods:
The Procurement, Transport, and Preparation of Specimens
Abstract	 This guideline has been drafted to address the growing number of molecular diagnostic methods used inroutine medical laboratories worldwide. A variety of biological specimens with varying characteristics
can be analyzed by several molecular diagnostic methods. Specimens of different origins require
adequate pretreatment before nucleic acid extraction through one of several protocols, depending on thespecimen’s characteristics and eventual use. The guidelines describe the general principles and basic
methods of collection, storage, transport, and preparation of specimens for the molecular analysis of
specific sequences for pathogens, somatic cells, and germ line cells. Each section discusses the proper
methods to assure appropriate specimen collection and processing, covers inappropriate specimen
conditions, their possible causes, and concludes with the presentation of various techniques that help toeither eliminate or avoid these errors.

Japanese Committee for Clinical Laboratory Standards (JCCLS).
48 Higashi-matsushita-machi, Kanda, Chiyoda-ku, Tokyo 101-0042 Japan

	
 

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JCCLS MM5-A1(2011.12.1)

Contents
1. Introduction
2. Scope
3. Storage and Transport of Specimens for Molecular Methods
a. Storage temperature for nucleic acids
b. Storage containersc. Transport of specimens
3.1 Storage and Transport of Specimens in Molecular Methods for Pathogens
3.1.1 Serum and Plasma
3.1.2 Urine
3.1.3 Sputum
3.2

Storage and Transport of Specimens in Molecular Methods for Somatic Cells
3.2.1 Tissue and Tissue Slice Fragments
3.2.2 Whole Blood (WBC)
3.2.3 Urine and Stool

3.3

Storage and Transport of Specimens in Molecular Methods for Germ Line Cells

3.3.1 Outsourcing of Genetic Testing

4. Preparation of Specimens for Molecular Methods
4.1 Preparation of Specimens in Molecular Methods for Pathogens
4.1.1 Serum
4.1.2 Plasma
4.1.3 Sputum
4.1.4 Stool
4.1.5 Urine
4.1.6 Whole Blood (WBC) and Bone Marrow
4.1.7 Pleural Effusion, Ascites, Pericardial Fluid, Pancreatic Fluid andBroncho-Alveolar Lavage Fluid (BALF)
4.1.8 Lymph Nodes and Solid Tissues (Biopsy or Surgery)
4.2 Preparation of Specimens in Molecular Methods for Somatic Cells
4.2.1 Lymph Nodes and Solid Tissues (Biopsy or Surgery)4.2.2 Formalin-Fixed Paraffin Embedded Tissue
4.2.3 Whole Blood (WBC) and Bone Marrow
4.2.4 Pleural Effusion, Ascites, Pericardial Fluid, Pancreatic Fluid,
Broncho-Alveolar Lavage Fluid (BALF), Urine Sediment and Sputum
4.2.5 Cultured Cells
4.3 Preparation of Specimens in Molecular Methods for Germ Line Cells
4.3.1 Whole Blood (WBC)

	
 

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JCCLS MM5-A1(2011.12.1)
4.3.2 Oral Mucosa and Fluid
4.3.3 Hair
4.3.4 Nails
4.3.5 Dried Blood Spots
4.3.6 Umbilical Cord

5. Collection of Specimens for Molecular Methods
5.1 Collection of Specimens in Molecular Methods for Pathogens
5.1.1 Viral Infection
5.1.1.1 Hepatitis Virus
5.1.1.2 Virus Causing Acute Respiratory Infection
5.1.1.3 Virus Causing Diarrhea
5.1.1.4 Encephalitis Virus
5.1.1.5 Aseptic Meningitis Virus
5.1.1.6 Epstein‐Barr virus: EBV
5.1.1.7 Cytomegalovirus: CMV
5.1.1.8 Human Parvovirus B19
5.1.1.9 Human Papilloma Virus: HPV
5.1.1.10 HTLV‐I (Human T‐Lymphotropic Virus Type I)
5.1.1.11 HIV (Human Immunodeficiency Virus)
5.1.2 Bacterial Infection
5.1.2.1 Tuberculosis and Other Mycobacteria
5.1.2.2 Legionella
5.1.2.3 Mycoplasma pneumoniae
5.1.2.4 Chlamydia pneumoniae/psittaci
5.1.2.5 Chlamydia trachomatis
5.1.2.6 Neisseria gonorrhoeae
5.1.2.7 MRSA (Methicillin‐Resistant Staphylococcus aureus)
5.1.3 Fungal infection
5.1.3.1 Pathogenic Fungi
5.1.3.2 Pneumocystis jiroveci
5.2 Collection of Specimens in Molecular Methods for Somatic Cells
5.2.1 Solid Tissue Tumors
5.2.1.1 Pancreatic Carcinoma
5.2.1.2 Lung Carcinoma
5.2.1.3 Colon Carcinoma
5.2.1.4 Gastrointestinal Stromal Tumor (GIST)
5.2.1.5 Breast and Stomach Carcinomas
5.2.2 Hematological Neoplasms
5.2.2.1 Leukemias
5.2.2.2 Malignant Lymphomas

	
 

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5.3 Collection of Specimens in Molecular Methods for Germ Line Cells
5.3.1 Genetic Testing
5.3.2 Single Gene Disorders
5.3.3 A pharmacogenomic test

6. References
7. Appendix A
8. Appendix B

	
 

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1. Introduction
This guideline is drafted to address growing number of molecular diagnostic methods used in routine
medical laboratories worldwide. Molecular diagnostic methods facilitate the analysis of specific nucleic
acid sequences for pathogens, somatic cells, and germ line cells. Prior to analysis, there are many stagesof specimen handling, including collection, storage, transport, and preparation. These preanalytic factorsof molecular diagnostics greatly impact the quality of an assay and its subsequent results. A variety of
biological specimens can be used for molecular diagnostics. They have varying physical, chemical, andbiological features, which may determine the quality of the assay, and therefore greatly influence theyield and the quality of the extracted nucleic acids. Each specimen type requires the appropriate
treatment prior to nucleic acid extraction, depending on the specimen’s characteristics and eventual
purpose of use. In order to assure the quality of assay results, it is extremely important to standardize thepre-analytic process, including the condition and treatment of specimens. However, it is difficult to
optimize the process of collection, storage, transport and preparation of specimens. One reason for this isdue to the number of professional and non-professionals, including patients, who are involved in thecritical procurement process: collection, storage, and transport of specimens. Furthermore, the knowledge
and skills involved in sample processing can be highly variable. A more critical explanation is that themethods involved in the collection, storage, transport, and preparation of patient samples are notuniversally common among different facilities and laboratories. Although several guidelines exist on this
topic, they are not suitable for practical and standardized use. We therefore identified the need for such a
guideline which is both standardized and effective. The descriptions provided herein deal with thecurrent status of specimens that have been subjected to differing methods of molecular diagnosis invarious types of medical laboratories. These discussions have varying levels of evidence. Many of thedescriptions are derived from both the literature and manufacturer’s instructions, and several are based on
the experience of experts in this field.
This guideline is intended to use by all professionals who are involved in some aspect of the collection,
storage, transport, and/or preparation of patient or tissue specimens. The appropriate handling ofspecimens ensures the high quality of such assays while also providing more reliable results, thus
contributing to better patient diagnostics and an improved quality of healthcare.

2. Scope
This guideline describes the principles and basic methods of sample procurement: namely, the collection,
storage, transport, and preparation of specimens for methods of molecular diagnosis to measure specificsequences for pathogens, somatic cells, and germ line cells. Each section discusses the proper methods toassure appropriate specimen conditions, and also discusses the inappropriate conditions of specimens,
possible causes of inaccurate results, and also suggests how to avoid these problems.

3. Storage and Transport of Specimens for Molecular Methods
It is desirable that specimens for molecular detection and measurement methods be used
immediately following collection, as in other testing protocols. In the healthcare setting,
	
 

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however, the specimens must sometimes be either refrigerated or frozen for later delivery to
the laboratories or facilities where the molecular analyses will be conducted. In addition,
tested specimens are stored for a certain time in case there is a need for retesting. During
this time, there is a possibility that the target microbes or cell condition may be altered,affecting the analyses and thus the results of gene-based testing. Therefore, it is requiredthat specimens be appropriately stored until tests are completed, and that extracted nucleic
acids be stored together with their corresponding specimens.
In many cases, gene-based testing is used in research laboratories and involves theextraction of high-purity nucleic acids (DNA/RNA). In addition, high-purity DNA can be
stored at relatively stable conditions, even at an ambient temperature. However, in the fieldof clinical testing, in which promptness is critical, nucleic acids are not always purified tothe highest degree, and are used as measurement tools with relatively low-purity yields.
Conversely, RNA stability is highly dependent on the extraction conditions.
Noncompliance with storage conditions, which are specifically set for individual extraction
conditions, can greatly affect the test results (values). It is also known that extracted RNA
has a very poor stability over long-term storage.
When in vitro diagnostic reagents are used as extraction reagents, it is important to extractand store the specimens properly in accordance with manufactures’ instructions. When
research reagents are used, it is important to follow the optimal extraction and storage
conditions specified in the manufacturers’ instructions, but the independent evaluation of
the optimal storage conditions for nucleic acids can improve RNA yield and purity.
a. Storage temperature for nucleic acids
For the storage of extracted high-purity nucleic acids, the appropriate storage methods must
be chosen depending on the type of nucleic acids and their eventual use. RNA in particular
is easily degraded and unstable and must be stored at ultra-low temperatures (-70 ˚C orbelow) for both short- and long-term storage.
For high molecular weight genomic DNA, including human genomic DNA, it isrecommended that the DNA be dissolved in Tris-EDTA buffer and stored in a refrigerator
at 2-8 ˚C because repeated freezing and thawing can induce DNA strand nicks or breaks.
Extracted DNA stored in a refrigerator at 2 - 8 ˚C is stable for at least a year. When highmolecular weight genomic DNA is stored at ultra-low temperatures (-70 ˚C or below) for along time, it is recommended that the DNA be divided into sealed aliquots to avoid freeze
dehydration and repeated freeze and thaw cycles.
b. Storage containers
Small polypropylene tubes are recommended as storage containers, because absorbed
amounts of nucleic acids by its surface is only minimal. Even so, it is required that theinside of the tubes be coated with silicon or some other protective materials when extremely
small amounts of nucleic acids are stored. Ordinary disposable tubes are not considered tobe contaminated with DNase (a DNA degrading enzyme) or RNase (an RNA degrading
enzyme) since they are thermoformed at high temperature, however, it is neverthelessrecommended that tubes be used which are clearly identified to be DNase and RNase free.
	
 

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This section mainly describes the stability of specimens in storage prior to extraction, andthe stability of nucleic acids in storage is addressed only for special cases in reference to theabove matters. When viruses, bacteria, and other species are measured, the target genestability in each specimen differs according to the individual microorganisms. In such cases,
handling depends on the properties of the microorganisms that are present.
c. Transport of specimens
When specimens for gene-based testing are sent to other laboratories, due care must be
taken to prevent any specimen delay by informing the personnel at destination facility inadvance to expect the arrival of these specimens. In addition, in order to prevent
misidentification, specimens must be clearly labeled with their own identification
information. The clearly identified specimens and the testing order forms should be shipped
together. The patient information associated with the specimens should be anonymized or
redacted as needed.
It is desirable that specimen transport be completed in as short a time as possible using theappropriate storage conditions for each specimen, as is the case with other testing. However,transport between facilities frequently occurs only over a long period of time. During theperiod of specimen transport, controlled temperature and storage conditions are essential.
Containers made of polystyrene foam or other appropriate materials that provide insulationand physical protection for the specimens are ideal for shipment.
The typical storage temperature conditions used for the specimen transport are frozen (-20
˚C or below), refrigerated (4-10 ˚C), and ambient (17-28 ˚C) temperatures. For frozen andrefrigerated conditions, dry ice and cooling gel, respectively, should be used as the coolingmedia. For ambient condition shipment, heat storage materials may be used when required.
The amount of temperature-controlled packing material should be adjusted by estimating
the temperature inside the containers when the specimens are packed and when they areunpacked. When transport takes a long time, as is the case when specimens are shippedabroad, the sender must anticipate this factor, and should consider consigning the transport
to carriers that can easily manage the specimen temperature by, for example, replenishingdry ice in the course of transportation.
The recipients of the specimens must store the specimens at the appropriate storage
temperatures immediately after confirming the specimen conditions: namely, the storage
conditions of the specimens during the transport, including the optimal longer-term storage
temperatures and any damage to the specimens, as well as specimen information, including
testing order forms.
3.1 Storage and Transport of Specimens in Molecular Methods for Pathogens
3.1.1 Serum and Plasma
To detect viruses, various molecular methods have been widely implemented because it isdifficult to culture targeted viruses, and, even when possible, is very lengthy andtime-consuming. For the hepatitis virus C (HCV) and the human immunodeficiency virus(HIV), it is recommended that the serum or plasma be separated within 6 hours after blood
	
 

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collection, and, for hepatitis virus B (HBV), it is recommended that this be performed
within 24 hours. It is well-known that virus measurement results are not significantly
affected within these time frames. After separation, serum and plasma specimens can be
stored for approximately 5 days under refrigeration (2-8 ˚C), but freeze preservation isrecommended for long-term storage. It is desirable to store specimens for DNA
measurement at -20 ˚C or below for HBV and at -70 ˚C or below for hepatitis virus A(HAV), HCV, and hepatitis virus E (HEV). Other viruses, including the cytomegalovirus(CMV) and the herpes simplex virus (HSV), are reported to be relatively stable in the blood.
Based on the above reports, it is considered that virus specimens are relatively stable inserum and plasma.
(1) Inappropriate and unsuitable conditions for testing
Formation of sediments, discoloration, and dehydration due to freezing(2) Causes
Possible causes include inappropriate storage conditions, such as long-term exposure tohigh temperature and repeated cycles of freeze and thaw.
(3) Troubleshooting
When specimens were stored under inappropriate conditions, including hightemperatures, new blood specimens are required for subsequent tests. If the extracted
nucleic acids have been appropriately stored, they can be used for re-testing. The
number of freeze and thaw cycles should be recorded and minimized.
(4) Measures to avoid inappropriate situations
It is important for laboratories to specify the storage temperature, the number of days
that the specimens can be stored, to label the storage date on specimens, and to store
them under appropriate temperature conditions.
3.1.2 Urine
Urine specimens (male) are used for nucleic acid testing for Chlamydia trachomatis andNeisseria gonococcus. It is desirable that urine specimens be stored with refrigeration (2-8˚C) and undergone the process of nucleic acid extraction within four days. The stability ofthe extracted nucleic acid depends on the extraction conditions. When extracted properlywith the standard methods, the DNA remains stable for one week or longer underrefrigerated conditions (2-8 ˚C). For long-term storage, freezing of the specimens isdesirable.
(1) Inappropriate conditions not suitable for testing
Urine specimens may have a cloudy appearance due to bacterial growth.
(2) Causes
Possible causes include failure to store the specimens under appropriate conditions,such as leaving specimens for a long time in collection containers under inappropriate
storage temperatures.
(3) Troubleshooting
For initial testing, it is required that new blood specimens be acquired. For re-testing,
	
 

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extracted nucleic acids can be used if they have been stored appropriately. The numberof freeze and thaw cycles should be carefully recorded.
(4) Measures to avoid the inappropriate situations
It is important for laboratories to specify the storage temperature and the number ofdays that specimens can be stored, to label the storage dates on specimens, and to store
them under the appropriate temperature conditions. Specimens that were stored underconditions other than indicated should not be used for measurements.
3.1.3 Sputum
Sputum specimens have commonly been employed in nucleic acid testing for
Mycobacterium tuberculosis and strains of atypical Mycobacterium . In the detection of M.
tuberculosis, it is critical to assure the quality of sputum specimens. (Refer to the Guideline
for Mycobacterial Examination by Ministry of Health, Labour, Welfare of Japan, 2007.)
The sputum quality is assessed by a macroscopic observation according to the classificationof Miller & Jones. For nucleic acid testing, sputum specimens are decontaminated with
NALC-NaOH and suspended in phosphate buffer. No detailed data have yet been reported
on the storage stability of the suspended specimens. It is empirically known thatsuspensions can be stored for a week under refrigeration (2-8 ˚C) and for longer periods
when frozen. It is recommended that the suspensions be dispensed into aliquots before
cryopreservation in order to avoid repeated freeze and thaw cycles. The stability of theextracted nucleic acids is highly dependent on the extraction conditions. When properly
extracted with standard methods, DNA is stable for one week or longer under refrigeration(2-8 ˚C). For long-term storage, freezing of the specimens is optimal.
(1) Inappropriate conditions not suitable for testing
Sputum specimens with poor viscosity may have been stored in inappropriate
conditions.
(2) Causes
Possible causes include inappropriate storage conditions, such as long-term exposure tohigh temperature and repeated cycles of freeze and thaw.
(3) Troubleshooting
For initial testing, the collection of new sputum specimens is required. For re-testing,extracted nucleic acids can be used if they have been stored properly. The number offreeze and thaw cycles should be recorded and minimized.
(4) Measures to avoid inappropriate sample handling
It is important for laboratories to specify the storage temperature and the number ofdays that the specimens can be stored, to label the storage date on specimens, and to
store specimens using the appropriate temperature conditions. Specimens that werestored using conditions other than those indicated should not be used for subsequent
measurements.
3.2 Storage and Transport of Specimens in Molecular Methods for Somatic Cells
	
 

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As the human genome analysis progresses, accumulating information is available forclinical applications. With the development of DNA microarray techniques, morecomprehensive types of gene analyses (gene expression and genotypic) may be performed.
As a result, genetic testing methods have been applied to the field of clinical research. RNA,the target of gene expression analyses in these tests, is unstable and can be degraded by
RNase (RNA degrading enzyme) contamination, which is usually derived from specimens
and/or the operators (examiners and researchers). Therefore, when handling RNA, carefulattention is critical.
RNA is purified to a relatively high-purity level by silica membrane adsorption in mostcases, including clinical testing. To assess the purity of purified RNA, conventional
methods include the A260/A280 spectrophotometric absorbance ratio and (denaturing)
agarose gel electrophoresis. In addition, recent simple methods have been developed andare widely used. Examples include confirming the 28S and 18S size distribution ofribosomal RNA (rRNA), as well as calculating the 28S–to-18S rRNA ratio using microchip
gel electrophoresis.
It is generally desirable to store the purified RNA specimens at -70 ˚C or below, but somefacilities store these samples at -20 ˚C or below. Therefore, personnel should be aware of
the performance of the freezer in which specimens are stored and sufficiently consider
storage conditions to avoid freeze and thaw cycles and freezing dehydration. It is generally
recommended to dispense RNA specimens into aliquots before storage to avoid freeze andthaw cycles. In practice, the specimen yield is extremely low, and such specimens are
cryopreserved without dispensing. In such cases, it is also recommended to reverse
transcribe a portion of the RNA sample to obtain the more stable cDNA prior to storage.
If the purpose of the testing is accurate quantification of gene expression levels, RNA must
be promptly extracted from the specimen under conditions at which minimum autolysis oforgans or cells occurs. The same is true for cases in which a high detection sensitivity isrequired, due to an extremely low expression level.
When specimens are to be used only after some time in storage, they should be stored for
maximum stabilization of the nucleic acids, depending on purpose of the testing, specimen
types, storage methods, the storage period, and the types of nucleic acid.
3.2.1 Tissues and Tissue Slice Fragments
Recently, a comparative analysis of gene expression profiles based on the DNA microarray
technique has been applied to clinical diagnoses, and some published reports have alreadydescribed the conditions of fresh tissue specimens used for these examinations. The mRNA
expression level for transcription factors and cytokines in bone marrow tissue are
significantly altered over time following the specimen collection, therefore requiring special
attention for the handling of bone marrow tissue. Recently, commercially available RNA
stabilizers which retain specimen RNA have come into use. It has been reported that,
although the total amount of recovered RNA and its molecular weight distribution patternremained unchanged with the addition of the stabilizer, some variations have been found at
	
 

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the individual gene level. In these tests, it is important to establish certain fixed conditions
that are applied from the time of specimen collection to nucleic acid extraction, and to
confirm in advance the changes that may be brought about in the target genes under these
conditions.
The handling of tissues obtained by biopsy or surgical removal is more complicated. A
small mass of tissue or the targeted parts of the tissue are cut into sections and are either
processed immediately for the extraction of nucleic acids or fresh-frozen in liquid nitrogenfor longer-term storage. In studies involving tissues, special attention should be paid to thespecimen storage conditions to avoid RNA degradation.
Cancer tissue sections are also used for molecular techniques. In the examination of cancercell-specific gene expression, tissue specimens are embedded in a matrix for the preparationof frozen tissue sections, which are used for microdissection (a method to obtain cancer
cells alone using microscopy). In implementing microdissection, it is important to properly
process tissue specimens in accordance with the treatment methods specific for each
instrument.
(1) Inappropriate conditions not suitable for testingSpecimens containing cells that are destroyed due to repeated cycles of freeze and thaw
are inappropriate for analyses. When a fixative is used for tissues in pathologicalexamination, nucleic acids may not be sufficiently recovered to a sufficient degree ofquantity and quality. When isolated RNA is used as a specimen for polymerase chain
reaction (PCR), careful attention is required to avoid cross-contamination from theprocess of tissue section preparation.
(2) Causes
Storage under inappropriate conditions, including failure to use appropriate procedures,such as freezing immediately, can contribute to the degradation of tissue samples.
Avoiding repeated cycles of freeze and thaw can minimize this damage.
(3) Troubleshooting
For initial testing, re-extraction from another aliquot of cryopreserved specimen or
re-collection of a tissue specimen is required. For re-testing, extracted nucleic acids canbe used if they have been stored appropriately.
(4) Measures to avoid inappropriate situations
It is important for laboratories to specify the storage temperature and the number ofdays that specimens can be stored, to label the storage date on specimens, and to store
these under the appropriate temperature conditions. Specimens that were stored underconditions other than those indicated should not be used for measurements.
3.2.2 Whole Blood (WBC)The nucleic acids (DNA/RNA) extracted from blood cells are used for the molecularanalysis of leukemic diseases. Whole blood, or the white cell fraction of the blood isolated
by density gradient centrifugation using Ficoll or by cell sorters, is primarily used. It has
been reported that peripheral blood contains circulating free-DNA and free-RNA, which
	
 

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appear to originate from degraded blood cells or tissue-derived cells. Recently, theapplication of these specimens to cancer diagnosis has been investigated. It has beenreported that these circulating nucleic acids are stable in vivo. Presumably, there are some
protective mechanisms for such nucleic acids released into the circulating blood. In contrast,
it is known that the nucleic acids are rapidly degraded when they are added to either theserum or plasma ex vivo.
(1) Inappropriate conditions not suitable for testingSpecimens in which cells have been destroyed are inappropriate for testing because of
their possible influence on nucleic acid extraction.
White blood cells can be destroyed by strong negative pressure in the syringe at thetime of collection, and they are particularly fragile when blood is removed from patients
undergoing treatment with anti-cancer chemotherapeutic agents.
(2) Causes
Possible causes include storage under inappropriate conditions, such as long-term
exposure to high temperatures and repeated cycles of freezing and thawing.
(3) Troubleshooting
Re-extraction from another aliquot of cryopreserved specimen or recollection of a blood
specimen is required. For retesting, extracted nucleic acids can be used if they havebeen stored appropriately.
(4) Measures to avoid inappropriate situations
1) It is important for laboratories to specify the storage temperature and the number ofdays that specimens can be stored, to label the storage date on specimens, and to
store them under appropriate temperature conditions. Specimens that were stored
under conditions other than those indicated should not be used for themeasurements.
2) Specimens should be transported in containers that will prevent damage of cells.
3.2.3 Urine and Stool
Urine contains DNA that can be used for cancer screening. Colorectal cancer screening
using DNA and RNA excreted into the stool has also been explored, but the stool contains
an abundance of food-derived components and various bacteria, which are reported toinhibit gene-amplification reactions. Therefore, when stool specimens are used forgene-based testing, sufficient attention is required for the pretreatment of the specimens.
(1) Inappropriate conditions not suitable for testing
Urine may appear cloudy due to bacterial growth.
(2) Causes
Possible causes include storage under inappropriate conditions, such as long-term
exposure to high temperatures or repeated freezing and thawing cycles.
(3) Troubleshooting
For initial testing, it is required that new specimens be acquired. For retesting, extracted
nucleic acids can be used if they have been stored appropriately. The number of freeze
	
 

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and thaw cycles should be recorded and minimized.
(4) Measures to avoid inappropriate situations
It is important for laboratories to specify the storage temperature and the number ofdays that specimens can be stored, to label the storage date on specimens, and to store
them under appropriate temperature conditions. Specimens that are stored underconditions other than those indicated should not be used for the subsequent analyses.

3.3 Storage and Transport of Specimens in Molecular Methods for Germ Line Cells
Recently, genes responsible for the so-called single gene disorders have been identified, and,to date, gene-based testing methods using a wide variety of techniques have been used. In
addition, genetic testing for single-gene diseases is being performed at various facilities,
and, in particular, testing for rare single-gene diseases is implemented primarily at researchfacilities. This therefore creates the necessity for transporting specimens from medical
facilities (consigners) to the research facilities (consignees).
White blood cells are the major specimen used for genetic testing. Oral mucosa is also used,
depending on the purpose of the genetic testing. Oral mucosa is collected using special
cotton swabs, but the amount of recovery of DNA differs among individuals and depends
on the sampling conditions. Therefore, proper specimen collection and transport underappropriate storage conditions is critical. For the transport of these biological specimens,due consideration is required to prevent sample contamination or damage.
3.3.1 Outsourcing of Genetic Testing
Because genetic testing may reveal important information about the subjects (patients) as
well as their family members and relatives, obtaining informed consent is absolutely
essential, and genetic counseling must be provided as required. When genetic testing is
consigned to other facilities, the appropriate security measures (organizational, human,
physical, and technical) must be taken to protect the individual’s genetic information. In
other words, patients’ names should be anonymized, and due attention must be paid to thehandling of specimens and the management of test information throughout all stages of theprocess, from specimen collection to the reporting of test results. In addition, an agreement
should be enacted between the concerned participants and subjects prior to testing
concerning the post-analytical handling of specimens and test results. The most common
specimen used for genetic testing is white blood cells from peripheral blood samples
collected from the subjects. After the identity of the subject has been confirmed, qualified
medical personnel collect the blood specimens.
Depending on the purpose of the testing, specimens that are collectable by the subjects
themselves, such as oral mucosa specimens, may also be tested. Even in such cases, dueconsideration is required, i.e., a responsible or accredited third person must attend during
the specimen collection to identify the subject and his/her specimen. Collected specimens
are anonymized and patient information redacted in a traceable fashion, and are stored
	
 

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under the most appropriate conditions. In order to prevent the loss of specimens or theiraccompanying information during transport, the specimens and testing order forms should
be sent together and strictly managed.
Responsibility for the transportation of the specimens between facilities is assumed directlyby the personnel concerned, or it may be consigned to common delivery systems including
door-to-door parcel delivery services, unless a special specimen transport system has been
established between the facilities. The means of specimen transport have to be selected
carefully and must comprehensively consider the safety, stability, reliability, required time,and other factors related to the transport system.
Specimens should be transported with the appropriate storage temperature suitable for thetype of specimen and be contained in polystyrene foam shipment boxes, which provide
excellent insulation and are resistant to crushing.
The facilities that perform the molecular diagnostics should confirm the receipt of thespecimen, acknowledge the content of the testing order form, and perform the analyses
based on the information in the order form. The examination processes and the subsequent
results should be accurately recorded, stored, and managed so as to be able to respond toinquiries during testing and after the test results are reported. In addition, the information on
analytical validity, clinical validity, and clinical utility of the implemented tests must be
provided to the consigner of the test, as required.
The results of the tests should be reported only to the consigner. To prevent leakage of thetest results to third parties, the report should be placed in a thick envelope so that the resultscannot be read from the outside, the envelope should be marked “CONFIDENTIAL,” andthe envelope should be sent to the facility that requested the test (consigner). Whencommon delivery systems, such as the postal service or door-to-door parcel delivery
services, are used, sender should request that tracking information and a delivery record beavailable upon request.
After the completion of testing and reporting the result, the specimens and the extracted
nucleic acids (DNA/RNA) should be properly discarded, retained or sent back to the facility
that requested the testing. In the interim, proper storage conditions should be maintained, atleast until the next proficiency testing to allow for identification of problems and corrective
re-testing, if necessary, in accordance with the agreement with the facility. Upon receipt of
the test results, the consigner of the test identifies the subject of the test and uses the results
to diagnose that specific subject’s condition.
This section summarizes methods to prepare specimens used for molecular methods.

4.Preparation of Specimens for Molecular Methods
4.1 Preparation of Specimens in Molecular Methods for Pathogens
The purpose of molecular methods for pathogens is to identify pathogens responsible fordiseases by specifically detecting the genomic DNA or RNA of foreign pathogens.
Therefore, it is important to select the most suitable testing material that is most likely toharbor targeted viruses or bacteria, based on the pathology, disease stage, or manner of
	
 

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infection. Due to widespread use of in vitro diagnostic reagent kits, specimens of theaffected lesions, including serum, plasma, sputum, urine, and a swab are the norm for themolecular diagnostics involved in the examination of pathogens.
During the last few years, an increasing number of molecular methods for pathogens usingstool specimens have been implemented, because the symptoms of vomiting and diarrhea
due to norovirus often occur and spread during the winter season. In addition, specimens
are collected from many different sources, depending on the nature of the pathogen and the
purpose of the testing. These specimens include peripheral blood, cerebrospinal fluid,
swabbing of affected lesions, broncho-alveolar lavage fluid (BALF), and biopsies ofaffected tissues.
When these specimens are collected, the quality of the specimen may be compromised
depending on the collection methods or conditions, and the establishment of standardized
preparation methods is ideal. In this section, the following are listed for each of the typicaltesting materials: 1) findings of inappropriate conditions of specimens that are often seen in
our daily testing procedures, 2) the possible causes of these conditions, 3) methods forrescuing specimens under poor conditions, and 4) how to avoid obtaining specimens inpoor conditions. In cases new specimens cannot be obtained other than inappropriate
conditions of those, a try for use of buffers or enzymes that may reduce inhibitory effects is
to be considered.
4.1.1 SerumThe specified amount of peripheral blood is drawn using a vacuum blood collection tube
that contains a serum separating gel. The tube is left at an ambient temperature for an hour
until coagulation is completed. The tube is immediately centrifuged (at 1,190 x g (gravity
acceleration) [2,8 00 rpm for the rotor with a radius of 170 mm], at 4 ˚C for 10 minutes). If
a coagulation accelerator is added to the collection tube, the time required for coagulation isshortened. The collection tube that contains a reagent for neutralizing heparin should not be
used, because of its inhibitory effects on nucleic acid amplification.
Only the amount of specimen required for molecular diagnostics for pathogens should betaken from the blood collection tube. Transferring the specimen into another container
should be avoided. When specimens are not going to be tested immediately after
centrifugation, the separated serum in the original blood collection tube should be directly
frozen (-20 ˚C or below) and stored until use.
(1) Inappropriate conditions not suitable for testing
Transparent rust-colored, red-colored, or yellow-green colored serum should not be
used for the molecular analyses.
(2) Causes
The rust or red color is due to hemoglobin contamination and hemolysis. The
yellow-green or green tea color is bilirubin contamination due to jaundice. If vacuum
blood collection tubes are used for blood specimen collection, hemolysis-induced
hemoglobin contamination is less susceptible to damage from physical forces, including
	
 

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JCCLS MM5-A1(2011.12.1)
intensive negative pressure at the time of blood collection. However, this contamination
may occur if the specimens were erroneously frozen before centrifugation and thenthawed prior to the analyses. It may also occur if the specimens were frozen before
allowing the red blood cells to settle to the lower layer of the separating gel, usually due
to insufficient centrifugation and low centrifugal load (g) or too short a spin time.
(3) Troubleshooting
Bilirubin contamination is attributable to the patient disease status at the time of bloodcollection, and even re-collection of a blood specimen will not improve this factor.
Therefore, efficient nucleic acid extraction methods that can remove impurities should
be used. The inhibitory effect of nucleic acid amplification reaction can be overcome byadsorbing the nucleic acids onto filters, membranes, or magnetic particles and washingout impurities, rather than extracting it with phenol/chloroform and precipitating withethanol or isopropanol. If an internal standard is included with the detection reagents,
the inhibitory effect against the amplification reaction can be assessed and can reduce
the frequency of false-negatives. When no internal standard is included, the effect of
inhibitory substances can be confirmed by evaluating the amplification efficacy after
adding a known amount of plasmid DNA to the DNA solution extracted from thespecimen or the cDNA.
(4) Measures to avoid inappropriate situations
Serum specimens allow stable nucleic acid amplification using basic operation procedures.
Specifically, the collection of a specified blood volume without creating negative pressurein the blood collection tubes, waiting until the coagulation reaction is completed, andcentrifuging the tube are the steps required for this process. When freezing blood collection
tubes commonly used in nucleic acid testing following serum separation, the color andturbidity of the serum layer immediately after the centrifugation must be noted, and mustalso be checked for the presence of remaining clots that have not completely sunk into thelower layer of the separating gel before storing these samples in the freezer. If the color of
the serum is off the empirically-determined standard range, collection of a new blood
specimen should be considered.
4.1.2 Plasma
EDTA should be used for plasma anti-coagulation. Separated plasma, for which ACD (acidcitrate dextrose) or CPD (citrate phosphate dextrose) are used, can also be used for nucleic
acid amplification tests similarly to those treated with EDTA.
(1) Inappropriate conditions unsuitable for testing
1) Fibrin precipitation is suspected as observed by either the presence of a clot or if thespecimen has a high viscosity.
2) Massive leukocyte contamination may be present according to the turbidity anddeposit status.
3) Heparin has contaminated the specimen.
(2) Causes
	
 

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JCCLS MM5-A1(2011.12.1)
1) If EDTA tubes are not immediately and repeatedly inverted and mixed following
vacuum blood collection, blood coagulation gradually progresses and fibrin
precipitation occurs some time after the plasma separates. In mild cases, the plasma
may be slightly viscous. Similar conditions are observed when blood is drawn
without the use of anti-coagulation agents and are immediately transferred into
EDTA tubes, because the coagulation system has already been activated by thistime.
2) Due to insufficient centrifugal conditions in terms of g (gravitational acceleration)or the time of centrifugation, lymphocytes are present at a low density and platelets
are distributed within the lower layer of the plasma. When the plasma fraction ispipetted, some of these leukocytes may be aspirated together with the plasma. Thisis especially problematic when plasma levels of RNA from viruses such as HIV,
which propagate through the infection of T lymphocytes, are monitored over time,since the incorporation of lymphocytes accurately quantifies RNA levels.
3) In essence, EDTA, ACD, and CPD are the anti-coagulants that can be used for the
preparation of plasma specimens for molecular diagnostics. However, nucleic acidamplification tests, including PCR, are frequently inhibited when blood is drawn
using heparinized vacuum tubes or heparinized syringes. In addition, patients with
IVH (intravenous hyperalimentation), retained catheters, or who on artificial
hemodialysis are often heparinized for the prevention of blood clot formation. In
such cases, depending on the concentration, heparin in the blood may directly affectnucleic acid amplification reactions. Even a trace amount of heparin contamination(0.05 units per reaction) inhibits DNA amplification in PCR.
(3) Troubleshooting1) In a plasma specimen, virus particles may unevenly distribute and become
entrapped among fibrin fibers, and as a result accurate quantification may becompromised. Recentrifugation of the plasma after plasma homogenization (making
the viral particles distribute as evenly as possible by stirring the plasma withdisposable pipette tip or homogenization) is followed by carefully pipetting thesurface layer to obtain the specimens for nucleic acid extraction.
2) Centrifuge the plasma again under appropriate conditions (at 1,710 x g (gravity
acceleration) [3,000 rpm for the appropriate rotor, with a radius of 170 mm], at 4 ˚C
for 10 minutes or longer), and pipette the plasma from the surface layer carefully so
as not to disturb the pellet with jarring or shock. Because the major part of therelated analysis using plasma specimens lies in the amplification of nucleic acids,
this strength of centrifugation is sufficient to precipitate blood cells without
affecting the results of the test.
3) Even if some inhibitory effects are detected in the nucleic acid amplification
reaction, it is difficult to attribute these effects to heparinization unless we observe
the blood collected in heparinized vacuum tubes or with other possible situations.
However, if an internal standard is included in the reaction mixture, it may actually
	
 

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JCCLS MM5-A1(2011.12.1)
demonstrate the inhibitory effect against the amplification reaction. When nointernal standard is included, the effect of inhibitory substances can be confirmed byevaluating the amplification efficiency, after adding a known amount of plasmid
DNA to the reaction mixture together with DNA extracted from the specimen ortranscribed cDNA.
When heparinized plasma is used as the specimen, viral nucleic acids should be
purified to eliminate the effect of heparin as much as possible using nucleic acidextraction reagents based on filters, membrane, or magnetic particle methods, sinceheparin, unlike blood cells, cannot be removed by repeated washes andcentrifugations. In addition, treating extracted nucleic acids with heparinase issometimes effective in removing heparin.
The simplest and easiest action to be taken is to confirm resolution of the inhibitory effectby diluting the nucleic acid specimens, although it should be noted that there is a risk thattargeted nucleic acid levels are also diluted to a level below the detection limit.
(4) Measures to avoid inappropriate conditions
1) EDTA vacuum tubes should be used for blood collection; the tubes should be gently
and repeatedly inverted 10 or more times to prevent blood coagulation.
2) A complete buffy coat layer is formed by centrifugation (at 1,710 x g (gravity
acceleration) [3,000 rpm for a rotor with a radius of 170 mm], at 4 ˚C for 10 minutes
or longer) with a low-speed, cooled centrifuge. The blood collection tubes should be
removed from the centrifuge gently to avoid vibration or jarring, and the minimum
required volume of the specimen from the middle layer of the plasma fraction
should be removed by pipetting, avoiding contamination of blood cells from thesurface layer.
3) EDTA-containing vacuum tubes should be used at all times. Avoid sharing blood
specimens with those for cellular immunology or chromosomal analyses, for whichblood specimens are collected in heparinized tubes. When the subject is heparinized
in a clinical setting, a new blood specimen should be collected after confirming thatthe effect of the heparinization has been eliminated. When blood is drawn throughan IVH catheter, the first several few milliliters (2-3 times the catheter volume)
should be discarded due to the potential effect of heparin, and blood drawn after thatshould be used as the specimen.
4.1.3 Sputum
Sputum specimens are collected either through expectorating from the bronchus at deep
coughing by the patients themselves or through aspirating with a suction catheter. Sputum
specimens are mainly used for the diagnosis of infectious diseases caused by tubercle bacilli,
atypical mycobacteria, and others.
(1) Inappropriate conditions unsuitable for testing1) Specimens comprised most partly of saliva, as judged by macroscopic observationof non-viscous (S(M), according to Miller and Jones’ classification) sputa.
	
 

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JCCLS MM5-A1(2011.12.1)
2) Specimens that have a large amount of blood with the sputa.
(2) Causes
1) Possible causes include insufficient expectoration by the patient or limited sputum
secretion.
2) Possible causes include hemorrhages from affected respiratory tracts, such as thebronchus.
(3) Troubleshooting
1) When too little sputum is contained in the specimen, targeted pathogens, including
acid-fast bacilli and non-tuberculous mycobacteria, may not be detected and may
give false-negative test results. Therefore, the collection of alternative specimens,
including pooled sputum, gastric juice, broncho-alveolar lavage fluid (BALF), andbronchial scratching, should be considered.
2) To eliminate red blood cells and hemoglobin, the specimen should be left for
approximately 15 minutes after the addition of 5-10 times its volume of sterile,
distilled water to induce hemolysis and centrifuged thereafter to obtain the
precipitate, which is used for nucleic acid extraction. However, it should be notedthat normal sputum is highly viscous and does not precipitate in this state, even after
centrifugation (at 1,190 x g (gravity acceleration) [2,500 rpm for the rotor with aradius of 170 mm], at 4 ˚C for 10 minutes) in many cases. After centrifugation, as
much supernatant as possible should be gently removed by aspiration. This
supernatant should be placed in another container rather than being discarded.
Dissolve the remaining fraction of sputum, which contains cellular components andbacteria surrounded by mucus, in NALC (N-acetyl-L-cysteine) or semi-alkaline
protease (SAP) (trade names: “Sputazyme,” “Presolve,” and others) to eliminate
viscosity. Then, the sputum must be washed with physiological saline or PBS,
centrifuged (at 1,190 x g (gravity acceleration) [2,500 rpm for the rotor with a radiusof 170 mm], at 4 ˚C for 10 minutes), and used for nucleic acid extraction. If
specimen pre-treatment procedures are indicated in a package insert for the reagents,
follow these instructions. If the color of precipitate after washing and centrifugationis nearly white, it is possible that inhibition of the reaction by hemoglobin will notoccur in the following procedures.
(4) Measures to avoid inappropriate situations
When a large amount of blood is mixed in the sputum, hemoglobin contamination of
the centrifugal precipitate of sputum is unavoidable, and the subsequent inhibition of
the nucleic acid amplification reaction in the PCR and TMA analyses is a serious
consideration. In such cases, it is desirable to collect additional sputum specimens, but it
is always necessary to take measures suitable to the subject’s disease state, especiallythose patients with respiratory diseases. This is particularly applicable to cases in which
only saliva was collected. If subjects have difficulty in expectorating sputum bythemselves, sputum collection by aspiration with suction catheters or utilization ofgastric juice as an alternative specimen should be considered.
	
 

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JCCLS MM5-A1(2011.12.1)

4.1.4 Stool
Recently stools have been increasingly used as the specimen for molecular methods for
detection of pathogens, primarily for diagnosing viral diarrhea. Because specimens arecollected at the peak of the diarrheal symptoms, most of these are watery in nature. To
evaluate the patient as an infection source to prevent secondary infections, the testing isrepeated several times following the first diagnosis until patient recovery to confirm
whether a virus is present. Stool specimens are also used for identification ofenterohemorrhagic Escherichia coli or the detection of verotoxin genes, although these
examinations are less frequent.
(1) Inappropriate conditions unsuitable for testing
The stool specimen may have been deteriorated in the quality if it is not frozen (at -20
˚C or below) within one day of sample collection. However, it is difficult to technicallydetect deterioration of specimen quality prior to analysis. There is always a possibility
that the detection sensitivity for the targeted pathogen of the nucleic amplification acidtesting has been reduced, because the deterioration of quality cannot be easily
recognized before testing.
(2) Causes
Leaving specimens in ambient temperature for a long time leads to the expansion of avariety of non-targeted bacteria, lowering the detection sensitivity for the targeted
pathogen and increasing the frequency of non-specific amplification events, eventually
resulting in false-negative results. It is also possible that viral nucleic acids are degradedby proteases and nucleases produced by bacteria, thereby leading to false-negative test
results.
(3) Troubleshooting
If the bacteria and viruses in the stool cannot be stabilized by rapid cryopreservation
within a few hours of defecation, it is practically impossible to regain the original stateof protein and nucleic acid quality. In this circumstance, it is impossible to estimate theextent of quality deterioration.
(4) Measures to avoid inappropriate situations
It is essential to properly explain the requirements for nucleic acid testing to subjects,
and that the subjects understand that stool specimens must be stored at refrigerated
temperatures for as short a time as possible, must be refrigerated as soon as possible,
and must be frozen for longer-term storage. In particular, if a stool specimen is collected
outside the medical facilities, the time of collection, the storage period, and thetemperature condition during the storage should be recorded.
4.1.5 Urine
Most molecular methods for pathogens in urine specimens diagnose sexually transmitted
diseases (STD), such as infections by gonococci, chlamydia, and other pathogens. Typically,
the initial morning voiding urine is used as a specimen. The initial voiding urine at birth is
	
 

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JCCLS MM5-A1(2011.12.1)
often used for the diagnosis of cytomegalovirus infection in the fetus or neonate.
(1) Inappropriate conditions not suitable for testing
Urine specimens suspected of hematuria or bilirubinuria and those with large amounts
of precipitates should not be tested.
(2) Causes
Hematuria is attributed to the inflammation of urethra or bladder, which are affected bymany pathogens. Precipitates are also formed when the specimens are refrigerated.
(3) Troubleshooting
To eliminate contamination with blood cell components prior to extracting and purifying
nucleic acids, the use of low-speed cooled centrifugation (at 430 x g (gravity acceleration)[1,500 rpm for a rotor with a radius of 170 mm], at ambient temperature for 5 minutes) is
effective. However, cryopreserved urine specimens are occasionally contaminated withhemoglobin, which is unavoidable because hemolysis occurs during the thawing process.
When the detection target is a virus, then centrifuged supernatant can be used for analysis,
but when the target is bacteria, then careful attention should be paid to the supernatant,because improper centrifugation may precipitate bacterial cells. Rather, it is more important
to make the specimen homogeneous by repeatedly inverting the specimen collection tubes.
Because deposits are frequently formed due to specimen refrigeration, the specimen must
be dissolved as much as possible by incubation at 37 ˚C for 30 minutes before pipetting andthe subsequent analysis. To minimize the effects of impurities, it is advisable to selectnucleic acid extraction reagents which utilize filters, membranes, or magnetic particles.
In addition, in order to monitor the actual inhibitory effect of impurities on nucleic acidamplification reactions, the internal standards included with the detection reagent kits, ifany, should be used. If such standards are not available, it is desirable to conduct anaddition-recovery test independently by using plasmid DNA of known copy numbers close
to the detection limit. When the effect has been confirmed, the nucleic acid amplification
reaction may be recovered by diluting the nucleic acid specimen. It should be noted that thisprocedure contains some risk, in that the targeted nucleic acid may be diluted to a level
below the limit of detection, thereby causing false-negative test results.
(4) Measures to avoid inappropriate situations
Because the major objective of this testing is to diagnose infectious diseases, it isimportant to collect urine specimens by selecting the appropriate timing so that thehighest probability exists for pathogenic genome DNA and RNA. It is best to collectinitial voiding in the morning, so as not to obtain false-negative results by the washoutof pathogens by urination over the course of the day. In such cases, the specimen may
be purulent with a large amount of white blood cells, red blood cells, and exfoliatedepithelial cells. When extracting nucleic acids, contamination of genome DNA andRNA derived from these human cells, which are not the target of detection in thistesting, should be prevented. However, a certain level of contamination is inevitablebecause collection of pathogens affected in the human cells is a primary concern in thistesting.
	
 

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JCCLS MM5-A1(2011.12.1)

4.1.6 Whole blood (WBC) and Bone Marrow
After a specified volume of blood is drawn into blood collection tubes containing ananti-coagulant (EDTA and others), the tubes should be immediately inverted and its
contents (the specimen) gently mixed. If nucleic acids are not extracted on the day the
specimens are collected, they should be refrigerated (2-8 ˚C). When blood collection tubes
containing anti-coagulants (EDTA and others) are used, there is no need to add a stabilizer.
However, in the case of tests to detect pathogens that are responsible for bacteremia or
sepsis, the long-term storage of specimens at an ambient temperature may reduce detection
sensitivity. Therefore, when cells for such tests are stored for a long time, they should be
stored at ultra-low temperatures (-70 ˚C or below). In the case of bone marrow specimens(bone-marrow aspirate), a specified volume should be stored in a special storing solution(cell culture medium containing FBS) with refrigeration (2-8 ˚C).
(1) Inappropriate conditions unsuitable for testing1) In specimens in which fibrin clots are observed, centrifugal collection of bacteria ishampered by the presence of fibrin, and sampling from the fluid fraction isprevented; this may result in a loss of accuracy in quantitative measurements. Fibrin
clots can be recognized by the poor fluidity of the tube contents or during pipetting
operations.
2) In analyses using PCR, heparin in the blood may cause poor or no amplification in
PCR, leading to “undetectable” or false-negative results because heparin is a potent
inhibitory substance for PCR. (Even a trace amount of heparin contamination (0.05
units) inhibits DNA amplification in PCR.)
Because heparin contamination cannot be detected visually, label information on thecollection tube should be confirmed carefully. In particular, careful attention must be paid
to bone marrow specimens, because they are often collected using heparin as ananti-coagulant.
3) When the quantity of bacteria responsible for bacteremia or sepsis is near thedetection sensitivity limit, the bacteria may not be detected.
(2) Causes
1) The possible causes of fibrin deposition include failure to use tubes containing
anti-coagulants (EDTA and others) for specimen collection and failure to invert the
tubes and mix the contents immediately after the collection.
2) The main cause of heparin contamination is considered to be blood collection using
heparin as an anti-coagulant. This applies to the case in which blood specimens
collected for other analytical purposes are used for molecular methods. It is also ofconcern that blood specimens collected from patients who are receiving heparin as apart of either hemodialysis or thrombolytic therapy may be contaminated withheparin.
3) The quantity of bacteria in the specimens depends on the pathological condition of
the patients at the time of specimen collection.
	
 

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JCCLS MM5-A1(2011.12.1)(3) Troubleshooting
1) Before extracting nucleic acids, the fibrin clots in the specimen must be physicallycrushed until they start dissociating and fibrin is sufficiently dispersed throughout the
specimen. Thereafter, the specimen and the fibrin should be tested. In the DNA
extraction process, addition of DTT (dithiothreitol) and the protein-digesting enzyme
Proteinase K promotes fibrin degradation.
2) The effects of heparin can be reduced by using nucleic acid extraction kits, which rely
on nucleic acids adsorption to filters, membranes, or magnetic particles with asubsequent washout of any impurities. However, it is impossible to completely
remove impurities. Heparin contamination can be resolved by completely digesting
the sample with heparinase.
3) In some of the specimens that contain a small quantity of the bacteria responsible forbacteremia or sepsis, treatment with proteinase K may improve the DNA extraction
efficiency and enable detection in some cases.
(4) Measures to avoid inappropriate conditions
1) Fibrin deposition can be prevented by repeatedly inverting blood collection tubes
containing anti-coagulants (EDTA and others) and by sufficiently mixing thecontents immediately after specimen collection.
2) Heparinized blood collection tubes should not be used in nucleic acid testing. In
heparinized patients, it is desirable that blood specimens be collected after heparin
has been eliminated from the blood. However, when heparin contamination is
unavoidable, as in cases in which the patients are on heparin therapy, thisinformation should be provided to the staff involved in testing by writing, “thepatient is heparinized” on the testing order form. This also applies to bone marrowspecimens. When nucleic acids are extracted from bone marrow specimens, caremust be taken to thoroughly wash the cells to remove any heparin that may havebeen present during specimen collection.
3) Because the quantity of pathogens responsible for bacteremia or sepsis in thespecimen depends on the condition of the patient at the time of specimen collection,
the test detection sensitivity should therefore be considered when test results areinterpreted.
4.1.7 Pleural Effusion, Ascites, Pericardial Fluid, Pancreatic Fluid and Broncho-Alveolar
Lavage Fluid (BALF)
Each specimen should be refrigerated (2-8 ˚C) after collection or should be frozen (-70 ˚Cor below) for long-term storage.
(1) Inappropriate conditions unsuitable for testing
1) When a large amount of cell components, including hemoglobin, is present in thespecimens due to hemolysis, there is a concern that the detection sensitivity will belowered due to the inhibition of PCR amplification. The extent of incorporation ofblood components can be confirmed by measuring the hemoglobin level, counting
	
 

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JCCLS MM5-A1(2011.12.1)
the number of white blood cells, or by semi-quantitation according to the color of
the specimen.
2) In specimens with fibrin clots, the centrifugal collection of bacteria is hampered,and accurate sampling cannot be performed, which may result in a lack ofquantitative performance in cases of quantitative analysis. Fibrin clots can bedetected by observing the fluidity of the tube contents or during pipetting
operations.
3) Purulent specimens contain vastly different bacteria types, and there is a possibility
that DNA extraction by alkaline heat treatment may lead to undetectable results.
This purulent condition can be confirmed macroscopically or microscopically.
(2) Causes
1) Incorporation of blood components at the time of specimen collection.
2) Incorporation of purulent discharge at the time of specimen collection.
(3) Troubleshooting1) In specimens contaminated with blood components, the inhibitory effect ofhemoglobin from red blood cells after PCR amplification may be eliminated byhemolysis by the addition of sterile distilled water to the specimen, followed bycentrifuging and subsequent extraction of DNA from these precipitates.
Alternatively, PCR can be performed using the extracted diluted DNA solution to
reduce the concentration of the inhibitory substances.
2) Before extracting nucleic acids, fibrin clots in the specimen must be physicallycrushed until they start dissociating and sufficiently disperse fibrin throughout the
specimen. Then, the whole specimen containing the fibrin is used in the analyses. In
the process of DNA extraction, the addition of DTT (dithiothreitol) and proteinase
K promotes fibrin degradation.
3) In order to prevent bacterial growth in the specimens after collection, instructions
for the storage conditions of the specimens should be strictly followed. The volume
of pretreatment solution used for nucleic acid extraction should be increased toremove impurities with surfactants and protein denaturing agents.
(4) Measures to avoid inappropriate situations
1) Incorporation of blood cell components at the time of specimen collection should be
avoided.
2) Containers containing sodium citrate or EDTA should be used for the collection of
pleural effusions, since pleural effusions sometimes coagulate after collection due tofibrin deposition.
3) Specimens should be collected when purulent discharge are not present.
4.1.8 Lymph Nodes and Solid Tissues (Biopsy or Surgery)Tissue specimens harvested by biopsy or surgery should be frozen immediately at ultra-low
temperatures (-70 ˚C or below) to prevent nucleic acid degradation due to tissue autolysis.
At this time, it is desirable to dissect only the affected lesions alone by removing them from
	
 

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JCCLS MM5-A1(2011.12.1)
the harvested tissue. For detection of tubercle bacilli, affected lesions can be confirmed bymorphological examination techniques.
(1) Inappropriate conditions unsuitable for testing
1) For large or oversized specimens, the proportion of the affected lesion (the target of
the test) to the whole specimen is smaller. The affected lesion can be diluted by thepresence of other tissues, which may reduce the detection sensitivity, eventuallyleading to false-negative results. For tests for tubercle bacilli, the affected lesions
can be confirmed by morphological examination via microscopy.
2) There is a concern that red blood cell contamination causes poor amplification in
PCR, which leads to false-negative results, in detection of Helicobacter pylori (H.
pylori) using gastric biopsy tissues and HCV (hepatitis C virus) and HBV (hepatitis
B virus) using hepatic biopsy tissues. The status of red blood cell contamination canbe confirmed by visually observing the rust color or by measuring the hemoglobin
concentrations.
(2) Causes
1) Tissue other than the affected lesion has not been removed from the whole tissue
harvested as the specimen.
2) The harvested tissue (specimen) contains blood.
(3) Troubleshooting
1) The morphological examination of tissue specimen is useful in distinguishing thetargeted cellular components from other components, so that they can be separated
and removed for testing. Laser microdissection allows accurate collection oftargeted cellular components from the prepared frozen tissue section on the glass
slide, but requires special equipment. The simplest and easiest method is tomanually collect targeted cellular components with a razor, excluding unnecessary
cellular components as much as possible from the paraffin embedded tissue section
on the glass slide.
2) The inhibitory effect of contaminated impurities following PCR amplification canbe reduced by using nucleic acid extraction kits based on a purification method inwhich nucleic acids are adsorbed to filters, membranes, or magnetic particles, andimpurities are washed out and removed.
(4) Measures to avoid inappropriate situations
1) At the time of specimen collection, affected lesions should be isolated from theunaffected areas and stored. Even when accurate differentiation under visualobservation is difficult, it is important to remove tissues other than the target tissue
as much as possible.
2) It is inevitable that solid tissues harvested by biopsy or surgery will contain blood
components.
4.2 Preparation of Specimens in the Molecular Methods for Somatic Cells
Molecular methods for somatic cells can clarify temporary genetic information that changes
	
 

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JCCLS MM5-A1(2011.12.1)
with medical conditions, such as DNA mutations and gene expression changes in cancer
cells. These methods are used for the diagnosis of malignant tumors in general, including
cancer, leukemia, and sarcoma, utilizing various approaches for the major purposes of, i)differentiating between malignant and non-malignant tumors, ii) evaluating the degree ofmalignancies, iii) detecting an early stage of malignant tumor cells, and iv) differentiating
between metastatic and multiple primary cancers.
The targets of molecular methods for somatic cells are those that represent pathologicalchanges, such as cancer cells. Therefore, the major specimens are blood cells and solid
tissue, but others which may contain pathological cells are also used: pleural effusions,
ascites, pericardial fluids, pancreatic fluid, sputum, broncho-alveolar lavage fluid (BALF),
and urine (sediment). When specimens are collected, it is not possible to separately collect
the targeted pathogenic cells. As a result, most of the specimens contain normal cells
surrounding the target ones. If a large quantity of normal cells is incorporated in thespecimen, there is a concern that the target cells are diluted so that the test could givefalse-negative results due to a failure to detect mutations in the cancer cells. Therefore, thetest results must be carefully interpreted. In cases new specimens cannot be obtained otherthan inappropriate conditions of those, a try for use of buffers or enzymes that may reduce
inhibitory effects is to be considered.
Recently, there has been an increase in the number of molecular methods for somatic cells
utilizing formalin-fixed, paraffin-embedded, tissue blocks which were used as specimens
for histopathological examinations. Because these tissue blocks can be stored for a longtime after histopathological examination, their use provides advantages for medical experts
in a retrospective study of the pathology and parallel use with morphological examinations.
It also provides many advantages for patients because it eliminates the need of additional
tissue collection every time a new test method is developed. On the other hand, a significant
percentage of the tests utilizing formalin-fixed, paraffin-embedded, tissue blocks ends up
giving “undetectable” results, because the DNA in most of the tissue blocks is fragmented
due to DNA degradation caused by formalin fixation or remaining DNase activity in thetissue, or because amplification is reduced by remaining impurities due to inappropriate
DNA extraction techniques. Therefore, some modification to be considered for
improvement in DNA or RNA detection from such tissue blocks, including using methods
that enable high-purity nucleic acid (a column method is preferable for RNA) extraction,
evaluation of the quality of the extracted DNA, or a focus on short DNA fragments as thetargets of detection. Since the formalin fixation protocols greatly affect DNA fragmentationand frequently cause false-negative results, it is desirable to establish a standardized fixation
method that does not affect pathological examination (morphological examination anddiagnosis) and that is less likely to cause DNA fragmentation. RNA integrity number(RNA), which is used for evaluation of RNA quality, constantly gives a low value forformalin-fixed, paraffin-embedded, tissue.
In order to perform Southern blotting or to create a DNA genomic library using DNA
extracted from fresh tissues, blood cells, or cultured cells, DNA with high molecular weight
	
 

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JCCLS MM5-A1(2011.12.1)
and high-purity must be prepared. Since high molecular weight DNA is easily damaged byphysical forces, including shear force, the DNA must be extracted from a fresh specimen ora freshly frozen specimen, without adding physical stirring.
4.2.1 Lymph Nodes and Solid Tissues (Biopsy or Surgery)Specimens harvested by biopsy or surgery should be frozen immediately at an ultra-low
temperature (-70 ˚C or below) and stored to prevent degradation of nucleic acids due toautolysis of the tissue. At this time, it is desirable to dissect the affected lesion from theharvested tissue before storage.
To accurately quantify gene expression, the pretreatment of specimens for measurement
should be implemented immediately (within 2 hours after specimen collection). Otherwise,
the specimen should be treated with an appropriate method to stabilize the RNA and should
be stored until the RNA is extracted. The method of RNA stabilization depends on the
purpose of the test, the specimen type, and the storage period. When RNA is extracted withguanidine isothiocyanate at a concentration of 5 ml/L to prevent denaturing of the specimen,
the specimen can be stored for approximately one week at ambient temperature. The
resulting extracted RNA should be stored at an ultra-low temperature of -70 ˚C or below.
Repeated freezing and thawing of extracted RNA should be avoided, because this cancontribute to the degradation of the RNA and thus may cause false-negative results.
(1) Inappropriate conditions unsuitable for testing1) The specimens that contain a large amount of cellular components may givefalse-negative results due to the dilution of targeted cells with non-targeted cells.
The proportion of the affected lesion in the specimens can be confirmed by themorphological examination of the frozen tissue sections.
2) When the tissues are either necrotic or apoptotic, the DNA in the tissue has alreadybeen degraded and may not render good quality DNA for subsequent testing.
Necrotic and apoptotic conditions can be confirmed by morphological approaches
utilizing H&E (hematoxylin-eosin) staining and the TUNEL (TdT-mediateddUTP-biotin nick end-labeling) method, respectively. Electrophoretic examinationof DNA extracted from necrotic tissues shows a smeared pattern, because the DNA
is completely degraded, and the DNA from apoptotic tissues gives a ladder-likepattern, because the DNA is degraded into segments according to the size of
nucleosomes (185 bp).
3) Tissues embedded in embedding media specific for the preparation of the frozen
section may not render appropriate testing results as in the PCR procedure, where
embedding media can have an inhibitory effect on PCR.
(2) Causes
1) When it is difficult to macroscopically differentiate between a targeted affectedlesion and non-targeted tissues at the time of specimen collection by biopsy or
surgery, it is sometimes unavoidable that the specimens contain tissues other than
the target tissue.
	
 

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JCCLS MM5-A1(2011.12.1)
2) The necrosis process or the apoptosis process may have already started in thespecimen tissue .
3) The specimen is embedded in media specific for the preparation of the frozen tissue
section.
(3) Troubleshooting
1) The detection sensitivity of these methods should be confirmed so that targeted genemutations can be specifically detected, even when the overall proportion of targeted
cells is low. Otherwise, the frozen tissue sections should be prepared and thetargeted cellular components collected using morphological examination andmicroscopy to prevent the incorporation of non-target cellular components. The
method of laser microdissection allows the accurate collection of targeted cellularcomponents from prepared frozen tissue sections on glass slides, but specialized
equipment is required. The simplest and easiest method is to manually collect thetargeted cellular components with a razor, excluding unnecessary cellularcomponents as much as possible from the tissue section on the glass slide. Whencellular components are collected based on morphological observations and aretested for the purpose of medical treatment, such as selecting patients for certain
treatments, the confirmation of target lesions by pathological experts is imperative.
2) In order to detect degraded DNA following necrosis or apoptosis, it is necessary todevise appropriate measures such as setting primers to detect shorter target DNA
fragments.
3) To remove the embedding media used for the preparation of the frozen section, thespecimens should be washed adequately with PBS (phosphate-buffered saline)
before the extraction of nucleic acids, as these fragments are soluble in PBS.
(4) Measures to avoid inappropriate situations
At the time of specimen procurement, the affected lesions for target detection
should be stored after being isolated from the unaffected area. Even when accurate
differentiation under visual observation is difficult, it is important to remove tissues
other than the detection target as throughly as possible.
When the tissues to be tested are already in the necrotic or apoptotic state in vivo,
their effect on the measurement cannot be avoided. Therefore, specimens in thesestates should not be used for testing.
When the tissue is scheduled for a molecular method, avoid the use of embedding
media for the preparation of the frozen tissue section. When embedding media has
been used for the preparation of the frozen tissue section, it is necessary to informthe involved professionals who are responsible for performing the molecularanalyses.
4.2.2 Formalin-Fixed Paraffin Embedded Tissue
Specimens used for pathological examination should be appropriately fixed by immersion
in a 10-20% formalin solution immediately after collection by biopsy or surgery. After
	
 

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JCCLS MM5-A1(2011.12.1)
fixation, the tissue is immediately embedded in paraffin to prepare a tissue block. Whenformalin-fixed paraffin-embedded blocks are to be used for molecular methods, tissue slices
5-10-µm thick must be cut using a microtome. To prevent cross contamination among
specimens, due consideration is required, including exchanging the microtome blade after
the preparation of each specimen.
Generally, the recommended fixing solution is 10% neutral buffered formalin, and thestandard fixing time at ambient temperature is between roughly 18 and 36 hours and 3 to 12hours, for surgically and biopsy obtained specimens, respectively. It has been reported thatfixation in 10% neutral buffered formalin delays progression of nucleic acid fragmentationcompared with fixation in 10-20% non-buffered formalin. In addition, in aparaffin-embedded tissue block stored at ambient temperature or at low temperatures (4 ˚C),
most nucleic acids can be preserved without further degradation for a long time. Tissue
slices can be used in 10 days after cutting without further degradation of RNA.
(1) Inappropriate conditions unsuitable for testing
1) Formalin fixation treatment is accompanied by the fragmentation of nucleic acids,
and therefore this treatment is generally not suitable for a molecular method that
requires high molecular weight DNA, such as Southern blotting. It may even affectthe results of measurement using PCR, which targets low molecular weight nucleic
acids. The results should be interpreted carefully, because inaccurate amplification
may occur using PCR. Simple methods to determine the test target for a specimen,
such as excessive or insufficient fixation, include morphological examination
utilizing H&E staining and immunostaining, which both allow a certain level of
estimation. The RNA purity with the A260/A280 over 1.8 may provide alikelihood of the success in amplification. In addition, methods to directly evaluate
the fragmentation status of DNA include direct confirmation by electrophoresis,
evaluation of the PCR amplification of a known length of DNA fragments(housekeeping genes, including β-globin and the vitamin D receptor), andevaluation of the amplification speed using real-time PCR. To evaluate RNA
fragmentation, methods include investigating the RNA purity and the A260/A280
ratio or directly measuring the RNA confirmation using denatured agarose gelelectrophoresis. Furthermore, the integrity of the extracted RNA can be evaluatedby the ribosomal RNA (rRNA) and housekeeping gene expression levels, including
glyceraldehyde-3-phosphate dehydrogenase (GAPDH ) and β-actin.
2) Specimens that contain a greater amount of cellular components (normal andnon-tumor cells) than the target cells may give false-negative results due to thedilution of the target cells by normal cell populations. The proportion of target cells
in the specimens can be confirmed using a morphological approach, including
histopathological examination using H&E staining.
(2) Causes
1) Nucleic acids in tissues can be fragmented by such factors as inappropriate handlingof the tissue before fixation (such as letting a long time pass before immersing the
	
 

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JCCLS MM5-A1(2011.12.1)
tissue in the fixation solution), autolysis by intrinsic nucleolytic enzymes duringformalin fixation, protein crosslink formation by formalin fixation, and the effect ofacidification due to the formation of formic acid in the fixation solution. The extent
of fragmentation attributable to formalin fixation is greatly influenced by the typesof fixation solution, time, and the temperature.
2) Paraffin-embedded tissue blocks used as a specimen may contain a greater amountof cellular components (normal and non-tumor cells) compared to target cells.
(3) Troubleshooting1) In order to detect nucleic acids that were degraded into small molecules due to theformalin fixation treatment, appropriate measures include setting primers to detect
shorter lengths of target DNA fragments. When using specimens in which thenucleic acids are severely fragmented, it is important to implement a molecularmethod only after fully understanding the characteristics of the specimens in orderto obtain highly reliable results. In particular, special attention is required when apreviously paraffin-embedded tissue block is used for more recent tests, such as in a
retrospective study.
2) The detection sensitivity of the molecular method should be high enough to
specifically detect targeted gene mutations, even when the proportion of targeted
cells is low. Otherwise, a method to visually collect targeted cellular components
from the section under morphological observation is useful to minimize theincorporation of cellular components other than the target cell population. The
method of laser microdissection allows the accurate collection of targeted cellularcomponents from the paraffin-embedded section on the glass slide, but special
equipment is required for these experiments. The simplest and easiest method is tomanually collect the targeted cellular components with a razor, excluding
unnecessary cellular components as much as possible from the paraffin-embedded
section on the glass slide. When collected cellular components are based onmorphological observations and are used in tests for the purpose of medical
treatment, such as selecting patients for certain chemotherapy treatments,
confirmation by pathological experts is imperative.
(4) Measures to avoid inappropriate situations
1) When a tissue is scheduled to be analyzed by molecular methods besides
pathological examination, it is important to separately secure these specimens bycryopreserving a portion of the affected lesion at ultra-low temperatures (-70 ˚C orbelow) to avoid the fragmentation of nucleic acids. When formalin-fixed tissues areused for molecular examination, it is desirable to perform the pre-fixation treatment
and formalin fixation at a low temperature in the shortest possible time. If thespecimen is also used for pathological examination, it is important to consider
balancing the requirements for specimens for pathological examination and for thesubsequent molecular analyses. For example, specimens for pathologicalexamination should be fixed for a certain period of time at an ambient temperature
	
 

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JCCLS MM5-A1(2011.12.1)
because of the requirements to maintain tissue morphology and structure, as well as
for reasons of occupational safety. Therefore, in such cases, a certain level offragmentation of nucleic acids is unavoidable, but it is possible to avoid completely
defective specimens by noting the following points: As a prefixation treatment,
large masses of tissue, such as those obtained by surgery, should be sliced into 4-5
mm thicknesses so that the fixation solution can easily penetrate throughout thetissue, thereby shortening the time required for fixation. In the fixation procedure,
the size of the tissue and the time for formalin penetration are key issues in avoiding
the fragmentation of the nucleic acids. It has been reported that 10% formalinusually penetrates the tissue at the rate of about 1 mm per hour under ambient
temperature, which can be referenced to set the fixation time for specimens obtainedby both biopsy and surgery.
2) If multiple numbers of paraffin-embedded tissue blocks are available for an
identical lesion, those with the largest overall proportion of affected cells should beselected as the specimen based on morphological examination utilizing H&E
staining. Even when only one paraffin-embedded tissue block is available, theexistence of affected cells should be confirmed by morphological examination
utilizing H&E staining or other methods prior to the molecular analysis.
4.2.3 Whole Blood (WBC) and Bone Marrow
After a volume of blood is drawn into the blood collection tubes containing anti-coagulant(EDTA), the specimen should be inverted and mixed immediately. If nucleic acidextraction will not be performed on the same day of specimen collection, the samples must
be refrigerated. If the purpose of the testing requires the separation of the cell componentsof the collected blood, the required procedure should be performed. For bone marrowspecimens, an indicated volume should be stored in special storing solution (cell culture
medium supplemented with FBS) under refrigeration (2-8 ˚C).
In case of gene expression analysis for accurate quantification, total RNA containing
mRNA should be extracted immediately (within two hours) after collection of the specimen.
When RNA extraction cannot be implemented immediately, the specimen should be treatedwith an appropriate method to stabilize the RNA and stored until the RNA is to be extracted.
The method that is chosen to stabilize the RNA depends on the purpose of the test, thespecimen type, and the storage period. When guanidine isothiocyanate at a concentration of
5 ml/L is used to prevent denaturing of the specimen, the specimen can be stored for abouta week under ambient temperature. The extracted RNA should be stored at an ultra-low
temperature of -70 ˚C or below. Repeated freezing and thawing of extracted RNA should be
avoided, because this can contribute to RNA degradation and thus may cause false-negative
results.
When DNA extraction is performed for gene mutation analysis, the specimen can be stored
at ambient temperature for as long as three days without causing any significant effect.
However, it is desirable that DNA extraction be performed within 24 hours for molecular
	
 

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JCCLS MM5-A1(2011.12.1)
methods that examine high molecular weight DNA, as is the case with Southern blotting,and otherwise within three days, as for PCR. For long-term storage, the specimens should
be stored at ultra-low temperature (-70 ˚C or below) after the appropriate procedures,
including separation of the buffy coat.
When DNA is extracted from whole white blood cells without separating blood cellcomponents, the specimen may be frozen and stored immediately after collection. However,
the amount of DNA recovered from this sample will decrease. Freezing and thawing cause
the degradation of nucleic acids, and these processes should not be repeated. For long-term
storage, specimens should be stored at an ultra-low temperature (-70 ˚C or below).
When specimens are collected from patients who have received pretreatment forhematopoietic stem cell transplantation (bone marrow transplantation) and who haveextremely low white blood cell counts, it should be noted that several times the indicatedvolume of blood may be needed to secure the required number of white blood cells for the
analyses.
(1) Inappropriate conditions unsuitable for testing1) Specimens that contain a greater amount of cellular components (normal cells andnon-tumor cells) than the target cells may give false-negative results due to thedilution of the targeted cells by normal cell populations. The proportion of targeted
cells in the specimens can be confirmed by the hemogram and myelogram analyses.
2) In specimens with fibrin clots, white blood cells are not evenly distributed in thespecimen, which may result in a lack of quantitative performance in the case ofquantitative analysis. Fibrin clots can be detected by observing the fluidity of thespecimen in the blood collection tubes or during pipetting.
3) If heparin exists in the specimens, poor PCR amplification will occur, resulting in“undetectable” or false-negatives, because heparin inhibits PCR. Because heparincontamination cannot be visually detected, the information on the tube label should
be carefully checked. Heparin is often used as an anti-coagulant when collectingbone marrow specimens, so careful attention is required for such specimens.
(2) Causes
1) It is difficult to separately collect targeted cells at specimen collection, and in manycases, contamination with normal cells is unavoidable.
2) The possible causes of fibrin deposition include the failure to use tubes that containanti-coagulants (EDTA and others) when collecting blood specimens, failure to use
a special storage solution (cell culture media supplemented with FBS) for the
storage of bone marrow, and insufficient tube inversion and mixing immediately
after sample collection.
3) The main cause of heparin contamination is considered to be the use of heparin as
an anti-coagulant during blood collection. This also applies to cases in which blood
specimens collected for other analytical purposes are used for molecularexamination. Heparin contamination is also a concern when blood specimens arecollected from patients who are receiving heparin in hemodialysis or thrombolytic
	
 

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JCCLS MM5-A1(2011.12.1)
therapy.
(3) Troubleshooting
1) Detection sensitivity of the method should be confirmed to be high enough to detecttargeted gene mutations, even when the proportion of targeted cells is low.
2) Before extracting nucleic acids, fibrin clots in the specimen must be physicallycrushed until they start dissociating and sufficiently dispersing fibrin throughout the
specimen. The whole specimen and the fibrin can be used for the subsequent
analysis. In the process of DNA extraction, the addition of DTT (dithiothreitol) andproteinase K promotes fibrin degradation.
3) The effect of heparin can be reduced by using nucleic acid extraction kits in which
nucleic acids are adsorbed onto filters, membranes, or magnetic particles, and thenimpurities are washed out and removed. However, it is impossible to completely
remove impurities. The effect of heparin can be resolved either by completely
digesting the heparin by the addition of heparinase, or by washing the white blood
cell fraction with physiological saline at least twice after fractioning of white blood
cells.
(4) Measures to avoid inappropriate situations
1) In the case of a specimen in which the proportion of targeted cells is low, thefractionation of targeted cells should be performed by density gradientcentrifugation using Ficoll, by the antibody method, or by the use of a cell sorter.
When leukemia has progressed, the mRNA from the tumor cells dominate thespecimen, so the mRNA derived from contaminating normal lymphocytes isgenerally minimized.
2) Fibrin deposition can be prevented by repeatedly inverting the blood collection
tubes (containing EDTA or other anti-coagulants) and sufficiently mixing theircontents immediately after collecting blood specimens, and by using a special
storage solution (cell culture media supplemented with FBS) when bone marrowspecimens are collected.
3) In principle, heparinized blood collection tubes should not be used for molecularmethods. In heparinized patients, it is desirable that the specimen be collected after
the effects of heparin have been eliminated from the blood. However, when heparincontamination is unavoidable, for example, when patients are on heparin therapy,
this information should be provided to the professionals performing the molecularanalysis by stating, “the patient is heparinized” on the testing order form. This also
applies to bone marrow specimens. When nucleic acids are extracted from bonemarrow specimens, a thorough washing of the cells is required to remove any
heparin that may have been present during specimen collection.
4.2.4 Pleural Effusion, Ascites, Pericardial Fluid, Pancreatic Fluid, Broncho-Alveolar
Lavage Fluid (BALF), Urine Sediment and Sputum
Each specimen should be stored under refrigeration (2-8 ˚C) after collection. If the purpose
	
 

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JCCLS MM5-A1(2011.12.1)of the testing requires separated collection of blood cell components, the requisite
procedure(s) should be performed.
For accurate quantification of gene expression analysis, total RNA and mRNA should be
extracted immediately (within two hours) after the collection of the specimen. When RNA
extraction cannot be performed immediately, the specimen should be treated with anappropriate method to stabilize the RNA and should be stored until RNA extraction. The
method that is chosen to stabilize the RNA depends on the purpose of the test, the specimen
type, and the storage period. When guanidine isothiocyanate is used at a concentration of 5
ml/L to prevent specimen denaturing, the specimen can be stored for approximately one
week in an ambient temperature. The extracted RNA should be stored at an ultra-low
temperature of -70 ˚C or below. Repeated freezing and thawing of extracted RNA should be
avoided, because this can contribute to RNA degradation and thus cause false-negative
results.
When DNA extraction is performed for gene mutation analysis, it is desirable that DNA
extraction be performed within 24 hours for the analyses that examine high molecularweight DNA, as is the case with Southern blotting, and otherwise within three days, as is
the case with PCR. For long-term storage, specimens for DNA tests should be stored asfollows:○ Pleural effusion, ascites, pericardial fluid, pancreatic fluid, broncho-alveolar lavage
fluid (BALF): After centrifugation (760 x g (gravitational acceleration) [2,000 rpm], at
an ambient temperature, for 10 minutes), the precipitates are washed with PBS. Afteran additional centrifugation, the supernatant is discarded and only the precipitates are
stored at ultralow temperatures (-70 ˚C or below).
○ Urine Sediment: After centrifugation (6,000 x g (gravitational acceleration) [8,200rpm], at an ambient temperature, for two minutes), discard the supernatant and store
only the precipitates at an ultralow temperature (-70 ˚C or below).
○ Sputum: Store at an ultra-low temperature (-70 ˚C or below).
(1) Inappropriate conditions unsuitable for testing1) Specimens that contain a greater amount of cellular components (normal cells andnon-tumor cells) than the target cells may give false-negative results due to thedilution of the targeted cells by normal cell populations. The proportion of targeted
cells in the specimens can be visually confirmed with morphological examination.
2) In specimens in which fibrin clots are observed, centrifugal collection of cells ishampered and sampling from consistent fluid is prevented, which may result in alack of quantitative performance for quantitative analysis. Fibrin clots can bedetected by observing the fluidity of the specimen in the tubes or during pipetting
operations.
(2) Causes
1) Because collection of pleural effusion, ascites, pericardial fluid, pancreatic fluid,
sputum, broncho-alveolar lavage fluid (BALF), and urine sediment are not methods
to directly harvest the affected lesion, contamination by cells not in the target area,
	
 

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JCCLS MM5-A1(2011.12.1)
including normal cells, is unavoidable in many cases.
2) Possible causes include the contamination of blood components at the time of
specimen collection.
(3) Troubleshooting
1) The detection sensitivity of these methods should be confirmed to be high enoughspecifically detect the targeted gene mutations, even when the proportion of targeted
cells is low.
2) Before extracting nucleic acids, fibrin clots must be physically crushed
in the specimen until fibrin begins to disaggregate and sufficiently disperses
throughout the specimen. Then, the specimen is sampled together with the fibrin. In
the process of DNA extraction, the addition of DTT (dithiothreitol) and proteinase
K promotes fibrin degradation.
(4) Measures to avoid inappropriate situations
1) In case of specimens in which the proportion of targeted cells is low, of targeted
cells should be fractionated by density gradient centrifugation using Ficoll, by the
antibody method, or by using a cell sorter.
2) Because pleural effusions and others may coagulate after collection due to fibrin
deposition, they should be collected in a container that contains sodium citrate or
EDTA.
4.2.5 Cultured Cells
After collecting cultured cells in the centrifuge tubes, they should be centrifuged (300 x g(gravitational acceleration) [1,250 rpm for a rotor with a radius of 170 mm], at an ambient
temperature, for five minutes). The precipitated cells should be stored at an ultra-low
temperature (-70 ˚C or below) and used for the extraction of nucleic acids.
To analyze the gene expression for accurate quantification, total RNA should be extracted
immediately (within two hours) after the collection of the specimen. When RNA extraction
cannot be implemented immediately, the specimen should be treated with an appropriate
method to stabilize RNA and then should be stored until the RNA is extracted. The
methods for stabilizing RNA differ by the purpose of the test, the specimen type, and thestorage period. When guanidine isothiocyanate (5 ml/L) is used to prevent specimen
denaturing, the specimen can be stored for approximately one week with in ambient
temperature. The extracted RNA should be stored at ultra-low temperatures of -70 ˚C orbelow. Repeated freezing and thawing of extracted RNA should be avoided, since it may
contribute to the degradation of RNA and thus cause false-negative results.
(1) Inappropriate conditions unsuitable for testing
It is necessary to consider the effects of the anti-freezing agent DMSO (dimethyl
sulfoxide) contained in the cryopreservation solution of the cultured cells. DMSO issometimes used to enhance the specificity of the PCR reaction, but when DMSO
remains in the solution of extracted nucleic acids (DNA/RNA), it may lower theamplification efficiency, or the fluorescent substance included in DMSO may raise the
	
 

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JCCLS MM5-A1(2011.12.1)
background level in the measurement of DNA concentration.
(2) Causes
Possible causes include residual DMSO in extracted nucleic acids. DMSO is ananti-freezing agent that is added for the purpose of preventing freezing of the cultured
cells during cryopreservation.
(3) Troubleshooting
Sufficiently wash cultured cells with PBS and others to remove the anti-freezing agent(DMSO) before nucleic acid extraction.
(4) Measures to avoid inappropriate situations
When the anti-freezing agent (DMSO) is used, the information should be provided tothe staff engaged in the molecular method by describing this issue in the testing orderform.
4.3 Preparation of Specimens in Molecular Methods for Germ Line Cells
Molecular methods for germ line cells can reveal permanent genetic information that does
not change over a long period of time, such as genetic testing for single-gene diseases. Thisis classified into the following categories: (1) genetic testing for confirmatory diagnoses
performed for patients (or the proband), carrier testing, presymptomatic testing, diseasesusceptibility testing, pharmacogennomic testing, prenatal testing, and neonatal screening;(2) genotype testing to clarify the HLA type of the donor and recipient (patient) for the
purpose of transplantation; and (3) DNA testing for the determination of parentage paternity
and personal identification.
In molecular methods for germ line cells, blood specimens are mainly used, but oral
(buccal) mucosa, hair, and nail specimens are sometimes used because their collection isless invasive. In addition, in the field of forensic medicine, blood stains and umbilical cords
that are kept as tokens of one’s birth are also used for to provide specimens for evidence
and to identify an individual, respectively.
When the test is performed for an individual who underwent either hematopoietic stem celltransplantation (bone marrow transplantation) or organ transplantation, it is potential thatthe specimen contains blood or tissue of the donor. Thus, before the collection of a
specimen, it should be confirmed whether or not the person has undergone organtransplantation. If the individual has received organ grafts, tissues that do not contain
donor-derived tissues should be used for testing.
In order to perform Southern blotting or to create a DNA genomic library using DNA
extracted from blood, high molecular weight and high-purity DNA must be prepared. Sincehigh molecular weight DNA can be easily damaged by physical forces, including shear
force, DNA must be extracted from a fresh specimen or from a freshly-frozen specimen
without physical stirring.
Because genetic testing can reveal genetic information about the subject (patient) as well as
the subject’s relatives, obtaining informed consent is essential prior to performing the tests.
Generally, blood and other specimens are collected at medical facilities, but some
	
 

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JCCLS MM5-A1(2011.12.1)
specimens, including oral mucosa and hair, can be collected by the subjects themselves.
However, in order to ensure personal identification, it is required that specimen collection
be conducted in the presence of a responsible or accredited third person.
4.3.1 Whole Blood (WBC)
After the specified volume of blood is drawn into blood collection tubes that contain ananti-coagulant (EDTA and others), the specimen should be inverted and mixed immediately
and refrigerated if nucleic acid extraction is not performed on the same day of specimen
collection. The specimens can be stored at an ambient temperature for as long as three days
without any significant effects. However, it is desirable that DNA extraction be performed
within 24 hours for molecular methods that require high molecular weight DNA, as is thecase with Southern blotting, and otherwise performed within three days, as for PCR. Whenblood specimens are cryopreserved after collection, they may be frozen immediately, but it
is important to avoid repeating the freezing and thawing process, which contributes to thedegradation of nucleic acids. For long-term storage, blood specimens should be stored at
ultra-low temperatures (-70 ˚C or below). In cases new specimens cannot be obtained otherthan inappropriate conditions of those, a try for use of buffers or enzymes that may reduce
inhibitory effects is to be considered.
When specimens are collected from patients who have received pretreatment forhematopoietic stem cell transplantation (bone marrow transplantation) and who haveextremely low white blood cell counts, it should be noted that several times the specified
volumes of blood specimens may be needed to secure the required number of white blood
cells for the test. Blood specimens should be collected by qualified medical personnel only
after the subject has been carefully identified.
(1) Inappropriate conditions unsuitable for testing1) In specimens with fibrin clots, fibrin prevents the even distribution of the whiteblood cells throughout the specimen. Fibrin clots can be detected by observing thefluidity of the specimen in the blood collection tubes or during pipetting operations.
2) In the PCR analyses, if heparin is present, PCR amplification will be poor and thus
an “undetectable” result will be obtained, because heparin is an inhibitory substancefor PCR. Because heparin contamination cannot be visually detected, theinformation on the tube label should be carefully checked.
(2) Causes
1) Potential causes of fibrin deposition include failure to use tubes containing
anti-coagulant (EDTA and others) and improper inversion and mixing of the tubes
immediately after the specimens are collected.
2) The main cause of heparin contamination is considered to be the use of heparin as
an anti-coagulant agent during blood collection. This also applies to the case inwhich blood specimens collected for other analytical purposes are used for genetic
testing. Heparin contamination is also a concern when blood specimens arecollected from patients who are receiving heparin for thrombolytic therapy.
	
 

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JCCLS MM5-A1(2011.12.1)(3) Troubleshooting
1) Before extracting nucleic acids, fibrin clots in the specimen must be physicallycrushed until they begin dissociating and sufficiently disperse fibrin throughout the
specimen. Thereafter, the whole specimen containing the fibrin are analyzed.
During the process of DNA extraction, the addition of DTT (dithiothreitol) andproteinase K promotes fibrin degradation.
2) It has been stated in the literature that the effects of heparin can be reduced by using
nucleic acid extraction kits, in which nucleic acids are adsorbed onto filters,
membranes, or magnetic particles, and impurities are washed out and removed.
However, it is impossible to completely remove the impurities. The effect ofheparin can be resolved either by completely digesting the heparin with heparinase
or by washing the white blood cell fraction with physiological saline at least twice
after fractioning of white blood cells.
(4) Measures to avoid inappropriate situations
1) Fibrin deposition can be prevented by repeatedly inverting the blood collection
tubes that contain anti-coagulant (EDTA and others) and sufficiently mixing thecontents immediately after collecting blood specimens.
2) In principle, heparinized blood collection tubes should not be used in genetic testing.
For heparinized patients, it is desirable that the specimen be collected after the
heparin effects have been eliminated from the blood. However, when heparincontamination is unavoidable, for example when patients are on heparin therapy,
information should be provided to the medical professionals performing themolecular analyses by stating, “the patient is heparinized,” on the testing orderform.
4.3.2 Oral Mucosa and Fluid
Oral mucosal specimens are collected by rubbing the inside of the cheek several times witha swab. Subsequently, the swab should be air dried immediately and stored at an ambient
temperature. After oral mucosa collection, if the swab is left in moist conditions, such as
stored in a sealed plastic bag or a test tube without drying, the DNA in the oral mucosa will
be rapidly degraded due to DNases in the sample.
Oral mucosal specimens can be collected by the subjects themselves, but in order to ensure
personal identification, the specimen collection must be conducted in the presence of a
responsible or accredited third person after confirming the subject’s identity.
(1) Inappropriate conditions unsuitable for testing
Regarding specimens collected just after eating, drinking, or breast-feeding, the effectsof the incorporation of foods or breast milk on DNA extraction or test results are ofconcern.
(2) Causes
If oral mucosa specimens are collected while food, drink, or breast milk is in the oralcavity, there is the possibility that they will be collected together with the oral mucosa
	
 

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JCCLS MM5-A1(2011.12.1)
by the swab.
(3) Troubleshooting
By using molecular methods that can specifically detect human DNA, the effect of
DNA derived from food and drink can be eliminated. Regarding the effect of breast
milk, the extent of incorporation of maternal DNA can be confirmed by personal
identification by genetic testing using the extracted DNA.
(4) Measures to avoid inappropriate situations
Because the effects of eating, drinking, or being breast-fed disappear in about 30
minutes, specimens should not be collected until 30 minutes after eating, drinking, orbeing breast-fed. In addition, the oral cavity should be rinsed with water before
specimen collection to remove any remaining effects of food, drink, or breast milk.
4.3.3 Hair
Hair specimens should be collected by removing the hair with tweezers. The specimens
should be dried and stored at an ambient temperature. Genomic DNA in hair exists mainlyin the root cells of the hair, and extremely small amounts exist in the shaft of the hair.
Therefore, for the detection of genomic DNA, the root cells of the hair should be confirmed
to be attached. The amount of recoverable DNA is much less from fallen hair than fromremoved hair. The use of fallen hair as a specimen should also be avoided, so that the originof the specimen is attributable to an individual.
Mitochondrial DNA can be detected from the shaft of the hair, but yields hetroplasmic
results more often than that from other tissues. Thus, the results should be carefullyinterpreted.
Hair specimens can be collected by the subjects themselves, but, in order to ensure personal
identification, it is required that specimen collection be conducted in the presence of a
responsible or accredited third person after confirming the subject's identity.
(1) Inappropriate conditions unsuitable for testing
1) Hair specimens to which hair dressing or hair dye has been applied may give poor
PCR amplification. A recent history of using hair dressings and hair dyes should bechecked with the test subject, and hair dye should also be confirmed by measuring
the amount of dye compound and by microscopic examination.
2) There is a possibility for poor PCR amplification due to the effects of melaninremaining in the extracted DNA.
(2) Causes
1) Some components in hair dressings may inhibit the PCR reactions. In addition,
DNA in the hair shaft may be fragmented by the oxidative effects of hair dyes.
2) Possible causes include insufficient melanin removal in the process of DNA
extraction.
(3) Troubleshooting
1) Before extracting DNA, hair specimens should be washed with ethanol to remove
any remaining hair dressings. In order to detect DNA that has been degraded into
	
 

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JCCLS MM5-A1(2011.12.1)
small molecules, it is necessary to devise appropriate measures such as setting
primers to detect shorter DNA fragments.
2) The amount of specimen (hair) used in one batch of the DNA extraction reaction
can be reduced to improve the efficiency of melanin removal during the DNA
extraction procedures. For protein removal during DNA extraction, methods that aresuitable for melanin removal should be used; for example, the NaI method has beenreported to be more suitable for melanin removal than the phenol method. In orderto reduce the inhibitory effect of melanin on PCR, extracted DNA should be diluted
and used for PCR.
(4) Measures to avoid inappropriate situations
1) The subjects of the test should be carefully interviewed concerning their recent useof hair dressings and hair dyes, and the subjects should provide information to thestaff through the testing order form.
2) If no DNA is detected, a new specimen should be obtained.
4.3.4 Nails
Before collection, carefully wash the areas between the nail and the finger, remove
fingernail scrapings and dirt to the maximum extent possible, and use nail polish remover toremove all nail polish. Cut a specimen from the excess white part at the tip of the nail (freeedge of the nail) with a nail clipper and store the specimen at ambient temperature. To
avoid mixing nail specimens, each patient must use a different, clean nail clipper.
Nail specimens can be collected by the subjects themselves, but in order to ensure personal
identification, it is required that collection be conducted in the presence of a responsible oraccredited third person after confirming the correct person’s identity.
(1) Inappropriate conditions unsuitable for testingIf large pieces of the nail are used for DNA extraction, they may not be completely
dissolved by the protein-digesting enzymes during the treatment process, and theamount of recovered DNA may be reduced.
(2) Causes
Possible causes include incomplete dissolution of the specimen, since the hard fibrous
protein (keratin), which is one of the main components of the nail, is not easily
dissolved by treatment with protein -digesting enzymes.
(3) Troubleshooting
If the specimen cannot be dissolved by the protein-digesting enzymes during the DNA
extraction process, the DNA extraction should be continued using only the dissolved
portion.
(4) Measures to avoid inappropriate situations
Specimens used for DNA extraction should be cut into small pieces (around 1 mm x 1mm) in advance to improve the digestion efficiency by protein-digesting enzymes.
4.3.5 Dried Blood Spots
	
 

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JCCLS MM5-A1(2011.12.1)
Blood should be dripped on a white filter paper, air dried, and stored under an ambient
temperature. In cases of specimens that will be used in the field of forensic medicine, theblood may be stained on various materials. If it is possible to collect a blood spot alone
from any of the materials, peel off the blood spot or collect blood components using swabs
that have been moistened with distilled water and dry them for storage under an ambient
temperature. If blood is absorbed in the material and it is impossible to collect blood
components alone, collect the stained material and dry and store it under an ambient
temperature. The conditions before and after the collection of the blood spot must berecorded.
(1) Inappropriate conditions unsuitable for testing
In the case of specimens used in the field of forensic investigations, blood which is
inseparably mixed with some substances may be a cause of poor amplification in PCRor a band shift in electrophoresis.
(2) Causes
In forensic investigations, blood is generally found where it has stained various
materials. Possible causes include that the specimen includes some substances thatcannot be removed in the DNA extraction processes.
(3) Troubleshooting
The efficiency in impurity removal in DNA extraction differs depending on thesubstance the DNA is mixed with. Thus, it is necessary to consider and selectappropriate DNA extraction methods for different specimens.
(4) Measures to avoid these situations
In forensic investigations, blood that has already stained material is used in these tests.
Accordingly, contamination with unknown substances is unavoidable. When swabs areused for sampling, special swabs that do not affect DNA extraction should be used.
4.3.6 Umbilical Cord
This section describes a dried umbilical cord kept as a token of one’s birth, and that wasused as a specimen for identification of an individual by genetic testing. The origin of theumbilical cord is the fetus, but because it is a colloidal connective tissue, little DNA can becollected from the umbilical cord itself. Instead, blood-derived cells in the umbilical cord
are used for DNA extraction. Information attached with the umbilical cord should be usedas the specimen collection record.
(1) Inappropriate conditions unsuitable for testing
Blood spots that are considered to be of maternal origin are found on the surface of theumbilical cord.
(2) Causes
Possible causes of maternal blood found on the surface include incomplete removal ofmaternal blood at the time of umbilical cord collection. However, this is an
uncontrollable situation because, at the time the umbilical cord was collected, there was
no intention that it would be used for DNA extraction.
	
 

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JCCLS MM5-A1(2011.12.1)(3) Troubleshooting
Whether there are blood spots on the surface of the umbilical cord or not, the outersurface of the umbilical cord should be washed with PBS or physiological saline prior
to DNA extraction. In case DNA types of more than one individual are detected by
DNA testing, DNA testing of the mother should be performed to identify the source ofthe detected DNA type.
(4) Measures to avoid inappropriate situations
It should be assumed that an umbilical cord that is kept as a token of one’s birth iscontaminated with tissues of maternal origin. The outer surface of the umbilical cordshould be washed with PBS or physiological saline before DNA extraction procedures,
even if no contamination is visually recognized.

5. Collection of Specimens for Molecular Methods
This section summarizes methods to collect specimens for molecular methods.
5.1 Collection of Specimens in Molecular Methods for Pathogens
5.1.1 Viral Infection
5.1.1.1 Hepatitis Virus
The following types of hepatitis viruses are known: hepatitis A virus (HAV), hepatitis B
virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), and hepatitis E virus(HEV). HDV requires the co-existence of HBV for replication. Other than these viruses,
the EB virus (EBV), cytomegalovirus (CMV), and herpes simplex virus (HSV) also
cause hepatitis. HAV and HEV are primarily orally transmitted, while HBV, HCV, andHDV are transmitted through the blood.
If viral hepatitis is suspected, typically serum or plasma separated from blood collectedwith EDTA is used as a specimen. It is desirable that specimens for molecular methods
for pathogens be stored at temperatures of -20 ˚C or below for the measurement for
HBV DNA, or at temperatures of -70 ˚C or below for the measurement of RNAs of
HAV, HCV, and HEV. It is of critical importance to avoid repeated freezing andthawing cycles. To confirm that viruses are completely eliminated after anti-viraltherapy, an ultra-sensitive nucleic acid amplification test may be performed using tissue
from a liver biopsy as the specimen. As the activity markers, such techniques asdetection of negative-strand RNA and cccDNA (covalently closed circular DNA) for
HCV and HBV, respectively, in the hepatic cells have been attempted in someinvestigational studies.
Acute hepatitis by HAV and HEV is transient, and does not usually have a chronic
course. Serum collected in the acute phase is used as the specimen. Stool is also used as
an important specimen, since the viruses that cause these diseases characteristically tend
to be excreted in the stool for a long time after they are eliminated from the blood.
5.1.1.2 Virus Causing Acute Respiratory Infection
	
 

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JCCLS MM5-A1(2011.12.1)
Several viruses responsible for acute respiratory infections include influenza virus, RSvirus, parainfluenza virus, coronavirus, enterovirus (non-polio), rhinovirus, andadenovirus.
The major specimens collected to identify causal viruses are listed below. The
collection methods of specimens for nucleic acid testing conform to those for virus
isolation.
1) Collection of throat swab specimens
Tubes containing 2 ml of PBS (-) with 0.5% BSA and cotton swabs with wooden shafts
that can be snapped with the fingers should be prepared. After rubbing the patient’s
throat with the tip of the swab, the cotton part should be dipped into the solution in thetube, the cotton part rinsed by vigorous agitation, and the solution squeezed out of thecotton part of the swab by pressing it against the inside of the tube, after which the swab
should be discarded. Alternatively, the swab with shaft can be left in the solution in the
tube after the shaft is snapped with the fingers.
2) Collection of nasal wash solution
The subject’s head should be tilted back by approximately 45 degrees. The tip of therubber bulb syringe containing 3-5 ml of physiological saline should be inserted into thenostril, and a squeeze of the bulb will inject the physiological saline into the nostril,
then upon release it will immediately collect the physiological saline in the nostril backinto the bulb due to the negative pressure. This solution is used as the specimen for thetesting. This procedure requires the cooperation of the subject and skillful technique,and it has some other disadvantages in that the physiological saline injected into thenostril may not be recovered sufficiently or the virus concentration may be reduced ifsome of the physiological saline remained in the bulb, thereby not being injected into
the nostril.
3) Collection of nasopharyngeal discharge
A vinyl catheter with a trap should be prepared between the vacuum source and the tip.
This catheter should be inserted into the innermost boundary of the nostril and thenasopharyngeal discharge aspirated. The nasopharynx is the place where the Eustachiantubes open, and infants and young children have extensive nasopharyngeal discharge inthis area. The isolation rates of influenza virus and RS virus are higher innasopharyngeal discharge specimens than in throat swab specimens, which makenasopharyngeal discharge a favorable specimen for nucleic acid testing. There is analternative method for collection of nasopharyngeal discharge, which utilizes a swab
with fine, flexible shaft, but not a hard one for the throat. The swab should be carefully
inserted along the lower edge of the nostril by imagining a plane that connects thenostril opening to the acoustic opening, reaching the innermost section of the nostril.
The swab is left there for several seconds, and then removed.
4) Collection of throat wash solution
The patient gargles with a minimal volume of PBS or physiological saline, and thissolution is used as the specimen. Only a minimal amount of solution should be used,
	
 

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JCCLS MM5-A1(2011.12.1)
otherwise it is difficult to extract viral nucleic acids from a large specimen volume,
which may affect the test results. Specimens should be collected in the acute phase andtested immediately after the collection. For storage, freezing is required.
5.1.1.3 Virus Causing Diarrhea
Viruses responsible for acute, infectious diarrhea include rotavirus, adenovirus, calicivirus,
astrovirus (norovirus and sapovirus), parvovirus, coronavirus, enterovirus, and influenza
virus. Gastroenteritis accompanying rotavirus and influenza as well as Calicivirus(oyster-related food poisoning) infections are most commonly seen during the winter, while
enteroviral infections occur more frequently during the summer. Adenovirus and sporadic
Calicivirus are detected in the stools of patients with diarrhea all the year.
Many of these viruses replicate in the intestinal mucosal cells and bring about severe
diarrhea and vomiting. An extremely large number of virus particles are excreted into thestool (several hundred million particles per 1 g of stool). For the detection of pathogens by
nucleic acid testing, stool specimens collected at the peak of the symptoms are used, butvomitus is sometimes used as the specimen in cases of norovirus infections. Because thenumber of viruses contained in the vomitus is lower than that of the stool, it is difficult to
detect pathogens by immunoassay, and requires the highly-sensitive PCR.
5.1.1.4 Encephalitis Virus
Viral encephalitis is classified into two types: primary encephalitis, which is developed bydirect viral infection of the brain tissue, and secondary (post-infection) encephalitis, whichdevelops secondarily to the infection of other organs of the body. Primary encephalitis
includes those by herpes simplex virus (HSV) encephalitis and Japanese encephalitis, andsecondary encephalitis includes those caused by measles (rubeola), epidemic parotitis(mumps), and rubella.
To diagnose these viral encephalites, pathogenic examinations, including the isolation ofviruses and the detection of viral nucleic acids, are important tools. In the case of acuteencephalitis, cerebrospinal fluid (CSF) is useful as a specimen, because the testing target isthe affected brain tissue lesion. CSF should be collected in the acute phase prior totreatment. Sufficient care is required to avoid blood contamination during the collection. If
the patient dies, the brain parenchyma is used as the affected lesion in the tests.
5.1.1.5 Aseptic Meningitis Virus
Viruses responsible for aseptic meningitis are represented by enteroviruses, including
echovirus and Coxsackie B virus. Aseptic meningitis is prevalent during the summer inJapan, as are other diseases caused by enteroviral infection, but can also occur during thefall and winter. Some meningitis cases are caused by other viruses, including the mumps
and herpes simplex viruses. The major symptom of meningitis caused by enterovirus isfever, which is sometimes accompanied by headache, nausea, and vomiting. Generally,
however, the patient prognosis is good. The serotype of prevalent enterovirus and the
	
 

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JCCLS MM5-A1(2011.12.1)
degree of the epidemic usually differs every year. The major route of infection is fecal-oraltransmission, and the incubation period is typically 4-6 days.
Stools, spinal fluid, and oral or throat swabs are used as specimens for molecular methods
for pathogens, as in the case with virus isolation. Recently, many reports have describedviral identification methods that use homology searches based on the base sequence of thevirus. In these methods, specimens with virus are inoculated and cultured in medium, andnucleic acids of the obtained virus are amplified and are used for the determination of thebase sequence.
Specimens should be collected as soon as possible after the onset of the symptoms andtested immediately. If stored, specimens should be frozen. However, test methods forenteroviruses have not been standardized. Depending on the purpose of the test, appropriate
specimens and appropriate procedures should be selected.
5.1.1.6 Epstein-Barr virus: EBV
EBV is a virus of the γ-herpesvirinae subfamily that primarily infects B lymphocytes andcertain epithelial cells; after that, it persists in a latent state. EBV is transmitted primarily by
the saliva. Primary infection during early adolescence and after may lead to infectious
mononucleosis, virus-associated hemophagocytic syndrome, and chronic active EBV
infection (CAEBV). In addition, EBV is considered a tumor virus, as it is involved in thedevelopment of nasopharyngeal cancer, Hodgkin’s lymphoma, NK/T lymphoma, gastriccancer, and opportunistic lymphoma.
Various specimens, including serum, blood, spinal fluid, and throat swabs, are used toconfirm the presence of EBV by molecular methods for pathogens. Quantification of EBV
DNA in peripheral mononuclear cells is useful for the diagnosis of CAEBV. In addition,affected tissues are also used as specimens. When a significant amount of the EB viralgenome is confirmed in the tested area, the clonality of the infected cells is analyzed bySouthern blotting.
5.1.1.7 Cytomegalovirus: CMV
CMV belongs to the β-herpesvirinae subfamily, and, after the primary infection, it remains
permanently latent in the spinal cord, peripheral mononuclear cells, andgranulocyte-macrophage precursor cells. When the primary infection occurs, it infrequently
causes infectious mononucleosis-like syndrome, but usually the infection is not apparent.
However, a primary infection during pregnancy is placentally transmitted to the fetus andmay lead to the cytomegalic inclusion disease, which is associated withsplenohepatomegaly, jaundice, hemorrhagic lesions, microcephaly, cerebral calcification,and chorioretinitis. In many such cases, the prognosis is poor. Even in the case of
non-apparent infection, the infant may develop hearing loss and intelligence disorder later,which requires long-term follow-up. In addition, such infectious diseases as interstitial
pneumonia, hepatitis, encephalitis, retinitis, and enteritis due to CMB are issues of great
concern in immunocompromised hosts, including children with primary immunodeficiency
	
 

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JCCLS MM5-A1(2011.12.1)
diseases, patients who have had a kidney transplantation or hematopoietic stem celltransplantation (bone marrow transplantation), and those with AIDS or malignant tumors.
Many specimens are useful for diagnosing a patient with CMV including serum, plasma,
cerebrospinal fluid, ocular aqueous humor, and amniotic fluid, and have great diagnostic
significance by providing good indicators of CMV infection activity. Recently, it has been
described that the quantification measurement of CMV DNA in peripheral blood is usefulfor the diagnosis of post-transplantation and congenital CMV infections. Other CMV
specimens include urine specimen collected within the first three weeks of life for the
diagnosis of congenital CMV infection, broncho-alveolar lavage fluid (BALF) andpulmonary tissue for the diagnosis of interstitial pneumonia, hepatic tissue for the diagnosisof CMV hepatitis, peripheral white blood cells and serum for the determination of activity
of systemic CMV infection, cerebrospinal fluid for the diagnosis of encephalitis, and ocularaqueous fluid for the diagnosis of retinitis.
When the nucleic acid amplification test is used for the diagnosis of CMV infection, it isimportant (i) to take the immune status of the patient into consideration, (ii) to use
specimens taken from the infected site as possible, and (iii) to implement quantitative
measurements as required.
5.1.1.8 Human Parvovirus B19
Human Parvovirus B19 is a causal virus for infectious erythema, which is accompanied bya rash on the cheek. Seven to 10 days before the development of the rash, a slight fever andcommon cold-like symptoms are observed in many cases, during which period the viremia
is occurring. The prognosis is generally good, but in patients with hemolytic anemia,
infection with this virus may cause severe anemic crisis, for which special caution is needed.
Otherwise, it is known that this virus causes persistent infection in patients with arthritis,
rheumatoid arthritis, thrombocytopenia, granulocytopenia, hemophagocytic syndrome, andimmune disorders. In addition, it should be noted that infection during pregnancy has been
linked to fetal anomaly (hydrops fetalis) and spontaneous abortion.
Because culture methods for this virus have not been established yet, nucleic acid testing is
performed to prove the presence of the pathogen. To perform nucleic acid testing by dot
blotting or Southern blotting, it is required that a blood specimen be collected at the periodof viremia. The PCR technique allows detection of viral DNA from the serum specimens, atleast during the period when the IgM against the virus is detectable. Compared with otherviruses, this virus is difficult to inactivate or remove by heat treatment or filtration, and thepotential contamination of various plasma-derived products cannot be excluded. In
response to each of the cases mentioned above, it is required that biological materials that
contain the greatest amount of the virus be collected at the appropriate time. Serum is thestandard specimen for the test, but, depending on the clinical symptoms, other specimens
including joint fluid, peripheral white blood cells, bone marrow fluid, amniotic fluid,
umbilical blood, and blood preparation products can be used.

	
 

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5.1.1.9 Human papilloma virus: HPV
HPV is currently classified into more than 100 types, based on the base sequence of the
HPV genome DNA, and each type can be classified into an epidermal or mucosal type,based on the site of infection. The mucosal type is further classified into the high-risk andthe low-risk groups. The high-risk group infects the mucosal epithelia of genitourinaryorgans and the head and neck area, and is involved in the development of malignant lesions
in these areas. The low-risk group is involved in the development of benign lesions,
including Condyloma acuminatum and papillomas.
Because HPV culture methods have not yet been established, the virus must be detected by
nucleic acid testing. In the high-risk group, specimens are collected together with thecytology specimen at the time of cervical cancer screening by rubbing the cervical wallwith a special sampling tool. In Europe and the U.S., liquid-based cytology (LBC) has
recently become the mainstream of the screening and is regarded as the most favorable
method for specimen preparation, that is, scraped cells are stored suspended in alcohol
fixative, and a cytological examination is performed using cytospin specimens preparedfrom the suspension. In order to examine the involvement of human papillomavirus in thedevelopment of cervical cancer, the analyses using PCR and in situ hybridization are
implemented, using tissue sections excised from cancerous areas. For the diagnosis of the
low-risk group, which includes causal viruses for verruca vulgaris and Condyloma
acuminatum, PCR and in situ hybridization are performed using biopsied skin tissues toconfirm the presence and types of the virus.
5.1.1.10 HTLV-I (Human T-Lymphotropic Virus Type I)
HTLV-I is a virus responsible for adult T-cell leukemia/lymphoma (ATLL), HTLV-I
associated myelopathy/tropical spastic parapalysis (HAM/TSP), and uveitis of the eye.
After the virus infects human T cells, its genome is integrated into the host cell genome in
the form of provirus DNA, at which point the infected person becomes a carrier of HTLV-I.
It is transmitted through provirus DNA in lymphocytes. One out of 1000 HTLV-1 carriers
develops ATL every year. Approximately three out of 100,000 carriers develop HAM
every year. The routes of infection are mainly from mother to the newborn infant throughbreast milk, but other routes include transfusion and, extremely rarely, between husband
and wife (mostly from male to female).
Carriers are generally detected by the measurement of serum HTLV-I antibody level, butwhen the result is unclear and difficult to judge, provirus DNA testing by PCR is performed
using genomic DNA extracted from peripheral white blood cells. Highly sensitive provirus
DNA testing with PCR is the only tool that can be used to determine whether infants havebeen infected through breast milk, especially during the period after maternal antibodies
have disappeared and until their own antibody titer has risen. However, caution is requiredin interpreting the results because there can be false-positive results due to contamination as
well as false-negative results due to insufficient collection of peripheral white blood cells or
inhibition of amplification. In order to examine the cause of such diseases as HAM and
	
 

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JCCLS MM5-A1(2011.12.1)
uveitis, which are induced by an increase in the number of infected T cells and in theamount of provirus DNA in some local regions, it is desirable to obtain specimens fromsites that reflect the affected lesions. Spinal fluid and ocular aqueous fluid can also be usedas specimens. In order to diagnose ATL, it is required to detect, by Southern blotting,proviral HTLV-I DNA that was inserted into the chromosomal genomic DNA of neoplastic
T lymphocytes and to prove monoclonal propagation of infected T cells based on its band
pattern. The specimens used for this purpose include DNA extracted from abnormal cells in
the peripheral blood, affected lymph nodes, or skin tissues.
5.1.1.11 HIV (Human Immunodeficiency Virus)
HIVs (HIV-1 and HIV-2) are the viruses responsible for AIDS (acquired
immunodeficiency syndrome). HIV is present in body fluids, including blood, seminal fluid,
and vaginal secretions, as well as the tissues or organs of infected people. As the majorroutes of infection, sexual contact with infected persons, contact with blood contaminatedwith HIV, and infected mother-to-child transmission are currently known.
Nucleic acid testing to determine the pathogen includes quantification of HIV RNA using
nucleic acid amplification techniques, proviral DNA detection, subtype identification, anddetection of drug-resistant genes. The quantification of HIV RNA in the blood of patients
infected with HIV is useful for the purpose of understanding patients’ pathologicalconditions, monitoring the effects of antiviral chemotherapy, and estimating the progressionof the disease. Serum or plasma is used as a test specimen. For the collection of plasma
specimens, sodium citrate or EDTA is used as an anticoagulant. The use of heparin should
be avoided because heparin inhibits the PCR amplification reaction. In addition, because
HIV infects low-density lymphocytes, if the plasma fraction is collected when the buffy
coat formation by centrifugation is incomplete, there is a risk for incorporating lymphocytesthat have not fully sedimented. Therefore, careful attention is needed when the quantity ofviruses is closely monitored. The HIV proviral DNA test is useful for the diagnosis ofneonates born from mothers who are HIV antibody-positive, and genomic DNA extracted
from peripheral white blood cells is used as the specimen for this test, as in HTLV-1 testing.
The plasma HIV RNA level is an indicator for viral replication, and higher RNA levels arelinked to the possibility that the number of CD4 positive lymphocytes decreases earlier andthat the condition progresses to AIDS development. However, this kind of viral activity
cannot be evaluated by a proviral DNA test alone. For drug resistance testing, viral RNA
extracted from viral particles in the blood is used.
It is extremely important to identify infected persons in the earlier stages of AIDS
development. The period during which the presence of the viruses cannot be detected, eventhough they are present and can be transmitted to other people through transfusion or othermechanisms, is referred to as the “window” period of infection. While this period is 22 and18 days for the antibody test and the simultaneous antigen and antibody detection tests,
respectively, it is calculated to be 11 days for the nucleic acid amplification test (NAT), thus
suggesting that the HIV RNA test and the HIV provirus DNA test are useful for early
	
 

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JCCLS MM5-A1(2011.12.1)
diagnosis.
5.1.2 Bacterial Infection
5.1.2.1 Tuberculosis and Other Mycobacteria
Accurate and rapid diagnosis and differentiation of tubercle bacilli and nontuberculous
mycobacteria are each extremely important for selection of the treatment, estimation of the
prognosis, and prevention of group infection and nosocomial infection. The smear test
allows rapid detection but has low sensitivity and is unable to differentiate between tubercle
bacilli and nontuberculous mycobacteria. Culture tests enable the identification of bacterial
species and drug resistance testing, but take a long time before the identification can be
made. Molecular methods for pathogens, when combined with conventional methods, areextremely useful for rapid diagnosis. Test methods include direct detection from specimens,
identification of isolated bacterial species, and identification of drug-resistant genes.
\Sputum is a major specimen, but an enormously diverse array of other specimens is also
used. These specimens include broncho-alveolar lavage fluid (BALF), aspirated bronchial
mucus, gastric juice, stools, spinal fluid, plural effusion, ascites, urea, purulent materials,
abscess walls, granulation tissues, and organs. For highly precise detection, it is importantto select the appropriate specimen and to control the quality of the specimen. The selected
specimen should be that which most closely reflects the lesion. Sputum is extensively used,and high quality sputum, which reflects a lesion in the lower respiratory tract, must becollected. The purulent portion should be collected while avoiding saliva incorporation.
Precautions include collecting the specimens at the time of awakening in the early morning
after sufficient gargling, if possible, and collecting specimens before anti-tuberculosis
treatments are started. In order to collect high-quality sputum, appropriate instructions forpatients and collaboration among the concerned medical staffs are important. At the time ofcollection, the medical staffs should take certain measures to prevent occupational infection,such as wearing a mask and gloves. Because pleural effusion and other specimens may
coagulate after collection due to fibrin deposition, they should be collected in a containerthat contains sodium citrate or EDTA. The incorporation of blood should be avoided,
because it is an inhibitory substance. Specimens collected in sterile containers should becompletely sealed to avoid dehydration and leakage to the outside.
5.1.2.2 Legionella
Legionnaires’ disease or legionellosis is a collective term for the infectious diseases caused
by bacteria belonging to the genus Legionella. These bacteria are roughly classified into the
Pontiac fever type, for which the prognosis is generally good, and the pneumonia type(Legionella pneumonia), which often cause severe symptoms. The Pontiac fever typepresents common cold-like symptoms including fever, chills, muscle ache, joint ache,fatigue, and headache, but most patients recover within several days. Conversely, thepneumonia type progresses rapidly. After an incubation period of 2-10 days, symptoms
begin with general fatigue, headache, and muscle ache in the early stages. Thereafter,
	
 

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respiratory disease symptoms appear several days later, including a fever exceeding 39 ˚C,
dry or sometimes wet coughing, chest pain, purulent sputum, and difficulty in breathing.
These symptoms become severe within 48 hours. Neurological symptoms, such as limb
tremors and clouded consciousness, may appear.
In molecular diagnosis of pathogens, such respiratory tract-derived specimens as sputum
collected before the administration of antibacterial drugs, broncho-alveolar lavage fluid(BALF), endotracheal aspirate, pleural effusion, and lung tissues are used as with bacterial
isolation cultures, but pericardial fluid, spinal fluid, and blood can also be used as the
specimens. Because a large amount of blood cells and other cells present in the specimens
can inhibit the amplification reaction, in general it is desirable to use broncho-alveolar
lavage fluid (BALF), pleural fluid, or endotracheal aspirate. For the identification of thesource of infection, specimens from the environment, including (concentrated) water fromcooling towers and bath water, and hot-water systems are also checked.
5.1.2.3 Mycoplasma pneumoniae
The major diseases caused by this bacterium are bronchitis and pneumonia. Together withchlamydial pneumonia, mycoplasma pneumonia represents a large proportion of primaryatypical pneumonia cases, at a frequency of approximately 30-40%. It occurs relatively
frequently in younger people, but it has been observed in almost all age groups. In Japan, it
used to be called the “Olympic Games pneumonia,” since its outbreak occurred every four
years in the country, but now such periodic outbreaks no longer occur. Its complications
include skin rash, hemolytic anemia, arthritis, otitis media, meningoencephalitis, peripheral
neuropathy, pericarditis, and constrictive pericarditis. When it is accompanied by central
nervous system complications, the disease may become severe.
The specimens that are most commonly used are throat swabs. However, if there are
symptoms of complications, other specimens may also be used for the tests, including
pleural fluid, spinal fluid, and cellular tissues. When collecting specimens with throat swabs,
the cells should be scraped from the posterior wall of the throat using sterile swabs. Because
this bacterium adheres to mucosal cells in the throat, it is important to scrape off as manymucosal cells as possible using swabs. This bacterium is susceptible to dryness andrefrigeration, so the specimens must not be allowed to dry out. For long-term storage, astorage temperature of -70 ˚C or below is recommended.
5.1.2.4 Chlamydia pneumoniae/psittaci
C. pneumoniae is a causal bacterium for bronchitis and pneumonia, but its links withbronchial asthma and coronary artery atheroma have also been reported. The prevalence ofantibodies in people increases drastically after the age of 4-5 years, and 50-60% of the adult
population is antibody positive. C. psittaci causes severe pneumonia (parrot fever, frombird) for which fetal cases have been reported. Based on antibody survey results, it isestimated that approximately 100 people develop this disease in Japan every year.
Specimens used for the detection of C. pneumoniae include nasopharyngeal/throat swabs,
	
 

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JCCLS MM5-A1(2011.12.1)
sputum, broncho-alveolar lavage fluid, and autopsied tissues. Among these, thenasopharyngeal/throat swab is the most commonly used. The specimen is scraped from thenasopharynx, or posterior wall of the throat, by rotating sterile swabs. For nucleic acidtesting, the swab is put in the tube as it is and transported at a temperature of 4 ˚C or isfrozen with dry ice. As for C. psittaci, human specimens used for nucleic acid testing are
similar to those used for C. pneumonia testing, while, for obtaining avian specimens,
cloacal swabs and feces are collected from living birds, whereas specimens are collectedfrom the liver, spleen, lungs, and intestinal tracts of dead birds that are suspected as thesource of infection.
5.1.2.5 Chlamydia trachomatis
Chlamydia trachomatis is a causal bacterium for sexually transmitted diseases (STD), andapproximately 40% of patients with STDs are infected with this bacterium. This bacteriumcauses urethritis, cervicitis, uterine adnexitis, and intrapelvic infection, as well as neonatal
conjunctivitis and pneumonia by birth canal infection. However, many adult patients are
asymptomatic, and several percentages of healthy pregnant women are positive for thisbacterium.
Specimens from women are collected using uterine cervical swabs. After wiping off thecervical mucus, the cervical cells are scrubbed by turning a sterile swab. After scrubbing,
snap the shaft of the swab, leave the swab in the sterile test tube, seal the tube, and transport
the specimen at a temperature of 4 ˚C or under frozen condition with dry ice for nucleic
acid testing. The range of infection is wide for females, and there is a possibility that nobacteria will be detected from the uterine cervix even though the patient has a peritoneal
infection. In such cases, it is necessary to detect the abnormality from the symptoms andfindings from pelvic examination. In the case of male patients, specimens should be taken
by scrubbing with a sterile swab inserted into the urethra. However, since nucleic acidtesting has a high level of sensitivity, the bacteria can be sufficiently detected even frommorning first-voiding urine. The urine specimens should be stored at a temperature of 4 ˚Cand centrifuged as soon as possible, or otherwise cryopreserved at a temperature of -70 ˚C.
When pharyngeal infection is suspected, a specimen should be collected with a throat swab.
As for neonatal pneumonia and inclusion conjunctivitis, use nasopharyngeal swabs and eye
mucosa swabs, respectively.
5.1.2.6 Neisseria gonorrhoeae
Neisseria gonorrhoeae is a causal bacterium for STD, and it mainly infects the urethra andthe uterine cervix in male and female patients, respectively. Because this bacterium requires
carbon dioxide to survive, it cannot survive in most environments. The main route of
infection is human-human as an STD. Infection with this bacterium is not restricted togenitourinary organs and pharynx, but is disseminated hematogenously and can cause
arthritis, hepatitis, pericarditis, and meningitis, which are referred to as disseminatedgonorrheal infections (DGIs).
	
 

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Specimens to detect this bacterium include purulent urine and discharge from patients withacute urethritis, as well as prostate discharge, urine voided after prostate massage, andvaginal discharge from chronic patients. In suspected DGI patients, bacteria can be detectedfrom blood (especially collected from eruption), knee joint fluid, pharyngeal mucus, andrectal mucus. Thus, specimens should be collected, considering the infectious pathology ofthis bacterium. Some methods or kits for throat swab testing show the cross-reaction withintraoral bacteria of the Neisseria family, so careful attention is required for the diagnosis ofgonorrheal pharyngeal infection.
5.1.2.7 MRSA (Methicillin-Resistant Staphlococcus aureus)
MRSA is Staphylococcus aureus that has become resistant to antibiotics. Although it issometimes isolated from medical staff, they remain as healthy carriers, and do not develop
symptoms. Patients who are susceptible to the development of this disease include elderly
people, immunodeficient patients, including premature babies and those with leukemia,
those who have undergone gastrointestinal or cardiac surgery, and those with underlying
medical conditions, including diabetes, those who are receiving prophylactic antibiotics, arecatheterized, or are hospitalized for a long time. When antibacterial agents are administered,substitution of microbial flora is induced. When this happens, MRSA that are colonized to
the nasal cavity or throat of the patients can propagate abnormally, sometimes leading tosuch infectious diseases as sepsis, respiratory infections, or MRSA enteritis. In thesepatients, bacterial testing is conducted using specimens of blood, sputum, or stools.
Otherwise, to identify the carrier state, the presence of nosocomial bacteria, including
MRSA and Pseudomonas aeruginosa, should be regularly checked. MRSA can bedifferentiated by confirming the presence of the mecA gene by PCR.
5.1.3 Fungus Infection
5.1.3.1 Pathogenic Fungi
Among fungal diseases, molecular methods are most often used for the diagnosis ofdeep-seated mycoses, for which ordinary isolation and culture of the causal fungi is difficult.
Most of the deep mycoses observed in Japan, including candidiasis, cryptococcosis,
aspergillosis, and zygomycosis, are caused by microbial flora in humans and animals or
saprophytes in the environment, which are of low pathogenicity. Therefore, blood, spinal
fluid, or biopsied tissues are used as specimens for the detection of these microbes. These
specimens are normally germ-free, and usually yield negative results in healthy individuals.
If nucleic acids of a certain fungus are detected in these specimens, the subject would be
diagnosed as having an overt infection due to the fungus. Most appropriate specimens vary
according to the type of disease, for example blood for fungemia, spinal fluid for fungal
meningitis, sputum for pulmonary infections, and biopsied liver tissue section for hepatic
abscesses. Various patterns of these deep mycoses and of superficial mycoses and deep
dermatomycoses can be diagnosed by the use of appropriate specimens, including tissue
sections and scrapes of a local lesion and purulent materials.
	
 

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5.1.3.2 Pneumocystis jiroveci
Pneumocystis pneumonia is a type of pneumonia caused by the yeast-like fungus
Pneumocystis jiroveci. In the past, it was called “carinii pneumonia,” since it was regarded
as caused by Pneumocystis carinii, which used to be classified in the protozoan category. It
is rarely observed in people with normal immune functions, and is an opportunistic
infection that occurs in immunocompromised people due to acquired immunodeficiency
syndrome (AIDS), chemotherapy, long-term internal steroid use, and other causes.
Specimens used to detect this fungus include biopsied lung tissues, BALF, and sputum.
Many people have non-apparent infections with this fungus. Therefore, even if this fungus
is detected by highly-sensitive PCR testing, it does not necessarily mean the person has
pneumonia. The results must be interpreted carefully. This fungus strongly adheres to thealveolar epithelium with its pseudopodia, and does not easily come out in the sputum. This
makes it difficult to detect the fungus by the conventional staining method. In such cases,
nucleic acid testing provides useful and supplementary information.
5.2 Collection of Specimens in Molecular Methods for Somatic Cells
5.2.1 Solid Tissue Tumors
5.2.1.1 Pancreatic carcinoma
It is known that nearly 90% of pancreatic cancers have a K-ras mutation, most of which arepoint mutations at codons 12, 13, and 61, especially concentrated around codon 12.
Therefore, this method has long been used as a test to complement imaging diagnostics andpancreatic fluid cytology, which have been used for the diagnosis of pancreatic cancer.
However, because the highly sensitive detection system also detects similar mutations in
non-tumorous lesions, including epithelial hyperplasia of the pancreatic duct, chronicpancreatitis, and pancreatic cysts, such molecular methods cannot be necessarily said tohave high clinical significance. Therefore, for clinical diagnosis the results of this testing
must be interpreted carefully, and clinical information and the results of other tests mustalso be considered. Pancreatic fluid collected by endoscopic retrograde
cholangio-pancreatography (ERCP) is usually used for the K-ras mutation analyses. In
contrast, surgically removed pancreatic cancer tissues are rarely used. In addition topancreatic fluid, other specimens that are used for the testing are bile, lavage fluid from theopening of the duodenal papilla, pancreatic duct brush cytology specimens, and fine-needle
biopsied pancreatic cancer tissue.
5.2.1.2 Lung Carcinomas
The EGFR gene and K-ras gene mutation analyses for lung cancer have been used as an
item for gene-based testing to identify malignant tumors. Currently, EGFR mutation
analysis for lung cancer is widely utilized to identify the best mode of chemotherapy, toselect a treatment method, and as an indicator for drug efficacy assessment. Approximately
40% of pulmonary adenocarcinomas have mutations in the EGFR gene. The mutations are
	
 

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JCCLS MM5-A1(2011.12.1)
concentrated around exons 18, 19, 20, and 21, which encode the tyrosine kinase active siteof the EGFR protein. Mutations in exons 18, 20, and 21 are primarily point mutations, andthe mutation in exon 19 is mainly a deletion. Regarding the relationship between mutations
and such molecular-targeted therapeutic agents as Gefitinib and Erlotinib, mutations inexons 18, 19, and 21 are related to chemotherapeutic sensitivity, while mutations in exon 20
are related to chemotherapeutic resistance.
Meanwhile, K-ras mutations in lung cancers primarily show point mutations in codons 12
and 13, which differ from EGFR mutations. However, it is known that drug efficacy isgreatly reduced when these mutations are detected. In lung cancers, molecular-targeted
therapies are applied for unresectable or recurrent cancers. Thus, surgical specimens
collected at the time of resection of the primary lesion or biopsied specimens are primarily
used as the specimens for the molecular methods. However, when surgical collection of
specimens is difficult, pleural effusions, BALF, pericardiac fluid, exfoliated cells forcytology, or formalin-fixed paraffin-embedded tissue can be used instead.
5.2.1.3 Colon Carcinomas
For many years, the analyses of point mutations and loss of heterozygosity (LOH) in p53
and K-ras genes in colorectal cancer have been conducted using various techniques incolorectal carcinogenesis models, in the evaluation of risk of hepatic metastasis, or as anindex for evaluating malignancies. Freshly resected cancer tissues are the most common
specimens for this analysis, but in most cases, the purpose of the analysis is investigationalrather than diagnostic. Furthermore, investigational attempts to detect these mutations asbiomarkers in colorectal cancer cells in patient stool samples have been conducted.
However, this method cannot compete with the fecal occult blood test in terms of costeffectiveness at present.
However, in addition to the fact that molecular-targeted therapy has come to be used forcolorectal cancer, it has been reported that the clinical effects of such molecular-targeted
therapeutics as Cetuximab and Panitumumab differ significantly, depending on thepresence or absence of K-ras mutations.
Surgically-resected cancer tissues and endoscopically-resected biopsy tissues have beenused as alternative specimens for mutational testing. However, in the future, formalin-fixedparaffin-embedded tissue is expected to be the only specimen for these analyses in manycases. Therefore, there are important challenges for the future, including the improvement
in analytical methods, more careful interpretation of test results, and the development andestablishment of new technologies.
5.2.1.4 Gastrointestinal Stromal Tumors (GIST)
C-kit mutation analysis is a gene-based testing method related to gastrointestinal stromal
tumor (GIST). Since the publication of a report entitled “c-kit mutation in GIST,” this test
has been widely utilized for making judgments on the application of therapeutic agents, forselecting treatment methods, and as an indicator for drug efficacy, as with “EGFR
	
 

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JCCLS MM5-A1(2011.12.1)
mutations in lung cancers.” Sixty to eighty percent of GIST cases have a c-kit mutation,
most of which (80-90%) lie in exon 11 in various mutational forms, including deletions,point mutations, and insertions. Multiple insertions in exon 9 and point mutations in exons
13 and 17 have also been observed.
Meanwhile, PDGFRα mutations are detected in approximately half of the GIST cases
lacking a c-kit mutation, and various gene alterations such as deletions, point mutations,insertions in exons 12 and 18, and point mutations in exon 14 have been found. GIST caseswith either of these mutations show good response to some molecular-targeted therapeutics,
including Imatinib and Sunitinib. In particular, Imatinib is known to show extremely highclinical efficacy in GIST cases with c-kit mutations in exon 11, and it is suggested that theefficacies differ according to the site and type of genetic alterations. It is also known thatthe clinical efficacy of this drug is best in GIST cases with c-kit mutations in exons otherthan exon 11, and the PDGFRα mutation is not as common as that in GIST cases with c-kit
mutation in exon 11.
As unresectable or recurrent cases are the target of chemotherapy, specimens used formutation analysis are primarily comprised of small amounts of biopsied tissues orformalin-fixed paraffin-embedded tissues. Because the site and type of genetic alterations
vary widely, as mentioned above, improved analytical methods/techniques should be
devised, and great care should be taken when the specimens are collected, because theanalysis is difficult without a sufficient amount of high quality DNA.
5.2.1.5 Breast and Stomach carcinoma
Tests for HER2 breast or stomach carcinoma include immunohistochemistry (IHC) whichmeasures the production of the HER2 protein by the tumor, and fluorescence or bright fieldin situ hybridization (BRISH) which uses fluorescence or dinitrophenyl (DNP)-labeledprobes, respectively, to look at the number of HER2 gene copies in a tumor cell. A result of
HER 2 positive is important, as it indicates that the cancer can be treated with trastuzumab,
in combination with other chemotherapy drugs. In metastatic cases with resistance totrastuzumab and HER2 positive, lapatinib can be used.
In preparation for formalin-fixed paraffin-embedded tissues, the recommended fixing
solution is 10% neutral buffered formalin, and the standard fixing time at ambient
temperature for surgically obtained specimens is between 6 and 48 hours (depending on thesize in cases of biopsied tissue). Tissue slices in 6 weeks after cutting can be used.
5.2.2 Hematological Neoplasms
5.2.2.1 Leukemias
Highly sensitive detection using real-time RT-PCR of chimeric mRNA formed bychromosomal translocation is frequently used for routine testing of hematopoietic organ
tumors, with BCR/ABL chimeric mRNA testing as a typical example. In these diseases,
because tumorigenesis occurs at the level of hematopoietic stem cells, bone marrow fluid
would be an ideal specimen to ensure the detection of MRD following treatment. However,
	
 

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JCCLS MM5-A1(2011.12.1)
considering the burden on the subjects, a highly invasive bone marrow puncture every time
the test is conducted should be avoided, and detection of MRD using peripheral blood as
the test specimen is more desirable and less invasive for the patient. In BCR/ABL chimericmRNA testing, almost the same level of results are obtained for CML from both bonemarrow and peripheral blood specimens, but it must be noted that peripheral blood
specimens are not suitable for acute lymphoblastic leukemia (ALL) following treatment.
5.2.2.2 Malignant Lymphomas
Detection of immune-related gene rearrangements by PCR or Southern blotting to diagnosemalignant lymphomas, acute lymphoblastic leukemias, and chronic lymphocytic leukemias,has been used.
The primary treatment for malignant lymphoma until recently was surgical resection or
chemotherapeutic reduction of tumor-forming lesions of lymph node and extra-nodal
tissues. Currently, in the cases of follicular lymphoma with BCL2/IGH translocation, theeffect of the molecular-targeted antibody Rituximab is a promising adjunct to the standard
anti-cancer chemotherapeutic regimen (CHOP chemotherapy). In particular, lymphoma
cells of this type characteristically flow into the peripheral blood stream. Thus, peripheral
blood can be used as the specimen for the detection of MRD in the efficacy evaluation.
5.3 Collection of Specimens in Molecular Methods for Germ Line Cells
5.3.1 Genetic Testing
In a broad sense, molecular methods for germ line cells are defined as “the tests to detectchanges in the germline DNA sequence or in the hereditable genome sequence, and it
includes genetic testing for definitive diagnosis, diagnosis of pathogenic conditions,pre-clinical diagnosis, carrier diagnosis for single gene disorders, a test to determine anindividual’s drug response, a test to determine an individual’s susceptibility to diseases(genetic predisposition or diathesis), and a test to determine common parentage in order to
identify individuals.”
Among these genetic tests, pre-clinical diagnosis and carrier diagnosis may be implemented
for asymptomatic individuals, and the results can be used to predict the individuals’ future
health conditions and have implications for the subjects and for their family members.
Therefore, when genetic tests are conducted, personal genetic information must be carefully
handled with the ethical, legal, and social challenges in mind. In addition, the genotypic
determination test results are permanently maintained as a part of the patient’s medical
records, usually without confirmatory retesting.	
  Thus, it is important to provide theappropriate levels of various supports to the subjects and their families before conducting
the tests and after the test results are returned. When various kinds of information areprovided as the core of the support, cooperation with experts such as medical geneticists orcertified genetic counselors will help meet the needs of the subjects, and these professionals
can help the patients in an informed manner.
As previously mentioned, for the implementation of genetic tests the specimens should be
	
 

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JCCLS MM5-A1(2011.12.1)
collected with due consideration of the characteristics of the test. In addition, various
analytical techniques are used in genetic testing, including genomic and gene analysis(nucleic acids; DNA/RNA), various chromosomal analytical technologies (e.g., the CGH
array method, the FISH method, and the SKY method), and genetic biochemical methods.
Other than DNA extracted from blood (white blood cells), other specimens are used forgenetic testing, including skin fibroblasts, amniotic fluid cells, villous cells, umbilical cord
blood, B lymphoblast-like cell line, oral mucosa, and oral fluid. The choice of the specimen
depends on the purpose of the test. In the field of genetic biochemistry, serum is also used
as a specimen.
This manual has covered the genetic tests that use nucleic acids (DNA/RNA), and it focuseson tests “to clarify the genetic information in the genome and mitochondria that theindividual inherently possesses and will essentially not change for lifetime (information thatcan be unraveled by germline genetic analysis), including genetic testing related to single
gene diseases, multifactorial disorder, efficacy / adverse effect / metabolism of drugs, andpersonal identification.”
5.3.2 Single Gene Disorders
Hereditary disease testing is a typical genetic test for single gene diseases and inborn errors
in metabolism. When these hereditary disease tests are conducted, the scientific validity ofthe tests (analytical validity, clinical validity, and clinical utility) and the ethical, legal, andsocial issues associated with the testing must be upheld. Therefore, when specimens for thetests are collected, the subject’s human rights and concerns must be considered. The mainmaterials used as specimens for hereditary disease testing include peripheral blood (whiteblood cells), cultured cells, and oral mucosa.
(1) Hereditary disease testing is implemented when the patient is suspected to have eitherof the following genetic diseases.
(i) Duchenne muscular dystrophy(ii) Becker muscular dystrophy(iii) Fukuyama-type congenital muscular dystrophy(iv) Dystrophic epidermolysis bullosa(v) Familial amyloidosis(vi) Congenital long QT interval syndrome(vii) Spinal muscular atrophy(viii) Congenital cerebral white matter atrophy(ix) Mucopolysaccharidosis type I(x) Mucopolysaccharidosis type II(xi) Gaucher’s disease(xii) Fabry’s disease(xiii) Pompe’s disease(xiv) Huntington’s disease(xv) Spinal and bulbar muscular atrophy
	
 

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JCCLS MM5-A1(2011.12.1)(2) The genetic disease tests listed from (i) to (viii) of (1) are performed by the PCR
method, by DNA sequencing, the FISH method, or by Southern blotting. Those listedfrom (ix) to (xiii) are performed by enzyme activity measurements, by DNA
sequencing, or the culture method. That listed in (xiii) of (1) is performed by the PCR.
(3) These tests must be conducted in compliance with the requirements specified in“Guideline for the Appropriate Handling of Personal Information by Medical andNursing Care Related Operators,” issued by MWHL in December 2004, and“Guideline on the Genetic Testing,” issued by concerned societies in August 2003.
5.3.3 A pharmacogenomic test
Invader UGT1A1 molecular assay is a pharmacogenomic test that identifies patientswith polymorphism associated with an increased risk of neutropenia after use ofCamptosar (irinotecan). This was approved for marketing and medical care charge inthe health insurance scheme by MHLW in June and November 2006, respectively.

6. References
1.Recovery of microarray-quality RNA from frozen EDTA blood samples; Johanna M.
Beekman, Joachim Reischl, David Henderson, David Bauer, Rainer Ternes, Carol Pena,
Chetan Lathia, Jurgen F. Heubach, Journal of Pharmacological and Toxicological Methods
2009, 59: 44-49
2. Time course analysis of RNA stability in human placenta; Fajardy E. Moitrot, A.
Vambergue, M. Vandersippe-Millot, P. Deruelle, H. Rousseaux, BMC Molecular Biology
692009, 10: 29
3. Characterization of the effect of sample quality on high density oligonucleotide
microarray data using progressively degraded rat liver RNA; K. L. Thompson, P. S. Pine, B.
A. Rosenzweig, Y. Turpaz and J. Retief, BMC Biotechnology 2007, 7: 57
4. Systematic method for determining an ideal housekeeping gene for real-time PCR
analysis; V. P. Mane, M. A. Heuer, P. Hillyer, M. B. Navarro and R. L. Robin, J.
Biomolecular Techniques 2008, 19: 342-347
5. American Society of Clinical Oncology/College of American Pathologists.
Guideline recommendations for human epidermal growth factor receptor 2 testing in breast
cancer; Wolff AC et al., J Clin Oncol 2007, 25: 118-145
6. Factors in tissue handling and processing that impact RNA obtained from formalin-fixed,
paraffin-embedded tissue; Chung JY et al., J Histochem Cytochem 2008, 56: 1033-1042 23.
RNA extraction from ten year old formalin-fixed paraffin-embedded breast cancer samples:
a comparison of column purification and magnetic bead-based thecnologies;
Alfredo Ribeiro-Silva, Haiyu Zhangl and Stefanie S Jeffrey, BMC Molecular Biology
2007,8: 118
7. Analysis of DNA in fresh and fixed tissue by the polymerase chain reaction; Beverly B R
et al., American Journal of Pathology, Vol. 136, No. 3, March 1990

	
 

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7. Appendix A
Terms and Abbreviations Used in this Report	 1) SNPｓ (single nucleotide polymorphisms)
2) PGｘ (pharmacogenomics)
3) HAV (hepatitis A virus)
4) HBV (hepatitis B virus)
5) HCV (hepatitis C virus)
6) HDV (hepatitis D virus)
7) HEV (hepatitis E virus)
8) EBV (epstein‐barr virus)
9) CAEBV (chronic active EBV infection)10) CMV (cytomegalovirus)
11) HSV (herpes simplex virus)
12) HPV (human papilloma virus)
13) HTLV‐I (human T‐lymphotoropic virus typeI)14) HIV (human immunodeficiency virus)15) MRSA (methicillin‐resistant Staphylococcus aureus)16) ERCP (endoscopic retrograde cholangio‐pancreatography)17) LOH (loss of heterozygosity)18) TUNEL：TdT‐mediated dUTP‐biotin nick end‐labeling19) GAPDH：Glyceraldehyde 3-phosphate dehydrogenase20) DTT：dithiothreitol21) G (gravitational acceleration)22) EMR (endoscopic mucosal resection)23) MRD (minimal residual disease)24) BALF (broncho‐alveolar lavage fluid)25) CSF（cerebrospinal fluid）
26) DGI (disseminated gonorrheal infection)

8. Appendix B
The member Lists of the Technical Committee on Standardization for Gene-based
Testing and its Working Group (As of March, 2011)
Technical Committee on Standardization for Gene-based Testing
Chairperson : Kiyoaki Watanabe, MD, PhD, International University of Health and Welfare,Tokyo, Japan.	
  	
  	
  	
  	
  	
  	
  	
  	
 
Vice-chairpersons: Hayato Miyachi, MD, PhD, Tokai University School of Medicine,
Kanagawa, Japan.
	
  	
  	
  	
 
Masayoshi Tsutsumi, SRL Inc., Tokyo, Japan.

	
 

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JCCLS MM5-A1(2011.12.1)
Members: Naotaka Hamasaki, MD, PhD, Nagasaki International University, Nagasaki,
Japan.
Kunihiro Ueda, MD, PhD, Kobe Tokiwa University, Hyogo, Japan
Tadashi Kawai, MD, PhD, International Clinical Pathology Center, Tokyo,
Japan.
Kiyoshi Kitamura, MD, PhD, The University of Tokyo, Tokyo, Japan.
Shinzi Kosugi, MD, PhD, Kyoto University, Kyoto, Japan.
Yoshiaki Tazawa, Roche, Diagnostics K.K., Tokyo, Japan.
Koichi Chiba, PhD, National Institute of Advanced Industrial Science andTechnology, Ibaraki, Japan.
Humio Nomura, MD, PhD, Chiba University, Chiba, Japan.
Yoshimitsu Fukushima, MD, PhD, Shinshu University School of Medicine,
Nagano, Japan.
Masato Maekawa, MD, PhD, Hamamatsu University School of Medicine,
Shizuoka, Japan.
Tadao Funato, MD, PhD, Tohoku Fukushi University, Miyagi, Japan.
Tomoshige Hori, PhD, Japan Bioindustry Association, Tokyo, Japan.
Koji Nakayama. PhD, Mitsubishi Chemical Medience, Tokyo, Japan
Hiromitsu Yokota, PhD, The University of Tokyo, Tokyo, Japan.
Hiroshi Yoshikura, MD, PhD, Ministry of Health Labor and Welfare, Tokyo,
Japan.
Mamoru Kubota, SYSMEX Corporation, Hyogo, Japan..

Working Group
Chairperson: Hayato Miyachi, MD, PhD, Tokai University School of Medicine, Kanagawa,Japan.
Vice-chairperson: Masayoshi Tsutsumi, SRL,Inc., Tokyo, Japan.
Members: Kiyohumi Kamiyama, SRL Inc., Tokyo, Japan.
Tadao Funato, MD, PhD, Tohoku Fukushi University, Miyagi, Japan.
Hiromitsu Yokota, PhD, The University of Tokyo, Tokyo, Japan.
Kazumi Ssawagami, Precision System Science Co., Ltd., Chiba, Japan.
Koji Nakayama, PhD, Mitsubishi Chemical Medience, Tokyo, Japan.
Toshimitsu Annaka, Eiken Chemical Co., Ltd., Tochigi, Japan.
	
  	
  	
  	
  Yutaka Hatanaka, PhD, Hokkaido University Hospital, Hokkaido, Japan.
Yasuhiro Izumisawa, Abbott Japan Co., Ltd., Chiba, Japan.
Hiroshi Ueyama, Fuso Pharmaceutical Industries, Ltd., Osaka, Japan.
Kiyonori Katsuragi, PhD, Otsuka Pharmaceutical Co., Ltd., Tokushima, Japan.
Katsuya Togawa, Sekisui medical Co., Ltd., Tokyo, Japan.
Naoyuki Nishimura, PhD, Shimadzu Corporation, Kyto, Japan.
Kumiko Nozima, Sakura Finetek Japan Co., Ltd., Tokyo, Japan.
	
 

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Mitsuharu Hirai, Arkray Inc., Kyoto, Japan.
Kunihiko Hayashi, Roche Diagnostics KK, Tokyo, Japan.
Satoko Nakajo, SRL Inc., Tokyo, Japan.
Makoto Nagano, BML Inc., Saitama, Japan.
Yasushi Hasui, SYSMEX Corporation, Hyogo, Japan.
Secretariat: Kazuo Fujihashi, Japanese Committee for Clinical Laboratory Standards,
Tokyo,Japan	 61