Biospecimen Collection, Processing, Storage, and Information Management

Introduction To Biospecimen Management

Biospecimens have been collected for various uses such as clinical trials, molecular epidemiology, and other research. It is important for specimen management to occur in a controlled environment. An environment where there are strict policies and guidelines in place that help ensure the quality and integrity of the specimen and data. With proper procedures, biorepositories are able to produce high quality biospecimens that are needed for research. Biospecimens are collected from donors for patient monitoring, care, and research studies. They have helped many medical advances such as those for cancer, heart disease, and AIDs. Due to the increased sensitivity and specificity of analytic techniques over the years, it is crucial that biospecimens are of the highest quality.  

Biospecimen Collection

There are various types of specimens that are required based on different research goals. Some examples include:

• Whole blood and blood fractions (red blood cells, serum, plasma, buffy coat)

• Tissue obtained from transplants, surgery, or autopsy

• Urine

• Buccal cells and saliva

• Bone marrow

• Placental tissue, cord blood, or meconium

• Feces

• Hair

• Semen

• Nail clippings

• Etcetera

Specimens should be collected, processed, and stored according to guidelines that take into account future analyses. The collection procedures will differ for different biospecimens and intended analyses. However, all procedures should be accurately documented.

a)       Blood Collection

Blood sample collection should be performed by a trained phlebotomist to reduce donor discomfort and to avoid compromising the quality of the sample. These standard protocols should be followed.

• Glass or plastic tubes with appropriate additives should be used

• Blood should be drawn in an orderly manner to avoid cross-contamination of additives.

• Serum should be separated from other components as soon as possible to reduce contamination. This is important as serum can be used for the improved analyses of nutrients, lipids, antibodies, and lipoproteins.

• Follow guidelines and recommendations for time elapsed between blood collection or removal from the storage unit and temperature for processing of blood specimens depending on the intended analyses.

• Avoid thaw and refreeze cycles.

• Since RNA and proteins are vulnerable to enzymatic degradation, follow necessary protocols that help ensure their integrity during the collection and processing phase.

b)      Tissue Collection and Fixation

Most tissues are obtained through surgery, biopsy, or autopsy. Generally, it would be best if the procurement of the specimen is performed by a trained pathologist. The time between the collection and stabilization process should be minimal. This means that the best approach is to collect, stabilize, and process the specimens rapidly. Detailed records regarding the timing for excision, fixation, or freezing should be kept. For autopsy specimens, it is vital to know the interval between death, collection, and processing of the specimen as tissues degrade rapidly after death. Tissues can be fixed using formalin, alcohol, and paraffin embedding as it has a relatively low cost when freezing or when storage facilities are unavailable.

c)       Urine Collection

Urine collection can be performed in some study designs and to achieve certain analytical goals. Examples of this include:

• Urine collected upon waking up in the morning

• Random specimens used for drug monitoring or cytology studies

• Timed urine collections

• Etcetera

Urine specimens should be kept refrigerated or kept on ice with or without a preservative.

d)      Saliva or buccal cell collection

Saliva along with buccal cells are a great source of DNA for genetic studies. Samples are easily collected by asking donors for self-collection. Methods include using cytobrushes, swabs, and a mouthwash protocol.

Preservation of Biospecimen Stability

As previously mentioned, it is important to minimize the interval between collection and stabilization. The temperature of biospecimens should be reduced when freezing is the endpoint. Control of processing time is necessary if fixation is the stabilization endpoint. Biobanks should utilize the method that preserves the highest number of analytes.

Biospecimen Processing

Biospecimens should be processed using the methods that preserve the analytes of interest or following the study design. For blood specimens, the processing method used should be based on the laboratory analyses. Tissues can be processed in the pathology suite or operating room once the specimen is resected. Buccal cells can be processed via centrifugation. For DNA extraction, the gold standard method is phenol-chloroform extraction. However, other methods can be used.

Storage of Biospecimen

Based on the intended laboratory analyses and requirements, biospecimens can be stored in various conditions. Examples include mechanical freezers, liquid nitrogen tank, room temperature, among others. Backup and alarm systems are necessary in case of mechanical failure. Staff should be trained for maintenance and repair of equipment. The labels used for biospecimens should be capable of withstanding the storage conditions.

Information Management and Specimen Tracking

Information management involves the collation and analysis of data associated with biospecimens as it helps support research. Since there are vast amounts of data, extensible and flexible informatics systems will be required. Biospecimens are documented and tracked using various forms of data management tools such as notebooks, multi-user software, and various automated information systems.

References

Vaught JB, Henderson MK. Biological sample collection, processing, storage, and information management. IARC Sci. Publ. 2011; 163:23-42. Accessed 10/25/2018. https://publications.iarc.fr/_publications/media/download/1398/68b153f74693289ae66d767a8cbe1ca667df4f1b.pdf

Pre-analytical Variables Affecting the Integrity of Human Biospecimens

Introduction

Biorepositories or biobanks function to collect, process, transport, and store biospecimens. The integrity of these biospecimens is crucial for the success of clinical trials and research. There are many factors that can influence the results within research such as:

• Pre-analytical environmental or biological variables

• Pre-analytical technical variables

• Analytical variables

• Post-analytical variables

Pre-analytical variables are defined as factors that can have an impact before the start of the analytical phase. It not only affects the integrity of the tissue samples, but also the results of the analysis. Pre-analytical variables are critical as the analytical integrity of the research can be jeopardized. Seeing as most errors in the laboratory can be attributed to pre-analytical errors this stage is of upmost importance.

Pre-analytical Factors in the Collection of Biospecimens

It is important to adhere to guidelines for general laboratory safety. The collection of biospecimens require a balance of:

• Accrual rate

• Types of biospecimens

• Sample size

• Costs

• Location

• Storage requirements

• Transport logistics

The biospecimens collected can be either invasive or noninvasive. Biospecimens that are collected through non-invasive methods may lead to an increase in sample size due to easiness of collection, reduced costs, and willingness of donor to participate. This method is especially important when dealing with pediatric biobanking. It is important that biological and environmental factors are standardized and documented when interpreting results as it can affect the downstream analysis. It is also vital to take measurements to observe the effects of intervention and the changes over time.

Pre-Analytical Factors That Affect the Collection of Blood Samples and Its Derivatives

The collection of blood samples should be performed by trained staff. Those that are involved in collecting samples from children should specialize in pediatric phlebotomy. The staff needs to be highly trained as this ensures the highest quality of specimens and prevents the donors from experiencing any kind of discomfort. Depending on the research requirements different additives may be required. Different types of additives are coded using different colored collection tubes. Some of the important pre-analytical factors to take note of include:

1. Using the same tube brand and the same lot number throughout the study. This would be ideal as different brands can have different anticoagulants, additives, and may introduce bias.

2. Another important factor is the expiration dates on the tubes as the vacuum in these tubes can decrease with age and negatively impact the blood draw and filling of the tube.

3. Using the same posture such as supine, standing, or supine as these can cause plasma volume changes that may lead to increased analyte levels.

4. Using the recommended needle gauge as a needle that is too thin can lead to hemolysis that distorts the potassium concentrations and hematological cell counts.

5. Using the recommended and same duration of tourniquet use as prolonged use can cause changes in analyte concentrations and hemoconcentration.

6. Avoid inadequate filling as this can result in inaccurate results due to the decrease in blood and additive ratio.

A general rule for common analyses is to use ethylenediaminetetraacetic acid for hematology, DNA, hemoglobin A1C, and a range of proteins. For plasma glucose, it is recommended to use sodium-fluoride tubes while lithium heparin plasma can be used for assays such as kidney function, iron parameters, liver enzymes, thyroid hormones, C-reactive protein, and more.

In remote sites that are resource-poor, capillary dry blood spot (DBS) are easy biospecimens that can be collected. Small volumes of capillary blood from the peripheries can be deposited onto specific paper cards and dried at room temperature for three to four hours. DBS can be used in many analyses. However, some of the pre-analytical variables to note are:

·         Type of collection paper used

·         Type of chemical used in the manufacturing of the paper

·         Thickness of paper

·         The volume of blood deposited

·         Environmental factors such as heat, humidity, sunlight, and moisture

In DNA and RNA collection, there are also biological factors that can affect the biospecimens. These include the donor’s:

·         Gender

·         Age

·         Body mass index

·         Tobacco consumption

Since RNA is more vulnerable to degradation, some of the preanalytical collection factors that can affect the integrity are:

• Tube additive

• Tube type

• Tube sterility

• Type of biospecimen

• The volume of blood collected

• Short-term storage temperature

• Lag time until extraction

In microRNA’s, the pre-analytical variables include:

• Diet

• Age

• Race

• Exercise

• Drugs

• Altitude

• Tobacco use

• Chemicals

• Hemolysis

• Coagulation times

• Temperature

Pre-analytical Factors That Affect the Collection of Urine and Saliva

Urine can be collected in many different ways as it can be used for measurements of many analytes. In urine biospecimens, the preanalytical requirements can be conflicting.  This may result in the requirement of multiple biospecimens. Some of the preanalytical variables for urine collection include:

• Collection method

• Environmental exposure

• Urine dilution

• Dipstick components

• Preservatives or additives used

For saliva, these biospecimens have many advantages as they are easy to collect and can be used in many situations especially if donors are afraid of needles. The preanalytical variables for this biospecimen include:

• The time of collection

• The temperature the specimen is stored

• The collection method

Conclusion

The factors mentioned are pre-analytical variables that affect the biospecimens during the collection phase. However, it is important to note that there are many more pre-analytical variables that can affect the integrity of the biospecimens during the processing, transport, and storage phase.

Human Blood Samples in Biobanks

What are Blood Samples?

Blood samples are most commonly obtained from the antecubital fossa where the veins are closest to the surface. The blood sample can be taken by anyone from a doctor to a phlebotomist or a nurse. Most blood sample collections will occur at a clinic, hospital, or at a pathology collection center. A tourniquet is first wrapped around the upper arm to slow down the blood flow while the area where the insertion area for the needle is cleaned with an antiseptic cloth. The needle is inserted, and the blood sample is transferred into containers or tubes. Proper dressing of the wound is then administered to prevent infection and to keep it clean. These tubes are then labeled with a unique identification number along with other important information. These samples are then transported to their respective destinations, such as laboratories or biorepositories.

An Introduction to Biorepositories

Biobanks and biorepositories assist in providing the materials required in clinical trials and research. There is a growing number of biobanks that help to collect data and samples from the population as a response to the increased demand of these services. The services provided by various biobanks also mean that acquiring biological materials can be guaranteed. The existence of biobanks has allowed the accumulation of biological samples from various resources. 

Biospecimens in Biobanks

Some of the examples of human biospecimens available through biorepositories include both normal and diseased states such as:

• Purified DNA

• Hair tissue

• Nail

• Whole blood

• Plasma

• Serum

• Red blood cells

• Platelet concentrates

• Platelet-rich plasma

• Saliva

• Semen

• Breast milk

• Organ tissue

• Etcetera

All specimens should be collected and processed according to the proper guidelines and procedures. 

Functions of Biorepositories

While collecting biospecimens, biorepositories also collect demographic data such as medical history, lifestyle habits, medications, and family history to create an accurate scientific database. These samples are only labeled with a unique code for identification purposes. These specimens are then maintained in the proper environment and equipment to ensure the highest quality. Another crucial point in the management of any biobank is the privacy and rights of the donors. This means biobank managers need to train their staff regarding the policies and standard operating procedures (SOPs) of the biobank.

Whole Blood and Blood Cells 

In human biospecimens, the buffy coat and whole blood are essential for biorepositories. Whole blood refers to a sample that consists of red blood cells, white blood cells, platelets, and plasma. The buffy coat describes the white blood cells and platelets that form the anti-coagulated blood sample. Both these samples are essential as they are the main source for cellular nucleic acids, construction of a DNA biobank, and achieving the maximum quality and quantity of germline DNA. 

Storage for Human Blood Cells

Blood is one of the most common biospecimens collected in human biobanks as it is a source for DNA and RNA. This is why anti-coagulated blood is a prerequisite for plasma-derived cell-free circulating nucleic acid molecules and genomic or mitochondrial DNA and RNA. One of the most commonly used anticoagulants is ethylenediaminetetraacetic acid (EDTA) for various protein assays and DNA based studies. However, citrate is more appropriate for white blood cell cultures. Storage conditions and quality of biospecimens are of vital importance as it determines the yield of extracted DNA and RNA from buffy coat or whole blood samples. 

Since RNA is easily degradable, the World Health Organization – International Agency for Research on Cancer (WHO-IARC) has suggested that it be stored in nitrogen storage below -130⁰C. Samples stored at -140⁰C by liquid nitrogen have been observed to keep the RNA in a functional state and intact for more than 50 months. To maintain the biospecimen’s reliability and preventing the possibility of multiple freeze-thaw cycles, DNA protection and stabilization can be done at room temperature which eliminates the costs for freezer storage and lowering maintenance costs for biobanks. While purified DNA can be stored at -20⁰C for months, both purified DNA and RNA are much more stable at -80⁰C in nuclease-free water or aqueous buffers for long-term storage. 

For plasma, anticoagulants such as lithium-heparin and EDTA can be used. Storage of both serum and plasma at -80⁰C have shown that there is adequate stability in the different biomolecules. The cycle of freezing and thawing should be avoided as it leads to the degradation of nucleic acids and proteins. 

Conclusion

Due to the increasing scientific developments in the past few years, it has increased the need for biological material in clinical trials and research. Biorepositories play a crucial role in supporting the researcher’s access to samples that meet their scientific criteria. It is important for biobanks to play their role in the management of data, collection, processing, and storage of biospecimens.  

References:

Mohamadkhani A, Poustchi H. Repository of Human Blood Derivative Biospecimens in Biobank: Technical Implications. Middle East J Dig Dis 2015;7:61-8.