Chapter 3.6. Cold chain free diagnostics


Lim Boon Huat, Wong Weng Kin, Foo Phiaw Chong, Armando Acosta

Art work
In a famine-fields
Uli Reinhardt
Developing customized diagnostic methods for specific populations, is an imperative. 
In a malnourished population, tests based on the immune response should be avoided, 
in contrast, very simple antigen-detection tests should be prioritized.
There is a deaf world, 
There is a rift by which the dead cross the border.
Tomas Tranströmer 
Winter solstice


Bio-industries have been utilizing cold-chain technology for transporting diagnostic kits to either prolong the shelf-life or to ensure the performance of the kits. Interestingly, the development of the cold chain free technology in diagnostics is beginning to gain momentum. The need to produce diagnostic kits that can be stored and transport without cold preservation facilities, yet meet the required test performance is especially pertinent in low-resource settings, where basic facilities are non-existence or merely rudimentary.

Cold chain technology can assist in delivering diagnostic tests to remote areas but transportation cost is usually a deterrent.  As such cold-chain free diagnostics can reduce the affordable factor of the ASSURED model.  Basically, development of cold-chain free diagnostics depends on three major technologies, namely thermostabilisation, freeze-drying and packaging.

•    Thermostabilisation

This technology focuses on stabilizing the heat-labile substances that are easily deteriorated caused by decrease in physical functionality at alleviated temperature. This technology is important in vaccine and antibiotic deliveries to aid and/or compensate the flawed cold chain transportation in rural areas. The stabilizers used for preserving these compounds include silk-film, sugars, protein and salts complexes [8, 16]. In diagnostic kits, the stabilizer is used to preserve compounds such as chromogenic substrate, enzyme, antibody, and oligo-nucleotides. However, the thermostabilised heat-labile ingredient has its shelf-life. Normally, shelf-life estimation is determined by using accelerated stability test. Nevertheless, estimating the actual period where a kit can be kept at room temperature is still pertinent [7].

•    Freeze-drying

This is a dehydration technology that removes water from biological ingredient through sublimation. In diagnostics, a few compounds are usually mixed together in liquid form to make a ready-to-use mix. The mixture can then be freeze-dried to reduce the water content at frozen state through sublimation. Technically, the compounds are well preserved in low temperature. The humidity of a freeze-dried ingredient is almost zero and such condition slows down most of the chemical reactions of the material, thereof preserved the original properties of the material. Besides, this technology also reduces the weight of the kit and makes it lighter and easier for transportation. [2, 3].

•    Packaging

Tight packaging with additional desiccant like silica gel is commonly used to protect diagnostics from light and moisture as some of the ingredients are light- sensitive and unstable in high moisture content. Other packaging technologies that could be applied to increase shelf-life of diagnostic kit include vacuum and inert air flushing technology to remove oxygen.

In addition to the high sensitivity and specificity concerns of a diagnostic kit, the design of the kit must be made accessible to end-users, especially the bottom billions of the world population where life-threatening tropical diseases are rampant. The World Health Organization recommends that diagnostic devices for the poor should be ASSURED: Affordable, Sensitive, Specific, User-friendly, Rapid and robust, Equipment-free, and Deliverable to end-users [13]. Immunochromatography strips that meet the ASSURED criteria are available commercially. A fine example is Brugia Rapid, a rapid serology dipstick test for detection of brugian filariasis [14]. However, nucleic acid amplification techniques still remain elusive to the bottom billions. The techniques are no doubt highly sensitive and specific but are often too expensive, not user-friendly, time-consuming, equipment dependant and rarely deliverable to remote areas with limited resources. In spite of these challenges, numerous innovations to the existing nucleic acid amplification techniques have been reported to be useful in meeting some of the ASSURED criteria [4, 6, 11, 12].

Cold-Chain Free PCR Diagnostics

Rapid diagnosis and timely treatment of patients will ensure patient survival [1]. Similarly, early detection of the causative pathogen is the first important step in controlling a potential outbreak, and the most sensitive detection technique available is the polymerase chain reaction (PCR) assay or its derivatives. Generally, cold-chain technology is used to create low enough temperature to protect the integrity of heat-sensitive reagents required for PCR diagnostics. However, a cold-chain free technology that can protect the integrity of heat-labile reagents, which are ready-to-use would be more practical for use in remote settings.

PCR, a revolutionary method developed by Kary Mullis in the 1980s has become one of the most useful molecular techniques that give precise diagnosis within a relatively short turnaround time. However, hindrances to PCR assay in low-resource settings include the need for cold chain transportation, cold storage, and skilled personnel in performing the test [4]. In most PCR protocol, the reagents used are stored at -20°C and multiple pipetting steps are required prior to the PCR run. Moreover, cold-chain transportation and/or storage dependent PCR materials incur extra cost to the users. Hence, a thermostabilised ready-to-use PCR premix is deemed possible to overcome these setbacks.

A dried-based reagent for PCR assay is technically cold-chain free. By merely adding DNA samples and PCR-grade (DNase-free) water to the dried reagent prior to a PCR run is an upright innovation. Chua et al [4] and Foo et al [5] reported the usefulness of such cold-chain free PCR diagnostic assay by using sugar as the preservative in the dried PCR mix. The integrity of the reagent was reported to be protected for a period of approximately 6 months at room temperature. The process of converting a cold chain dependent enzyme into a dried form is known as thermostabilisation. This process uses sugar to protect the active sites of the enzyme and maintain its protein structure, whereas glycerol acts as a preservative agent.

Although sugars have been commonly used as additives during thermostabilisation of enzyme, not all sugars provide the required properties to assist stability of proteins during dehydration and long term storage without cold-chain [10]. Trehalose, has been proven to have the characteristics of remaining amorphous during dehydration, where it formed hydrogen bonds with dried protein, and preserve native protein structure during and after the drying process [4]. Trehalose was reported to be the best stabiliser candidate that could maintain the structure and function of the proteins [9, 15]

The PCR mix was first thermostabilised to examine the difference in the amplicon bands intensities in agarose gel before and after dehydration. Optimization of the thermostabilisation was continued when the pre-dehydration and post dehydration produced same intensity of amplicon bands in agarose gel after PCR.

Thermostabilisation was conducted in a vacuum-dried condition with the presence of an enzyme stabilizer covering the DNA polymerase enzyme. The addition of enzyme stabilizer is to ensure that the activity of the DNA polymerase enzyme after total dehydration was maintained in a PCR reaction. The dehydration process involved preparation of the PCR mix with deglycerolized DNA polymerase enzyme together with the enzyme stabilizer, followed by vacuum-dried into a dry pellet. The dry pellet in the PCR tube was then stored in the presence of silica gel packs.

During the thermostabilisation of the DNA polymerase enzyme, the concentration of trehalose was optimized. The amount of enzyme stabiliser to be used was optimized together with three different DNA polymerase amounts to determine an optimal balance of both reagents that could still maintain the enzymatic activity after post-dehydration process. The amount of DNA polymerase was elevated intentionally to replace some loss of enzymatic activity which was expected during and after the vacuum-drying process. The amounts of enzyme stabiliser investigated were 3%, 6%, 9% and 12% w/v final concentration of stabiliser per reaction. The amounts of DNA polymerase investigated were based on 150% (1.125 U), 200% (1.50 U) and 250% (1.875 U) increments. A best combination of concentration of enzyme stabilizer and amount of DNA polymerase is vital to ensure the best protection and stability to the thermostabilised multiplex PCR mix.

Cold-chain free PCR diagnostic is at most as specific as its corresponding assay. However, the advantages it offers could ensure a wider appeal to users in low-resource settings by allowing them to avoid cold storage and also simplify the preparation process prior to PCR run.


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