Using the 'Shake Test' to Determine Vaccine Efficacy (Part 2)

April 24-30, 2016 is the World Health Organization’s (WHO) World Immunization Week 2016. According to the WHO Website, World Immunization Week – celebrated in the last week of April – aims to promote the use of vaccines to protect people of all ages against disease.

This will be second year of the WHO’s Close the Immunization Gap campaign and unlike 2015, 2016 will focus not only on children but adolescents and adults in their goal to reach vulnerable people across the globe. Part of a multiyear plan, Close the Immunization Gap will continue to educate people that immunization saves millions of lives and is widely recognized as one of the world’s most successful and cost-effective health interventions.

As discussed in the previous article, countries across the globe have been making free vaccinations available to mothers and children to help insure the health of future generations. And as is seen in studies about cold chain compliance for vaccines, both developed and developing countries have challenges ensuring vaccines administered to the recipients have not been damaged by freezing or overheating.

Some vaccines must be kept frozen until administered, stored and transported at -15°C or lower. A refrigerated vaccine must be stored between 2°C and 8°C. Refrigerated vaccines that experience temperatures above 8°C for an extended period of time may be damaged and rendered ineffective. A refrigerated vaccine that freezes is also likely to be damaged and rendered ineffective. As the WHO Study states, "when a vaccine is damaged by freezing, the potency lost can never be restored – the damage is permanent." One way to ensure a vaccine has been stored or transported properly is to use a thermometer and record the temperature at regular intervals. This can be done manually or with an automatic temperature monitoring or recording device.

How can medical professionals determine if a refrigerated vaccine they are about to administer has been kept at recommended temperatures? One way is to check cold chain temperature logs where available but unfortunately that is not often the case. Refrigerated vaccines that have been subjected to elevated temperatures may appear the same as those that were not. For refrigerated vaccines that have been frozen, there is a simple test, the Shake Test, that can help identify suspect vaccines. In a previous piece we described the history and development of the Shake Test.

The WHO Study describes the test: 

The shake test is designed to determine whether adsorbed vaccines have been affected by freezing. After freezing, the lattice (made up of bonds between the adsorbent and the antigen) in a vaccine is broken. Separated adsorbent tends to form larger, heavier granules that gradually settle at the bottom of the vial when this is shaken. When freezing and thawing cycles are repeated, the granules appear to increase in size and weight. In a typical demonstration of the shake test, two identical vials of a vaccine (i.e. from the same batch and the same manufacturer) that is suspected of having been exposed to freezing temperatures are selected; one of the two vials is purposely frozen and then thawed as the negative control, while the second vial serves as the vial to be “tested” against this negative control. The two vials are held together in one hand and shaken; they are then placed side by side on a flat surface. Provided the test vial has not been frozen, sedimentation is slower in the test vial than in the control vial that has been frozen and thawed. If the test vial has been frozen, the test and control vials will have similar sedimentation rates.  Figure 1 illustrates how the appearance of frozen (i.e. frozen and thawed, and therefore freeze-damaged) and non-frozen test vials compares to that of their frozen control vial, 1 minute and 28 seconds after shaking.

Figure 1. Visual difference in sedimentation rates after shake test for detecting freeze damage to adsorbed vaccines. Source: WHO

The WHO study’s purpose was to compare the Shake Test to the industry gold standard. The most appropriate gold standard for the shake test (apart from testing the vaccine in humans, which is impractical) is visual observation under a phase contrast microscope. Phase contrast microscopy is a confirmed method of identifying freeze damage in vaccines. Damaged vaccines contain large conglomerates (massed precipitates with amorphous, crystalline, solid and needle-like structures), whereas vaccines maintained within the optimal temperature range (2°C to 8 °C) show a fine-grain structure under phase contrast microscopy. In short, frozen vaccines clump together, vaccines that were not frozen don’t.

Figure 2. Phase contrast microscopy findings using study vials of hepatitis B vaccine kept at different temperatures shows clumping in partially and fully frozen vaccines. 

Source: WHO

Figure 3. Tabulated results from hepatitis B vaccine study compares partially and fully frozen vials using phase contrast microscopy vs. shake test.  

Source: WHO

The study showed, the Shake Test had 100% sensitivity, 100% specificity and 100% positive predictive value in this study, which confirms its validity for detecting freeze damage to aluminum-based, freeze-sensitive vaccines.

A video detailing the Shake Test is available at https://vimeo.com/8389435 and may help clarify the test procedure and interpretation. As noted in the video, patience is a key element to a proper determination.

Vaccine cold chain controls are critical to insure recipients receive safe and effective vaccines. Temperature monitoring devices are that record and provide cold chain data logs can help determine whether a vaccine has been frozen and rendered ineffective. Monitoring devices that provide real time alerts can even save vaccines from damage if corrective action is taken. In all cases medical professionals have a low cost, effective tool to test vaccine effectiveness: the Shake Test. The test requires no special equipment and can be performed anywhere. Performing the shake test on incoming vaccines as well as those that have been stored for extended periods provides medical professionals with the assurance they need to perform their duties with confidence.