Microscopic Study of Injectable Steroids: Effects
of Postmixing Time on Particle Aggregation
Authors: Orduña-Valls Jorge M. 
Published: March 2021

Background: Epidural steroid injection (ESI) is a common practice for pain treatment since 1953. In
2014, the FDA issued a warning about ESI. Studies have focused on the effect of the particle size and
their ability to generate harmful aggregates. Although steroid aggregates provide longer times for
reabsorption, therefore a longer anti-inflammatory effect, they are potentially harmful to the central
nervous system via embolic mechanisms.
Previous studies have established that steroidal aggregates with asizes over 100 μm are potentially
able to occlude blood vessels. Studies by Tiso et al and Benzon et al addressed the role of steroids
on CNS adverse events, with similar outcomes. The main difference was on the role of aggregates
with a size over 100 μm, which Benzon et al. attributed to the ability of certain steroid preparations
to rapidly precipitate and form large aggregates.
Objectives: Studying the effect of the time elapsed between mixing the steroid preparation and
injection on the number and size of aggregates with sizes above 100 μm.
Study Design: Original study in basic science.
Setting: Basic science
Methods: Steroids evaluated are commonly used in Spain for ESI: betamethasone, triamcinolone,
and dexamethasone. The size and number of the aggregates was determined for undiluted
commercial steroid preparations in the usual amount for a single and double dosage used for ESI.
Samples were examined with a Leica TCS-SP2 microscope at the first, the fifth and the 30th minute after
shaking the preparations. Aggregates observed in the different preparations were manually counted and
grouped in the following size range: 0-20, 20-50, 50-100, 100-300, 300-500 and > 500 μm.
Statistical analysis was carried out using the R software. Nonparametric techniques were used in the
comparison of aggregate size. Global comparison of the groups using the Kruskal-Wallis test and post-hoc
comparisons using the Wilcoxon test, adjusting P-values by the Holm method for multiple comparisons
Results: Aggregates present in triamcinolone and betamethasone samples were statistically larger
than in dexamethasone samples. Triamcinolone suspensions produced significantly larger aggregates
than betamethasone five minutes after mixing. Triamcinolone preparations produced greater particle
aggregates (> 500 μm), which were not present in dexamethasone and betamethasone preparations.
Limitations: Study how the human internal factors like blood elements and spinal fluid could
interact with steroids and influence the size of the aggregates formed.
Conclusions: This study demonstrates that the size of the particles injected depends on the type
of steroid and the time allowed between mixing and injecting. The results demonstrate that waiting
longer than 5 minutes between mixing and injecting can predispose the formation of potentially
harmful aggregates in triamcinolone and betamethasone samples. The presence of greater particle
aggregates (> 500 μm) may occlude some important vessels and arteries with serious adverse results.
Vigorous shaking of the injectable could prevent such events.
Key words. Epidural steroid injection. Triamcinolone. Betamethasone. Dexamethasone. Steroid
aggregates.