Fresh Paraformaldehyde Preserves Thrombus Biochemistry - Infrared Spectroscopy and Secondary Ion Mass Spectrometry Investigation of Pulmonary Artery Thrombi
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Background
Biochemical analyses of pulmonary embolism derived thrombi depend critically on fixative quality.
Objective
To quantify the impact of paraformaldehyde (PFA) shelf life on thrombus molecular integrity using attenuated total reflectance Fourier transform infrared (ATR–FTIR) spectroscopy and time-of-flight secondary ion mass spectroscopy (SIMS) combined with principal component analysis.
Methods
Ten pulmonary artery thrombi were fixed in either aged PFA (6 months; n = 6) or freshly prepared 4% PFA (n = 4); an in-vitro plasma clot control received the same fresh PFA. Triplicate ATR–FTIR spectra (400–4000 cm −1 ) were collected and vector-normalized to the Amide I band. ToF-SIMS measurements were performed using a 30 keV Bi₃⁺ primary ion beam Fixative chemistry was assessed by the carbonyl absorbance at 1700 cm −1 .
Results
Freshly fixed thrombi displayed narrow Amide I (~1650 cm −1 ) and Amide II (~1540 cm −1 ) bands with a higher signal-to-noise ratio (SNR; median 41.8) than aged-PFA samples (18.2; P < .01). Carbonyl absorbance at 1700 cm −1 was markedly higher in fresh PFA. Aged PFA introduces chemical variances of biological samples in low-mass molecule fragments.
PCA showed clear separation of fresh versus aged specimens and alignment of fresh thrombi with plasma controls across spectral windows.
Conclusions
PFA solutions older than three months markedly deteriorate thrombus biochemical fidelity. ATR–FTIR and ToF-SIMS offer a rapid quality-control assay prior to molecular analyses.
Key points
PFA stored ≥3 months loses >70% of reactive aldehydes, yielding broad Amide envelopes, attenuated lipid bands and a two fold SNR drop.
Aged PFA introduces chemical variances of biological samples in low-mass molecule fragments.
PCA of fingerprint, protein or lipid windows cleanly separates aged from fresh fixations (PC1 up to 80% variance).
A rapid QC workflow (carbonyl absorbance ≥0.25 a.u. plus PCA verification) safeguards molecular integrity irrespective of patient age, DVT history or clinical severity.
Adopting this workflow will harmonise multi centre clot biorepositories and enhance the reproducibility of proteomic, lipidomic and imaging studies.
