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. 2024 Sep 24;25(19):10235.
doi: 10.3390/ijms251910235.

Utility of an Archival Dried Blood Spot (DBS) Collection from HIV-Infected Individuals with and without Cancer in a Resource-Limited Setting

Rongzhen Zhang  1   2 , Paige M Bracci  2   3 , Alan Leong  1   2 , Cassandra Rapp  1   2 , Michael S McGrath  1   2
Affiliations

Utility of an Archival Dried Blood Spot (DBS) Collection from HIV-Infected Individuals with and without Cancer in a Resource-Limited Setting

Rongzhen Zhang et al. Int J Mol Sci. .

Abstract

The frequency of virus-associated cancers is growing worldwide, especially in resource-limited settings. One of the biggest challenges in cancer research among people living with HIV (PLWH) has been understanding how infection with both HIV and Kaposi sarcoma-associated herpesvirus (KSHV) promotes the pathogenesis of Kaposi sarcoma (KS), the most common cancer among PLWH worldwide and a significant public health problem in regions with high prevalence of HIV such as Sub-Saharan Africa (SSA). The AIDS and Cancer Specimen Resource (ACSR) provides samples for research, including dried blood spots (DBS) that were collected from large clinical epidemiology studies of KSHV and KS in PLWH conducted more than a decade ago in SSA. Here, we validated the quality of DNA derived from DBS samples from SSA studies and provided evidence of quantitative recovery of inflammatory cytokines using these DBS samples through comparison with paired frozen plasma. Significant differences in DNA, protein yields, and inflammatory biomarker levels were also observed between PLWH with/without KS. Establishing the fitness of DBS samples for studies of KS pathogenesis extends the number of projects that can be supported by these ACSR special collections and provides evidence that DBS collection for future KS research is a practical option in resource-limited settings.

Keywords: AIDS and Cancer Specimen Resource (ACSR); DNA; HIV; Kaposi sarcoma (KS); Kaposi sarcoma-associated herpesvirus (KSHV); archival dried blood spot (DBS); inflammatory cytokines.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
DNA yield comparison between UARTO and ARKS cohorts. Box-and-whisker plots depict the DNA yields from DBS for UARTO and ARKS cohorts. Significantly higher DNA yields from people living with HIV (PLWH) with confirmed Kaposi sarcoma (KS) (ARKS, n = 12, in green) as compared to non-KS PLWH cohort (UARTO, n = 12, in blue) (unpaired t-test, p = 0.003).
Figure 2
Figure 2
(a) Gel image example of DNA from DBS from the Agilent 2100 Bioanalyzer. Lane L, ladder; Lanes 1, 10 and 11, DNA extraction controls isolated from empty/blank spots (ES Ctrl.); Line 2–9, DNA samples isolated from DBS. (b) Histogram example of empty/blank spot control samples (Line 1, ES Ctrl.). (c) Histogram example of DNA samples isolated from DBS (Line 2, DNA from DBS).
Figure 2
Figure 2
(a) Gel image example of DNA from DBS from the Agilent 2100 Bioanalyzer. Lane L, ladder; Lanes 1, 10 and 11, DNA extraction controls isolated from empty/blank spots (ES Ctrl.); Line 2–9, DNA samples isolated from DBS. (b) Histogram example of empty/blank spot control samples (Line 1, ES Ctrl.). (c) Histogram example of DNA samples isolated from DBS (Line 2, DNA from DBS).
Figure 3
Figure 3
Protein yield comparison between UARTO and ARKS cohorts. Box-and-whisker plots depict the protein concentrations from DBS for UARTO and ARKS cohorts. Significant higher protein concentrations were observed in people living with HIV (PLWH) with confirmed Kaposi sarcoma (KS) (ARKS, n = 12, in green) as compared to the non-KS PLWH cohort (UARTO, n = 12, in blue) (unpaired t-test, p = 0.01).
Figure 4
Figure 4
The direct relationship between DNA yield and protein concentration from the same DBS samples (correlation analysis, r = 0.492, p = 0.01, n = 24).
Figure 5
Figure 5
Examples of relationships between DBS extraction and paired plasma biomarker levels. A positive correlation was observed between log-transformed biomarker levels from DBS extraction supernatants and paired plasma samples (correlation analysis, n = 24); MIG (r = 0.417, p = 0.04), CRP (r = 0.910, p < 0.0001), RANTES (r = 0.552, p = 0.005), INF-g (r = 0.524, p = 0.009), IL-8 (r = 0.450, p = 0.03), IL-17 (r = 0.820, p < 0.0001).
Figure 6
Figure 6
High levels of biomarkers observed in DBS extraction supernatants. Box-and-whisker plots depict log-transformed biomarker levels for DBS extraction supernatants (n = 24, in blue) and paired plasma samples (n = 24, in green). There were significantly higher levels of four biomarkers in DBS extraction supernatants (paired t-test), RANTES (p < 0.0001), IL-1b (p < 0.0001), IL-2Ra (p = 0.01), and IL-8 (p < 0.0001).
Figure 7
Figure 7
Log-transformed levels of inflammation-associated biomarkers from DBS extraction supernatants were increased in ARKS compared to UARTO participants. Box-and-whisker plots depict the log-transformed biomarker levels of DBS extraction supernatants for UARTO (n = 12, in blue) and ARKS (n = 12, in green). Levels of RANTES (p = 0.03), MIG (p = 0.03), INF-g (p = 0.05), and IL-1b (p = 0.03) were significantly higher in DBS extraction supernatants from the ARKS than from UARTO (unpaired t-test).
Figure 8
Figure 8
Log-transformed levels of KS/inflammation-associated biomarkers were higher in ARKS than in UARTO plasma samples. Box-and-whisker plots depict log-transformed biomarker levels in UARTO (n = 12, in blue) and ARKS (n = 12, in green) plasma samples. The six log-transformed biomarkers with statistically significantly higher levels in ARKS than in UARTO are MCP-1 (p = 0.02), MIG (p = 0.03), TNF-a (p < 0.001), IL-10 (p = 0.01), IL-2 (p = 0.002), IL-8 (p = 0.007) (unpaired t-test).
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