An inkjet-printed polysaccharide matrix for on-chip sample preparation in point-of-care cell counting chambers.

On-chip sample preparation in self-contained microfluidic devices is a key element to realize simple, low-cost, yet reliable in vitro diagnostics that can be carried out at the point-of-care (POC) with minimal training requirements by unskilled users. To address this largely unmet POC medical need, we have developed an optimized polysaccharide matrix containing the reagents which substantially improves our fully printed POC CD4 counting chambers for the monitoring of HIV patients. The simply designed counting chambers allow for capillary-driven filling with unprocessed whole blood. We carefully tailored a gellan/trehalose matrix for deposition by inkjet printing, which preserves the viability of immunostains during a shelf life of at least 3 months and enables controlled antibody release for intense and homogeneous immunofluorescent cell staining throughout the complete 60 mm2 image area within 30 min. Excellent agreement between CD4 counts obtained from our fully printed CD4 counting chambers and the gold standard, flow cytometry, is demonstrated using samples both from healthy donors and HIV-infected patients.

All-printed cell counting chambers with on-chip sample preparation for point-of-care CD4 counting.

We demonstrate the fabrication of fully printed microfluidic CD4 counting chips with complete on-chip sample preparation and their applicability as a CD4 counting assay using samples from healthy donors and HIV-infected patients. CD4 counting in low-income and resource-limited point-of-care settings is only practical and affordable, if disposable tests can be fabricated at very low cost and all manual sample preparation is avoided, while operation as well as quantification is fully automated and independent of the skills of the operator. Here, we show the successful use of (inkjet) printing methods both to fabricate microfluidic cell counting chambers with controlled heights, and to deposit hydrogel layers with embedded fluorophore-labeled antibodies for on-chip sample preparation and reagent storage. The maturation process of gelatin after deposition prevents antibody wash-off during blood inflow very well, while temperature-controlled dissolution of the matrix ensures complete antibody release for immunostaining after the inflow has stopped. The prevention of antibody wash-off together with the subsequent complete antibody release guarantees a homogeneous fluorescence background, making rapid and accurate CD4 counting possible. We show the successful application of our fully printed CD4 counting chips on samples from healthy donors as well as from HIV-infected patients and find an excellent agreement between results from our method and from the gold standard, flow cytometry, in both cases.

Changes in lymphocyte subsets in workers exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

OBJECTIVES: 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is known to have toxic effects on the haematopoietic system in animals but epidemiological studies in humans have shown inconsistent results. In this cross-sectional study we investigated changes in peripheral blood cell counts and lymphocyte subsets among workers from a Dutch historical cohort occupationally exposed to chlorophenoxy herbicides and contaminants including TCDD. METHODS: Forty-seven workers who had been exposed to high levels of TCDD in the past and 38 low-exposed workers were included in the current investigation. Complete blood counts and differential and major lymphocyte subsets were analysed. Current plasma levels of TCDD (TCDD(current)) were determined by high-resolution gas chromatography/isotope-dilution high resolution mass spectrometry. TCDD blood levels at the time of last exposure (TCDD(max)) were estimated using a one-compartment first order kinetic model. RESULTS: Cell counts and lymphocyte subsets were similar between high- and low-exposed workers, except for a non-dose dependent increase in CD4/CD8 ratio among high-exposed workers. Interestingly, most lymphocyte subsets, in particular the B cell compartment, showed a decrease with increasing levels of both TCDD(current) and TCDD(max). CONCLUSIONS: Overall, our study showed that plasma TCDD levels had no effect on white blood cell counts and major subsets. However, a non-significant decrease in most lymphocyte subsets was noted, with the strongest effect for B cells. The latter finding may suggest that dioxin exposure might have an adverse impact on the haematopoietic system and lends some support to B cell lymphoma induction by dioxin.