Multitest reagents

For the validation of the CD4 count, a custom-built prototype instrument was used. The instrument uses the same type of LED excitation and optical filters as used in the laboratory setup, but with a different lens (TC23009, 1× telecentric lens, OptoEngineering) and a different camera (MR655MU-BH, Ximea), resulting in an image area of 60 mm².
All experiments were performed in compliance with the Dutch Medical Research Involving Human Subjects Act (WMO), approved by the Medical Ethics Review Committee (METC) of the University Medical Center Utrecht. Leftover blood samples from HIV-infected patients (who had given prior written informed consent for the use of their anonymized samples for research purposes) at the UMC Utrecht that had been drawn by venipuncture into EDTA blood collection tubes and been used for routine analysis (including a CD4 count), were used to evaluate the performance of our approach. One slide with two printed CD4 counting chambers on each, as depicted in Fig. 1 and S2a,† was used per patient sample. The slides were stored over silica gel at 4 °C (4sg). The CD4 counts obtained using our chambers were compared with the reference counts from flow cytometry, using MultiTest™ reagents and MultiSet™ four color automated immunophenotyping software (Becton Dickinson) on a FACS Canto II v3.0 (Becton Dickinson) flow cytometer at the UMC Utrecht.

An 8 ml blood sample was obtained by venipuncture to measure CD4+T cell count, HIV-1 viral load, hematology, glycaemia and blood lipids. The blood samples were collected and analyzed following biosafety procedures. The CD4+T cell count was performed on whole blood using a FACS-Calibur^TM instrument with MultiTest reagents, TruCOUNT tubes and MultiTEST software (all from Becton Dickinson, USA). HIV-1 viral load was determined using a COBAS^®TaqMan system 48 Analyser with COBASTaqMan HIV-1 test reagents (Roche Diagnostics, Germany) for automated amplification and detection of nucleic acid with limit of detection above 20 copies/ml. Haematological parameters were measured within six hours after blood sample collection using an automated hematology analyser, the Sysmex KX21 (Sysmex Corporation, Japan), and glycaemia and lipid profiles were analyzed by using the Selectra Junior (Vital Scientific NV, The Netherlands). All testing were performed at the laboratory of the Instituto Nacional de Saúde, Ministry of Health, Mozambique.