Sample collection and storage
Anonymized blood samples were obtained from patients with immunodeficiency and from adult controls (CTRL) without pathologies referring to the ASST Spedali Civili of Brescia hospital for routine diagnostic testing. Blood samples were collected using ethylenediaminetetraacetic acid (EDTA) tubes. Two types of nylon FS (FLOQSwab™ Genetics, Copan, Brescia, Italy) were tested: a single FS with an integrated active drying system in the tube stopper (4N6FLOQSwab code 4504C) and a dual FS without the drying system (4N6FLOQSwab code 4511C). All FS were immersed in blood for a time ranging from 10 to 60 s; the first type of FS was immediately inserted into containers, while the other FS, without the integrated drying system, were left to dry for at least 2 h and then inserted back into containers. FS were stored at room temperature until DNA extraction. For sample stability evaluation, DNA was extracted after 1 week, and then after 1, 3, 6, 9 and 12 months from FS preparation.
Blood aliquots were also used to prepare PBMC by Ficoll-Hypaque gradient centrifugation which were kept in liquid nitrogen; other aliquots were directly subjected to DNA extraction. Simultaneously, fresh blood drops were placed on Nucleic-Cards™ (Copan Italia, Brescia, Italy), which were stored at room temperature until processing. Discs of 2 mm were punched from each card and subjected to DNA extraction. DNA extracted from HeLa cell line, which does not contain TRECs or KRECs, was used to assess dPCR specificity and to establish background signals.
DNA extraction
DNA from FS was extracted either with QIAamp DNA Blood Mini Kit and with Gentra Puregene Blood Kit according to manufacturer’s instructions (Qiagen, Hilden, Germany). Red blood cell lysis was not performed with the Gentra kit. In addition, we developed the homemade protocol for DNA extraction as detailed below. FS shafts were snapped at the molded breakpoint, inserted in the Nucleic Acid Optimizer (NAO™) basket (Copan), placed in microtubes and filled with 300 µL of saline solution, 300 µL of ATL buffer and 7 µL of Puregene Proteinase K 20 mg/mL (Qiagen). After an overnight incubation at 56 °C on a shaker at 1400 rpm, samples were centrifuged at 13,000 rpm for 5 min. During this centrifugation the grid bottom of the basket allows the passage of sample elution buffer from the FS. FS and baskets were removed from the microtubes and protein precipitation was obtained by incubating samples on ice for 20 min with 233 µL of ammonium acetate 7.5 M. After centrifugations at 13,000 rpm for 10 min, supernatants were transferred to new microtubes containing 600 µL of isopropanol and 1 µL of 5 mg/mL glycogen (Ambion Inc., Austin, TX), mixed gently until DNA was visible as threads or clumps, and then incubated for 10 min at room temperature. Vortexing was avoided to minimize fragmentation of the extracted DNA [22]. After centrifugation at 13,000 rpm at 4 °C for 20 min, supernatants were discarded and pellets washed with 500 µL of ethanol 70%, incubated at room temperature for 5 min and centrifuged at 13,000 rpm at 4 °C for 10 min. Dried pellets were subsequently dissolved using 40 µL of TE (10 mM Tris, pH 8.0, 0.1 mM EDTA, pH 8) and incubated at 65 °C for 10 min on a shaker.
DNA from 300 µL of WB and from six 2-mm punch discs of dried blood on cards (corresponding to 8.4 µL of blood) was extracted using the Gentra Puregene Blood Kit following the manufacturer’s instructions. DNA from 3 × 106 of PBMC and HeLa cell line was extracted using the QIAamp DNA Blood Mini Kit.
DNA from FS and cards without absorbed blood were used to test eventual cross contaminations during extraction procedures.
Quantity, purity, and integrity of DNA extracted from FS
DNA extracted from FS, cards, WB, PBMC, and Hela cells was analyzed spectrophotometrically (Nanodrop 2000c, Thermo Fisher Scientific, Waltham, MA) at 260 nm to evaluate DNA concentration and by measuring the 260/280 nm absorbance ratio to estimate DNA purity.
The level of precision of the homemade method for DNA extraction from FS was determined in several replicates by evaluating intra- and inter-assay variation. Yield was expressed as percentage of recovery that is the proportion of extracted over expected DNA. The expected DNA was calculated based on the white blood cell (WBC) count and a mean DNA content of 6.6 pg/cell.
For DNA integrity and size range analysis, 500 ng of DNA were subjected to electrophoresis on a 0.8% agarose gel in 1× TBE buffer (0.089 M Tris base; 0.089 M boric acid; 0.002 M EDTA, disodium salt dihydrate; final pH 8.3) containing ethidium bromide. The DNA size marker was Lambda DNA Hin
d
III Digest (Sigma-Aldrich, Saint Louis, MO). To further evaluate genomic DNA integrity and to exclude the presence of any inhibitory material interfering with amplification reaction, 50 ng of DNA were amplified in a 50 μL mix containing 500 nM of primers specific for a fragment of albumin gene (forward: 5′-TGA AAC ATA CGT TCC CAA AGA GTT T-3′ and reverse: 5′-CTC TCC TTC TCA GAA AGT GTG CAT AT-3′), 0.2 mM deoxynucleotide triphosphates, 1.5 mM MgCl2, and 1.5 U AmpliTaq Gold DNA polymerase with reaction buffer II (Applied Biosystem™, Austin, TX). A no-template control was used to check for contamination. The amplification program consisted of one step at 95 °C for 7 min, followed by 30 cycles of 95 °C for 30 s, 60 °C for 30 s, 72 °C for 30 s and a final extension at 72 °C for 10 min. Amplified products were separated on 2% agarose gel containing ethidium bromide; the DNA size marker was DNA molecular weight marker VIII (0.019–1.11 kbp; Roche Diagnostics, Basel, Switzerland). Gels were photographed by G:Box™ gel documentation system (Syngene, Cambridge, UK).
Quantification of TRECs and KRECs
TRECs and KRECs were measured after amplification with both TaqMan qRT-PCR and dPCR assays. The well established “reference” qRT-PCR was carried out as previously reported, using DNA extracted from PBMC, with a triple plasmid containing fragments of TRECs, KRECs, and TCR constant alpha gene created in our laboratory and used to prepare the standard curve [7]. With this method, the number of lymphocytes plus monocytes (which are the cells obtained in PBMC preparation) contained in 1 mL of blood was used to calculate the number of TRECs or KRECs per milliliter of blood (copies/mL), that is = (TRECs or KRECs per 1 × 106 PBMC) × (lymphocyte plus monocyte count in 1 mL of blood)/106 [7].
dPCR was performed using micro-well- chip-based QuantStudio® 3D Digital PCR (Applied Biosystems™, Thermo Fisher Scientific, Waltham, MA), following the manufacturer’s instructions and the same primers and probes for the TaqMan assay, with the exception of 5′ reporter dye of KREC probe labelled with VIC instead of HEX. Four microliter of DNA (corresponding to about 500 ng depending on sample concentration) were added to a mixture consisting of 8 µL of 2× QuantStudio® 3D Digital PCR master mix v2, 900 nM of both TREC and KREC forward and reverse primers and 200 nM of both TREC FAM-TAMRA and KREC VIC-TAMRA probes, and 0.29 μL of BSA (2 mg/mL). Using automatic chip loader, 14.5 μL of this mix were added on each QuantStudio® 3D Digital PCR 20K Chip v2. Chips were loaded on ProFlex™ 2× flat PCR System and cycled according to the following parameters: 95 °C for 8 min, followed by 45 cycles of 62 °C for 1 min and 95 °C for 15 s, and a final extension at 62 °C for 2 min.
Singleplex dPCR was performed amplifying 4 μL (500 ng) of DNA in a mixture consisting of 8 µL of 2× QuantStudio® 3D Digital PCR master mix v2, 900 nM of forward and reverse primers for TRECs (or KRECs) and 200 nM of probe for TRECs FAM-TAMRA (or KRECs VIC-TAMRA), and 0.29 μL of BSA (2 mg/mL).
Absolute quantification was determined using Quantstudio® 3D Digital PCR System and analyzed with QuantStudio® 3D AnalysisSuite Cloud Software (Thermo Fisher Scientific).
Sensitivity and specificity of dPCR platform were evaluated with the plasmid created for qRT-PCR diluted in the DNA from HeLa cell line. The copy number was estimated according to the molecular weight, amount, and length of plasmid. Plasmid dilutions were set up in 125 ng/µL of DNA from HeLa cells; the plasmid copy numbers charged on chip were 0, 5, 10, 25, 50, 100, 500, 1000, and 2500 and the amount of charged DNA was 500 ng. The sensitivity of dPCR was compared with that of qRT-PCR by testing, with the two methods, progressive dilutions of plasmid DNA copies.
The reproducibility of dPCR assay was determined by intra- and inter-assay variation experiments, performed starting from the DNA obtained from FS prepared using the same blood sample.
Direct quantification of TRECs and KRECs per mL of blood
When amplified DNA is extracted from blood, either in liquid form or absorbed and dried onto a FS, TRECs and KRECs per mL of blood can be calculated according to the following equation, which takes into account the variable recovery of DNA due to an inevitable DNA loss during extraction:
$$\left[ {{\text{Target}}/(\upmu{\text{L}})} \right] = {\text{QTY}}_{\text{target}} \times \frac{{{\text{DNA}}_{\text{extracted}} ({\text{ng}})}}{{{\text{DNA}}_{\text{loaded }} \left( {\text{ng}} \right)}} \times \frac{1}{{{\text{Vol }}\left( \upmu{{\text{L}}} \right)}} \times \frac{1}{\text{Rec}}$$
where: QTYtarget = copy number of target sequence in the loaded DNA, as determined by qRT-PCR or dPCR (in the case of dPCR, this is the copy number per chip, obtained by multiplying the copy number per partition by the number of filled partitions), DNAextracted (ng) = total amount of extracted DNA determined after measuring the DNA concentration using the spectrophotometer, DNAloaded (ng) = actual amount of DNA loaded for the PCR reaction, Vol (μL) = volume of WB used for DNA extraction from blood in liquid form or absorbed onto a FS, Rec = proportion of DNA recovered after the extraction.
Rec can be calculated as:
$${\text{Rec}} = \frac{{{\text{DNA}}_{\text{extracted}} \left( {\text{ng}} \right)}}{{{\text{DNA}}_{\text{tot}} \left( {\text{ng}} \right)}}$$
The DNAtot (ng) is the theoretical total DNA content in the liquid blood volume used for the extraction/blood absorbed onto the FS, and can be estimated as follows:
$${\text{DNA}}_{\text{tot}} \left( {\text{ng}} \right) = {\text{WBC}}/\left( \upmu {{\text{L}}} \right) \times {\text{Vol}}\left( \upmu{{\text{L}}} \right) \times {\text{DNA}}_{{1{\text{cell}}}} ({\text{ng}})$$
where: DNAlcell = mean DNA content in human cells (6.6 × 10−3 ng).
After substituting these terms in the first equation, we obtain the following formula:
$$\left[ {{\text{Target}}/(\upmu {\text{L}})} \right] = {\text{QTY}}_{\text{target}} \times \frac{{{\text{WBC}}/\upmu ({\text{L}}) \times 6.6 \times 10^{ - 3} ({\text{ng}})}}{{{\text{DNA}}_{\text{loaded}} \left( {\text{ng}} \right)}}$$
Alternatively, because 1 mL = 103 μL, we can directly obtain [Target/(mL)] as:
$$\left[ {{\text{Target}}/({\text{mL}})} \right] = {\text{QTY}}_{\text{target}} \times \frac{{{\text{WBC}}/(\upmu {\text{L}}) \times 6.6}}{{{\text{DNA}}_{\text{loaded}} \left( {\text{ng}} \right)}}$$
where the final estimation of the number of targets depends neither on the exact volume collected by the FS (which varies in distinct FS), nor on the estimation of DNA recovery; it only depends on the amount of loaded DNA and on the number of WBC/mL of blood, assuming that the absorption process does not alter the concentration of cells present in the sample (i.e. absorbed blood WBC concentration being equal to the circulating blood WBC concentration).
Statistics
The percentage of DNA recovery was analyzed by nested ANOVA, thus accounting for the presence of replicates and for the different days in which the experiments were performed. The variance components estimated by the nested ANOVA were employed to calculate the coefficients of variation, following the Clinical and Laboratory Standards Institute EP05-A3 document suggestions (http://shop.clsi.org/method-evaluation-documents/EP05.html). Correlations were analyzed with the Pearson coefficient of determination. TREC and KREC changes with age were assessed by linear regression. Results were considered significant if p < 0.05.
