Abstract
CMV infection is an established risk factor in patients undergoing hematopoietic stem cell transplantation (HSCT). Hence, the status of the donor is carefully considered, especially for high risk groups such as CMV seronegative recipients.
The incidence of CMV neonatal infection is low thus, CB is an advantageous graft alternative to other HSC sources. Screening of CB donors is performed by testing the maternal sample for total Antibodies to CMV (CMV-Ab), while some CB banks, in addition, screen CB for CMV-DNA. Since CB units that have CMV viremia are not recommended for use in HSCT, their characteristics have not been fully evaluated. The aim of this study was to assess the impact of congenital CMV infection on CB cells, specifically on HPC concentration and their CFU functionality, in relation with the viral load in the CB and the donor's mother.
Methods:
To quantify CMV-DNA in CB a multiplex Real Time (RT) PCR assay was developed and validated using the Step One PlusTM RT PCR system (Applied Biosystems). The amplification target was located within the Glycoprotein B conserved sequence and a CMV standard control (SRM2366; National Institute of Standards and Technology) was used for validation experiments. To confirm the correct detection of different CMV genotypes and rare mutants, lyophilized synthetic oligonucleotides (SIGMA-ALDRICH) were used. Saliva culture and a validated qualitative PCR (PCR) were performed as described previously (Albano et al, Blood 2006;108). CB counts were measured in a Sysmex XE2100 analyzer, CD34+/CD45+ cells were evaluated using single platform, 3-color flow cytometry with 7-AAD, with ISHAGE gating strategy. The Colony Forming Unit (CFU) assay was performed using the National Cord Blood Program (NCBP) CFU strategy with High Resolution Digital Imaging (Albano et al, ASH 2008). CB was collected and all samples tested following NCBP approved procedures and donor consent.
Results:
We evaluated the incidence of CMV viremia in two groups of CB units donated to the NCBP. Initially, we screened prospectively 30,308 neonates for CMV infection by saliva culture (period 1/1995 - 3/2007, Group 1) and found 41 positive cases (0.14% incidence in CB donors), all from mothers with positive serum CMV antibodies at delivery.
Based on this observation, subsequently, CMV-DNA was screened by PCR retrospectively in 4,712 newborns from CMV-Ab positive mothers (period 3/2007- 3/2016, Group 2). Eighteen cases had CMV-DNA detected (0.38%). Thus, a total of 59 newborns had CMV infection. Viremia was detected by RT-PCR in all of them (average viral load: 20.6 x104 vc/mL; range 110-2.3 x10^6 vc/mL). None of the neonates had clinical symptoms at birth. Screening by the Obstetric Hospital independently diagnosed CMV congenital infection in 2 of the infants.
Only 7 of the 59 neonates had mothers who also carried CMV-DNA in blood at the time of delivery (range 300-5740 vc/mL) however, with no association to newborns' viral load.
Potency evaluation, including HPC function (CFU), CD34+/CD45+ cells, and Total Nucleated Cells (TNC) was performed prospectively in a cohort of CMV positive (N=19) and control (N=76) CB samples collected and tested during the period 2006-2012. Control cases which were matched for birth date and hospital collection site; race, gender and delivery route were comparable to the CMV positive group. Birth weight and platelet counts were slightly lower in the group with CMV viremia, without reaching statistical significance. Interestingly, TNC and CD34+ cells were significantly higher in the CMV positive group, as well as HPC function (measured by Total CFU; Table). Across cellular progenitor lineages, CFUs showed a trend for high CFU-GM and CFU-GEMM with significantly elevated CFU-E (2843 +/-1949 x 10^3 vs 1389 +/- 1316 x 10^3; P= 0.029). The strong association seen between CD34+ and CFU content (R2=0.79; P<0.001) was consistent with all NCBP data. Notably, there was no association between these two parameters and CMV viral load at birth.
Conclusions:
The test developed, RT-PCR, is valid to detect and quantify CMV-DNA in CB.
Maternal CMV-Ab status or maternal viremia at birth are poor predictors of CB CMV infection. The observations of this study suggestthatcongenital transmission of CMV as shown by CMV viremia at birth, impacts HPC, CD34+/CD45+ cells and erythrocytic precursors towards higher proliferation. Factors responsible for this effect need to be evaluated in future studies.
No relevant conflicts of interest to declare.
