Digital polymerase chain reaction strategies for accurate and precise detection of vector copy number in chimeric antigen receptor T-cell products.

BACKGROUND AIMS: Vector copy number (VCN), an average quantification of transgene copies unique to a chimeric antigen receptor (CAR) T-cell product, is a characteristic that must be reported prior to patient administration, as high VCN increases the risk of insertional mutagenesis. Historically, VCN assessment in CAR T-cell products has been performed via quantitative polymerase chain reaction (qPCR). qPCR is reliable along a broad range of concentrations, but quantification requires use of a standard curve and precision is limited. Digital PCR (dPCR) methods were developed for absolute quantification of target sequences by counting nucleic acid molecules encapsulated in discrete, volumetrically defined partitions. Advantages of dPCR compared with qPCR include simplicity, reproducibility, sensitivity and lack of dependency on a standard curve for definitive quantification. In the present study, the authors describe a dPCR assay developed for analysis of the novel bicistronic CD19 × CD22 CAR T-cell construct. METHODS: The authors compared the performance of the dPCR assay with qPCR on both the QX200 droplet dPCR (ddPCR) system (Bio-Rad Laboratories, Inc, Hercules, CA, USA) and the QIAcuity nanoplate-based dPCR (ndPCR) system (QIAGEN Sciences, Inc, Germantown, MD, USA). The primer-probe assay was validated with qPCR, ndPCR and ddPCR using patient samples from pre-clinical CAR T-cell manufacturing production runs as well as Jurkat cell subclones, which stably express this bicistronic CAR construct. RESULTS: ddPCR confirmed the specificity of this assay to detect only the bicistronic CAR product. Additionally, the authors' assay gave accurate, precise and reproducible CAR T-cell VCN measurements across qPCR, ndPCR and ddPCR modalities. CONCLUSIONS: The authors demonstrate that dPCR strategies can be utilized for absolute quantification of CAR transgenes and VCN measurements, with improved test-retest reliability, and that specific assays can be developed for detection of unique constructs.

A quantitative polymerase chain reaction test to enumerate leukocytes in allograft tissue and the implications for donor eligibility testing.

A country-to-country analysis of infectious disease screening requirements for donated tissues or cells reveals they are not often harmonized. Transmission of one such infectious disease, human T-lymphotropic virus (HTLV), is related to the transfer of HTLV-infected, viable leukocytes of sufficient number. The ability to characterize allograft tissue as being absent of leukocytes, or containing relatively few leukocytes, by using a specific test has not been previously investigated. A quantitative polymerase chain reaction (qPCR) test was developed to interrogate protein tyrosine phosphatase, receptor type C (PTPRC) gene expression in tissue samples and was able to determine the number of leukocytes present in a tissue. The impact of a qualified leukocyte tissue testing method should be significant and lead to changes in donor eligibility regulations in certain countries. Human leukapheresis samples were used as a control to establish the amount of PTPRC in leukocytes. That value was used as a comparator to determine the number of leukocyte equivalents in tissues of interest. The qPCR test measured tissue leukocyte equivalents and the results were consistent with the relative abundance of leukocytes predicted for each tissue. Using qPCR to calculate leukocyte equivalents based upon PTPRC gene expression can be successfully employed to estimate the number of leukocytes in a tissue or allograft. This method could be used as a screen to rule out tissues that do not meet the criteria of being leukocyte rich and, therefore, do not need direct HTLV testing.

Fractures of the thoracolumbar spine complicating ankylosing spondylitis.

OBJECT: In this clinical review of ankylosing spondylitis, the authors emphasize the fragility of the spine and the degree of neurological deficit that can result from minor injury. The management of thoracolumbar fractures complicating ankylosing spondylitis is discussed. METHODS: Six thoracic and five lumbar fractures were identified in a review of patients in whom ankylosing spondylitis had been diagnosed. Nine fractures were associated with an extension deformity. The mechanism of injury was regarded as major in three and minor in eight. The mean age in patients suffering fractures after minor traumatic injury was 59.6 +/- 14.5 years (+/- standard deviation), whereas the mean age in patients suffering fractures after major traumatic injury was 49.3 +/- 10.1 years. Two patients were treated with bedrest and brace therapy, and nine underwent instrument-assisted fusion. Postinjury neurological deficits were demonstrated in six patients, in three of whom neurological improvement was observed. CONCLUSIONS: Patients with ankylosing spondylitis are at risk of injury, particularly extension fractures of the thoracic and lumbar spine. The incidence of trauma-induced neurological deficit is by no means negligible. Management consists of surgery in patients with vertebral instability and neural compression, and recumbency in those with stable fractures.

In vitro biomechanical comparison of pedicle screws, sublaminar hooks, and sublaminar cables.

OBJECT: Three types of posterior thoracolumbar implants are in use today: pedicle screws, sublaminar titaniumcables, and sublaminar hooks. The authors conducted a biomechanical comparison of these three implants in human cadaveric spines. METHODS: Spine specimens (T5-12) were harvested, radiographically assessed for fractures or metastases, and their bone mineral density (BMD) was measured. Individual vertebrae were disarticulated and fitted with either pedicle screws, sublaminar cables, or bilateral claw hooks. The longitudinal component of each construct consisted of bilateral 10-cm rods connected with two cross-connectors. The vertebral body was embedded in cement, and the rods were affixed to a ball-and-socket apparatus for the application of a distraction force. The authors analyzed 1) 20 vertebrae implanted with screws; 2) 20 with hooks, and 3) 20 with cables. The maximum pullout (MPO) forces prior to failure (mean +/- standard deviation) for the screw, hook, and cable implants were 972 +/- 330, 802 +/- 356, and 654 +/- 248 N, respectively (p = 0.0375). Cables allowed significantly greater displacement (6.80 +/- 3.95 mm) prior to reaching the MPO force than hooks (3.73 +/- 1.42 mm) and screws (4.42 +/- 2.15 mm [p = 0.0108]). Eleven screw-implanted vertebrae failed because of screw pullout. All hook-and-cable-implanted vertebrae failed because of pedicle, middle column, or laminar fracture. CONCLUSIONS: These findings suggest that screws possess the greatest pullout strength of the three fixation systems. Sublaminar cables are the least rigid of the three. When screw failure occurred, the mechanism was generally screw back-out, without vertebral fractures.

In vitro biomechanical studies of an anterior thoracolumbar implant.

After L1 corpectomy in T11-L3 human cadaveric spine, anterior thoracolumbar instrumentation with strut grafting restores spinal stability. T12-L2 angular rotation was measured in response to moments of 0.0, 1.5, 3.0, 4.5, and 6.0 Nm in flexion, extension, lateral bending, and axial rotation, respectively. The spines were tested: 1) intact; 2) after partial L1 corpectomy, grafting, and instrumentation (Profile plate, DePuy-AcroMed, Raynham, MA), with the wooden dowel graft screwed to the plate; 3) without graft screw fixation; and 4) after flexion-extension cyclic fatiguing for 5000 cycles at a load of +/-3.0 Nm. Before and after fatiguing, the instrumented spine was significantly (p

Factors affecting the pullout strength of self-drilling and self-tapping anterior cervical screws.

STUDY DESIGN: A biomechanical comparison of 12-, 14-, and 16-mm self-tapping and self-drilling screws in cadaveric cervical spines was performed. OBJECTIVE: This study compared self-drilling and self-tapping screw designs used in anterior cervical locking plates in terms of insertion torque and pullout strength. In addition to screw design, the effects of screw length (12, 14, and 16 mm) and bone mineral density were examined. METHODS: A total of 201 screws were implanted and tested. Nineteen cadaver spine specimens (C2 through C7) were radiographed and their bone mineral density measured. In each vertebral level, two screws of the same length were implanted and their insertion torque measured. One of each screw design (self-drilling or self-tapping) was randomly placed in each vertebra for side-by-side comparison. Vertebrae then were individually potted and mounted on an MTS machine for accurate measurement of maximum pullout strength. RESULTS: Pullout strength was strongly related to screw length ( = 0.0002). There was no significant difference in pullout strength between the self-drilling and self-tapping screw designs at any length ( = 0.9352). A significant correlation (