Cost-effective and scalable DNA extraction method from dried blood spots.

BACKGROUND: Dried blood spot (DBS) samples have been widely used in newborn screening (NBS) for the early identification of disease to facilitate the presymptomatic treatment of congenital diseases in newborns. As molecular genetics knowledge and technology progresses, there is an increased demand on NBS programs for molecular testing and a need to establish reliable, low-cost methods to perform those analyses. Here we report a flexible, cost-efficient, high-throughput DNA extraction method from DBS adaptable to small- and large-scale screening settings. METHODS: Genomic DNA (g.DNA) was extracted from single 3-mm diameter DBS by the sequential use of red cell lysis, detergent-alkaline, and acid-neutralizing buffers routinely used in whole blood and plant tissue DNA extractions. We performed PCR amplification of several genomic regions using standard PCR conditions and detection methods (agarose gel, melting-curve analysis, TaqMan-based assays). Amplicons were confirmed by BigDye® Terminator cycle sequencing and compared with reference sequences. RESULTS: High-quality g.DNA was extracted from hundreds of DBS, as proven by mutation detection of several human genes on multiple platforms. Manual and automated extraction protocols were validated. Quantification of g.DNA by Oligreen® fluorescent nucleic acid stain demonstrated a normal population distribution closely corresponding with white blood cell counts detected in newborn populations. CONCLUSIONS: High-quality, amplifiable g.DNA is extractable from DBSs. Our method is adaptable, reliable, and scalable to low- and high-throughput NBS at low cost ($0.10/sample). This method is routinely used for molecular testing in the New York State NBS program.

Fresh osteochondral allografting for steroid-associated osteonecrosis of the femoral condyles.

BACKGROUND: Osteonecrosis is a complication of corticosteroid therapy with limited treatment options in young, active patients. These options include debridement, core decompression, osteotomy, allografting, and partial or total knee replacement. Few studies exist regarding the use of osteochondral allografts for treatment of steroid-associated osteonecrosis. QUESTIONS/PURPOSES: We asked if fresh osteochondral allografts would (1) heal to host bone in the presence of osteonecrosis, (2) provide a clinically meaningful decrease in pain and improvement in function, and (3) prevent or postpone the need for prosthetic arthroplasty. PATIENTS AND METHODS: Twenty-two patients (28 knees) who underwent osteochondral allografting for high-grade, corticosteroid-associated osteonecrosis were evaluated. Their average age was 24.3 years (range, 16-44 years). The mean graft surface area was 10.8 cm(2) (range, 5.0-19.0 cm(2)). Evaluation included a modified (for the knee) D'Aubigné and Postel (18-point) score, International Knee Documentation Committee (IKDC), and Knee Society function scores. The minimum followup was 25 months (mean, 67 months; range, 25-235 months). RESULTS: Five knees failed. The graft survival rate was 89% (25 of 28). The mean D'Aubigné and Postel score improved from 11.3 to 15.8; 19 of 25 (76%) had a score greater than 15. The mean IKDC pain score improved from 7.1 to 2.0, mean IKDC function score from 3.5 to 8.3, and mean Knee Society function score from 60.0 to 85.7. CONCLUSIONS: Our data suggest osteochondral allografting is a reasonable salvage option for osteonecrosis of the femoral condyles. TKA was avoided in 27 of the 28 of knees at last followup. LEVEL OF EVIDENCE: Level IV, case series. See Guidelines for Authors for a complete description of levels of evidence.

Fresh osteochondral allografting for osteochondral lesions of the talus.

BACKGROUND: Osteochondral lesions of the talus (OLT) are relatively common sequelae of traumatic injuries involving the talus. We report on clinical outcomes of osteochondral allografting (OCA) of the talus for refractory, symptomatic OLT. MATERIALS AND METHODS: OCA was performed in 12 ankles in 11 patients with OLT. All involved partial, unipolar grafts of the talar dome, implanted through an anterior approach without osteotomy, under temporary distraction. Clinical evaluation was performed utilizing the Olerud-Molander Ankle Score (OMAS). Subjective outcome measures included patient questionnaires evaluating pain, function, and satisfaction. Eleven patients (seven males, four females) had a mean age of 35.5 (range, 26 to 57) years. One patient had bilateral involvement. Six OLT affected the medial, six the lateral talar dome. Patients had an average of 1.8 previous surgeries (range, 1 to 5). Mean followup was 38 (range, 24 to 107) months. RESULTS: Mean OMAS improved from 28 to 71 points (p < 0.05). Three had further surgery; overall graft survival rate was 10/12. Of surviving grafts, 30% recorded excellent (OMAS: 100 to 91 points), 20% good (OMAS: 90 to 61 points), 30% fair (OMAS: 60 to 31 points), and 20% poor (OMAS: 30 to 0 points) outcomes. All non-failing patients completed questionnaires; 90% were satisfied, 80% reported reduced pain, and 60% improved function. CONCLUSION: OCA achieved good to excellent results in five of 12 patients, resulting in significant improvement in function and pain with good patient satisfaction. All but one patient avoided arthrodesis. Partial talus OCA is a reasonable treatment option for appropriately selected patients with unipolar OLT.

Fresh osteochondral allograft transplantation for bipolar reciprocal osteochondral lesions of the knee.

BACKGROUND: Osteochondral allograft (OCA) transplantation is an effective treatment option for chondral and osteochondral defects of the knee. HYPOTHESIS: Patients treated with OCAs for reciprocal bipolar lesions of the knee would demonstrate significant clinical improvement. STUDY DESIGN: Case series; Level of evidence, 4. METHODS: Between 1983 and 2010, OCAs were implanted for bipolar chondral lesions in 46 patients (48 knees). The 21 male and 25 female patients averaged 40 years of age (range, 15-66 years). Thirty-four lesions were tibiofemoral, and 14 were patellofemoral. Forty-two knees (88%) had undergone a mean of 3.4 previous surgeries (range, 1-8). The mean allograft area was 19.2 cm(2). Clinical evaluation included the modified Merle d'Aubigné-Postel (18-point), International Knee Documentation Committee (IKDC) pain and function, and Knee Society function (KS-F) scores. Further surgeries on the operative joint were documented. RESULTS: Survivorship of the bipolar OCA was 64.1% at 5 years. Thirty knees underwent further surgery; 22 knees (46%) were considered failures (3 OCA revisions, 14 total knee arthroplasties, 2 unicondylar arthroplasties, 2 arthrodeses, and 1 patellectomy). Among patients whose OCA was still in situ at follow-up, the mean follow-up was 7 years (range, 2.0-19.7 years). The mean 18-point score improved from 12.1 to 16.1; 88% (23/26 knees) of surviving allografts scored ≥15. The mean IKDC pain score improved from 7.5 to 4.7, and the mean IKDC function score improved from 3.4 to 7.0. The mean KS-F score improved from 70.5 to 84.1. CONCLUSION: Osteochondral allograft transplantation is a useful salvage treatment option for reciprocal bipolar cartilage lesions of the knee. High reoperation and failure rates were observed, but patients with surviving allografts showed significant clinical improvement.

Fresh osteochondral allograft transplantation for isolated patellar cartilage injury.

BACKGROUND: The treatment of patellofemoral cartilage injuries can be challenging. Osteochondral allograft (OCA) transplantation has been used as a treatment option for a range of cartilage disorders. PURPOSE: To evaluate functional outcomes and survivorship of the grafts among patients who underwent OCA for patellar cartilage injuries. STUDY DESIGN: Case series; Level of evidence, 4. METHODS: An institutional review board-approved OCA database was used to identify 27 patients (28 knees) who underwent isolated OCA transplantation of the patella between 1983 and 2010. All patients had a minimum 2-year follow-up. The mean age of the patients was 33.7 years (range, 14-64 years); 54% were female. Twenty-six (92.9%) knees had previous surgery (mean, 3.2 procedures; range, 1-10 procedures). The mean allograft area was 10.1 cm(2) (range, 4.0-18.0 cm(2)). Patients returned for clinical evaluation or were contacted via telephone for follow-up. The number and type of reoperations were assessed. Any reoperation resulting in removal of the allograft was considered a failure of the OCA transplantation. Patients were evaluated pre- and postoperatively using the modified Merle d'Aubigné-Postel (18-point) scale, the International Knee Documentation Committee (IKDC) pain, function, and total scores, and the Knee Society function (KS-F) score. Patient satisfaction was assessed at latest follow-up. RESULTS: Seventeen of the 28 knees (60.7%) had further surgery after the OCA transplantation; 8 of the 28 knees (28.6%) were considered OCA failures (4 conversions to total knee arthroplasty, 2 conversions to patellofemoral knee arthroplasty, 1 revision OCA, 1 patellectomy). Patellar allografting survivorship was 78.1% at 5 and 10 years and 55.8% at 15 years. Among the 20 knees (71.4%) with grafts in situ, the mean follow-up duration was 9.7 years (range, 1.8-30.1 years). Pain and function improved from the preoperative visit to latest follow-up, and 89% of patients were extremely satisfied or satisfied with the results of the OCA transplantation. CONCLUSION: OCA transplantation was successful as a salvage treatment procedure for cartilage injuries of the patella.