Data reported was collected from patients treated during August 2005-July 2007 as part of medical practice at the Nanshan Affiliated Hospital of Guangdong Medical College. All patients were free of: 1) prior history of severe allergic reactions; 2) history of, or active, malignancy; 3) active systemic or severe focal infections (including HIV and syphilis); 4) active cardiac, pulmonary, renal, hepatic or gastrointestinal disease; 5) coagulopathy or any other contraindication for lumbar puncture; 6) gastrostomy, tracheostomy or noninvasive ventilatory support - as these could influence the prognosis and end-point measurements; 7) any severe psychiatric disorder and 8) any immunodeficiency disease or condition.
Age range was 15 to 68 and the male:female ratio was 1.6:1 (70 males, 44 females). In terms of diagnosis, 4 patients had multiple system atrophy (MSA), 23 patients had ataxias, 42 patients were paraplegic, 19 patients had multiple sclerosis, 12 patients had Amyotrophic Lateral Sclerosis (ALS) and 14 patients had other diagnoses (Table ). The local institutional review board of the Nanshan Affiliated Hospital of Guangdong Medical College, under the auspices of the National Ministry of Heath, approved application of the technique and consent forms were obtained from each patient before initiation of treatment.
Patients treated by condition
Umbilical Cord Blood (100~ 150 mL) was collected from healthy unrelated donors (signed an informed consent) in accordance with the sterile procurement guidelines for cord blood in each hospital. After collection, each blood sample was tested for communicable diseases such as HBV, HCV, HIV, ALT, and Syphilis. Cord blood was diluted with saline in the ratio 2:1 and 30 mls of the diluted blood was then added to 15 mls of Ficoll in every 50 ml centrifuge tube and then centrifuged (750 g × 22 minutes). Mononuclear cells were collected and washed twice in saline. Contaminating erythrocytes were lyzed with lysis buffer comprising of injection grade water.
Cell density was adjusted to 2 ~ 6 × 106/ml and seeded in DMEM/F12 culture medium with bFGF and EGF at a concentration of 20 ng/ml. Culture media was mixed with 2% v/v B-27 Stem Cell Culture Supplements. Cells were cultured at 37°C with saturated humidity and 5% CO2 by volume. At this stage, all relevant information about the initial culture is entered in the batch information record including test results for sterility, mycoplasma and endotoxin. Cell growth was regularly monitored and the inspection records updated accordingly. Cells were harvested for clinical application after one week of cultivation with cell quantity ≥1 × 107 and viability ≥95%.
To ensure the quality of the UCB-derived mononuclear cells, a number of parameters are confirmed before use. These are as follows: 1) Raw material control: Tests (HBV, HCV, HIV, ALT and Syphilis) for communicable diseases for UCB units are carried out before any processing begins. Testing was performed by third party laboratory under local government-monitored conditions.
2) In-process control: Non-qualifying cells were eliminated in accordance with Beike's cell counting and morphology standards which include cell quantity ≥1 × 107 and the highly homogeneous cells possessing a round shape and non-adherence to the culture flask.
3) Culture control: Any contaminated cell suspensions or unhealthy cells were eliminated upon discovery. Non-contamination was determined as lack of sterility, mycoplasma, and lack of visible microorganisms by microscopy. Furthermore samples had to have an endotoxin level≤0.5 EU/ml and be negative for free DNA.
4) Finished product control: This incorporates a final cell count (≥1 × 107, containing 1.0-2.0% CD34+ cells as determined by flow cytometry), cell viability (≥95%) and sterility test.
Intrathecal injection by lumbar puncture was combined with intravenous infusion and repeated four or five times - depending on the patient's condition. Treatments were separated by one week intervals. Lumbar puncture was performed in the lateral decubitus position, prepped and draped in sterile fashion, and the needle placed in the lumbar cistern. Two mls of Cerebro-Spinal Fluid (CSF) was removed and replaced by 2 mls of cell suspension containing 1-3 × 107 cells. A 30 ml intravenous infusion of cell suspension was given through an intravenous catheter in 15-20 minutes.
Adverse events were analyzed for all 114 cases, and are presented as percentage values. For analysis of laboratory parameters, the continuous variables were compared using Student t-test with alpha set at 0.05 by group. When the data set did not conform to the normal distribution, logarithmic transformation was used. Inter-quartile-range (IQR) computation and boxplots were used to detect outliers. The outliers were firmly believed to be data errors or data entry errors and were removed from the data analysis. The SPSS 13.0 statistical package was applied for statistical analysis.