Autism Research and Treatment are categorized as autistic. In addition, scores falling within the autistic range can be divided into two categories—mild- to-moderate autism and severe autism. Decreased scores on this rating scale reﬂect improvement in symptoms. 2.3.4. Clinical Global Impression-Improvement. The investi- gator evaluated each child using the clinical global impres- sion score (CGI-I) at baseline, six weeks, and twelve weeks. The CGI is designed to reﬂect changes in core autism symptoms and behaviors over multiple examinations [16]. Decreased scores on this rating scale reﬂect improvement in symptoms. 2.3.5. TNF-α Assay. CSF and serum TNF-α were measured up to 21 months prior to baseline. Serum TNF-α was obtained again at the 12-week visit. CSF TNF-α was also measured in children whose parents opted for a lumbar puncture. The lumbar puncture was conducted after patients were admitted into the pediatric outpatient treatment unit of Sutter Memorial Hospital (Sacramento, CA) and the parent or guardian signed an additional consent granting permission to perform the procedure. An anesthesiologist monitored and sedated the child with propofol to allow a safe and painless procedure for obtaining CSF for cytokine values. A topical anesthetic was applied at the site of the puncture and the puncture site was cleaned with gauze pads soaked in antiseptic solution. After the anesthetic took eﬀect the patient had the procedure performed and 5–10 mL of CSF was removed for analysis as above, and the child was monitored until safely released from the procedure per the attending anesthesiologist. The CSF cell count, protein, and glucose were obtained simultaneously so that no other inﬂammatory process was present. Patients where the CSF was obtained more than 12 months prior to baseline (n = 2) had the option to repeat the CSF levels. Both CSF levels were repeated for both patients. All patients completing the treatment trial had CSF levels and serum levels for TNF-α repeated per protocol (n = 4). 2.4. Specimen Collection and Preparation. The CSF and serum samples were frozen after collection and analyzed by Inter Science Institute (ISI), Inglewood, CA (CLIA certiﬁ- cation 05D0549406) using a solid phase ELISA technique with a monoclonal antibody speciﬁc for TNF-α (R&D systems/Quantikine). Three in-house controls with high, medium, and low concentrations were included in the assay. As part of standard preanalytical procedures, any CSF samples that were observed to contain sediment were centrifuged at 1300 g for 5 minutes to remove any particulate matter. The assay was otherwise performed according to the standard protocol. Specimens were diluted 1 : 1 and 1 : 10 for ﬁve patients at baseline and four patients at 12 weeks, except for two patients who had only the lower diluted values, to establish comparative values for TNF-α. Serial dilution linearity had been previously validated up to 1 : 32. A standard point was added by an additional dilution to extend the lower range of reporting. The CSF and serum of patients were analyzed and ratios were calculated. 3 2.5. Analytical Sensitivity and Reproducibility of ISI’s TNF- α Assay. Sensitivity of the ISI assay was established over 10 replicates. The mean sensitivity was 0.298 pg/mL with an LOD of 0.3133 pg/mL. Interassay and intra-assay variation were established using high, medium, and low control materials. In 20 consecutive assays, the target values for interassay variation of the control materials were 21.7, 323.5, and 628.2 pg/mL with coeﬃcient of variation (CV) of 9.2, 3.5, and 1.7%, respectively. The intra-assay variation had target values of 21.8, 333.3 and 621.0 pg/mL, respectively, with CV’s at 9.8, 2.7 and 1.5% respectively. The linearity of the assay had an r2 value of 0.9999 with an r of 0.9999 and a slope of 0.9903; the recovery observed values had similar r- values with a slope of 0.9992 and average recovery of 102%. 2.6. Pharmacokinetic Testing. At the baseline visit, patients were checked into an outpatient pediatric center for the day and given a brief physical. Subject’s parents were given the choice to have a saline lock for blood draws or to have four venipunctures for the pharmacokinetic samples to be drawn. The samples were collected at 1, 2, 4, and 8 hours after ﬁrst study drug dose. Blood samples were collected in a 6 mL K2-EDTA tube. Samples were immediately immersed in ice. Within 15 minutes of collection time, sample was taken to the laboratory and placed in refrigerated centrifuge at 1500 g for 10 minutes at 4◦C. Plasma was aliquoted into 2 prelabeled cryovials. Within 30 minutes of blood collection time, the cryovials were placed in −20◦C freezer. The samples were kept frozen and sent for analysis in batches. The validated HPLC-MS/MS methods were used to determine lenalidomide concentrations in human plasma samples. Lenalidomide was analyzed after the liquid-liquid extraction (with methyl tert-butyl ether (MtBE): ethyl acetate, 50 : 50, v/v) from 0.2 mL of human plasma samples. The reference standard of lenalidomide and stable isotope-labeled internal standard [13C5]-CC-5013 were synthesized by Celgene Cor- poration (Summit, NJ). HPLC was performed on a Synergy Polar-RP 4-μm column (Phenomenex, Torrance, CA, USA). A triple quadrupole mass spectrometer (API 4000, Applied Biosystems, Foster City, CA, USA) was used for mass analysis and detection. Quantiﬁcation was performed using multiple- reaction monitoring (m/z of 260.3/149.2 for lenalidomide and 265.1/149.2 for the IS) with electrospray ionization in the positive ion mode. The lenalidomide standard curve was linear (r2 > 0.99) from 1 to 200 ng/mL in plasma. The lower limit of quantitation was 5 ng/mL in plasma. Validation quality control samples at 4 concentrations (1, 3, 60, and 180 ng/m) were used to determine assay accuracy and precision. Interday assay accuracy ranged from −1.3 to 6.5% deviation from the nominal concentration. Interday precision did not exceed 8.0% CV in the method validation. All samples were analyzed by QPS (Newark, DE, USA). 2.7. Treatment. Lenalidomide 2.5 mgs was given daily for 12 weeks. Subjects had a weekly study visit after administration of the ﬁrst dose. A parent was given instructions on how to administer lenalidomide including precautions for medication handling. Subsequent doses were administered