NEUROLOGY/2005/101881
Appendix (E)A-1:
Detailed description of methods for ascertaining outcome and safety measures.
1. Columbia Neurological Score (CNS): A semi-quantitative tool was developed to summarize the clinical evaluations of patients with mitochondrial disease for the following domains: (1) height, weight, and head circumference; (2) general medical exam; (3) fundoscopic exam; (4) cranial nerves; (5) stance and gait; (6) involuntary movements; (7) sensation; (8) cerebellar function; (9) muscle bulk, tone and strength; (10) tendon reflexes, (11) Babinski signs; and (12) other findings. Results of these domains were scored as normal or abnormal and summarized in the Columbia Neurological Score (CNS), ranging from 0 to 76, with 76 being perfect. We have previously shown that the instrument has good inter-rater reliability and correlates with other measures of disease severity(1).
2. Neuropsychological Assessment (NP): Areas of cognitive function were assessed with a brief global mental status examination and specific tests to assess the following domains: (1) General cognitive functions/abstract reasoning (modified mini mental status examination assessing a wide range of functions including orientation, memory, calculations, language, naming, construction,attention(2), and Wechsler Adult Intelligence Scale – Revised (3)); (2) Memory (selective reminding test to assess verbal memory(4); the Benton Visual Retention Test to test visual memory (5)); (3) Language (Boston naming test (6); controlled word association for verbal fluency (7), animal naming subtest of the Boston Diagnostic Aphasia examination testing category fluency (8)); (4) Executive functions (Trail Making Test (9), Odd Man Out test assessing the ability to switch mental set (10)), (5) Visuo-Spatial abilities (Hooper Visual Organization Test(11)).Each test of the neuropsychological battery was scored according to standard guidelines. The raw score, age-scaled score, z-score, and performance percentile were recorded. Depending on the percentile range of the results a score was assigned for each domain (0 = percentile within range 17-99%, 1 = percentile within range 5-16%, 2 = percentile less than 5%). The mean of all seven domain scores was calculated to obtain a global score, ranging from 0 to 2(4, 12-15).
3. Health related event inventory (HREI): We counted migraine, stroke and seizure episodes, as well as non-routine medical encounters in the three months preceding each visit. The sum of health related events is expressed per 3 months.
4. Karnofsky Score: We used this established instrument to evaluate daily living functional abilities. The scale rates performance in activities of daily living and independence of an individual on a semi-quantitative scale. Someone able to work, without complaints and without evidence of disease is given a score of 100, for example. Someone who is moribund or with a rapidly progressing fatal process is given a score of 10. Within this range of 0-100,the investigators assign a score in increments of 10. The instrument has a descriptive statement next to each numeric level, e.g. a score of 50 describes someone who is moderately disabled, dependent, and who requires considerable assistance and frequent care (16).
5. MRI of the Brain: Brain MRI of each subject was performed at 1.5 Tesla using routine clinical pulse sequences; these included axial T1-weighted spin echo, axial T2-weighted fast spin echo, and axial Fluid Attenuated Inversion Recovery (FLAIR) images. The FLAIR (TR=10,002 ms; TE=152/Ef; TI=2200) images were semi-quantitatively scored for severity and extent of signal abnormalities using an MRI scoring system. Scored brain regions included the right and left insulae, basal ganglia, cerebellum, frontal, parietal, temporal, and occipital lobes, as well as the midbrain. Signal abnormalities were scored in each region (3= normal; 2=mild; 1=moderate; and 0=severe) and summated to produce a total MRI score (45 for perfect score and lower values for abnormal studies). The inter-rater reliability for this scoring instrument was good (kappa statistics between radiologists were 0.88-0.98).
6. 1HMR spectroscopic imaging (MRSI): Following T1-weighted localizer images, multi-slice proton magnetic resonance spectroscopic imaging (1HMRSI) was performed on each subject using methods previously described(17). The data analyzed in this study were obtained from two 15-mm axial-oblique brain sections. The lower of the two slices traversed and encompassed the bodies of the lateral ventricles. The following acquisition sequences were used: TE/TR 280/2300 ms, FOV 240mm, 32x32 phase-encoding steps with circular k-space, and 256 sample points. Each MRSI voxel had a nominal size of 0.75x0.75x1.5cm, or 0.83cc. The strong pericranial lipid resonances were suppressed using octagonally tailored outer volume pre-saturation pulses. Since there are no neuronal structures in the lateral ventricles, the levels of N-acetylaspartate, creatine, and choline are expected to be undetectable. The lactate peaks are therefore quantitated as the ratio of the lactate signal area over the mean square root of the background noise, and are given in institutional units [i.u.]. We chose the ventricular lactate (derived from a voxel placed over the lateral ventricle) as a biological marker in this trial, since it is the most sensitive 1H MRSI marker in our experience(1, 18).
7. Venous and CSF lactate: Venous and CSF lactate were measured in the hospital laboratory according to standard procedures and evaluated against reference ranges derived from a normal control population (0.5-2.2 mM/l for venous and 0.6-2.2 mM/l for CSF lactate).
8. Nerve conduction studies: Nerve conduction studies were performed using standard equipment and procedures. To minimize patient discomfort we did not perform electromyography and we limited our investigation to the following nerve conduction studies: ulnar and peroneal motor studies, and radial and sural sensory studies. Skin temperature was carefully monitored during the testing. Patients underwent unilateral testing, or in some cases, when indicated and tolerated, bilateral testing.
9. Safety laboratory measures: Complete hematological, endocrine, and chemical profiles were measured according to standard methods in the Clinical Chemistry Laboratory of Columbia University Medical Center and compared to normal values established in that laboratory.
References:
1.Kaufmann P, Shungu DC, Sano MC, et al. Cerebral lactic acidosis correlates with neurological impairment in MELAS. Neurology 2004;62:1297-1302.
2.Teng EL, Chui HC. The Modified Mini-Mental State (3MS) Examination. J Clin Psychiatry 1987;48:314-318.
3.Wechsler D. Wechsler Adult Intelligence Scale-Revised. New York: The Psychological Corporation, 1981.
4.Buschke H, Fuld PA. Evaluating Storage, Retention, and Retrieval in Disordered Memory and Learning. Neurology 1974;24:1019-1025.
5.Benton AL. The Visual Retention Test. New York: The Psychological Corporation, 1955.
6.Kaplan E, Goodglass H, Weintraub S. Boston Naming Test. Philadelphia PA: Lea and Febiger, 1983.
7.Benton AL. FAS Test. Victoria, BC: The University of Victoria, 1967.
8.Goodglass H, Kaplan D. The Assessment of Aphasia and Related Disorders. Philadelphia PA: Lea and Febiger, 1983.
9.Reitan RM, Wolfson D. The Halstead-Reitan Neuropsychological Test Battery: Theory and Clinical interpretation. Tuscon AZ: Neuropsychology Press, 1985.
10.DATATOP: AMulticenter Controlled Clinical Trial in Early Parkinson's Disease. Parkinson Study Group. Arch Neurol 1989;46:1052-1060.
11.Hooper HE. The Hooper Visual Organization Test Manual. Los Angeles CA: The Western Psychological Services, 1958.
12.Benton. The Visual Retention Test. In: The Psychological Corporation New York, 1955.
13.Benton. A FAS Test. In: Spreen O BA, ed. Neurosensory Center Comprehensive Examination for Aphasia. Victoria BC: University of Victoria, 1967.
14.Folstein MF, Folstein SE, McHugh PR. "Mini-Mental State". A Practical Method for Grading the Cognitive State of Patients for the Clinician. J Psychiatr Res 1975;12:189-198.
15.Kaplan E, Goodglass H, Weintraub S. Boston Naming Test. In. Philadelphia, PA: Lea and Febiger, 1983.
16.Karnofsky KA, Burchenal JH. The Clinical Evaluation of Chemotherapeutic Agents in Cancer. In: MacLeod CM, ed.: Columbia University Press, 1949: 196.
17.Duyn JH, Gillen J, Sobering G, van Zijl PC, Moonen CT. Multisection Proton MR Spectroscopic Imaging of the Brain. Radiology 1993;188:277-282.
18.Shungu DC, Mao X, Kaufmann P, et al. Comparison of In Vitro and In Vivo CSF Lactate in A3243G MELAS Patients: AViable Method for Absolute Quantitation of CSF Lactate by 1H MRSI. Proc Int Soc Magn Reson Med 2002;10:695.
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