Abstract:
A method for diagnosing disorders in a subject organism, in which fluid samples from normal and abnormal organisms are analyzed to generate electrical signal patterns representative of molecular constituents of the samples. A data base of electrical signal patterns representative of frequency distribution of sample constituents from the abnormal organisms having known categories of disorders and control samples from normal organisms are created, and a fluid sample taken from the subject organism is analyzed by comparing it to the data base for conformity to the electrical signal patterns representative of the frequency distribution. The invention has particular applicability to assisting in the diagnosis of degenerative diseases such as Alzheimer&#39;s Disease, Parkinson&#39;s Disease, Huntington&#39;s Disease, schizophrenia, amyotrophic lateral sclerosis and Progressive Supernuclear Palsy.

Description:
This is a continuation of application Ser. No. 07/643,541 filed on Jan. 18, 1991 (now abandoned). 
     This application is in part a continuation of my application Ser. No. 07/274,505, filed Nov. 11, 1988 now U.S. Pat. No. 5,104,639, which in turn is a divisional application of my Ser. No. 06/797,615, filed Nov. 13, 1985 (Now U.S. Pat. No. 4,863,873), which is in turn a continuation of my application Ser. No. 06/670,483, filed Nov. 13, 1984 (now abandoned), which in turn is in part a continuation of my application Ser. No. 06/579,401, filed Feb. 17, 1984 (now U.S. Pat. No. 4,511,659), which is in part a continuation of my application Ser. No. 06/472,387 filed Mar. 4, 1983 (now abandoned), and in part a continuation of my application Ser. No. 06/425,183 filed Sep. 28, 1982 (now abandoned), which is in turn a continuation of my application Ser. No. 06/111,917 filed Jan. 14, 1980 (now U.S. Pat. No. 4,404,065). 
    
    
     This invention relates to analytical and mathematical methods for diagnosing or categorizing disorders. The invention has particular utility for diagnosing or categorizing disorders in living animals from analysis profiles of biologically active materials such as neurotransmitters and other neurochemical substances in brain tissue, cerebrospinal fluid, plasma, serum, saliva, blood containing platelets, nasal mucosa, urine and the like, such as catecholamines, their precursors, cofactors and their metabolites. The invention is uniquely capable of differentiating a large number of compounds of biological, diagnostic and/or pharmaceutical significance and of using such differential for diagnosing disorders and will be described in connection with such utility although other uses are contemplated. 
     There is an extensive body of literature relating abnormalities in neurotransmitters, precursors, and metabolites to degenerative, neuropsychiatric and behavioral disorders, hypertension and certain carcinomas. See, for example, Schildkraut et al in  The Brain, Biochemistry and Behavior , Proceedings of the Sixth, Arnold O. Beckman Conference in Clinical Chemistry, pages 47-68. Although the potential role of these compounds in a number of significant disorders has been established, their routine analysis has not yet achieved widespread clinical use. Two problems in the clinical utility of neurotransmitter measurements are related to the economic and technical limitations of current technology. First, there is felt to be a high degree of interlaboratory and intersample uncertainty in quantitative values. Second, it has been difficult to measure enough of the known metabolically related compounds of a particular neurotransmitter to fully describe its biochemical significance in an individual sample, or to detect, identify and measure unusual neurotransmitters—an important aspect of basic research in various disease states that is presently very expensive and specialized. 
     While a number of interlaboratory technique intercomparisons for a variety of neurotransmitters have been carried out, there has been no comprehensive study within and among different techniques and laboratories for neurotransmitters in typical samples of interest. In the absence of such studies, given the complexity of the analytical problem and the historically wide variation whenever an analyte has been subjected to rigorous interlaboratory testing, the current values for normal and abnormal neurotransmitter levels must be taken with unspecified and probably wide limits of confidence. 
     Although the analysis of single neurotransmitters or metabolites from a complex biochemical pathway has been shown to correlate with a number of disorders utilizing statistical analysis over a large number of samples, the analytical level of a single neurotransmitter in an individual sample, with a few exceptions, has had relatively low clinical diagnostic utility. Essentially the state of the field of biochemical correlates of disorders is that while between large populations of normal and abnormal individuals a correlation generally can be determined for a particular biochemical, the scatter that results from both analytical and biochemical phenomena typically does not permit the level of a particular biochemical to be utilized diagnostically for a particular single individual. Nor may a single biochemical value be utilized for the rational prescription or development of a pharmaceutical for that individual. This is not particularly surprising in that both the levels and effects of a particular neurotransmitter are modified by a number of other neurotransmitters, in the same, or parallel metabolic pathways. If, for instance, 5-HT (serotonin) is to be used as a diagnostic tool for depression, suicidal tendencies, or schizophrenia, it would be necessary and perhaps provide a route to definitive diagnosis and pharmaceutical specification or development, to simultaneously determine the approximately 40 other compounds that derive from tryptophan and significantly effect the indolaminergic neuronal system&#39;s activity. 
     In recent years, LCEC (Liquid Chromotography with Electrochemical Detection) has become a common tool for the determination of catecholamines biogenic amines and their metabolites in biological fluids. Because of sensitivity limitations (typically 20-50 pg) and the complexity of biological samples, both separation and concentration steps typically have been necessary. Heretofore, plasma catecholamine analysis typically required three steps. First, the sample is collected and the catecholamines separated and concentrated, for example, using the alumina extraction procedure of Anton and Sayre (See A. H. Anton and D. F. Sayre, J. Pharmacol, Exp. Ther., 138 (1962), p. 360-375). The analytes, norepinephrine, epinephrine and dopamine, along with the internal standard DHBH (dihydroxybenzylamine), then are separated chromatographically, and finally detected electrochemically. Typical sample size requirements are 1.0 ml plasma or serum. In routine clinical use, there have been numerous problems with conventional techniques (alumina absorption, ion exchange and extraction), due to a large number of poorly understood variables, in the overall analysis system of sample acquisition, storage, preparation and sensor response. These problems have quite likely confused the relationships that may exist between levels and distribution of the catecholamines and various physiological and behavioral phenomena and disease states. 
     In the analysis of complex biological materials such as blood, serum and cerebrospinal fluids which may contain numerous different constituents, the important (e.g. abnormal) metabolites such as neurotransmitters to be identified may be present in only parts per trillion. While a chromatographic column can achieve macro separation of the various constituents, it may not provide adequate spatial (in time) separation of the extremely small portion of metabolites of interest from the much larger percentage of the many other compounds coeluted from the column at the same time as the metabolites of interest. Many of these interfering coeluted materials are electrochemically active but electrochemically irreversible, while many metabolites such as neurotransmitters are both electrochemically active and electrochemically reversible. It has been found that the analytical problems of reliable measurements of neurochemicals and related compounds are complicated by the fact that interferences with conventional or prior technologies are disorder related. This was discussed in my prior publication, (Matson et al, Clinical Chemistry, Vol. 30, No. 9, 1984) (see U.S. Pat. No. 4,511,659) for dopamine, dopac and seratonin measurements in directly analyzed brain extract anti cerebrospinal fluid for normal, schizophrenics and Alzheimers. Recent work has indicated that even for the widely used and accepted technique of alumina extraction for plasma catecholamines that interferences may be disorder specific. Higher values for Norepinephrine (NE) and Epinephrine (EP) were observed following alumina extraction and analysis of a single energy conventional electrochemical detector than for a three cell redox detector on neonatal stress blood samples. Analysis of the neonate extracts on the sixteen channel chemical imaging system revealed several unexpected compounds that are potential interferences including dihydroxyphenylacetic acid (DOPAC), 3 hydroxykynurenamine (3-OHKYA) and 3-hydroxy-anthranilic acid (3-OHAN). These compounds have not been detected in normal adult plasma alumina extracts. 
     In my aforesaid U.S. Pat. No. 4,511,659, there is provided an electrochemical detection system comprising a plurality of coulometrically efficient electrochemical cells, in series, for sequentially oxidizing and reducing selected substances in a sample solution under controlled conditions prior to measurement on a downstream testing electrode or electrodes. More specifically, in accordance with the invention provided in my aforesaid U.S. Pat. No. 4,511,659, a sample solution (e.g. a body fluid) is passed through a suitable chromatographic column and the eluant is streamed in contact with a series of electrochemically isolated, in-line coulometric electrodes operated under conditions so as to establish a series of “gates” for the sequential oxidation and reduction of substances in the sample solution whereby to screen (remove) selected interfering and electrochemically irreversible substances contained in the sample solution, while passing selected electrochemically reversible products for detection and measurement on a downstream electrode. The gate electrode series is follows in-line by one or more, preferably an array of six or more coulometric measuring electrodes, each formed of porous electrode base material such as fritted graphite, fritted carbon or other conductive fritted material, for detecting and measuring the electrochemically reversible compounds of interest (e.g. neurotransmitters). 
     As reported in my aforesaid U.S. Pat. No. 4,511,659, there are several beneficial effects of this approach to electrochemical analysis. Long-term drift in response is effectively eliminated by acquiring essentially 100% of the signal. The capability of analyzing essentially 100% of a material allows the assay of compounds of unknown purity by relating them to the basic principles of electrochemical reaction embodied in Faraday&#39;s Law. Poisoning of the electrode, a dominant problem with electrochemical sensors, is effectively eliminated by the use of a much larger relative surface area for reaction. And, finally, and most important to the eventual development of array and gate cells, a coulometric electrode by virtue of its essentially 100% efficiency allows sequential oxidation and/or reduction of compounds at successive-in-line detectors. The improved sensitivity of the detection system as discussed in my aforesaid U.S. Pat. No. 4,511,659, particularly where two or more active testing electrodes follow the screening electrodes has given the ability to do direct injections of serum filtrates and has also allowed the generation of reproducible patterns of compounds with catecholamine like electrochemical behavior of a large number of resolvable components. This provides the possibility of performing pattern recognition for the diagnosis or perhaps even predictive diagnosis, of various disorders or disease states. 
     In my copending application Ser. No. 797,615 and its parent U.S. Pat. No. 4,863,873, I describe a system for resolving and detecting hundreds of compounds in a single sample at femtogram levels whereby to provide a small molecule inventory or metabolic pathway pattern of an individual. As taught in my aforesaid U.S. Pat. No. 4,863,873, the small molecule inventory may be considered to reflect the underlying activity and distribution of the enzymatic pathways of an individual and hence reflect an operational measure of the genome determining those enzymes. The small molecule inventory of an individual may thus be used to determine the health state of the individual and/or to diagnose disease states. Correlation of the patterns from a plurality of individuals provides an understanding of the mechanisms of disorders or disease states or conditions and, in turn, provides a rational route to pharmacological development leading to treatment, cure or suppression of such disorders, disease states or conditions. 
     The present invention is an improvement in the invention described in my aforesaid U.S. Pat. No. 4,863,873. More particularly, in the practice of my invention as described in my U.S. Pat. No. 4,863,873, I have observed that the biochemical analysis profiles of “normal” or healthy individuals may vary quite widely, while the biochemical profile analysis data of individuals having disorders is far less chaotic. More particularly, I have observed that the frequency distribution of certain biochemical compounds or ratios of compounds in individuals suffering from a disorder are far less chaotic than “normal” or healthy individuals. This leads to a general protocol for diagnosing, categorizing or differentiating individuals based on comparisons of biochemical analytical data of small molecule inventory against data bases of known or previously diagnosed cases. By way of example the process of the present invention may advantageously be employed in the differentiation of neurological degenerative dementing or affective disorders such as Alzheimer&#39;s disease, Huntington&#39;s disease, Parkinson&#39;s disease, schizophrenia, or Progressive Supernuclear Palsy from each other and neurologically normal controls. Moreover, by suitable selection of variables, the process of the present invention also is applicable to classification of tumors, carcinomas, cardiovascular abnormalities and other disorders. Similarly, the process of the present invention advantageously may be utilized to select therapy based on categories of known successful vs. unsuccessful outcomes. 
     While not wishing to be bound by theory, the two fundamental hypotheses underlying the process of the present invention are: 
     1. The underlying genetic makeup or predis-position of an individual will reflect through the proteins, enzymes, and other factors it determines in patterns of small molecules. Individual components within these patterns will be affected by environmental effects such as diet, stress or chemical inset; however, the overall pattern of relationships will reflect the underlying operation of the genome or the interference of a particular disorder. Among the small molecules are the transmitters, cofactors and metabolites that regulate neuronal and endocrine functions and the interactions of somatic and central nervous system processes. Thus, the compounds such as purines, tyrosine and tryptophan derived neurotransmitters, peptides, pterin and vitamine cofactors are highly relevant to the effect or etiology of neurological disorders, cardiovascular disfunction and certain tumors or carcinomas. 
     2. The relationships of these biochemical patterns from a disorder are less chaotic or more regular than those from healthy controls. All of the biochemical systems of small molecules are interconnected and interrelated in a complex web of feedback and response. These interactions are highly nonlinear and thus, depending on subtle differences in initial conditions, the response of individual components in a biochemical pattern will be highly variable. The overall system will thus behave in a mathematically chaotic fashion. In a disorder, elements within the biochemical pattern are over or underregulated, thus reducing the degrees of freedom or overall variability. Consequently, the presence of a disorder implies more regulated or less chaotic variability of compounds or relationships among compounds in patterns from disordered individuals. 
     These two fundamental hypotheses provide an approach to diagnostic categorization of disorders using frequency distributions of compounds and relationships from large data bases (which may be of epidermiologically significant size). 
    
    
     DESCRIPTION OF THE DRAWINGS 
     For a fuller understanding of the nature and objects of the present invention, reference should be had to the following detailed description taken in combination with the accompanying drawings wherein: 
     FIGS. 1 a - 1   d  are plots showing frequency measurement distributions; 
     FIG. 2 is a plot showing sequential calculations; 
     FIG. 3 is a plot showing probability analysis; and 
     FIG. 4 is a plot similar to FIG. 3, and showing probability analysis corrected for error. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Methodology for Sample Analysis and Data Base Creation 
     280 cerebrospinal fluid (CSF) samples from the categories Alzheimer&#39;s Disease—AD (61 samples), Parkinson&#39;s Disease—PD (60 samples), schizophrenia—SC (60 samples), Huntington&#39;s Disease—HD (20 samples), Supernuclear Palsy—PSP (13 samples) and neurologically normal controls—C (68 samples), were electrochemically analyzed in accordance with the teachings of my aforesaid U.S. Pat. No. 4,863,873. Samples from normal and diseased individuals were prepared and flowed through a chromatographic column, and detected in an electrochemical cell using an NCA Chemical Analyzer, Model No. CEAS available from ESA, Inc., Bedford, Mass. All samples were from 7th or 8th mL aliquots of nostril caudal gradients. Pools were created for each category utilizing small subaliquots of the samples, and pools of all samples were created for analytical quality control and evaluation of unknowns. Samples were run under a variant of a standard reverse phase gradient procedure (Table 1) in the repetitive sequence Control Standard, Pool, 7 Samples, Control Standard, Pool, . . . as set forth in Table I: 
     
       
         
               
             
               
               
               
               
               
               
             
               
             
               
               
               
             
               
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
             
               
               
             
           
               
                 TABLE I 
               
               
                   
               
             
             
               
                 REAL TIME SETUP REVIEW 
               
             
          
           
               
                   
                 TIME 
                 DEVICE 
                 FUNCTION 
                 VALUE 
                 TOTAL 
               
               
                   
               
               
                 1 
                 0.00 
                 FLOW 
                 %B 
                 5 
                 1.20 
               
               
                 2 
                 0.20 
                 CLEAN CELL 
                 ON 
                 960  
               
               
                 3 
                 0.50 
                 CLEAN CELL 
                 OFF 
               
               
                 4 
                 7.00 
                 AUTO ZERO 
                 ON 
               
               
                 5 
                 7.20 
                 FLOW 
                 %B 
                 5 
                 1.20 
               
               
                 6 
                 7.58 
                 AUTO SAMPLER 
                 INJECT 
               
               
                 7 
                 8.00 
                 FILE 
                 START 
               
               
                 8 
                 66.00 
                 FLOW 
                 %B 
                 94  
                 1.06 
               
               
                 9 
                 66.00 
                 FLOW 
                 %B 
                 5 
                 1.20 
               
               
                 10  
                 70.00 
                 FILE 
                 STOP 
               
               
                 11  
                 70.00 
                 FLOW 
                 %B 
                 5 
                 1.20 
               
               
                 12  
                 70.00 
                 AUTO SAMPLER 
                 STEP 
               
               
                 13  
                 70.00 
                 END 
                   
                 2 
               
               
                 14  
                 70.00 
                 METHOD 
                 M147 
               
               
                   
               
             
          
           
               
                 REVIEW OF LIVE METHOD 
               
             
          
           
               
                   
                   
                 Autorange 
               
               
                 Full Scale Current 
                 P&#39;stats 
                 On 
               
               
                   
               
             
          
           
               
                 10 uA  
                 100 uA  
                 10 uA 
                 10 uA  
                 −40 mV 
                  25 mV 
                  90 mV 
                 155 mV 
                   
               
               
                 1 uA 
                 1 uA 
                 10 uA 
                 1 uA 
                 220 mV 
                 285 mV 
                 350 mV 
                 415 mV 
                 Floor 
               
               
                 1 uA 
                 1 uA 
                  1 uA 
                 1 uA 
                 480 mV 
                 545 mV 
                 610 mV 
                 675 mV 
                 100 nA 
               
               
                 1 uA 
                 1 uA 
                  1 uA 
                 1 uA 
                 740 mV 
                 805 mV 
                 870 mV 
                 910 mV 
               
             
          
           
               
                 Cell Box Temp: 35° C. 
               
               
                   
               
             
          
           
               
                   
                   
                 UPPER LIMIT 
                 LOWER LIMIT 
               
               
                   
                   
               
               
                   
                 PUMP A: 
                 350 
                 0 
               
               
                   
                 PUMP B: 
                 350 
                 0 
               
             
          
           
               
                   
                 VALVE: POS1 
               
               
                   
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
             
               
             
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE II 
               
             
             
               
                   
               
               
                 Sat. Nov. 18 17:15:22 1990 
               
             
          
           
               
                   
                 Standard  std0001 
               
             
          
           
               
                 Table: NOY8STD 
               
               
                 Study: CSFRUX01 
               
               
                 # Identified Compounds in Standard: 38 
               
               
                 # Missing Compounds in Standard: 0 
               
               
                   
               
             
          
           
               
                   
                 File 
                 Name 
                   
                   
                   
                   
                   
                   
                   
               
               
                 # 
                 RT 
                 Conc 
                 1 
                 2 
                 3 
                 4 
                 5 
                 6 
                 7 
               
               
                   
               
               
                 1 
                 std0001 
                 ASC 
                 1.717 
                 1.700 
                 1.725 
                 1.725 
                 1.750 
                 1.733 
                   
               
               
                   
                 1.725 
                 10000.00 
                 3872279 
                 9625125 
                 1.27e+07 
                 1.60e+07 
                 1.50e+07 
                 1.14e+07 
               
               
                 2 
                 std0001 
                 CYS 
                   
                   
                   
                   
                   
                   
                 2.267 
               
               
                   
                 2.283 
                 1000.00 
                   
                   
                   
                   
                   
                   
                 129467 
               
               
                 3 
                 std0001 
                 URIC 
                   
                   
                 2.333 
                 2.342 
                 2.350 
                 2.350 
               
               
                   
                 2.350 
                 0000.00 
                   
                   
                 5672 
                 1324197 
                 3193191 
                 1019128 
               
               
                 4 
                 std0001 
                 XAM 
               
               
                   
                 3.058 
                 1000.00 
               
               
                 5 
                 std0001 
                 HX 
               
               
                   
                 3.125 
                 100.00 
               
               
                 6 
                 std0001 
                 VMA 
                   
                   
                   
                 4.883 
                 4.867 
                 4.875 
                 4.875 
               
               
                   
                 4.875 
                 10.00 
                   
                   
                   
                 387 
                 4959 
                 16674 
                 13957 
               
               
                 7 
                 std0001 
                 GSH 
                   
                   
                   
                   
                   
                   
                 4.875 
               
               
                   
                 4.900 
                 500.00 
                   
                   
                   
                   
                   
                   
                 13957 
               
               
                 8 
                 std0001 
                 NE 
                 5.625 
                 5.642 
                 5.642 
                 5.650 
                 5.650 
               
               
                   
                 5.642 
                 10.00 
                 334 
                 18928 
                 17463 
                 1507 
                 117 
               
               
                 9 
                 std0001 
                 MHPG 
                   
                   
                   
                   
                 6.075 
                 6.075 
                 6.075 
               
               
                   
                 6.075 
                 10.00 
                   
                   
                   
                   
                 749 
                 13983 
                 5545 
               
               
                   
               
             
          
           
               
                   
                 File 
                 Name 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 # 
                 RT 
                 Conc 
                 8 
                 9 
                 10 
                 11 
                 12 
                 13 
                 14 
                 15 
                 16 
               
               
                   
               
               
                 1 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 2 
                 std0001 
                 CYS 
                 2.267 
                 2.258 
                 2.267 
                 2.275 
                 2.283 
                 2.292 
               
               
                   
                 2.283 
                 1000.00 
                 251043 
                 528682 
                 1298734 
                 1601456 
                 2250767 
                 1062584 
               
               
                 4 
                 std0001 
                 XAM 
                   
                 3.042 
                 3.033 
                 3.058 
                 3.067 
                 3.067 
                 3.108 
               
               
                   
                 3.058 
                 1000.00 
                   
                 47171 
                 1393667 
                 3304889 
                 2039171 
                 350667 
                 110609 
               
               
                 5 
                 std0001 
                 HX 
                   
                   
                   
                   
                   
                   
                 3.108 
                 3.117 
                 3.125 
               
               
                   
                 3.125 
                 100.00 
                   
                   
                   
                   
                   
                   
                 110609 
                 154070 
                 161138 
               
               
                 6 
               
               
                 7 
                 std0001 
                 GSH 
                 4.875 
                 4.875 
                 4.875 
                 4.892 
                 4.900 
                 4.988 
               
               
                   
                 4.900 
                 500.00 
                 28075 
                 51532 
                 117237 
                 133772 
                 197987 
                 85618 
               
               
                 8 
               
               
                 9 
                 std0001 
                 MHPG 
                 6.075 
                 6.083 
                 6.092 
               
               
                   
                 6.075 
                 10.00 
                 1828 
                 1718 
                 1044 
               
               
                   
               
             
          
           
               
                   
                 File 
                 Name 
                   
                   
                   
                   
                   
                   
                   
               
               
                 # 
                 RT 
                 Conc 
                 1 
                 2 
                 3 
                 4 
                 5 
                 6 
                 7 
               
               
                   
               
               
                 10 
                 std0001 
                 HGA 
                 6.392 
                 6.408 
                   
                   
                   
                   
                   
               
               
                   
                 6.392 
                 10.00 
                 16607 
                 4976 
               
               
                 11 
                 std0001 
                 G 
               
               
                   
                 7.942 
                 100.00 
               
               
                 12 
                 std0001 
                 GR 
               
               
                   
                 9.150 
                 500.00 
               
               
                 13 
                 std0001 
                 LD 
                 9.325 
                 9.283 
                 9.283 
                 9.292 
                 9.275 
               
               
                   
                 9.283 
                 10.00 
                 165 
                 10336 
                 14158 
                 1156 
                 562 
               
               
                 14 
                 std0001 
                 MET 
               
               
                   
                 9.650 
                 500.00 
               
               
                 15 
                 std0001 
                 AM 
                   
                   
                   
                 10.008 
                 10.008 
                 10.017 
                 10.000 
               
               
                   
                 10.008 
                 100.00 
                   
                   
                   
                 33745 
                 118573 
                 17512 
                 2128 
               
               
                 16 
                 std0001 
                 EPI 
                 10.167 
                 10.142 
                 10.150 
               
               
                   
                 10.142 
                 10.00 
                 367 
                 14152 
                 9783 
               
               
                 17 
                 std001 
                 EPI 
                 A 
               
               
                   
                   
                 10.217 
                 100.00 
               
               
                 18 
                 std0001 
                 DOPAC 
                 11.825 
                 11.833 
                 11.833 
                 11.842 
                 11.850 
                 11.833 
               
               
                   
                 11.833 
                 10.00 
                 247 
                 7794 
                 9749 
                 1412 
                 445 
                 251 
               
               
                   
               
             
          
           
               
                   
                 File 
                 Name 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 # 
                 RT 
                 Conc 
                 8 
                 9 
                 10 
                 11 
                 12 
                 13 
                 14 
                 15 
                 16 
               
               
                   
               
               
                 10 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 11 
                 std0001 
                 G 
                   
                 7.942 
                 7.942 
                 7.950 
                 7.958 
                 7.958 
               
               
                   
                 7.942 
                 100.00 
                   
                 2395 
                 122100 
                 53287 
                 4420 
                 975 
               
               
                 12 
                 std0001 
                 GR 
                   
                   
                 9.142 
                 9.133 
                 9.125 
                 9.142 
                 9.150 
                 9.175 
                 9.192 
               
               
                   
                 9.150 
                 500.00 
                   
                   
                 859 
                 26558 
                 194121 
                 430593 
                 1054244 
                 189936 
                 111915 
               
               
                 13 
               
               
                 14 
                 std0001 
                 MET 
                   
                   
                 9.642 
                 9.642 
                 9.650 
                 9.650 
                 9.650 
                 9.667 
                 9.675 
               
               
                   
                 9.650 
                 500.00 
                   
                   
                 986 
                 6461 
                 26013 
                 33548 
                 107832 
                 86595 
                 31459 
               
               
                 15 
               
               
                 16 
               
               
                 17 
                 std001 
                 EPI 
                 A 
                   
                   
                   
                   
                   
                   
                 10.250 
                 10.217 
               
               
                   
                 10.217 
                 100.00 
                   
                   
                   
                   
                   
                   
                   
                 26046 
                 36349 
               
               
                 18 
               
               
                   
               
             
          
           
               
                   
                 File 
                 Name 
                   
                   
                   
                   
                   
                   
                   
               
               
                 # 
                 RT 
                 Conc 
                 1 
                 2 
                 3 
                 4 
                 5 
                 6 
                 7 
               
               
                   
               
               
                 19 
                 std0001 
                 30HAN 
                   
                 13.225 
                 13.200 
                 13.217 
                 13.242 
                 13.250 
                   
               
               
                   
                 13.200 
                 10.00 
                   
                 1194 
                 29905 
                 8215 
                 2107 
                 1401 
               
               
                 20 
                 std001 
                 30HXY 
                   
                 13.225 
                 13.200 
                 13.217 
                 13.242 
                 13.250 
               
               
                   
                 13.200 
                 10.00 
                   
                 1194 
                 29905 
                 8215 
                 2107 
                 1401 
               
               
                 21 
                 std0001 
                 4HPLA 
               
               
                   
                 13.450 
                 100.00 
               
               
                 22 
                 std0001 
                 XMN 
                   
                   
                   
                   
                 13.683 
                 13.692 
                 13.683 
               
               
                   
                 13.692 
                 10.00 
                   
                   
                   
                   
                 1593 
                 14780 
                 3917 
               
               
                 23 
                 std0001 
                 4HBAC 
               
               
                   
                 13.992 
                 200.00 
               
               
                 24 
                 std0001 
                 TYR 
                   
                   
                   
                   
                   
                   
                 15.250 
               
               
                   
                 15.233 
                 1000.00 
                   
                   
                   
                   
                   
                   
                 232 
               
               
                 25 
                 std0001 
                 5HTP 
                   
                   
                 15.583 
                 15.592 
                 15.617 
               
               
                   
                 15.592 
                 10.00 
                   
                   
                 6994 
                 16297 
                 463 
               
               
                 26 
                 std0001 
                 DA 
                   
                 17.342 
                 17.342 
                 17.358 
                 17.367 
                 17.375 
               
               
                   
                 17.342 
                 10.00 
                   
                 17722 
                 6301 
                 305 
                 144 
                 8313964 
               
               
                 27 
                 std0001 
                 SHIAA 
                   
                 17.850 
                 17.892 
                 17.892 
                 17.908 
                 17.925 
               
               
                   
                 17.892 
                 50.00 
                   
                 233 
                 9330 
                 119759 
                 14604 
                 549 
               
               
                   
               
             
          
           
               
                   
                 File 
                 Name 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 # 
                 RT 
                 Conc 
                 8 
                 9 
                 10 
                 11 
                 12 
                 13 
                 14 
                 15 
                 16 
               
               
                   
               
               
                 19 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 20 
               
               
                 21 
                 std0001 
                 4HPLA 
                   
                 13.442 
                 13.450 
                 13.467 
                 13.458 
               
               
                   
                 13.450 
                 100.00 
                   
                 13364 
                 125434 
                 43118 
                 2233 
               
               
                 22 
               
               
                 23 
                 std0001 
                 4HBAC 
                   
                   
                   
                 13.983 
                 13.992 
                 13.992 
                 14.008 
               
               
                   
                 13.992 
                 200.00 
                   
                   
                   
                 75738 
                 207495 
                 311864 
                 170423 
               
               
                 24 
                 std0001 
                 TYR. 
                 15.242 
                 15.225 
                 15.233 
                 15.250 
                 15.250 
                 15.292 
               
               
                   
                 15.233 
                 1000.00 
                 3866 
                 102584 
                 1181673 
                 776764 
                 117510 
               
               
                 25 
               
               
                 26 
               
               
                 27 
               
               
                   
               
             
          
           
               
                   
                 File 
                 Name 
                   
                   
                   
                   
                   
                   
                   
               
               
                 # 
                 RT 
                 Conc 
                 1 
                 2 
                 3 
                 4 
                 5 
                 6 
                 7 
               
               
                   
               
               
                 28 
                 std0001 
                 4HPAC 
                   
                   
                   
                   
                   
                   
                 18.083 
               
               
                   
                 18.100 
                 200.00 
                   
                   
                   
                   
                   
                   
                 80 
               
               
                 29 
                 std0001 
                 KYA 
               
               
                   
                 18.117 
                 50.00 
               
               
                 30 
                 std0001 
                 30MD 
                   
                   
                   
                 21.575 
                 21.608 
                 21.608 
                 21.600 
               
               
                   
                 31.608 
                 10.00 
                   
                   
                   
                 58 
                 2183 
                 11535 
                 2176 
               
               
                 31 
                 std0001 
                 5HTOL 
                   
                 22.167 
                 22.200 
                 22.200 
                 22.217 
               
               
                   
                 22.200 
                 10.00 
                   
                 47 
                 1668 
                 19401 
                 1931 
               
               
                 32 
                 std00l 
                 HVA 
                   
                   
                   
                 23.925 
                 23.925 
                 23.917 
                 23.917 
               
               
                   
                 23.917 
                 200.00 
                   
                   
                   
                 225 
                 13994 
                 237705 
                 163964 
               
               
                 33 
                 std0001 
                 KYX 
               
               
                   
                 24.258 
                 100.00 
               
               
                 34 
                 std0001 
                 TYRA 
               
               
                   
                 24.492 
                 80.00 
               
               
                 35 
                 std0001 
                 5HT 
                   
                 30.742 
                 30.783 
                 30.792 
               
               
                   
                 30.792 
                 10.00 
                   
                 231 
                 6955 
                 9627 
               
               
                 36 
                 std0001 
                 3HT 
                   
                   
                   
                 31.967 
                 31.975 
                 31.975 
                 31.967 
               
               
                   
                 31.975 
                 100.00 
                   
                   
                   
                 143 
                 11501 
                 33026 
                 2425 
               
               
                   
               
             
          
           
               
                   
                 File 
                 Name 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 # 
                 RT 
                 Conc 
                 8 
                 9 
                 10 
                 11 
                 12 
                 13 
                 14 
                 15 
                 16 
               
               
                   
               
               
                 28 
                 std0001 
                 4HPAC 
                 18.117 
                 18.092 
                 18.100 
                 18.125 
               
               
                   
                 18.100 
                 200.00 
                 1143 
                 37258 
                 461257 
                 287050 
               
               
                 29 
                 std0001 
                 KYA 
                   
                   
                   
                   
                   
                 18.075 
                 18.117 
                 18.167 
                 18.175 
               
               
                   
                 18.117 
                 50.00 
                   
                   
                   
                   
                   
                 59412 
                 112086 
                 54548 
                 38306 
               
               
                 30 
                 std0001 
                 30MD 
                 21.592 
               
               
                   
                   
                   
                 143 
               
               
                 31 
               
               
                 32 
                 std0001 
                 HVA 
                 23.925 
                 23.925 
                 23.950 
               
               
                   
                   
                   
                 47372 
                 8852 
                 2293 
               
               
                 33 
                 std0001 
                 KYX 
                   
                   
                   
                 24.267 
                 24.258 
                 24.283 
                 24.300 
                 24.308 
                 24.308 
               
               
                   
                 24.258 
                 100.00 
                   
                   
                   
                 101620 
                 108998 
                 55261 
                 39928 
                 21718 
                 4609 
               
               
                 34 
                 std0001 
                 TYRA 
                   
                 24.475 
                 24.492 
                 24.517 
               
               
                   
                 24.492 
                 80.00 
                   
                 2368 
                 28935 
                 5390 
               
               
                 35 
               
               
                 36 
                 std0001 
                 3MT 
                 31.975 
               
               
                   
                   
                   
                 171 
               
               
                   
               
             
          
           
               
                   
                 File 
                 Name 
                   
                   
                   
                   
                   
                   
                   
               
               
                 # 
                 RT 
                 Conc 
                 1 
                 2 
                 3 
                 4 
                 5 
                 6 
                 7 
               
               
                   
               
               
                 37 
                 std0001 
                 TPOL 
                   
                   
                   
                   
                   
                   
                 41.500 
               
               
                   
                 41.467 
                 10.00 
                   
                   
                   
                   
                   
                   
                 375 
               
               
                 38 
                 std0001 
                 TRP 
                   
                   
                   
                   
                 45.350 
                 45.342 
                 45.388 
               
               
                   
                 45.292 
                 700.00 
                   
                   
                   
                   
                 615 
                 8371 
                 23358 
               
               
                   
               
             
          
           
               
                   
                 File 
                 Name 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 # 
                 RT 
                 Conc 
                 8 
                 9 
                 10 
                 11 
                 12 
                 13 
                 14 
                 15 
                 16 
               
               
                   
               
               
                 37 
                 std0001 
                 TPOL 
                 41.475 
                 41.458 
                 41.467 
                 41.492 
                   
                   
                   
                   
                   
               
               
                   
                 41.467 
                 10.00 
                 2006 
                 7109 
                 9905 
                 4810 
               
               
                 38 
                 std0001 
                 TRP 
                 45.300 
                 45.275 
                 45.292 
                 45.325 
                 45.350 
                 45.350 
               
               
                   
                 45.292 
                 700.00 
                 248497 
                 967515 
                 2056846 
                 1090677 
                 187013 
                 109610 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
             
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE III 
               
             
             
               
                   
               
               
                 Tue Dec 18 11:43:57 1990 
               
             
          
           
               
                   
                 Sample Report 
                 Page 1 
               
             
          
           
               
                 Sample: POOL37 
               
               
                 Standard: 
               
               
                 Table: POOL19A 
               
               
                 Study: CSF19A 
               
               
                 # Compounds identified: 22 
               
               
                 # Known Compounds Not Found: 0 
               
               
                 # Unknown Peak Clusters: 745 
               
               
                 Compounds Identified 
               
             
          
           
               
                 Com- 
                   
                   
                   
                   
                   
               
               
                 pound 
                 Conc 
                 RT 
                 RT Error 
                 Height 
                 Ratio Accuracy 
               
               
                   
               
             
          
           
               
                 *p01 
                 104.413971 
                 2.617 
                 0.100 
                 167971 
                 
                   
                     
                       
                         
                           7 
                           / 
                           6 
                         
                         0.999 
                       
                     
                             
                     
                         
                     
                   
                 
               
               
                   
               
               
                 XAN 
                 973.750732 
                 2.900 
                 0.117 
                 756026 
                 
                   
                     
                       
                         
                           
                             10 
                             / 
                             11 
                           
                           0.861 
                         
                          
                         
                           
                             12 
                             / 
                             11 
                           
                           0.959 
                         
                       
                     
                             
                     
                         
                     
                   
                 
               
               
                   
               
               
                 *p02 
                 101.869156 
                 3.108 
                 0.125 
                 208822 
                 
                   
                     
                       
                         
                           15 
                           / 
                           16 
                         
                         0.666 
                       
                     
                             
                     
                         
                     
                   
                 
               
               
                   
               
               
                 p03 
                 131.687439 
                 5.733 
                 0.275 
                  2747 
               
               
                   
               
               
                 p04 
                 106.228569 
                 8.242 
                 0.325 
                 142085 
                 
                   
                     
                       
                         
                           
                             13 
                             / 
                             14 
                           
                           0.977 
                         
                          
                         
                           
                             15 
                             / 
                             14 
                           
                           0.891 
                         
                       
                     
                             
                     
                         
                     
                   
                 
               
               
                   
               
               
                 TYR 
                 979.260254 
                 13.567 
                 0.692 
                 1375195  
                 
                   
                     
                       
                         
                           
                             9 
                             / 
                             10 
                           
                           0.987 
                         
                          
                         
                           
                             11 
                             / 
                             10 
                           
                           0.977 
                         
                       
                     
                             
                     
                         
                     
                   
                 
               
               
                   
               
               
                 *P05 
                  99.553802 
                 14.633 
                 0.583 
                  52515 
                 
                   
                     
                       
                         
                           13 
                           / 
                           14 
                         
                         0.949 
                       
                     
                             
                     
                         
                     
                   
                 
               
               
                   
               
               
                 P09 
                  89.990860 
                 15.117 
                 0.650 
                  7632 
                 
                   
                     
                       
                         
                           
                             9 
                             / 
                             10 
                           
                           0.850 
                         
                          
                         
                           
                             11 
                             / 
                             10 
                           
                           0.923 
                         
                       
                     
                             
                     
                         
                     
                   
                 
               
               
                   
               
               
                 P07 
                  92.967072 
                 15.275 
                 0.600 
                  55664 
                 
                   
                     
                       
                         
                           14 
                           / 
                           15 
                         
                         0.951 
                       
                     
                             
                     
                         
                     
                   
                 
               
               
                   
               
               
                 P08 
                  96.659180 
                 16.133 
                 0.417 
                  29579 
                 
                   
                     
                       
                         
                           10 
                           / 
                           11 
                         
                         0.976 
                       
                     
                             
                     
                         
                     
                   
                 
               
               
                   
               
               
                 *P10 
                  96.603462 
                 21.875 
                 0.942 
                  42516 
                 
                   
                     
                       
                         
                           9 
                           / 
                           10 
                         
                         0.949 
                       
                     
                             
                     
                         
                     
                   
                 
               
               
                   
               
               
                 HVA 
                 195.139450 
                 22.475 
                 0.800 
                  30508 
                 
                   
                     
                       
                         
                           
                             5 
                             / 
                             6 
                           
                           0.987 
                         
                          
                         
                           
                             7 
                             / 
                             6 
                           
                           0.921 
                         
                       
                     
                             
                     
                         
                     
                   
                 
               
               
                   
               
               
                 P11 
                 102.689194 
                 24.892 
                 0.717 
                  16233 
                 
                   
                     
                       
                         
                           16 
                           / 
                           15 
                         
                         0.892 
                       
                     
                             
                     
                         
                     
                   
                 
               
               
                   
               
               
                 P12 
                  91.103188 
                 25.150 
                 0.850 
                 10652 
                 
                   
                     
                       
                         
                           9 
                           / 
                           10 
                         
                         0.951 
                       
                     
                             
                     
                         
                     
                   
                 
               
               
                   
               
               
                 *p18 
                  98.121086 
                 26.850 
                 1.308 
                   470 
               
               
                 *p19 
                 121.804512 
                 27.192 
                 1.142 
                   324 
               
               
                   
               
               
                 P13 
                  96.415916 
                 28.158 
                 0.883 
                  53718 
                 
                   
                     
                       
                         
                           12 
                           / 
                           13 
                         
                         0.960 
                       
                     
                             
                     
                         
                     
                   
                 
               
               
                   
               
               
                 P14 
                  97.315338 
                 29.542 
                 1.108 
                  24489 
                 
                   
                     
                       
                         
                           13 
                           / 
                           14 
                         
                         0.969 
                       
                     
                             
                     
                         
                     
                   
                 
               
               
                   
               
               
                 P15 
                  94.104630 
                 29.642 
                 1.100 
                  10631 
                 
                   
                     
                       
                         
                           9 
                           / 
                           10 
                         
                         0.916 
                       
                     
                             
                     
                         
                     
                   
                 
               
               
                   
               
               
                 P16 
                  88.826569 
                 32.425 
                 0.767 
                  2937 
                 
                   
                     
                       
                         
                           10 
                           / 
                           11 
                         
                         0.761 
                       
                     
                             
                     
                         
                     
                   
                 
               
               
                   
               
               
                 P17 
                  96.782722 
                 37.992 
                 1.133 
                  22787 
                 
                   
                     
                       
                         
                           13 
                           / 
                           14 
                         
                         0.958 
                       
                     
                             
                     
                         
                     
                   
                 
               
               
                   
               
               
                 TRP 
                 674.284607 
                 42.150 
                 1.583 
                 277999 
                 
                   
                     
                       
                         
                           
                             9 
                             / 
                             10 
                           
                           0.979 
                         
                          
                         
                           
                             11 
                             / 
                             10 
                           
                           0.966 
                         
                       
                     
                             
                     
                         
                     
                   
                 
               
               
                   
               
             
          
         
       
     
     The analysis records were linked by a unique identifier to clinical data of clinical diagnosis, diagnostic criteria, age, pharmaceutical history, sex and race. Pools analyzed as samples against standards for known values were utilized to assess the precision of known compound values in the data base. Standards sequentially analyzed against identical standards were used as a measure of instrumental performance and pools sequentially analyzed against identical pools were utilized as a measure of the precision of unknown peaks. 
     Validation of the Data 
     Control standards analyzed against sequential control standards yielded precision values ranging from ±1%-±4% CV (coefficient of variation) with no outlying values. Pools analyzed as samples gave precision values ranging from ±2-±7% CV for compounds present at the 0.5 ng/mL level or greater and typically ±25-30% for compounds present at 2× the detection limit of 0.02-0.03 ng/mL (e.g. 5HT, EPI). Pools analyzed against sequential pools for unknowns gave values of ±3-±15% coefficient of variation. Typically, the coefficient of variation of the pools was 5-25 fold less than the coefficient of variation of analytes in a group of samples. Essentially, the contribution of assay variability to the results is minimal. 
     The data base, upon completion, contained 280 samples by 57 analytes (17,000 records). Of these, 163 were null either because no peaks were detected at the sensitivity limits of the assay, or because a signal detected did not meet the qualitative criteria for purity. 
     Regression Analysis 
     Linear regression analysis and stepwise regression analysis were used in a preliminary evaluation of the data. Both raw and mean corrected data was evaluated. 
     Regression comparison of the AD group (61) vs. controls (60) setting AD=1 and C=0 gave a categorical separation regression equation with an S (standard error of estimate) value=0.39 and p (the probability that the sample belongs in one group and not in another) value=0.0041 for 27 of the most significant known compound variables identified in stepwise regression (Table I). Inclusion of 7 of the most significant variables (labeled with asterisks in Table II) from the pool analysed unknown peak data base gave values of S=0.382 and p=0.0037. Assuming a clinical diagnostic error rate in the order of 10%, seven AD samples with regression calculated values (from −1.2 to 0.01) were removed from the calculation. The regression characteristics were then S=0.352 and p=0.0031. 
     Regression of the AD group with AD=1 vs. all others (219)=0 for the same variable group yielded an equation with S=0.481 and p=0.0013. 
     Observations: Although the AD group is separated from other groups with a high degree of probability, there is too high a degree of overlap for a simple linear regression algorithm to accurately categorize an individual sample. 
     Cluster Analysis Procedures 
     Cluster analysis procedures using nearest neighbor arid furthest neighbor approaches were applied to the data base. With both these approaches, the AD group tended to cluster, but controls were scattered relatively evenly, both outside and inside the AD region. Thus, the cluster analysis approach is not suitable as a categorization tool for this type of data. 
     Observations: The behavior of the data under cluster analysis protocols, and the observations that the standard deviations of compound values and of precursor/product ratios across metabolic pathways within a disorder group are smaller than within control groups is consistent with the hypothesis that the biochemical response of controls or normal individuals is more chaotic than that of disordered individuals. 
     Frequency Distribution Probability Analysis 
     The observations on the nature of the data distributions coupled with the technical ability to run large numbers of samples and variables offers an approach to categorization based on differences in the frequency distributions of variables in different disorder categories. This approach relies in basic probability considerations without any assumptions on the shape of a distribution curve of linearity of relationships. 
     The simplest question that I have investigated for the preliminary data base is that given an unknown sample, what is the probability (p) that that sample belongs in one group and not another. 
     For one variable, the question takes the form:        P   =         f        (     V   1     )       A           f        (     V   1     )       A     +       f        (     V   1     )       B                                
     where F(V n ) A  or f(V n ) B =the frequency with which an unknown sample value (V n ) occurs in category A or category B. 
     For multiple compounds, the expression expands:          P   =             f        (     V   1     )       A     ·       f        (     V   2     )       A                     …                     f        (     V   n     )       A                 f        (     V   1     )       A     ·       f        (   V   )       A                     …                     f        (     V   n     )       A       +           f        (     V   1     )       B     ·       f        (     V   1     )       B                     …                     f        (     V   n     )       B                                               
     If all frequencies are the same, the P value is 0.5 or a 50/50 chance that the unknown sample is A and not B. A positive answer compresses the expression to a 1 and a negative answer to 0. 
     Like cluster procedures and unlike regression, the use of the algorithm is independent of the number of variables used. 
     Implementation of the Procedure: 
     The implementation of the procedure is by the following steps: 
     1. Frequency distributions (shown in FIGS. 1 a - 1   d ) were created by using a smoothing algorithm based on a 3 point polynomial expansion function that treats each point in the sparse data distribution with equal weighting as the means of a distribution with a width at half height proportional to its value. The use of smoothing functions is a necessary assumption until the number of samples in a particular category reaches approximately 300-400. The procedure used was to divide all data in the categories by the maximum value among categories X 85, apply the polynomial expression algorithm, and normalize the data distributions for the number of samples in each category. The frequency distributions in each category are then organized into look up tables for each variable (Table IV) 
     
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE IV 
               
             
             
               
                   
               
               
                 DISTRIBUTION TABLE 
               
             
          
           
               
                   
                 HVA 
                   
                   
                 MHPG 
                   
               
               
                   
                 AD 
                 CONTROL 
                   
                 AD 
                 CONTROL 
               
               
                   
               
             
          
           
               
                 1 
                 0.010377 
                 0.000205 
                 1 
                 1.2E-10 
                 0.000000 
               
               
                 2 
                 0.045259 
                 0.001702 
                 2 
                 0.000000 
                 0.000003 
               
               
                 3 
                 0.126131 
                 0.007945 
                 3 
                 0.000000 
                 0.000026 
               
               
                 4 
                 0.242295 
                 0.025793 
                 4 
                 0.000000 
                 0.000140 
               
               
                 5 
                 0.379120 
                 0.064431 
                 5 
                 0.000000 
                 0.000576 
               
               
                 6 
                 0.503888 
                 0.129976 
                 6 
                 0.000001 
                 0.001932 
               
               
                 38 
                 0.798418 
                 0.938551 
                 38 
                 1.117459 
                 0.742461 
               
               
                 39 
                 0.761982 
                 0.889793 
                 39 
                 1.160736 
                 0.801194 
               
               
                 40 
                 0.730955 
                 0.836392 
                 40 
                 1.196534 
                 0.855713 
               
               
                 83 
                 0.006846 
                 0.088021 
                 83 
                 0.225452 
                 0.069938 
               
               
                 84 
                 0.005088 
                 0.081885 
                 84 
                 0.208132 
                 0.064130 
               
               
                 85 
                 0.003699 
                 0.075893 
                 85 
                 0.190170 
                 0.041177 
               
               
                   
               
             
          
         
       
     
     2. A sample record such as that shown in Table 5 
     
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE V 
               
             
             
               
                   
               
               
                 DATACASE RECORD 
               
             
          
           
               
                   
                   
                 C 
                 DISTRIBUT 
               
               
                   
                   
                 AT0022 
                 RANGE 
               
               
                   
                   
               
             
          
           
               
                   
                 MT3 
                 0.018080 
                 1 
               
               
                   
                 OHAN3 
                 0.018080 
                 3 
               
               
                   
                 OHKY3 
                 0.090402 
                 4 
               
               
                   
                 3OMD 
                 1.970763 
                 44 
               
               
                   
                 HVA 
                 58.03809  
                 56 
               
               
                   
                 MHPG 
                 8.714754 
                 62 
               
               
                   
                 12 
                 2911604 
                 22 
               
               
                   
                 P01 
                 5725159 
                 9 
               
               
                   
                 P02 
                 1359827 
                 49 
               
               
                   
                 P03 
                 1808040 
                 1 
               
               
                   
                   
               
             
          
         
       
     
     is then inserted into the look up table. Individual values are divided by the range value X 85 and the frequencies for each variable for category A and B are sequentially calculated in the algorithm after subtracting the effect of that sample from the frequency table. The effect of a sequential calculation across a group of variables is shown in FIG. 2 for 3 AD and 3 C cases from a group of 61 AD and 44 controls. One of the major features of the algorithm is that no single variable predominates as a differentiation among a large group of samples. 
     Testing the Algorithm on AD vs. Controls 
     For an initial test, conditions were set up such that each individual sample was evaluated as if the data base were set up without its contribution. The results of the initial scoring are shown in FIG.  3 . The scoring of five of the 61 AD cases as controls (p=less than 0.01 that the sample is an AD and not a control) is not surprising given the probable diagnostic error rate in AD. The scoring of 4 of the controls as AD are of concern. 
     One possible explanation is that the AD data base is in effect contaminated by five cases that clearly do not match the overall AD profile and are probably not AD. When these five samples are removed from the AD data base and all samples, including the 5 removed, are scored, the control and AD groups are uniquely separated as shown in FIG.  4 . The five samples that were removed from the AD scoring data group distribute in an equivocal region from 0.1 to 0.9. In subsequent application of the procedure and algorithm to AD samples vs. all other samples (PD, SC, HD, PSP and C) in the data base asking the question is this sample in the AD distribution and not in the distribution of all others yielded similar results scoring AD samples with p values=0.98 or greater. The distribution of scores of all others was scattered from 0.001 to 0.8 including the 5 AD samples which previously scored in this region vs. controls. 
     The invention has been described for use in diagnosing Alzheimer&#39;s Disease from CSF patient samples. It will be understood, however, that the invention advantageously may be used to diagnose and characterize other neurological, degenerative or defective disorders such as Huntington&#39;s Disease, Parkinson&#39;s Disease, schizophrenia, progressive supernuclear palsy, ALS amyotrophic lateral sclerosis (Lou Gehrig&#39;s Disease) and senile dementias. The invention also advantageously may be used to classify and diagnose tumors, carcinomas, cardiovascular abnormalities and other disorders, or for selection of therapy based on categories of known successful vs. unsuccessful outcomes. Moreover, both treatment protocols and new pharmaceuticals may be evaluated. 
     Still other changes and advantages will be obvious to one skilled in the art.