Technical Report No. IRL 1158

CYTOCHEMICAL STUDIES OF PLANETARY MICROORGANISMS
EXPLORATIONS IN EXOBIOLOGY

Status Report Covering Period January 1, 1972 to December 31, 1972
For

National Aeronautics and Space Administration
Grant NGR-05-020-004

 

Instrumentation Research Laboratory, Department of Genetics
Stanford University School of Medicine

Stanford, California 94305
Report to the National Aeronautics and Space Administration

"Cytochemical Studies of Planetary Microorganisms ~ Explorations in Exobiology

NGR 05-020-004

Summary Report Covering Period January 1, 1972 to December 31, 1972

Instrumentation Research Laboratory, Department of Genetics
Stanford University School of Medicine

Stanford, California

  
  

shua Lederberg Y
rincipal Investigator

 

 

Elliott C. Levinthal, Director ‘*

Instrumentation Research Laboratory
TABLE OF CONTENTS

A. INTRODUCTION

B. PROGRAM RESUME

I.

II.

Ifl.

IV.

VI.

VII.

VIII.

IX.

Chlorination of DNA Bases

Mass Fragmentography

Mass Spectrometry

Urine Analysis

Analysis of Natural Products by Mass Spectrometry
Dendral

A. Computer Science

B. Computer Aided Research Instrumentation
Cell Separation

A. High Speed Fluorescent Cell Sorter
Mariner Mars 1971

Viking Lander Imagery

C. REPORTS AND PAPERS
A. INTRODUCTION

This report covers the activities of the Instrumentation Research
Laboratory during the calendar year January 1, 1972 to December 31,

1972.

The main support of the IRL activities during this period continued
to be the NASA grant NGR 05-020-004. Some funds have come from other
grants, other agencies, and in some cases private institutions. This
report includes all the activities of the laboratory which relate to
or have benefited from NASA support regardless of whether or not they

were primarily supported by this NASA grant.

The Dendral project receives direct support from NIH grant GM 00612
and indirectly from the ACME grant NIH RR 00311 and Professor Feigenbaum's

ARPA grant SD-183.

Our work in the area of cell separation was supported in this period

by NIH grant GM 17367.
The efforts on image processing for the 1971 Mars Mariner are a
joint effort with the Artificial Intelligence Laboratory of the
Computer Science Department under Professor John McCarthy. It receives
support from NASA grant NGR-05-020-508 and JPL Contract 952489.
Our work on Viking lander camera imagery receives support under Langley

contract NAS 1-9682.

During the last six months of this report period we have had extensive
discussions with the Manned Spacecraft Center at Houston on ways to
exploit our experience in analytical methodology using: gas chromat ography
and mass spectroscopy for the purpose of improved physiological
monitoring of astronauts. In particular the work we have been doing

on the analysis of the metabolic constituents of urine has led to a
specific proposal. This proposal has been funded (NGR-~05-020-632) for

one year starting May 15, 1973.

We have continued to collaborate with Professor Marvin Chodorow of the
Applied Physics Department on a preliminary evaluation of the application
of microwave acoustic scattering to problems of cell discrimination

and detection.
The general areas of the Program Resume, Part B of the report, are:

II.

IIt.

IV.

VIIl.

IX.

Chlorination of DNA Bases

Mass Fragmentography

Mass Spectrometry

Urine Analysis

Analysis of Natural Products by Mass Spectrometry
Computer Aided Research (TENDRAL)

Cell Separation

Mariner Mars 1971 Orbiter Photography

Viking Lander Imagery
B. PROGRAM RESUME

I. Chlorination of DNA Bases

The study of the action of aqueous hypochlorous acid under physiological
conditions upon the bases present in DNA has been continued. Under

these conditions thymine yields 5-chloro-6-hydroxy-5, 6~—dihydrothymine
while with one and two equivalents of reagent 6-methyluracil is

converted into 5-chloro-6-methyluracil and 5,5-dichloro-6-hydroxy-5,
6-dihydro-6-methyluracil respectively. 1,3-Dimethyluracil reacts with
both one and two equivalents of hypochlorous acid to form 5,5-dichloro-6-

hydroxy-5 ,6-dihydro-1,3-dimethyluracil.

DNA bases containing the purine ring system, for instance guanine and
adenine, react with hypochlorous acid to yield parabanic acid. Xanthine
also yielded this product while the N-methylated purines caffeine and
theophiline afforded N-methyl parabanic acid. Mechanistically these
observations suggest that parabanic acid is derived from the six-membered

ring of the purine system.

Our chlorination studies were extended to the nucleosides, cytidine
and deoxycytidine and the nucleotide cytidine-5'-monophosphate. The
products from all three compounds were the corresponding 4-N-chloro
compound. This was determined from physical measurements including

mass spectrometry and especially from their NMR spectra which
exhibited a characteristic 0.42-0.52 ppm diamagnetic shift for the

chemical shift of the C-6 proton.

In addition the following papers on chlorination by hypochlorous acid
have been either published or accepted for publication:

Chlorination Studies. I. The Reaction of Aqueous Hypochlorous
Acid with Cytosine. By W. Patton, V. Bacon, A. M. Duffield,

B. Halpern, Y. Hoyano, W. Pereira and J. Lederberg.

Biochem. Biophys. Res. Commun., 48, 880 (1972).

 

Chlorination Studies. II. The Reaction of Aqueous Hypochlorous
Acid with a-Amino Acids and Dipeptides. By. W. E. Pereira,

Y. Hoyano, R. Summons, V. A. Bacon and A. M. Duffield.

Biophys. Biochem. Acta (in press).
II. Mass Fragmentography

Methods for the detection and quantitation of amino acids in varied
environments by mass spectrometry is being pursued. As the approach
used involves mass fragmentography we require the preparation of a
suitable derivative which must enhance the mass spectral identification
and in addition must possess suitable gas chromatographic properties.
The derivative of choice for these purposes would appear to be the amino
acid O-butyl ester, N-trifluoroacetate. Quantitation of the amino acid
levels present in crude soil extracts has been achieved by the addition
of an internal standard consisting of a known quantity of the deuterated
amino acids to be analyzed. Using the quadrupole-mass spectrometer-
computer system in the technique of mass fragmentography we have been
able to simultaneously quantitate up to ten of the amino acids present
in soil. The experimental details of this novel method await publication,
see "The Simultaneous Quantitation of Ten Amino Acids in Soil Extracts
by Mass Fragmentograph" by W. E. Pereira, Y. Hoyano, W. E. Reynolds,

R. E. Summons and A. M. Duffield, Anal. Biochem., in press.

We have also extended this quantitative technique to the measurement

of phenylalanine in plasma (i.e. phenylketonuria). See "The Determination
of Phenylalanine in Serum by Mass Fragmentography" by W. E. Pereira,

V. A. Bacon, Y. Hoyano, R. Summons and A. M. Duffield, Clinical Biochem.,

in press.
III. Mass Spectrometry

Phenothiazines represent a class of drugs commonly prescribed in
medicine as tranquilizers. Although the mass spectra of this group

of compounds have been investigated in several laboratories no definitive
study of their mass spectral decomposition processes using deuterium
labeling has appeared in the literature. Suitable deuterated derivatives
of promazine and promazine sulfoxide were prepared and from their mass
spectra the types of rearrangements occurring in the mass spectral
fragmentation processes of promazine and its sulphoxide were identified.
See "A Study of the Electron Impact Fragmentation of Promazine Sulphoxide
and Promazine using Specifically Dueterated Analogs," by M. D. Solomon,
R. Summons, W. Pereira and A. M. Duffield, Austral. J. Chem., 26, 325

(1973).
IV. Urine Analysis

Work is progressing on the organic chemical constituents of the urine

of premature babies hospitalized in the Pediatrics Ward of the Stanford
University Medical Center. Premature infants were selected for this

study because they are on strict diets and their urinary metabolites
should reflect body metabolites rather than food artifacts. Each urine
specimen is processed for their free acid and amino acid constituents

and these assays are repeated following hydrolysis of the urine. The
complex series of compounds contained in each of the four fractions is
separated by gas chromatography and the constituents of each chromatographic
peak identified (where possible) by their mass spectra signatures.
Currently our system can identify organic compounds present in derivatized

extracts below the microgram level.

As an example of the application of GC-MS to biomedical problems we
can cite preliminary studies on approximately 80 urine samples from a
total of 11 premature or "small for gestational age" infants. This
project was undertaken to investigate the phenomenon of late metabolic
acidosis. This condition is characterized by low blood pH levels and
poor weight gain and, as distinct from respiratory acidosis, occurs
after the 2nd day of life. Its incidence is higher in infants whose
birthweight is less than 1750 g (one study shows 92% incidence for
these children) than in infants with birthweight greater than 1750 g

(282%).
Of the 11 patients studied we were able to observe 6 closely and
continuously for periods ranging from 6 to 8 weeks from day 3 of life.
Three of these infants had birthweights below 1000 g and the other
three were born weighing less than 1500 g. Of the 6, five showed
symptoms corresponding to late metabolic acidosis and the other showed
normal and even development. The five infants showing the acidosis
all excreted very large amounts of p-hydroxyphenyllactic acid
together with smaller amounts of p-hydroxyphenylpyruvic acid and
p-hydroxyphenylacetic acid. After reaching a peak, the occurrence

of these compounds in the urine gradually diminished and were almost
completely absent at the time blood pH and weight gain had returned

to normal. The infant who did not show symptoms of acidosis only
excreted minute amounts of these compounds during the period of

observation.

The occurrence of large amounts of these compounds in the urine
indicates a temporary defect in phenylalanine - tyrosine metabolism
and dietary factors such as protein and vitamin intake can be shown to
affect the incidence and the severity of the condition. It is hoped
that further studies will result in a clearer picture of relationships
between the condition and diet and hence lead to a reduction in its

occurrence.
Vv. Analysis of Natural Products by Mass Spectrometry

During the past year research has continued on the structural analysis
by mass spectrometry of natural products isolated from plant, animal
and marine sources. A list of publications resulting from this
experimentation follows:

E. Ali, P. P. Gosh Dastidar, S. C. Pakrashi, L. J. Durham and
A. M. Duffield. "Studies on Indian Medicine Plants - XXVII
Sequiterpene Lactones of Enhydra Fluctuans Lour. Structures
of Enhydrin, Fluctuanin and Fluctuadin." Tetrahedron 28,
2285 (1972).

A. M. Duffield and 0. Buchardt. "Thermal Fragmentation of
Quinoline and Isoquinoline N-Oxides in the Ion Source of a
Mass Spectrometer." Acta Chem. Scand., 26, 2423 (1972).

A. N. H. Yeo and C. Djerassi. "Mass Spectrometry in Structural
and Stereochemical Problems. CCX. Evidence for Transition
States of Different Ring Sizes in the Loss of C,H, from

Phenyl n-Butyl Ether in the Mass Spectrometer." J. Am. Chem.
Soc. 94, 482 (1972).

B. A. Brady, W. I. O'Sullivan and A. M. Duffield. "The
Electron-Impact Promoted Fragmentation of Aurone Epoxides."
Organic Mass Spectrometry 6, 199 (1972).

P. Sedmera, A. Klasek, A. M. Duffield and F. Santavy.
"Pyrrolizidine Alkaloids. XIX. Structure of the Alkaloid
Eruotfoline." Coll. Czech. Chem. Commun., 37, 4112 (1972).

 

P. Perros, J. P. Morizur, J. Kossanyi and A. M. Duffield.
"Spectrometrie de Masse VIII. Elimination d'can Induite par

Impact Electronique dans le Tetrahydro-1,2,3,4-Naphtal-ene-diol-1,2.
Org. Mass Spectrom. 7, 357 (1973).

Y. M. Sheikh, R. J. Liedtke, A. M. Duffield and C. Djerassi.
"Mass Spectrometry in Structural and Stereochemical Problems
CCXVII. Electron Impact Promoted Fragmentation of O-Methyl
Oximes of some a,8-Unsaturated Ketones and Methyl Substituted
Cyclohexanones." Canad. Jour. Chem. 50, 2776 (1972).

10
VI. Dendral

A. Computer Science

Recent Progress in the Dendral Artificial Intelligence Project is

summarized in the following report.

il
B, Computer Aided Research Instrumentation

1. Objectives and Background

The objectives of this work are to develop and
demonstrate computer techniques for automating complex
laboratory instrumentation and procedures. Within these
objectives, three main areas of investigation are of
interest including: 1) methodologies for computer modeling
and control of instrument performance, 2) the integration of
intelligent data analysis and interpretation programs into
closed loop systems to optimize and economize problem
solutions, and 3) the organization of computing and hardware
resources needed to implement automated systems. We have
chosen gas chromatography/mass spectrometry (GC/MS) as a
specific environment in which to develop and test ideas.
This choice is based on the importance of GC/MS to on-going
research in medicine, biochemistry, ‘and computer science
described elsewhere in this report, as well as a genuine
need for system automation because of severe data volume and

analysis complexity problems.

2. Progress

Since the last report, we have made progress in
developing aspects of computer-aided GC/MS instrumentation

as summarized below,

12
a) MASS SPECTROMETER DATA SYSTEM AUTOMATION

Concentrating initially on the MAT-711 high resoluttion
mass spectrometer, we have made progress toward a reliable,
automated data acquisition and reduction system for scanned
low and high resolution spectra. This system is largely
failsafe and requires no operator support or intervention in
the calculation procedures. Output and warnings to the
operator are provided on a CRT adjacent to the mass
spectrometer. The system contains many interactive features
which permit the operator to examine selected features of
the data at his leisure. The feedback currently provided to
the operator to assist in instrument set-up and operation
can just as well be routed to hardware contro! elements for
these functions thereby allowing computer malntenance of

optimum instrument performance.

Progress in this area is an integration of our efforts

in hardware and software improvements:

HARDWARE - The basic system consists of the mass
spectrometer Interfaced to a PDP-11/20 computer for data
acquisition, pre-filtering, and time buffering fnto the ACME
time-shared 360/50. The more complex aspects of data
reduction are done in the 360/50 since the PDP-11 has
limited memory and arithmetic capabilities. New interfaces
for mass spectrometer operation and control have been
developed. The interfaces can handle (through an analog

13
multiplexer) several analog inputs and outputs which require
that the PDP-11 computer be relatively near the mass
spectrometer. We now have the capability for the following

kinds of operation through the new interfaces.

1) Computer selection of digitization rate

ti) Computer selection of data path (interrupt mode or

direct memory access (DMA)

iif) Direct memory access for faster operation In the

data acquisition mode,

iv) Computer selection of analog input and output

channels.

v) Sensing of several analog channels through a

multiplexer (e.g., ion signal, total ton current).

vi) Magnet scan control. This control can be exercised
manually or set by the computer. {ft controls both time
of scan and flyback time. Coupled with selection of scan

rate, any desired mass range can be scanned at any

desired scan rate,

vii) The computer can monitor the mass spectrometer's
mass marker output as additional information which will

be used to effect calibration.

Another development has been a signal conditioner for

14
the fon signal which incorporates a box-type Integrator to
sum the jon signal between A/D converter readings. This
modification makes successive intensity readings independent
of each other because the integrator is reset after each
reading. It also provides for low pass filtering the fon
current signal with a bandwidth automatically adjusted
correctly for different sampling rates and hence lessens
intensity measurement uncertainties caused by external

noises.

SOFTWARE - Automatic instrument calibration and data
reduction programs have been developed to a high degree of
sophistication. We can now accurately model the behavior of
the MAT-711 mass spectrometer over a variety of scan rates
and resolving powers. Our instrument diagnostic routines are
depended upon by the spectrometer operator to tndicate
successful operation or to help point to instrument
malfunctions or set-up errors. Some features of these

programs are described below.

1) Data Acquisition, Programs have been written which
permit acquisition of peak profile data at high data rates
using the PDP-11 as an intermediate data fliter and buffer
store between the mass spectrometer and ACME. This allows
data acquisition to proceed even under the time constraints
of the time-sharing system. Storage of peak profiles rather

than all data collected has greatly reduced the storage

15
requirements of the program and saves time as the hackground
data (below threshold) are removed in real-time. An
automatic thresholding program is in operation which
statistically evaluates background noise and thresholds
subsequent data accordingly. Amplifier drift can thus be
compensated. We have developed some theoretical models of
the data acquisition process which suggest that high data
acquisition rates are not necessary to maintain the
integrity of the data. Demonstration of this fact with
actual data has helped relieve the burden of high data rates
on the computer system, particularly as tmposed by GC/MS
operation, and permits more data reduction to he
accomplished in real-time or alternatively reduces the

required data acquisition computer capacity.

ii) Instrument Evaluation. A high resolution mass
spectrometer operating in a dynamic scanning mode is a
complex instrument and many things can go wrong which are
difficult for the operator to detect in real-time. !n order
for the computer to assist fn maintaining data quality, ft
must have a model of spectrometer operation on the basis of
which data quality can be assessed and processing suitably
adapted as well as instrument performance optimized. We have
developed a program which monitors the state of the mass
spectrometer. This preliminary program checks the following

items:

16
1) Data acquisition parameters such as scan range and
time constants, background threshold, a dynamic peak
model to determine resolution and threshold acceptance
levels for peak width and intensity, the number of peaks

collected, and data storage utilization statistics.

2) Calibration of the mass/time relation to be used as a
model for subsequent spectra, output of the mass range
over which the scale is calibrated, calibration peaks
missed, if any, and a graph of extrapolation error versus
mass. Any irregularities in this output point to scan

problems.

3) The dynamic resolution versus mass is determined and
Output as a graph. This allows the operator to adjust to

more constant resolution over the mass range.

iff) Data Reduction. A program has been written which
allows automatic reduction of high resolution data based on
the results of the prior instrument evaluation data.
Converston of peak positions tn time to the corresponding
mass values is effected by mapping each spectrum Into the
calibration model developed previously. The interpolation
algorithm between reference calibration points incorporates
a quadratically varying exponential time constant to account
for the second order character of a magnet discharging
through a resistance and a capacitance. It also takes into

account a mass offset at infinite time which affects

17
accuracy in determining low masses and which results from

residual magnetization at the end of a scan.

Perfluorokerosene (PFK) peaks, Introduced Into high
resolution mass spectra for internal mass calibration, are
distinguished from unknown peaks by a pattern recognition
algorithm which compares the relationships between sequences
of reference peaks in the calibration run with the set of
possible corresponding sequences in the sample run. The
candidate sequence is selected which best approximates
calibrated performance within constraints of tnternally
consistent scan model variations. This approach minimizes
the need for selection criteria such as greatest negative
mass defect for reference peaks, the validity of which
cannot be guaranteed. Excellent performance results from
using sequences containing 10 reference peaks,

Unresolved peaks are separated by a new analytical
algorithm, the operatton of whfch is based on a calculated
model peak derived from known singlet peaks rather than the
assumption of a particular parametric shape fe.g.,
triangular, Gaussian, etc.) This algorithm provides an
effective increase in system resolution by a factor of three
thereby effectively increasing system sensitivity. By
measuring and comparing successive moments of the sample and
model peaks, a series of hypotheses are tested to establish

the multiplicity of the peak, minimizing computing

18
requirements for the usually encountered simple peaks.
Analytic expressions for the amplitudes and positions of
component peaks have been derived in the doublet case in
terms of the first four moments of the peak complex. This
eliminates time consuming iteration procedures for this
important multiplet case. Iteration fs still required for

more complex multiplets.

Elemental compositions are calculated from high
resolution mass values with a new, efficient table look-up
algorithm developed by Lederberg (ref. 1) and appended

herewith.

Future work will extend these ideas to a system for the
acquisition of selected metastable information as well as to
include the quadrupole system used in the. routine low

resolution clinical work.

b) GAS CHROMATOGRAPHY/HIGH RESOLUTION MASS SPECTROMETRY

We have recently verified the feasibility of combined
gas chromatography/ high resolution mass spectrometry
(GC/HRMS). Using the programs described above we can acquire
selected scans and reduce them automatically, although the
procedures are slow compared to "real-time" due to the
limitations of the time-shared ACME facility. We have
recorded sufficient spectra of standard compounds to show

that the system is performing well. A typical experiment

19
which illustrates some of the parameters Involved was the
following. A mixture (approximately 1 microgram/ component)
of methyl] palmitate and methyl stearate was analyzed by GC
under conditions such that the GC peaks were well separated
and of approximately 25 sec. duration. The mass spectrometer
was scanned at a rate of 10.5 sec/decade, and a resolving
power of 5000. The resulting mass spectra displayed peaks
over a dynamic range of 100 to 1 and were automatically
reduced to masses and elemental compositions without
difficulty. Mass measurement accuracy appears to be 10 ppm
over this dynamic range. A more definitive study of mass
measurement accuracy will. be carrfed out shortly to

accurately determine the performance of the system,

We have begun to exercise the GC/HRMS system on urine
fractions containing significant components whose structures
have not been elucidated on the basts of low resolution
spectra alone. Whereas more work is required to establish
system performance capabilities, two things have become
clear: 1) GC/HRMS will be a useful analytical adjunct to our
low resolution GC/MS clinical studies to assist in the
identification of significant components whose structures
are not elucidated on the basis of low resolution spectra
alone, and 2) the sensitivity of the present system limits
analysis to relatively intense GC peaks. This sensitivity
limitation is Inherent in scanning instruments where one
gives up a factor of 20-50 in sensitivity over photographic

20
image plane systems in return for on-lfne data read-out.
This limitation may be relieved by using television read-out
systems in conjunction with extended channeltron detector
arrays as has been proposed by researchers at the Jet
Propulsion Laboratory. The development of such a sensor
system is beyond the current scope of our effort. We can
nevertheless make progress in applying GC/HRMS techniques to
accessible effluent peaks and can adapt the improved sensor

capability when available,

Recent experiments in operation of the mass
spectrometer In conjunction with the gas chromatograph have
also shown that the present ACME computer facIlity cannot
provide the rapid service required to acquire repetitive
scans at either high or low resolving powers. We can,
however, acquire scans on a periodic basis, meaning most GC
peaks In a run can be scanned once at high resolving power,
We are presently implementing a disk on‘the PDP-11 to act as
a temporary data buffer between the mass spectrometer and
ACME. This disk will allow acquisition of repetitive scans,
while data reduction must be deferred to completion of the

GC run,

c) AUTOMATED GC/MS DATA REDUCTION

The application of GC/MS techniques to clinical
problems has made clear the need for automating the analysts

of the results of a GC/MS experiment. Previous paragraphs

21
dealt with the problems of reducing raw data in preparation
for analysis. At this point the data must be analyzed with a
minimum of human interaction in terms of locating and
identifying specific constituents of the GC effluent. The
subsequent problem of identification is addressed by the
library search and DENDRAL mass spectrum interpretation
programs. The problem of locating effluent components in the
GC/MS output involves extracting from the approximately 700
spectra collected during a GC run, the 50 or so representing
components of the body fluid sample. The raw spectra are In
part contaminated with background "column bleed" and in part
composited with adjacent constituent spectra unresolved by

the GC.

We have begun to develop a solution to this problem
with very promising results. By using a unfque disk oriented
matrix transposition algorithm developed for image
processing applications, we can rotate the entire array of
700 spectra by 500 mass samples per spectrum to gain
convenient access to the "mass fragmentogram" form of the
data. This form of the data, displayed at a few selected
mass values, has been used at Stanford, MIT, and elsewhere
for some time to evaluate the GC effluent profile as seen
from these masses. Mass fragmentograms have the important
property of displaying much higher resolution in localizing
GC effluent constituents. Thus by transposing the raw data

to the mass chromatogram domain we can systematically

22
analyze these data for baselines, peak positions, and
amplitudes, and thus derive tdealized mass spectra for the
constituent materials free from background contamination and
influences of adjacent GC peaks unresolved in the overal]
gas chromatogram. These spectra can then be analyzed by

library search techniques or first principles as necessary.

The results of this work can also lead to reliable
prescreening analysis of GC traces alone by having available
a detailed list of GC effluent positions and expected
amplitudes for say a urine fraction. By dynamically
determining peak shape parameters for detected GC singlet
peaks, interpretation of more complex peaks can be made to
determine if unexpected constituents or abnormal amounts of

expected constituents are present,
d) CLOSED-LOOP INSTRUMENT CONTROL

In the long term, It would be possfble for the data
interpretation software to direct the acquisition of data in
order to remove ambiguities from interpretation procedures
and to optimize system efficiency. The achievement of this
goal is a long way off but we feel the above developments
along with progress in the computer interpretation of mass
spectra (DENDRAL) represent important preliminary steps

toward closed-loop control.
The task of collecting different types of mass spectral

23
information (e.g., high resolution spectra, low ionizing
voltage spectra and selected metastable Information) under
closed loop control during a GC/MS experiment is difficult
and may not be realizable with current technology. We are
studying this problem fn a manner which will allow the
system to be used for Important research problems (e.g.,
routine analysis of urine fractions without fully closed
loop control) while aspects of instrument control strategy

are developed in an incremental fashton.

The essence of this approach is to develop a multi (two
or three)-pass system which permits collection of one type
of data (e.g., high resolution mass spectra) durfng the
first GC/MS analysis. Processing of these data by DENDRAL
will reveal what additional data are necessary on specific
GC peaks during a subsequent GC/MS run to‘uniquely solve the
Structure or at least to reduce the number of candidate
structures. This simulated closed-lo@p procedure wil]
demonstrate the ability of DENDRAL type programs to examine
data, determine solutions and propose additional strategies,
but will not have the requirement of operating in real-time,
although some parameters in the acquisition of metastable

data will require change between consecutive GC peaks,

Studies such as these will identify in some detail the
feasibility and necessity of closed-loop automation as well

as the portions of the procedure which must be improved to

24
meet the time constraints imposed by limited sample
quantities and GC/MS operation. We have already identified
the problem of the rate at which resolution can be changed
and have determined a potential solution. Additional
problems under study are those of instrument sensitivity and

strategies for metastable fon measurement.

3. Future Plans

Our future plans represent extensions of the on-going
work described above under "PROGRESS" as well as the
continued routine maintenance of the GC/MS systems.
Specifics are briefly summarized below. A significant Impact
will occur with the termination of NIH support of the ACME
computing facility in July 1973. We now perform most of our
data reduction processing on the ACME 360/50. The follow-on
facility to ACME will be an unsubsidized, fee-for-service
facility mounted on a 370/158 computer along with other

Stanford Hospital administrative computing functions.

We are in the process of implementing interim,
stand-alone support for our GC/MS work on avallable
PDP-11/20 machines. We have a number of proposals pending
with NIH which would support longer term solutions, either
through augmented stand-alone PDP-11 capabilities or through

funds to operate on the fee-for-service 370/158,

a) MASS SPECTROMETER DATA SYSTEM AUTOMATION

25
Future efforts will include the transition of the
existing ACME-based system to a follow-on system, the
character of which will depend on NIH funding approval for
one of the alternatives. We will adapt previously developed
concepts for use in the Finnigan low resolution GC/MS system
being used for routine urine analysis. We will develop data
system extenstons for the MAT-711 system which allow
semi-automated acquisition and reduction of metastable
information to support fragmentation pathway studies,
Heuristic DENDRAL program development, and closed-loop
simulation. This metastable system will ftncorporate
calibration procedures and automated peak detection and
resolution procedures based on the high resolution system.
The existing hardware interface will be used to control
source or electrostatic analyzer voltages [n conjunction
with the magnet scan to measure specific parent-daughter ion

relationships.
b) GAS CHROMATOGRAPHY/HIGH RESOLUTION MASS SPECTROMETRY

We will complete the intermediate disk buffer in
conjunction with the follow-on computing system transition
to allow routine collection and filing of sequential
spectra. We will exercize the system on body fluid samples
in support of our clinical applications and the development
of interpretation programs. As developments occur which

Improve sensitivity, we will incorporate these to extend the

26
power of the system.
c) AUTOMATED GC/MS DATA REDUCTION

The approach described above is still in the formative
stage. We will complete the development and implementation
of these ideas, test them In the clinical application domain
and produce an automated system suitable for routine use by

the blochemist.
d) CLOSED-LOOP INSTRUMENT CONTROL

With the development of a more automated method for
acquiring information on metastable peaks (under subtask (a)
plans), we will develop and exercise the strategy planning
aspects of the Heuristic DENDRAL programs in connection with
managing a urine analysis GC/MS run. This. will be a
simulation of closed-loop operation intended to demonstrate
the feasibillty and need for an actual Implementation of
these ideas. In support of these closed-loop simulations we
will investigate the feasibility of Instrument mode
switching and simple control function such as fon source and

electrostatic analyzer potentials and magnet scan.

27
REFERENCE

1) Lederberg, Joshua, "Rapid Calculation of Molecular
Formulas from Mass Values," Journal of Chemical Education,

Vol. 4&9, Page 613, September, 1972.

28
VII. Cell Separation

While this work was initiated by the subject grant, most of the support
is now coming from NIH Grant GM 17367. The work has obvious applications

in the medical field as well as possible applications for exobiology.

A. High Speed Fluorescent Cell Sorter

This unit is designed to measure the fluorescence of cells in a jet of
liquid, break-up the jet into uniform drops and collect the drops in a
series of containers, with all drops containing cells with similar

fluorescent characteristics collected in the same container.

Our last annual report for the period ending December 31, 1971, described
a multichannel cell separator which had just been completed and upon
which testing was just commencing. The new instrument simultaneously
measures fluorescence and scattering cross section of each cell. Both
Signals are used as sorting parameters. Much of our recent efforts

have been devoted to evaluating, operating and improving this new

instrument.

Installation of a second, more powerful laser (4 watts) has improved
the sensitivity and made it possible to operate the new system completely
independently of the old instrument which continues to be frequently

used. Many modifications to the fluid and sample handling components

29
of the system have resulted in more reliable operation, accurate
monitoring of sample flow rate, rapid flushing and sample changing, and
fast recovery from nozzle blockages. The electronic signal processing
logic has been replaced with improved circuitry that permits independent
specification of scattering and fluorescence signal limits for each

of two separated fractions. Detected cells that do not satisfy the
criteria for sorting are positively isolated to the undeflected fraction,
thus preventing contamination of the wanted cells. Purity of the
separated fractions has also been improved by identifying pairs of cells
too closely spaced to properly sort, and also isolating these cells to
the undeflected fraction. These improvements also keep most empty
droplets and much unwanted debris from either separated fraction. Cells
are now processed at rates of several thousand per second with separated
fraction purities routinely between 90% and 99%. Further refinement of
the decision making circuitry is expected to increase both processing

rate and fraction purity.

Programs for processing and plotting data from the two parameter 1024
channel pulse height analyzer have been written. A storage indicator
and circuitry for displaying two-parameter "scatter plots" of cell

parameters has been added to the system.

The minimum detectable number of molecules of rhodamine or fluorescein
has been determined using radioassay techniques and conconavalin A

(Con A), a protein from jack bean meal which binds to carbohydrate

30
residues on the cell surfaces. The Con A was labelled with both the
fluor and with 23, The p25 count on cell suspensions was used to
determine the number of Con A molecules, and thus of fluor molecules
bound. The number of fluor molecules bound was reduced until it was
just possible to detect a signal above background noise in the cell

separator. This minimum number seemed to be less than 4000 for either

fluor.

In most of our work fluors are conjugated with immunoglobulins.
Considering that each immunoglobulin molecule will usually contain more
than one fluor molecule, and that further amplification can be obtained
using the immunofluorescence "sandwich" technique, it appeared that cells
with on the order of a few hundred active sites on their surface should
be separable under ideal conditions using this unit. This was confirmed
by tests on thymocytes carrying a few hundred molecules of synthetic
polypeptide, T, G, A----- L, labelled with yi5 as a marker for
autoradiography, reacted with a fluorescent antibody to T, G, A----- L

and separated. The practical separation limit is usually set by nonspecific

adhesion of fluor-containing molecules to the cells.

A major effort has been devoted to achieving sufficiently asceptic
operation that separated fractions can be cultured. This has involved
basic redesign of the flow system and much more care in sample and
machine treatment. The work has been successful and several cultures

have been grown, indicating anew that cells are viable after passing

31
through the unit.

The scatter channel has proved more useful than anticipated. The signal
it provided with various red blood cell samples was compared to that
from a Coulter counter. The variation with volume appeared to be
somewhat less than linear. However larger cells gave larger signals,
and the resolution of neighboring signals was significantly better in

the scatter channel than in the Coulter.

This equipment makes many desirable but previously impossible biological
experiments not only possible but relatively easy. The following are
among the many applications which have been made:
1. Rabbit lymphocytes bearing different membrane antibody light
chain markers have been separated and shown to give rise

preferentially to plasma cells bearing the same markers.

2. Spleen cells from mice immunized with two different antigens
were incubated with one of the antigens after the latter had
been made fluorescent. These fluorescent cells were then
separated. The non-fluorescent portion retained all of its
antibody titer to the second antigen but lost most of its
titer to the first, indicating that the activity was on

different cells.

3. Populations of different apparent sizes have been separated
from both mouse spleen and mouse thymus lymphocyte suspensions.

Preliminary indications are that the spleen lymphocytes giving

32
the smaller scatter signal are dead, but the smaller thymus
fraction appears to be functionally different than the larger,
or at least to react differently to hydroxycortisone treatment
of the animal. Experiments in this area, and in those discussed

below are continuing.

Electron micrographs of antigen reactive
cells have been made showing the location of molecules of

antigen on the cell surface.

Preliminary results on treatment of separated cells with mitogens
like PHA (phytohemagglutinin) and PWM (Pokeweed mitogen)

indicate these reagents can successfully stimulate division in

T but not B cells. Interferon is also produced in T but not B
cell cultures, in contradiction to earlier speculations that

B-cells are also interferon producers.

A series of experiments was conducted to,determine the minimum
leakage of Rh+ fetal cells into an Rh- maternal circulation
which could be detected. In these tests Rh+ antibody was added
to suspensions of Rh- cells containing various proportions of
Rh+ cells, and the suspension then treated with fluorescent goat
antihuman gamma globulin. Results showed that Rh+ cells could
be detected easily by the separator at dilutions of 107°, and

with care as low as 10°,

33
Work has started on using the separator to fractionate fetal
lymphocytes from maternal peripheral blood using HLA antigens
as markers. Preliminary experiments indicate that a sandwich
technique, in which the cells are treated with antiserum to the
father's HLA antigens, then with fluoresceinated rabbit anti-
human gamma globulin, and run through the separator should be
able to provide considerable enrichment of the fetal cells.

It may be possible eventually to use this technique to provide

fetal cell cultures for use in prenatal diagnosis of chromosome

abnormalities.

34
VIII. Mariner Mars 1971 Orbiter Photography

The image processing work being carried out in cooperation with the
Stanford Artificial Intelligence Project has resulted in about 500 image
difference operations on MM'72 photos. Thirty-one images of the
martian moons, Phobos and Deimos, were also processed to enhance their
contrast and high frequency information. Late in the reporting period
special emphasis was put on differencing images of the proposed landing

sites for Viking 1975.

The image differencing operation which is described in detail in
Technical Report IRL 1123 has represented our major interest. Candidate
images for differencing are selected by ourselves and members of the
MM~71 Image Team (primarily C. Sagan and J. Veverka). The necessary

image data and navigation data is read from magnetic tapes supplied by

Jet Propulsion Laboratory. The scientist requesting a particular set of
image differences then has the opportunity to observe the work in progress
and make decisions while the processing is being done. This interactive
approach is possible through the availability of the Stanford AI time-
sharing system operating on a PDP-10 computer and the associated image

processing equipment.

Work has also been done in the area of image information management. An
information retrieval capability has been implemented at the Stanford
AI project which enables us to quickly review the planet coverage of the

MM-71 TV Mission. It is primarily oriented toward revealing the extent

35
of repeated TV coverage of any area specified by latitude and
longitude. It enables the user to quickly determine if an area has
been photographed, and if so, how many times, on which orbits, by which
camera, and by which pictures within an orbit. On a display screen is
shown the disk of the planet, the footprint of the images, and vectors
indicating view and sun angles (See Figure 1). Since more than seven
thousand images exist, the need for such a system is obvious. This
capability could prove quite useful in picture targeting and landing

site refinement for the Viking mission.

It is important to note that this is an interactive system oriented
towards the needs of the scientists. Its success depends on its ability
to present data in a manner consistent in format and organization with

the way the experimenters view the object under investigation.

The above mentioned capability actually represents the initial phase

of the process for the projection and differtncing operation. With the
identifiers and footprints of all the images before the user the list
can be pruned until just the footprints of interest are present. The

user can then proceed directly to the projection and differencing steps.
The above capacity, when combined with a disc based storage system gives

the scientist a significant degree of flexibility to review the image

data and the processing carried out on it.

36
 

 

 

 

 

 

 
A total of forty-nine picture differences were made which covered the
areas at or near nine of the ten candidate landing sites for the Viking
1975 mission. One such difference can be seen in Figure 2. This
example shows the area around Viking Landing Site three. The upper

left image was taken on orbit 168 and the upper right on orbit 209 forty
days later. During this time significant clearing took place, the
differences left minus right and right minus left are shown in the

lower left and right respectively.

A portion of the Viking related work was presented at a Viking landing

site selection meeting in November 1972.

Projects for the next reporting period include a comprehensive study of
the albedo changes which occurred between the MM-6 and MM-7 period and
the MM-9 mission, completing work on a catalog of enhanced Phobos and

Deimos images and a limited number of additional MM-9 differencing.

38
ae

@ EDR: NPROF AVG ORTHO : , VW1ié EDR: NPRCF AWG ORTHO
eae aioe | toa

4 : ; : :
rerabvceedceantesaetacridveridecssetserridane

chathareebersi dare

seaepereepetesparaerer

111811 DAB 16 EDR: MPROF AY eRe: b uel cme! EBR: MPROF AWG
QRTHO ALIGN ia el!) ORTHO ALIGN = EDR: MPRCF
AVG ORTHO CUSTER CUSTER STRETCH cele CUSTER TER STRETCH

Le

 

Figure 2

39
IX. Viking Lander Imagery

During the last year, Drs. Levinthal and Liebes, as members of the

Lander Camera Science Team have assumed team responsibility for the
Science Operations Requirements Document (SORD), the Software Functional
Descriptions (SFD's) derived from SORD and the more detailed Software
Requirements Documents (SRD's) that, in turn, are derived from the SFD's.
In addition they are part of the ad hoc Viking Image Processing System
Steering Committee which is planning the hardware configuration to meet
the requirements for the imaging science component of Mission Operations.
All of the above activities, while separately supported by Langley, relate
to, and benefit from previous efforts in connection with the Mariner

Mission and some of the basic work supported by this grant.

40
C. REPORTS AND PAPERS

This section lists reports and papers not referred to in preceding

sections of the Program Resume.

REPORTS

1.

Annual Report for Period July 1, 1971 to June 30, 1972. JPL
Contract 95289. Instrumentation Research Laboratory, Genetics
Department, Stanford University. ''Mariner Mars 1972". IRL Report
1143 (1972).

PUBLICATIONS

1.

A. M. Duffield, W. E. Reynolds, D. A. Anderson, R. A. Stillman, Jr.,
and C. E. Carroll. "Computer Recognition of Metastable Ions."

Proc. Nineteenth Annual Conference on Mass Spectrometry and Allied
Topics, D4, 63-67 (1971).

W. E. Pereira, M. Solomon, B. Halpern. ''The Use of (+)-2,2,2-Trifluoro-
1-Phenylethylhydrazine in the Optical Analysis of Asymmetric Ketones
by Gas Chromatography." Aust. Jour. Chem. 24, 1103 (1971).

W. A. Bonner, H. R. Hulett, R. G. Sweet and L. A. Herzenberg.
"Fluorescence Activated Cell Sorting." Rev. Sci. Instr. 43, 404
(1972).

M. D. Solomon, W. E. Pereira, and A. M. Duffield. "The Determination
of Cyclohexylamine in Aqueous Solutions of Sodium Cyclamate by
Electron-Capture Gas Chromatography." Analytical Letters, 4(5),

301 (1971).

B. G. Buchanan, A. M. Duffield, A. V. Robertson. "An Application
of Artificial Intelligence to the Interpretation of Mass Spectra."
In "Mass Spectrometry: Techniques and Appliances" edited by George
W. A. Milne, Published by John Wiley & Sons, Inc. 1971, pp. 121-178.

J, Cymerman Craig, W. E. Pereira, B. Halpern and J. W. Westley.

"Optical Rotatory Dispersion and Absolute Configuration-XVII
a-Alkylphenylacetic Acids." Tetrahedron 27, 1173 (1971).

41
7.

10.

ll.

12.

13.

14.

15.

L. H. Quam, S. Liebes, Jr., R. B. Tucker, M. J. Hannah, B. G. Eross.
"Computer Interactive Picture Processing." Stanford Artificial
Intelligence Report Memo AIM-166, STAN-CS~72-281. (1972).

L. G. Brookes, M. A. Holmes, I. S. Forrest, V. A. Bacon, A. M.
Duffield and M. D. Solomon. "Chlorpromazine Metabolism in Sheep II.
In Vitro Metabolism and Preparation of 3H-7-Hydroxychlorpromazine."'

Agressologie 12, 333 (1971).

W. E. Pereira and B. Halpern. "The Steric Analysis of Aliphatic
Amines with Two Asymmetric Centres by Gas Liquid Chromatography of
Diastereoisomeric Amides." Aust. J. Chem. 25, 667 (1972).

A. Buchs, A. B. Delfino, C. Djerassi, A. M. Duffield, B. G. Buchanan,
E. A. Feigenbaum, J. Lederberg, G. Schroll and G. L. Sutherland.
"The Application of Artificial Intelligence in the Interpretation of

Low Resolution Mass Spectra." Advances in Mass Spectrometry, Vol. 5
314 (1972).

 

 

E. Steed, W. E. Pereira, B. Halpern, M. D. Solomon and A. M. Duffield.
"An Automated Gas Chromatographic Analysis of Phenylalanine in Serum."
Clinical Biochemistry, in press.

V. Tortorella, G. Bettoni, B. Halpern, P. Crabbe. "Optical Properties
of Dimedonyl Derivative of Aromatic Amines and Amino Acids." Tetrahedron
28, 2991 (1972).

E. C. Levinthal, W. B. Green, J. A. Cutts, E. D. Jahelka, R. A.
Johansen, M. J. Sander, J. B. Seidman, A. T. Young and L. A.
Soderblom. "Mariner 9 - Image Processing and Products." Icarus,
in press.

T. A. Mutch, A. B. Binder, F. 0. Huck, #. oC. Levinthal, E. C. Morris,
C. Sagan, and A. T. Young. 'Imaging Experiment: The Viking Lander"
Icarus, in press.

C. Sagan, J. Veverka, P. Fox, R. Dubisch, J. Lederberg, E. Levinthal,

L. Quam, R. Tucker, J. B. Pollack, and B. Smith. "Variable Features
on Mars: Preliminary Mariner 9 Television Results." Icarus, in press.

42