fluorescent excitation analysis in medicine

Journal of Radioanalytical Chemistry, Vol. 43 H9.78) 321-346

FLUORESCENT EXCITATION ANALYSIS IN MEDICINE

L. KAUFMAN,* D. C. PRICE,* M. A. HOLLIDAY,* B. PAYNE,* D. C. CAMP,** J. A. NELSON,*** F. DECONNINCK****

*University of California, San Francisco (USA) **Lawrence Livermore Laboratory (USA)

***University o f Utah (USA) * ** * Vrije Universiteit, Brussels (Belgium)

(Received August 8, 1977)

The fluorescent excitation analysis equipment and techniques developed at the Univer- sity of California, San Francisco are reviewed and the recent advances and their impact on extending the applicability are discussed.

Introduction

In the biological sciences, radiotracer techniques have become a potent tool for the study of physiologic processes and for diagnostic purposes. X-ray fluorescence is a technique that has achieved a considerable degree of success in identifying and quantitatirtg elemental composition of biological, mineral and processed samples. Under certain conditions it is possible to realize the accuracy and simplicity of radiotracer methodology through fluorescent excitation analysis (FEA). This assay technique for stable tracers has become the method of choice for performing a number of important clinical and investigative studies, and is slowly diffusing through the medical community. We review here the FEA equipment and techniques developed at the University of California, San Francisco, and discuss recent advances and their impact on extending the applicability of FEA.

F E A in medicine

The use of stable tracers assayed by fluorescent excitation analysis (FEA) offers a number of advantages over use of radionuclides in clinical and investigative applications. Principally, radiation exposure to patients and attending personnel is either drastically reduced or eliminated, thus allowing certain tests to be extended

J. Radioanal. Chem. 43 (1978) 321

L. KAUFMAN et al.: FLUORESCENT EXCITATION ANALYSIS

to serial studies in normals, pregnant women and children. Also, the technique is fast and accurate. Since the need to purchase and handle (what are often) short- lived and expensive radioisotopes is avoided, FEA is a cost-effective means of im-

plementing a number of diagnostic and research studies. In one of its first and most succesful clinical applications, FEA has been .devel-

oped as a tool to image the~ distribution of naturally occurring stable iodine in the

thyroid gland} In our investigations we have emphasized the replacement of radioactive tracer methodology with FEA.

The most complete review of the medical work performed up to 1971 with this technique is found in Semiconductor Detectors in the Future of Nuclear Med-

icine, z The Proceedings of the Symposium on Semiconductor Detectors in Medi- cine, 3 provide a review of later work.

The principle of FEA

When an element in a sample is exposed to X- or gamma-rays, the energy of which is higher than the binding energy (BE) of the K-shell electrons in that element, vacancies can occur in these shells. An L- or M-shell electron will then oc- cupy the K-shell vacancy. In this process, the atom will emit a photon of energy Ea or E/3, given by

Ea = B E ( K ) - BE(L)

and E/~ = B E ( K ) - BE(M).

These photons are called the Ka and K/~ characteristics radiations and are unique to each element. Similarly, lower energy characteristic X-rays are obtained by ion- ization of L-shell electrons. Since the excitation probability decreases rapidly as the

energy of the exciting photons increases (~E -3) it becomes desirable to use pho-

tons close in energy to BE(K) or BE(L) depending on whether K-shell or L-shell excitation is desired. However, if the energy is too close to the binding energy, Compton scattered photons overlap the characteristic X-rays and the signal-to-background ratio decreases. Compton scattering results in secondary photons of an energy lower than that of the exciting beam, file exact value of that energy depend-

ing on the scattering angle. Multiple Compton scattering also produces a diffuse background of lower energy X-rays. Scattered photons have two deleterious effects on the accuracy with which tracer concentrations are measured:

(a) They produce background counts in the characteristic X-ray channel of interest, thus degrading the signal-to-noise ratio; and

(b) They limit the speed with which characteristic X-rays can be detected.

322 J. Radioanal. Chem. 43 (1978)


Compton-scatter Compton-sca'der

-~ JCharacteristic X-rags)

Energg Backgrounds

Fig. 1. Typical spectrum obtained by 24 ~Am FEA of an iodine-containhlg solution, with Si(Li) detector collimated at 90-deg to the excitation beam

Limitations due to background

Referring to Fig. 1, in which a schematic drawing o f a typical fluorescent spectrum of a tracer-containing sample is shown, we notice the following features: co- herent scatter (no energy loss) from the source; Compton-scatter from the sample;

the effects of these latter photons undergoing further Compton-scatter in the detector; a degraded energy multiple-scatter region (mostly from inside the detector); the characteristic X-rays from the tracer; and background, i.e., non-fluorescence photons in the characteristic X-ray energy window.

We have chosen to measure the tracer background in absolute terms, in parts per million (ppm), and it is obtained as follows: defining NK as the counts in the

Ka characteristic X-ray channel, NC as the counts in an arbitrary region of the Compton-scattef spectrum, B as NK/NC for a sample of water, and W the concentration of tracer in sample, then

where K is a constant obtained from measuring NK and NC for a sample of known concentration W. From Eq. (1), the background in the units of K is KB, and is corrected for dead-time and Sample-volume variations. This quantity can be thought of as the tracer concentration necessary to double the counts NK when compared

r

to NK for water alone.

It is usual to state that the statistical validity of counting is determined by the counts in the channels of interest, thus, the percent error would be A = 100/x/N---K.


L. KAUFMAN et al-: FLUORESCENT EXCITATION ANALYSIS

Since NK is linear in time, reproducibility would then increase as x~. However, this is only true if KB "~ W, that is, if the background is negligible compared to the concentration of interest. For the case where KB > W, it is easy to show from Eq. (1) that A is multiplied by a term x / ~ + KB/W), that is, the statistics are worsened by a term containing the ratio of background to concentration. Thus reduction of background is of paramount importance in obtaining an accurate quan-

titation of very low level tracer concentrations.

Count rate limitations

Since the statistics of counting any one sample can be improved by obtaining more counts, it would seem desirable to use stronger sources for excitation, as well as large area, high efficiency detectors, to increase the count rate and statistics in

a fixed time. In practice, currently available amplifiers give rise to degraded detec-

tor resolution at count rates above 20 K-counts/second. For low concentrations of tracer, in the best detector systems, no more than 1 out of every 1,000 detected

events is in the Ka channel. Thus, fluorescence sources cannot be made more in- tense, since Compton scattered X-rays will eventually set a limit on data rates.

Detectors for FEA

Since the tracer signal may represent less than 0.1% of all detected events, high resolution detectors must be used in FEA. We have investigated the use of lithium- drifted silicon, Si(Li), and high purity germanium, I-IPGe, for this purpose, and have found that, Nven a fixed source - sample - detector configuration and essentially equivalent energy resolution, background among different detectors show variations over a twenty five-fold range. Since this is a key parameter in the quantitation of low-level tracers, careful consideration of the detector is critical in attain- ing sensitive systems for medical applications.

The use of HPGe was evaluated for FEA of iodine. Although the absolute detection efficiency of this material is superior to that of silicon, it is as high in the 30 keV region where, the iodine K X-rays are, as at the exciting beam energy

(60 keV). Thus dead-time introduced by detection of Compton scattered photons decreased the effective detection efficiency of characteristic radiation..In addition, the background was observed to rise drastically in three HPGe detectors obtained from two different manufacturers (Fig. 2).

Because of their excellent energy resolution, good signal-to-background ratios, and ready availability, Si(Li) detectors are preferred. Since the main limitation of the technique in terms of its applicability to medicine is one of minimal quantitat-

324 J. Radioanal. Chem. 43 (1978}


Fig. 2. A comparison of the spectra obtained under identical conditions for a sample with 1 mg/cm a of iodine, counted for 100 seconds using (a) an 80 mm 2 , 5 mrn deep HPGe detector, and (b) an 80 mm 2 , 3 mm deep Si(Li) detector. (Ordinates are shown in logarithmic scale). Notice the lower counts in the iodine K peak when the HPGe detector is used, as well as the higher background observed in this detector

able levels, we have sought to reduce detector-generated sources of background

through improved designs. In collaboration with the KeVex corporation, systematic

improvements have been realized, and systems with iodine background levels as

low as 8 ppm are now available.

In-vitro FEA analysis

An automated FEA analyzer for in-vitro work has been recently completed 4

(Fig. 3). It consists of a "low-background" KeVex detector, a KeVex 4510P am-

plifier, and Tennelec NIM bin and detector bias supply. Data is accumulated in an

Ino-Tech Ultima 2,000-channel programmable pulse-height analyzer (MCA). This unit incorporates a minicomputer behind the front panel. A program tape sets up

automatic operation. For in-vitro analysis the operator enters by teletype or front panel the NK and NC limits, and the constants B and K. Accumulation modes of

constant time or constant NK can be selected, but because of the wide dynamic range of the system a preferred mode sets minimum and maximum count time,

with accumulation otherwise completed when a preset integral is reached. Printout

includes NK and NC, counting time, the concentration W and the "statistical error"

KX/-N-K-/NC, where term are defined as in Eq. (1). Physiologic parameters of inter-

est can also be obtained by entering the appropriate ancillary data. The MCA con-

trois a sample changer developed in collaboration with the Lawrence Livermore

Laboratory. It holds 48 2-cc disposable vails, and includes a sample mix mode

that can be activated for whole-blood samples. The unit may also be operated

manually, or can be set up to do repeated assays of the same sample load. s

Z RadioanaL Chem. 43 (1978) 325


Fig. 3. Automated FEA system. Sample changer and Si(Li) detector with 2 41Am source are on the left and programmable PHA on right. Disposable vials are shown on PHA shelf

System performance

It is customary to define performance in terms of "sensitivity", the tracer con-

centration that yields a net signal equal to three times the square root o f the

counts for a tracer-free sample, both accumtdated in the characteristic X-ray chan-

nel in a counting time of 1,000 sec. Clearly sensitivity is a measure of the con-

fidence of detectabil i ty of a tracer. In terms of the medical applications discussed

here, such a measure has little significance. Maximizing sensitivity does not neces-

sarily maximize the accuracy of the result obtained from Eq. (1). For instance,

Fig. 4 shows that the maximum sensitivity occurs at an NK window with a width

of l o ( l o = FWHM). On the other hand, maximum accuracy of quanti tat ion is

dependant on the tracer concentration range of interest. Fig. 5 shows opt imum

NK window as a function of W/KB, the ratio of tracer concentration to absolute

326 J. Radioanaf_ Chem. 43 (t978j


E

e-0.8 c ,g

~ 0 . 6

>

go4 t~

0.5 I L I J J

1.0 1.5 2.0 2.5 3 0 NK window width~x5

Fig. 4. System sensitivity for iodine as a function of NK window width (1 ~ =FWHM)

m 10

5

0 w

10 1 5 2.0 Optimum NK window width, x 6

Fig. 5. Optimum NK window width for various iodine concentrations. Since in our system KB = 8ppm, these are the units of the ordinate axis

background, indicating the need for a variable window. In practice another para-

meter must be taken into account: Given the drift specifications of present analog

to digital converters, it is unavoidable to have random drifts o f at least one chan-

nel per 2,000 channels of memory. In our system this represents a drift o f 0.0830.

Fig. 6 shows the error introduced by shifts of one and two channels. As can be

seen, a minimum NIL window width of 1.5o should be used to remain reliably

within a 2% margin of error.

In a complex peak such as the K~ line of iodine, which contains a Kal line at

28.610 keV and a Ka2 line at 28.315 keV, the NK window should be further

broadened to compensate for the separation between the two lines.



-- 12

t ,

tu 10

8

6

2 -

0 1 2 3

NK w indow width~ x 0

Fig. 6. Systematic errors introduced in quantitation by energy digitization shifts of one and two channels

Because o f the ratio of NK/NC is used to obtain W [Eq. (1)], results are inde-

pendent of dead-time effects, and in first order, of sample volume and positioning.

The response of the system is linear for elements such as bromine, iodine and ce-

sium concentrations determinations over a broad concentration range, from less

than 1 ppm to over 2 -4% concentration. The upper range is limited by self ab-

sorption and electronic degradation in performance at very high counting rates, and

the lower limit is set by statistics. This large dynamic range is of importance in

medical work, where low concentration biologic samples have to be compared to

higher concentration injectates. Table 1 summarizes some system performance para-

meters. The accuracy o f concentration determinations for the elements of interest

are shown in Tables 2, 3 and 4.

Table 1 System performance parameters (April 1975)

Excitation Net count rate, Background, "Seqsitivity", Tracer source counts/sec/ppm ppm ppm

Bromine

Iodine Cesium

1 o 9 Cd

24~Am 24~Am

0.209

0.382

0.373

13.3

8.1 12.7

0.75

0.44

0.55

328 Jr. Radioanal. Chem. 43 (1978)


Table 2 Bromine

Counting Concentration Reproducibility, time range, ppm %

10s

30 s

l m

2 m

1 5 m

40 000-1 400

40 0 0 0 - 2 200

1 000

190

100

6

a < 2

o < 1

a----1

o ~ 2

a ~ 2

a ~ 5

Table 3 Iodine

Counting Concentration Reprodtieibilit y, time range, ppm %

10s

30 s

l m

5 m

30 m

l h

20 000-1 700

20 0 0 0 - 550

300

75

30

~3

-1

o ~ 1

o ~ 1

o ~ 2 - 3

cr ~ 3 - 4

Counting time

10s

30 s

l m

1 5 m

30 m

l h

Table 4 Cesium

Concentration range, ppm

20 000-1 600

750

370

100

6

3

1

Reproducibility, %

o~-1

a < l

o~=1

o~-2

a~-3

a~-6

a = 1 0

J. Radioanal. Chem. 43 H978) 329

L KAUFMAN et al.: FLUORESCENT EXCITATION ANALYSIS

Applications of in-vitro FEA

Established applications of in vitro FEA fall into two main areas: The study of

body compartments and the study of the kinetics of X-ray contrast agents.

Body compartments

The size of a body compar tment is usually assessed by measuring the volume

of distribution of a tracer known to distribute in that compartment. Typically, a

known weight of tracer is administered and blood samples obtained after equilib-

ration are assayed to obtain tracer concentrations. For instance, bromide (in the

form of NaBrTL cesium-labeled red cells ~ and iodinated albumin are used to meas-

ure extracellular fluid volume (ECFV), red cell volume (RCV) and plasma volume

(PV), respectively (Table 5).

Kinetics of iodinated contrast agents

Organ function can be assessed by measuring the time dependence o f the con-

centration of a tracer in tissues such as blood, plasma, urine, bile and cerebrospinal

fluid. For instance, Conray 60 (meglumine iothalamate) is a common iodinated

contrast agent that can be used to measure glomerular fdtrat ion rate (GFR), an

indicator of kidney function 9 (Fig. 7).

The pharmacokinetics of biliary contrast agents is being extensively studied with

FEA. These studies are leading to improved pharmaceuticals, more effective tech-

niques to obtain , opacification, and to an improved understanding of hepatophy siology, t O-12

Table 5 Summary of clinical in-vitro FEA tests

Test FEA label Conventional label Difference +_1 S.D. Subjects

Extra cellular Fluid volume

Red cell

volume Glomerular

Filtration rate

Plasma volume

Stable bromine 1-2% accuracy

Stable cesiura-labelled red cells

Conray 60 (stable meglumine

iothalamate)

Stable iodi- hated albumin

B 2 Br, 2.6% accuracy

s t Cr labelled

red cells Inulin (constant infusion)

1 ~ s I lothalamate

(single injection)

,2 s I albumin

0.15•

0.9 t8 .0 3.6 • 0.24• 6.3 •

1.1 •

0.4 + 7.6

Rats, human

Humans Rabbits Humans

Rats

Humans

Rabbits

330 J. RadioanaL Chem. 43 (1978)


E &

o

c

"5

300

200

100

80

60

40

20

--1~ �9 Conreg 60 g o 1251 iothalamate

8

- - I I �9 8

I.H., 4.71 mr/rain

R.G.~ 76.1 mr/rain

I I I I L ~,_ 50 100 150 200 250

t t rain

Fig. 7. Fraction of injected dose of iothalamate found in plasma of two patients, comparing and stable iodine labels. Overlap was exact where only one point is shown

z2s[

With the advent of X-ray computed tomography there has been a renewed in,

terest in understanding and quantitating the distribution of X-ray contrast agents

in normal and pathologic tissues and the ability of FEA to assay off-the-shelf contrast agents makes it an important tool for this kind of work. 13

In clinical use, the techniques described above present common advantages over

radioisotopic studies or chemical analysis of stable tracers: Measurements that be-

cause of considerations of radiation dosage to critical patient populations (child-

ren, pregnant women, normal volunteers and serial studies in the same subject) are

sometimes omitted, can be performed with safety, convenience and accuracy, at

costs that are comparable to or below those of other procedures.

In animals, the availability of FEA for assay has permitted a large number of

experiments at low costs. Accuracy is as high as or better than when radioisotop-

ically labeled agents are used, and counting times are less. Consequently, experiments in which over 100 samples per animal need to be analyzed become feasible.

The wide dynamic range of FEA has allowed the use of dosages that range from

Z Radioanal. Chem 43 (1978) 331 3


tracer to pharmacologic saturation levels. In addition, the costly quarantine procedures needed for maintenance of animals used in chronic studies, can be avoided,

and off-the-shelf tracers can be used.

New directions

FEA clearly lacks the sensitivity (vis-a-vis tracer concentration) of radioisotopic

techniques. Presently quantitation is not practical in unprepared samples that contain tracer levels of under a microgram per gram. In contrast, 10 -13 g of carrier-

free 12sI in a lg sample will yield 1% counting statistics in a time of 5 min. Even when the material is not carrier-free, the weight sensitivity to lZSI exceeds that of

FEA of stable iodine by over 4 orders of magnitude. Thus, broad-based utilization of FEA is dependent on improvements in the technique, i.e., in reducing back-

ground and increasing effective counting rates. We have demonstrated that excitation with polarized X-rays reduces by an order

of magnitude the counting time needed to achieve a certain quantitation accuracy for samples with low levels of tracer. 14 The polarized beam is obtained by select-

ing X-rays that have scattered at 90 degrees from a low atomic number target. Although the scattering process is quite efficient (~25%), the collimation associ- ated with this process reduces the beam to approximately 1% of the intensity available for direct excitation. This makes it impractical to use radioactive sources for excitation purposes. 15 On the other hand, X-ray tubes yield primary intensities

that are sufficient for achieving a practical source of polarized X-rays, including appropriate filtration. While the fluorescence efficiency of a photon beam is independent of its polarization state, Compton scattering is suppressed along the direc- tion parallel to the polarization vector. Thus, a detector collimated along this vector sees an improved fluorescence to scatter ratio (Figs 8, 9). Since in FEA count rates are limited by the pulse processing electronics, and at low tracer levels most

of the counts are from the scatter peak, polarized FEA allows for increases of the effective rates at which fluorescent X-rays can be counted. Since background is

mostly dependent on the intensity of Compton-scattered photons reaching the detector, polarized FEA achieves lower background levels. The degree of improvement realizable is not directly proportional to the increase in the fluorescent to

scattered photon ratio because even with heavy filtration the excitation beam is not monochromatic, and multiple-scattering from the sample becomes a significant component of background (Fig. 10).

We had originally determined that if the spectrometer-generated component of background were to be reduced or eliminated a reduction of a factor of 10 in

332 J. Radioanal. Chem. 43 (1978}


[ ~ X-rag source

�9 I . " ' ~ Fitters

s I t",~\',\ ? ,~\ \ " "

Polarize

J

Sample

/

Detector

Fig. 8. The component of an unpolarized X-ray beam which scatters at 90-deg is polarized on the x, y plane. This secondary beam will not scatter at 90-deg on its own plane of polarization. Thus, a detector placed along the y axis sees little or no Compton-scattered radiation from the sample

counting time would be possible. Following this the KeVex Corporation has been successful in obtaining detectors with a 50% average reduction in background levels. Thus, there exists the potential to gain another factor of five in background

reduction (and counting time). Recent developments have encouraged us to believe

that a major fraction of this potential gain can be achieved. Unfortunately, be-

cause of the higher multiple scatter component from the sample when polarized

excitation beams are used, the combined gain from polarization and improved spectrometers will not be as large the sum of the gains achieved by each develop-

ment individually. In the near term future, decreases of a factor of at best 2 0 - 3 0

in counting time, compared to current performance, are achievable. Alternatively,

with the current counting times, minimum quantitable levels could be reduced by

a factor of five at best.

Can this be of significance in extending the applicability o f FEA to new areas?

In general, it is desirable to reduce the minimum working tracer levels to minimize

possible toxicity problems, and to relax constrainst on compound labeling effici-

ency. For instance, the iodinated albumin used to obtain the results shown in

J. Radioanal. Chem. 43 (1978) 333 3*


t&2 ppml

Fig. 8. FEA spectra of a sample containiilg 14 ppmI, obtained with polarized excitation (left) and 241 Am excitation (right), shown in logarithmic (top) and linear (bottom) scales. The energy spanned along the horizontal axis ranges from 17 to 60 keV. Counting time was 600 seconds in both modes

Fig. 10.

.~z lo o48--.~.__~.. oo ._ . ~ %.

0.581~., _ 8

a 0.69 . . . . . . . . . . . ,,\%"..

~ 7 -- t m m Zr, O.15mmCu ~ , " " 6 - - \ " . �9 %%

4 -- Te I Cs Bo Lo ~ C e

1 3 i _ _ l i p _ 30 35

E, keY

Improvement factor over source-exited system for polarized excitation of tracers of interest, using three different filter thicknesses and 150 kV tube voltage



Table 5 has 10 iodine atoms per molecule of albumin. This is needed to minimize the amount of albumin injected. It is considerably simpler to prepare albumin with just one or two iodine atoms per molecule, which would be adequate in the more

sensitive FEA system. More importantly, a new area would open up to FEA with extended sensitivity:

The radioactive microsphere method, developed by RUDOLPH and HEYMANN in 196716 is today the preferred method of measuring flows to organs and regions

with organs, a7 The microspheres are made of polystyrene, are spherical (9 -50 /~m

diameter), have a specific gravity of about 1.3, and have the radionuclide incorpo-

rated in them so that it does not leach out. When microspheres are injected into the left atrium (in adult animals) or into the venae cavae (in fetal animals) they are distributed to various organs and regions within organs in proportion to the blood flow at that time. At the end of the experiment the animal is killed, and the organs to be examined are removed and cut up into small pieces which are then assayed for radioactivity.

As originally described, five radionuclides were chosen: 12sI, 141Ce, SlCr, 8SSr

and 9SNb. The reason for choosing these was that their spectral energy distribu- tions as measured by NaI(T1) detectors allowed relatively easy separation of the contributions of each nuclide in a mixture.

Restriction of the number of nuclides per experiment to no more than five is both wasteful and efficient of time and money. The preparation of the acute and chronic animal experiments often take several hours and sometimes ends in the animal's death before the actual physiologic measurements can be made. Once the elaborate preparation is ready it would be very valuable to be able to make 8 to 16 measurements rather than the one control and 4 experimental measurements

that are all that can now be made. As a result, many of the experiments now performed with this method rely on between animal comparisons rather than the- more efficient within animal comparisons.

Pilot tests carried out with barium-, cesium and lanthanum-loaded 15 /~m micro-

spheres, comparing them to simultaneous injection of radioactive microspheres,

have shown good agreement between stable and radioactive lables as well as inter- nalty between the stable tracers (Fig. 11). Unfortunately, the present systems re-

quires counting times of the order of 30 min per sample. With the improvements described above, practical counting times will be achieved.

FEA of stable microspheres would provide easily separable tracers, eliminate shelf life problems and obviate expensive animal quarantine and disposal needs.

,This technique could realize a dramatic decrease in the cost of performing research with microspheres, while simultaneously increasing the quality of the data available to the investigator. Thus, it alone justifies continued efforts to improve the technique.

J RadioanaL Chem. 43 (1978] 335


Fig. 11.

.c 5 E

E

2

o�9 �9 �9

"/2 1 2 3 4 5

S tab le ba r i um

Correlation of local blood f~ow in tissue samples of 0-5 -1.5 g weight, excised following simultaneous injection of stable Ba- and Ce- loaded microspheres, measured by FEA

In-vivo FEA

An attractive feature of FEA is that withdrawal of the excitation source instant- aneously stops the emission o f fluorescent radiation and leaves no remaining activ-

ity in the sample. Because of this, the technique lends itself to studies that necessi- tate periodic measurements over long periods (many days or even months), since the radiation dose is delivered to the subject only during those times when a measurement is being performed, (typically of the order of a few minutes to hours). In addition, because the tracer element is non-radioactive, radiation exposure in in- vivo studies is confined to regions of the body where the study is being conduct-

ed. Thus, with FEA we can limit the "where" and "when" of dose administration to the subject, markedly reducing radiation doses.

Applications of FEA techniques arise in the context of measuring:

(1) Slowly varying tissue concentrations of certain elements, where tissue sam- piing is not practical;

(2) Blood flow, where a relative value curve is obtained by external counting, with a timed blood sample yielding the absolute calibration scale; and

(3) Blood flow obtained by continuous sampling of blood in a catheter. We describe some examples of slowly varying functions measured by FEA.

336 J. Radioartal. Chem. 43 {1978/


Absolute iodine quantitation

During our work with cholecystographic agents it became desirable to obtain an absolute determination of iodine concentration in the liver in-vivo to complement bile measurements. Because the liver is a relatively deep organ, the in-vitro method could only be extended if a means of obtaining a correction for tissue attenuation of the fluorescent radiation could be developed. 17

Modifications must be made in Eq. (1) if in-vivo measurements are to be made in organs, since varying amounts of tissue are interposed between sampling point

and detector. In such a case, a correction can be effected by obtaining NKr the

counts in the Kt3 characteristic X-ray channel. Because of the energy difference between the K~ and Ks characteristic X-rays, their ratio varies as a function of the amount of tissue present between the source or radiation and the detector.

It can be shown 18 that the attenuation correction factor (ACF) is given by the expression

ACF=[ NK~ ]I~ NK-~a-Mo

where NKa, NK~ - background-subtracted values;

].LO~ - - / d C

K 2 - - - - ,

Mo = NK#/NKa for a "thin sample".

Eq. (1) can then be written as

NK,x[ NK~ ] K ~ W = K , ~ NK~-Mo (2)

In the work reported here we used a 0.6 Ci 241Am source with an area of

1 cm 2, collimated at 90-deg to the detector collimator. The Si(Li) detector was 80mm 2 X 3mm deep. The system was calibrated with samples having iodine concentrations from 0.049 mg/g to 6.47 mg/g, and tissue-equivalent absorption thicknesses up to 5 cm of lucite. The constants K1 and K2 were calculated from these measurements. Counts were obtained over intervals appropriate for current studies,

which measure relatively slowly varying phenomena; for instance, in-vivo liver measurements are performed using counting intervals of 200 seconds each during the first 15 to 30 minutes when the count rate is highest, and 500 seconds-long intervals during the remainder of an up to 4 hour study. The accuracy of the tech-

J. RadioanaL Chem. 43 (1978) 337


Table 6 Measured iodine concentration with attenuation correction

Standard, Measured 500 sec, A.D., or,% mg/cm 3 mg/cm 3 %

6.47 2.71 0.766 0.049

6.30 2.55 0.818 0.047*

-2.6 12 -5.9 9 +5.4 5 +4.0 12

*The ACF for the higher value was used here.

nique is primarily dependent on the statistics of the data, and, as such, is mainly

limited by the count rate in the Ka channel (about 20% of that in the Ks chan-

nel). As shown in Table 6, absolute quantitation with an accuracy of +10% is

readily attainable. For an experiment in which the concentration varies as a function of time, but the particular geometry is constant, the ACF can be obtained as

a weighted average for the full experiment, and then used to correct each meas-

urement point. This extends the useful range of the technique to lower concentration levels.

Absolute iodine quantitation was tested in-vivo in a systematic study in dogs, 19

The FEA apparatus was placed over the dog's abdomen in such a way that the areas defined by source and detector intersected within the dog's liver. FEA counts

were taken following an intravenous injection of 130 mg of meglumine iodipamide

per kilogram of body weight. Timed blood, urine and bile samples were also col-

lected (Fig. 12). Animals were sacrificed at varying times after administration of

the iodinated contrast agent in order to provide livers with a range of final iodine

concentrations. Liver samples from within the sensitive region, peripheral and cen- tral regions, and liver homogenates were assayed in-vitro, the results are consistent with those expected from considerations Of liver variability and ACF accuracy. 19

FEA is also suited to the in-vivo measurement of the natural iodine content of

the thyroid. Since this gland is near the surface of the body, the attenuation cor-

rection is smaller, and more accurate results can be obtained. Quantitation can also be performed as part of an imaging procedure. 2~

We have found this technique to be of great interest in the study of thyroid

disease. The correspondence of in vitro evaluation of the patient's thyroid to later

pathological examination of excised tissue was particularly good; in two patients

in whom pathology was evaluated in this fashion both evaluations of excised tissue differed from that of previous in vivo evaluation by less than 1%.



20 [~ .-~...,.~ Bile I / ~' [ \ . "'~ ..........

d,,...~,, ...~"'" ...... "a . . . . . . ~ . . . .

m

c

o -o

0 2 ~ e,,,. [

0.1 ~

Time of socr i f ice

o 2o ~,o 6o Bo ~oo ~2o

Minutes after drug odmin is t ro t ion

Fig. 12. Iodine concentration in a dog's liver (in-vivo), blood, and bile (in-vitro) after intravenous injection of meglumine iodipamide, 130 mg/kg body weight. Liver homogenate was measured in vitro

In the scanning mode (1), fluorescent excitation is useful in evaluating the etiol-

ogy of thyroid nodules. Nodules which contain low or no iodine have a 25% probability of being malignant. Moreover, the scan may be useful in demonstrating sup-

pressed thyroid tissue in the presence of hyper-functioning nodules. This technique is now being applied in a number of studies. =1

Clearance o f heavy metals from the lungs

Insuffiated powdered tantalum is being used for roentgenographic outlining of airways. 22 Measurement of the clearance of insuffiated tantalum and barium sul-

fate powder from the lung is important in the evaluation of tantalum as a roent-

genographic contrast agent, as well as in the investigation of lung clearance mech- anisms.

Tantalum powder adheres to the airway mucosa and provides excellent detail

of airway structure. In addition, alterations in the normal clearance pattern of tantalum from the airways provide information ha the diagnosis of carcinoma, bronchi-

tis, and bronchiectasis. Roentgenographic methods have been used to evaluate clearance both in experimental animals and man, but are only qualitative and are

influenced by variability in X-ray exposure and film response. Even relative assess-

J RadioanaL Chem. 4J (1978) 339


ments become difficult as the material disperses in the terminal air spaces of the lung.

lS2Ta can be used to evaluate clearance, but because both its biologic and phys- ical half-lives are long, the hazard of radiation precludes its use in humans. Special quarantine procedures are required when tS2Ta is used in animal studies.

Measurement by means of fluorescent excitation has a number of advantages.

It allows for quantitation of the tantalum present in a specific region of the lung. Radiation exposure is localized anatomically and limited to the time during which measurements are performed. Nonradioactive tantalum can be administered with no special precautions needed in handling and keeping the animals. We have applied this technique to characterize the lung clearance rate of tantalum and to compare it with the clearance rate of barium sulfate. 23

Excitation of tantalum was produced by the 97 keV and 103 keV gamma-rays from a 2-Ci lS3Gd source. This source is of particular value in this study because lSaGd decays to europium, which emits K X-rays suitable for the simultaneous excitation of barium (another contrast agent). The in-vivo FEA method used to quan-

titate tantalum and barium was similar to the technique used for iodine. Absolute

determinations in the range of 0. i g to 1 g of tantalum and 0.05 g to 0.5 g of ba-

rium were tested with phantoms, and were found to be linear with a standard de- viation of +8.5% for 200 sec measurements. Since it is difficult to reposition the animal exactly, we used a wide field of view (~10 cm) collimator. Displacing phantoms by up to 2 cm produced no significant variations in the results. We found that after the first few days, the material spreads and becomes less visible in the radiographs, suggesting clearance, but the amount determined by fluorescent excitation remains unchanged (Figs 13, 14). This pattern was characteristic through our study, indicating permanence times considerably in excess of those expected from roentgenographic assessment.

Blood flow measurements by external counting: cardiac output determinations

We have investigated the feasibility of studying blood flow through fluorescent excitation of intravenously injected, non-radioactive X-ray contrast media. Initial efforts were directed toward developing a technique to measure cardiac output

(C. 0.) in dogs, 24 since a comparison technique was readily available using indocyanine green.

A 1 cm 2 excitation beam of X-rays was directed at the left precordium previ- ously localized by fluoroscopy. A Si(Li) detector, also collimated to 1 cm 2 was placed so that the intersection of this sensitive region and the X-ray beam defined a small volume of blood within the heart. A tracer dose of 5cm 3 of iothalamate



rig. ]3.

60

4ol I 1 I t I 1 I [ o 5 ~0 ~s 20 2s 3o 35 40

Day

Clearance o f barium f rom a dog's lung. Al though the barium spreads and becomes less

noticeable in the radiographs, the total amount does not change significantly

Fig. 13.

A 700)-

50O

400 ~ _

~0o~~176 --~ . . . . . . ~ . . . . . . . . . . ~ ~- . . . . . . . . . . . t- , 0 0 - , - - - ~ 7 - - - , - - ~ . . . . I . . . . ~ . . . . , . . . . 7 - , . _

5 10 15 20 25 30 35 40 4.5 Dog

Clearance of tantalum from a dog's lung. Following rapid initial clearance, the amount of tantalum remains constant

J. Radioanal . Chem. 4 3 (1978 ) 341


(Conray 60), containing 282 mg/cm 6 of iodine, was injected as a bolus into a peripheral vein. Maximizing the count rate in the iodine K s window required filtration of both the X-ray beam and the detector, which increase the ratio of K s characteristic iodine photons to the total number of events registered by detector. This higher ratio increased the effective count rate during the experiment. The detector output was fed into two single channel analyzers that selected the iodine Ka peak

and another region of the scattered spectrum at 45 keV, the counts accumulated at 0.2 sec intervals. Data from the 45 keV window were used for normalization purposes, thus compensating for variation in X-ray tube output and dead-time losses.

Data were collected over the precordium for 30 seconds following intravenous

injection of the iothalamate. A background blood sample and simultaneous 20 second precordial count rate were obtained immediately prior to injection. This pro-

cedure was repeated 5 minutes after injection, after intravascular mixing, the data

required to convert the precordial count rate into actual units of concentration.

Prior to and at the completion of the fluorescence excitation study, a standard indocyanine green determination of C. O. was performed.

Calculation of C. O. from the fluorescence excitation data was performed by a modification of the Stewart-Hamilton dye dilution formula.

D Rs C. O. = (3)

C5 f R( t )d t

where D - injected dose of iodine in mg; Rs - precordial count rate in counts/second at 5 minutes following in-

jection;

Cs - blood concentration of iodine (also at 5 minutes following injection) in mg/cm 3 ;

R(t) precordial count rate in counts/second during the first 30 seconds following injection.

Typical precordial curves immediately following injection are shown in Fig. 15. The regular rapid fluctuations in count rate were due to movement of cardiac

blood out of the sensitive volume as a result of ventricular contraction. Variations in curve shape in this dog with deteriorating cardiovascular status are quite apparent.

Table 7 compares the estimates of C. O. by both the fluorescence excitation and indocyanine green methods in four dogs. The results are quite similar over a wide range of output values.



c 5 0 0 ~

400 ~- (J

3 0 0 - -

200

100

400

o 300

200

I 7 0t /rain ~ c 500 t

o ~ 4oo~ o

3 0 0 ~

a) 100

4 8

I

~176176 30o 7

20o r b) 1 0 0 ~ c)

/

~ , i n , : I ~ [ . i : . . . . . ~ i i , r , I _ _ _ l ~ , . 4 8 0 4 8 12 16

;00 0

Tim% sec T~me~ sec

~" 200 ta

d) o ~ t j

4 B 12 16 Tim% sec

Timej s e c

- - - Background

e}

; ~ , q I I ! LDm~_ ' ~ ' 8 ' ~ ,~ ~o

Tim % sec

Fig. 15. Cardiac output count rate curves obtained by in-vivo X-ray fluorescence of Conray 60 in the dog heart accumulated in 0.2 sec intervals. The animals's cardiovascular status deteri- orated over the five hour period which these data span

Table 7 Cardiac output results

2

4

Dog, No.

Study A

Study B

Study A

Study B

Fluorescent, l/min

2.5

2.3

1.4

2.3

3.5

2.0

lndocyanine green, l/min

Pre P

2.6 2.1551

2.6 2

1.5 1.7

2.2 2.9

2.9 3.3

2.0 2.0

Notes

Consecutive output deter-

minations 30 minutes

apart

Consecutive output deter-

minations 20 minutes

apart



The main problem with radioactive tracers is the difficulty in estimating a calibration factor required to convert the precordial count rate into a concentration. This problem has been resolved with the fluorescent excitation technique. Since the sensitive volume of blood inside the precordium was the only volume of tissue excited by the X-ray source and at the same time sensed by the detector, and because no non-linearities existed in the data, the ratio (calibration factor required) of precordial count rate to blood iodine concentration remained essentially constant through- out the study and until intravascular mixing of the iothalamate was complete.

This method of C. O. determination has additional advantages over radioactive techniques in that it is independent of counting geometry, and that the radiation exposure is confined solely to the area of interest rather than to the total region of tracer distribution.

Blood flow measurements by continuous sampling in small vessels

The methods discribed above are not suitable for measuring blood flow in small, difficult to localize vessels. For instance, in the study of iodinated biliary contrast agents, it is of importance to correlate hepatic blood flow, extraction fraction and drug clearance. As an alternative, a catheter can be placed in the sample point so that blood is extracted at a constant rate. The catheter passes within the sensitive volume of the in-vitro set-up, and is analyzed "on-line" by storing the characteristic and Compton channel data in a multi-scale mode. The excitation source can be 241Am or a fluoroscopy X-ray tube operated at about 110 kVp, filtered with 0.15 mm of tungsten. The system can be easily calibrated by filling the catheter with two samples: pure water and a known concentration of iodine in water.

When an agent that is not extracted by the liver is injected, indicator dilution formulation [Eq. (3)] can be used to obtain total hepatic flow. If extraction occurs, the same formulation is used, but the injected dose (D) is effectively lowered, giving artifically low values of flow. Comparison with true flow then yields the desired extraction fraction. In experiments in dogs data rates of up to 103/sec have been registered in the Ks channel (Fig. 16). Impractically large amounts of radioactive tracers would have to be administered to obtain equivalent "on-line" counting rates

_in the small volumes of blood being assayed. Generalization of this powerful methodology is certain to have an impact on the study of a number of physiologic systems.

Conclusions

FEA is a tool which has usefulness in laboratory and clinical practice. In terms of accuracy and cost it compares favorably to the techniques it replaces. Its main limitation is one of sensitivity. New techniques such as the use of polarized radia-

344 J. RadioanaL Chem. 43 (1978)

Fig. 16.


i m 10 3

3 o

O 0

.,"%... ,%

% %

O ~ OgiO

Background

J I I J I I I~_ 10 20 30 40 50 60 70

t~ s e c

Count rate curve obtained by onqine fluorescence of blood obtained from sampling the hepatic vein of a dog injected with 3cc of Cholografin 50. Data is accumulated in 1 sec intervals

t ion for exci ta t ion and improved spectrometers , promise fur ther improvements in

sensitivity. More impor tan t ly , the use o f physiological ly compat ib le tracers is a

strong compensat ing factor that is jus t n o w in an early stage o f explorat ion.

Where applicable, F E A has been shown to be a powerfu l and rewarding tool.

These investigations are supported in part by Grant GM 21017, by Training Grant GM 01271, by Career Development Awards GM 70598, GM 70304 and GM 70582 all from the NIGMS, by Training Grant HD-00182 from the NICHHD and by grants from the Academic Senate and the Research and Allocations Committee, University of California, San Francisco. The authors wish to acknowledge the collaboration of Mr. RICHARD FRANKEL and Dr. ROLF WOLDSETH of the KeVex Corporation.

References

1. E B. HOFFER et al., Radiology, 90 (1968) 342. 2. P. B. HOFFER, R. N. BECK, A. GOTTSCHALK (Eds), Semiconductor Detectors in the

Future of Nuclear Medicine, Society of Nuclear Medicine, New York, New York 1971. 3. L. KAUFMAN, D. C. PRICE, ,,Semiconductor Detectors in Medicine" U.S. Atomic Energy

Commission, CONF 730321, 1973. 4. L KAUFMAN et al., Invest. Radiol., 11 (1976) 210. 5. D. C. CAMP et al., Chem. Instrum., 7 (1976) 47.



6. L. KAUFMAN, C. J. WILSON, J. Nucl. Med., 14 (1973) 812. 7. D. C. PRICE et al., J. Nucl. Med., 16 (1975) 814. 8. D. C. PRICE et al., J. Lab. Clin. Med., 87 (1976) 535. 9. P. GUESRY et al., Clinical Nephrology, 3 (1975) 134.

10. J. A. NELSON et al., Invest. Radiol., 8 (1973) 126. 11. J. A. NELSON et al., Invest. Radiol., 9 (1974) 438. 12. R. J. HERZOG et al., Invest. Radiol., 11 (1976) 32. 13. D. R. ENZMANN et al., Acta Radiologica (in press). t4. L. KAUFMAN et al., IEEE Trans. Nuct. Sci., NS-24 (1977) 525. 15. L. KAUFMAN, D. C. CAMP, Adv. X-ray Anal., 18 (1975) 247. t6. A. M. RUDOLPH, M. A. HEYMANN, Circulation Res. 21 (1967) 163. 17. Bibliography: 3M Brand Tracer Microspheres. J-BIB (46.5) R1, The 3M Company, April

1976. 18. L. KAUFMAN et al., Invest. Radiol., 8 (1973) 167. 19. R. E. KOEHLER et al., Invest. Radiol., 11 (1976) 134. 20. D. W. PALMER et al., Radiology, 119 (1976) 733. 21. M. OKERLUND et al., Clin. Res., 25 (1977) 105A. 22. J. A. NADEL et al., New Eng. J. Med., 283 (1970) 281. 23. L. KAUFMAN G. GAMSU, IEEE Trans. Nucl. Sci. NS-21 (1974) 721. 24. L. KAUFMAN et al., Invest. Radiol., 7 (1972) 365.

346 J. Radioanal. Chem. 43 {1978)

fluorescent excitation analysis in medicine

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