Radiometry in medicine and biology

Kie B. Nahm1*, Eui Y. Choi

2

11Dept. of Physics,

2Dept. of Biomedical Sciences

Hallym University, Chunchon, Kangwon, 200-702, Korea 1,2

Boditech Med Inc., Chunchon, Kangwon, 200-883, Korea**

ABSTRACT

Diagnostics in medicine plays a critical role in helping medical professionals deliver proper diagnostic decisions. Most

samples in this trade are of the human origin and a great portion of methodologies practiced in biology labs is shared in

clinical diagnostic laboratories as well. Most clinical tests are quantitative in nature and recent increase in interests in

preventive medicine requires the determination of minimal concentration of target analyte: they exist in small quantities

at the early stage of various diseases. Radiometry or the use of optical radiation is the most trusted and reliable means of

converting biologic concentrations into quantitative physical quantities. Since optical energy is readily available in

varying energies (or wavelengths), the appropriate combination of light and the sample absorption properties provides

reliable information about the sample concentration through Beer-Lambert law to a decent precision. In this article, the

commonly practiced techniques in clinical and biology labs are reviewed from the standpoint of radiometry.

Keywords: Optics in Clinical Laboratory, ELISA, Spectrophotometry, Immuno-Fluorescence, Hematology

1. INTRODUCTION

Medical professionals these days rely heavily on factual data unlike their predecessors centuries ago when they trusted

their subjective judgment on the health and the disease of their patients. Although this practice of delivering medical

services based on personal training and experience deserves certain credits with frequent minor ailments, we came to

expect medical testing as a part of sound medical diagnostic process. Medical tests not only enhances the credibility of

the diagnosis by providing solid evidences for the call, but also reduces the potential bias if the call were based on

experiences and subjective judgment.

Samples for clinical tests come mostly from body fluids: blood, urine, saliva etc. and the blood is the most important

sample format for hundreds of tests. Mammalian body, in conjunction with the immune system, secretes/produces

characteristic substances into the blood stream when the body experiences abnormality for various causes. Increased

number of white blood cells against infection, resulting in fever and inflammation, is a good example of such a

mechanism. Medical scientists have established “reference ranges” for a long list of such substances indicative of

corresponding abnormalities in our bodily metabolism. Clinical tests are ordered to provide information on those

pertinent parameters.

Most components for medical testing exist in relatively low concentration in blood except for a few like blood sugar

(~102mg/dL), cholesterol (~10

2mg/dL) and hemoglobin (~10

1mg/dL). Prostate Specific Antigen (PSA) level generally

stays below 4 ng/mL for healthy males. Detecting such low a concentration reliably and econometrically requires

creative minds and sound scientific and engineering support. There is a horde of solutions to this formidable task, yet the

ultimate one that meets the practical restrictions is radiometric or optical ones.

Simplest kind of optical system used in this trade may be the turbidimeter, where the radiometric energy that passed

through the sample is used to estimate the concentration of the substance under test in the sample solution.

Nephelometers are just as useful. Spectrophotometers are the most widely employed systems for various testing

instruments in the day-to-day volume processing work flow as well as in the high precision testing environment.

Fluorescence instruments take care of about one half the load of the spectrophotometer-based analyzers.

Chemiluminescence pushes the technical limit of the single photon counting technique to achieve the ultimate sensitivity.

* kbnahm@hallym.ac.kr.

** www.boditech.co.kr

Tribute to William Wolfe, edited by Mary G. Turner, Proc. of SPIE Vol. 848384830C · © 2012 SPIE · CCC code: 0277-786/12/$18 · doi: 10.1117/12.978848

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I

2. RADIOMETRIC PRINCIPLES IN ACTION

2.1 Spectrophotometer

The spectrophotometer is not readily visible in clinical laboratories: it is embedded as an integral part of the

fully automated testing system. The very dominant automatic system in clinical laboratories is the chemistry

analyzer. As the name implies, this system employs a robotic and fluidics handling systems to processes the

batch samples autonomously according to the pre-programmed chemical steps. The chemical process is the

automated duplication of manual steps one would employ in biological research labs. Samples containing

target molecules are treated with various chemicals and enzymes (assay process). At the end of the process,

the target molecules are chemically and/or enzymatically transformed into materials that will display

characteristic absorption profiles.

The sample, contained in a clear cuvette with well-defined parallel surfaces and separation, is exposed to light

with selected wavelength. The attenuation of the radiation as modeled with the Beer-Lambert’s law will help

determine the concentration of the target substance. This law, as described by

( ) cx

oI x I e , (1)

where , c and x stand for the absorption coefficient, the concentration and the beam travel distance in the

media respectively, is in reality but an approximation and is applicable to practice with certain limitations.

One should pay attention to conditions where this law fails, such as working concentration ranges, presence of

particulate scatters and fluorescence or phosphorescence.

Figure 1 A representative automatic chemistry analyzer. Quantification is done by optical engines (Wikipedia)

Figure 1 is a representative automatic chemistry analyzer used in most major clinical laboratories and Figure 2 shows the

absorption spectrum of Bovine Serum Albumin (BSA) processed with a BCA assay. The same protein, if processed by

differing assay procedure, would have produced different set of spectrum. Table 1 is the summary of wavelengths with

differing assay method.

2.2 Optical absorptiometry in ELISA

Enzyme-linked immunosorbent assay (ELISA) is a straight-forward and robust format for telling the presence and the

concentration of molecules that can be manipulated via either the solid-phase enzyme immunoassay or ligand binding

assays. In spite of its high reliability and accuracy, ELISA requires minimal amount of hardware investment other than

the reader where the radiometry takes on the quantification duty.

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2.5

2.0

0.5

0.0

400 450 500 550 600 650

Wavelength (nm)

700 750

Figure 2 Absorbance spectra for BSA standards in BCA protein assay. BSA standards are 0, 125, 250, 500, 750, 1000, 1500

and 2000 µg/mL respectively. (Thermo Scientific)

Table 1 List of wavelengths for Protein Assay

http://www.invitrogen.com/site/us/en/home/References/Molecular-Probes-The-Handbook/tables/A-

comparison-of-reagents-for-detecting-and-quantitating-proteins-in-solution.html

First, one would immobilize “capture antibody” on the substrate (a). Immobilization means these antibodies will be

bound to the substrate and will endure subsequent procedures including washing and other immuno/binding reactions.

In the second step, the sample containing target substance/protein is added to these immobilized antibodies. Capture

antibodies and target substance are chosen in such a way that they bind to each other with a high specificity (b). At the

Assay Method (ex/emission) in nm Measurement range

Quant-iT protein assay 470/570 0.5 ~ 4 µg/mL

NanoOrange protein assay 470/570 10 ng/mL ~ 10 µg/mL

CBQCA protein quantitation assay 450/550 10 ng/mL ~150 µg/mL

EZQ protein quantitation assay 280 and 450/618 50 µg/mL ~ 5 mg/mL

Bradford assay 595 1 µg/mL ~ 1.5 mg/mL

BCA method 562 0.5 µg/mL ~ 1.2 mg/mL

Lowry assay 750 1 µg/mL ~ 1.5 mg/mL

Fluorescamine 390/475 0.3 µg/mL ~ 13 µg/mL

OPA 340/455 0.2 µg/mL ~ 25 µg/mL

UV absorption 280 10 µg/mL ~ 50 µg/mL or

50 µg/mL ~ 2 mg/mL

Figure 3 Running ELISA tests

2000 μg/ml

Capture Ab

(a)

Sample

(b) Enzyme conjugate

(c) Coloring

(d)

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A)

MkreplMewall

Mabk Insehpren

Laser B)

Paniclesscatter lightInto sphere

Solubility change

Light detector

MakinSphere

Light trap

third step, another antibodies tagged with enzymes are applied to form sandwich type conjugates. The surface density of

these conjugates is proportional to the concentration of target substance applied in the step (b). At the last stage, the

enzyme is activated biologically to render characteristic color and saturation. The higher the concentration of the target

substance, the more saturated color develops. The depth of the color saturation, as measured by optical transmission or

reflection is used to provide information on the concentration of the target substance. ELISA runs with known

concentration are used as the calibration reference. Figure 4 shows an ELISA result performed on a 96 well-plate.

Figure 4 ELISA result performed on a 96 well-plate and a read-out system. http://www.biology.arizona.edu/immunology/activities/elisa/elisa_intro.html,

http://www.bmglabtech.com/application-notes/nephelometry/microbial-growth-nephelometry-nephelometer-125.cfm

2.3 Turbidimetry and Nephelometry

The Beer-Lambert’s law (1) is applicable to solutions containing absorbing substance in liquid or molecular phase. If

there should exist solid phase substance in the solution, light scattering and attenuation get in the way of this relation and

the theoretical prediction goes awry.

One application in immunodiagnostic practice utilizes these characteristics. Protein molecules in water-based buffer

solutions are clear and transparent and significant light scattering is not observed, as in the egg white. If one can get

protein molecules together to form sizable clusters, one do observed light scattering. One such an application is Latex

Agglutination. Here an antibody is coated on the surface of latex spheres, which will bind to the incoming conjugate

antigen. Since the latex sphere is much larger than antibody molecule, a single sphere can bind to tens of thousands of

antigen molecules. The milky latex suspension behaves more like a Lambertian scatterer before agglutination takes place.

With the formation of agglutination and with a proper filtration process, the solution becomes rather granular and one

observes increased transmission and decreased scattering. Turbidimetric and nephelometeric principles are applicable for

the quantification of the target antigen in this case.

(a) (b)

http://www.cdc.gov/meningitis/lab-manual/chpt06-culture-id.pdf

Latex

suspension

light

Turbidimetry

Nephelometry

Figure 5 (a) Agglutination on a microscope slide, (b) optical arrangement for read-out

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Primary AntibodyN4,

DirectImmunofluorescence

FluorochromeA

IndirectImmunofluorescence

Secondary Antibody

Latex agglutination is a widely adopted technique in clinical diagnostic field. These were utilized to detect over 100

kinds of infectious diseases. These can be performed manually as well as automatically in various modified formats.

Figure 6 is an example of such a modified application.

Figure 6 An example of an automated Latex agglutination-turbidimeter system. The turbidimeter window is shown by the number

4. DCA 2000 system, SIEMENS

2.4 Chemiluminescence and Chemifluoroscence

In biology and the related research and diagnostic trades, one often wishes to trace and quantify target molecules and

substances. Since cells and proteins are primarily colorless and clear, one needs to tag visible markers to targets. The

process of tagging such markers is called “labeling”. Chemiluminescence takes advantage of the chemical reaction

occurring between an enzyme, such as horse radish peroxidase (HRP) and a chemiluminescent molecule, such as luminol.

Chemifluorescence labeling utilizes amine coupling between the protein and the fluorophore. Depending on the nature of

the tagged component, one can utilize radioisotope labeling, fluorescence labeling and enzyme labeling for example.

Substances labeled with fluorochromes will emit fluorescence when illuminated with radiation with appropriate

wavelength. Ones with enzyme will emit luminescence if subjected with substrates (nutrients). The amount of radiant

luminescent energy is used in both cases to provide quantitative information about the concentration of the tagged

substances. Chemiluminescence is more widely recognized and applied for it does not require excitation energy source.

Yet both are capable of delivering detection limit of femtograms. Figure 8 shows an example of chemifluorescence

adopted in a diagnostic instrument.

Figure 7 Direct and indirect fluorescence labeling

http://www.dako.com/08002_03aug09_ihc_guidebook_5th_edition_chapter_10.pdf

2.5 Lateral Flow Immunochromatographic Assay (LFIA) and fluorescence

Lateral tests were originally intended to detect the presence of the target substances in simple and easy steps without

resorting to costly laboratory equipment. The home pregnancy test kit is a good example employing this concept and

technology. This format is designed in such a way to accumulate target substances by the immune reaction. The target is

usually labeled with visible markers such as gold nanoparticles or colored dye molecules. For increased sensitivity and

quantitative analysis, one can adopt fluorescent molecules as well.

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Emission / Excitation

Lateral Flow Assay Architecture

Antibodies conjugated toAnalyte Gold Nanoparticles

SCapillary Flow

Sample ConjugateMembranePad Pad

Test Line Control Line(Antibody) (e.g. anti -IgG Antibody

Wicking Pad

J,Test Line Control Line(Positive) (valid Test)

Figure 9 explains the structure and working principle of a typical visible lateral flow assay. This type has a special

section where the antibodies conjugated to gold particles are pre-dispensed. The sample containing the antigen (analyte)

is added to the “sample pad”. The mixture then migrates to the adjacent section where the antigen binds with pre-

dispensed gold-conjugated. It keeps moving by the capillary force to the “capture” line, where another set of antibodies

(detection antibody) await. These waiting antibodies bind to incoming antigens that carry along gold nanoparticles. Gold

particles accumulated on this “band” produce dark line, indicating the presence of antigens in the sample. No antigen in

the sample, no dark line here. If the antigen were pregnancy related one like human chorionic gonadotropin (hCG), this

dark line would mean pregnancy.

LFIA has been and still is used extensively for qualitative applications. Its simplicity found users in many applications as

in medicine (malaria, HIV, inflammation etc.), environmental monitoring ( toxoplasma, water pollution etc.) and a horde

of other areas. With the increasing adaption in medical diagnostics, there came the need for the quantitative assay and the

fluorescence provided the solution. Those gold particles as shown in Figure 9 were replaced with fluorescent dye

molecules and we have the test line fully developed yet invisible to the naked eye. Boditech Med introduced a LFIA

system that takes advantage of the sensitive fluorescence quantification (Figure 10) in a compact format.

Figure 9 Typical lateral flow Immunochromatographic assay strip. (http://www.cytodiagnostics.com/lateral-flow-

immunoassays.php)

Figure 8. An embodiment of chemifluorescence based diagnostic system (miniVIDAS from Biomerieux). The

cartridge shown on the right has the fluorescence cell as indicated.

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4

Photosensor

-I

Filter

Emission Filter

Dichroic Mirror

V v

jCVN

Í7

Laser

<

NC membrane

NA >0.8

Fluorescence+ scattering

Figure 10. A quantitative fluorescence LFIA system and its fluorescence sensing optics (www.boditech.co.kr)

Once target substances are captured by the capture antibodies on the test line of the NC membrane, the strip is scanned

under laser illumination to generate fluorescence signal. The optical sensor module has the similar optical arrangement to

the one adopted in the fluorescence microscope.

3. CONCLUDING REMARKS

The above summary is an abridged description of the role that radiometry plays in the medical and biological

laboratories. Hematologic instruments can utilize optical sensors. Spectrophotometers (chemistry analyzers) and

fluorescence readers (in immuno and chemifluoroscence analyzers) take about 50% of the work load in the clinical

facilities.

New and novel approaches to achieve even higher sensitivity and specificity are under development and published daily.

Surface enhanced Raman Scattering (SERS), Surface Plasmon Resonance (SPR) techniques for example are getting

more attention at the research level, but are not fully integrated in routine work protocols. One particular aspect of

clinical application of radiometry could be that, on top of the undisputable credibility to the test result, the operation

should be economically competitive. Any technical solution that would incur excessive operation costs would not be

accepted among users.

A new trend is getting foothold in clinical diagnostics. Point of care testing (POCT) contrasts with the conventional test:

the test is performed on the spot in about 10 minutes. LFIA is an excellent competitor in this segment and radiometry

would be an integral part of the new diagnostic paradigm.

In view of all these and other factors, one would expect the role of radiometry and optical ideas to stay in the core

position in new diagnostic modalities to come in the future as well as in conventional test procedures.

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