Spectroscopic Analysis for biological samples : towards in situ sample analysis of body fluids

Gilwon Yoon September 27, 2006 Seoul National University of Technology

Spectra of water, Hb(RBC), albumin, glucose from visible to NIR (water compensated)

0.5 mm pathlength, temperature control, 37 hemoglobin, 16.9 g/dl
0.15

3

water

absorbance

0.10

0.05

1

albumin, 8 g/dl
0.00 400 600 800

glucose, 5g/dl
0 1000 1200 1400 1600 1800 2000 2200 2400

wavelength (nm)

absorbance, water

2

Absorption spectrum of water

Involved Key Technologies

Interfering substances Visible/IR Light source target component inhomogeneous medium Prediction of concentration Statistical analysis Spectroscopic detection Light interaction with tissue High S/N electronic detection Chemometrics Clinical test

Spectroscopic Analysis – Statistical Methods

Influence of measurement setup : Transmission or reflection measurement  Influence of red blood cell (hemoglobin) in partial least squares regression (PLSR) analysis  Independent Component Analysis (ICA) – a method without calibration process

I. Influence of measurement setup : Transmission or reflection measurement

sli t

Ligh t so ur ce

Mo no ch romato r Detec to r mple Sa

Detec to r

(a )

(b)

Comparison between reflectance and transmittance

wavelength region [nm] 1100-2500 diffuse reflectance (10mm thick sample) 1100-1850 1850-2500 diffuse transmittance (1mm thick sample) 1100-1800 2064-2338 1100-1800 2064-2338 diffuse transmittance (2mm thick sample) 1100-1830 2050-2392 1100-1830

SEC [mg/dl] 27.38 15.91 30.57 3.22 2.88 4.50 26.46 40.47

SEP [mg/dl] 275.44 437.54 192.00 24.69 26.77 43.51 39.07 83.58

Jeon, Hwang, Hahn, Yoon (2006), 11:1:014022, Journal of Biomedical Optics

Diffuse reflectance between 1100 and 1850 nm (a) SECV with respect to the optimal number of factor, (b) Loading vector of calibration model, (c) Regression vector of calibration model, (d) Prediction of glucose illustrated with the intralipid concentrations of sample solutions.
400 350

(a) loading vector [a.u.]

0.15 0.10 0.05 0.00 -0.05 -0.10 -0.15 -0.20 1000 1600 1400 1200 1000 800 600 400 200 0 0 200 400 600 800
s4.16 s4.08 s4.16 s4.08 s4 s4.16 s4.08s4 s4 s4.16 s4.08 s4 s4.16 s4.08 s4

(b)

SECV [mg/dl]

300 250 200 150 100 50 0 0 40000 2 4 6 8

factor1 factor2 factor3

10

1200

1400

1600

1800

2000

factor (c)
20000

wavelength [nm] predicted glucose [mg/dl]
SEP= 437.54 mg/dl CV= 98.8%
s4.08 s4.16 s4 s4.16 s4.08 s4

regression vector [a.u.]

0

-20000 -40000

(d)
ref prediction
1000

-60000 1000 1200 1400 1600 1800

2000

wavelength [nm]

reference glucose [mg/dl]

Diffuse transmittance with 1 mm thick samples (a) SECV with respect to the optimal number of factor, (b) Loading vector of calibration model, (c) Regression vector of calibration model, and (d) Prediction of glucose concentrations.

400 350

(a) loading vector [a.u.]

0.25 0.20 0.15 0.10 0.05 0.00 -0.05 -0.10 -0.15

SECV [mg/dl]

300 250 200 150 100 50 0 0 2 4 6 8 10

factor1 factor2 factor3

(b)

-0.20

1000 1200 1400 1600

2200

2400

factor
8000

wavelength [nm] predicted glucose [mg/dl] (c)
1200 1000 800 600 400 200 0
s4.16 s4 s4.08 s4.16 s4 s4.08 s4.16 s4.08 s4 s4.16 s4 s4.08

6000

regression vector [a.u.]

4000 2000 0 -2000 -4000 -6000 -8000 -10000 1000 1200 1400 1600 1800 2200 2400

ref prediction
s4.08 s4 s4.16

s4.08 s4.16 s4

(d) Prediction 1mm path length SEP= 24.69 mg/dl

s4.16 s4.08 s4

0

200

400

600

800

1000

wavelength [nm]

reference glucose [mg/dl]

II. Influence of red blood cell (hemoglobin) in partial least squares regression (PLSR) analysis

Biological Samples in the near infrared (1000 – 2500 nm)     Absorption becomes much stronger towards longer wavelengths Dominance of hemoglobin Interferences among the substances in blood or extracellular fluid Effect of preprocessing methods

2.4 2.1 1.8 1.5 1.2 0.9 0.6 0.3 0.0 1.0

1888

2044

a

whole blood

correlation coefficient (r)

a) Whole blood spectra of 98 samples and saline spectrum, b) Whole blood spectra are correlated with hemoglobin and glucose concentrations at each wavelength and computed correlations coefficients are shown.

absorbance (a.u.)

Saline
1200 1400 1600 1800 2200

b
Hemoglobin

0.8 0.6 0.4 0.2 0.0 1200 1400 1600 1800 2200

Glucose

wavelength (nm)

What is a maximally achievable accuracy ?
Calibration set Hbcal Hbhigh Hbmid Hblow mean value of glucose 228 213 221 207 221 207 213

Prediction set Hbpre Hbmid Hblow Hbhigh Hblow Hbhigh Hbmid

SEPa (rPreb) 25.5 (0.9764) 23.1 (0.9817) 48.7 (0.9279) 39.3 (0.9465) 46.9 (0.9328) 74.2 (0.8672) 33.8 (0.9603)

VCPrec [%] 11.2 10.8 22.0 19.0 21.2 35.8 15.9

Kim and Yoon (2006), 11: 041128, Journal of Biomedical Optics

The standard error of glucose prediction was 25.5 mg/dl and the coefficient of variation in prediction was 11.2%.

III. Independent Component Analysis (ICA) – method without calibration process

 Identification of pure, or individual, absorption spectra of constituent components from the mixture spectra without a priori knowledge of the mixture.  This method was tested with a two-component system consisting of aqueous solution of both glucose and sucrose, which exhibit distinct but closely overlapped spectra.  ICA combined with principal component analysis was able to identify a spectrum for each component, the correct number of components, and the concentrations of the components in the mixture. This method does not need calibration process.
Hahn and Yoon (2006), in print, 45:32, November, Applied Optics

Pure, or individual, water-subtracted absorption profiles of Glucose (G) and Sucrose (S)

G

Absorbance

S

960

980

1000

1020

1040

1060
-1

1080

1100

1120

Wavenumber (cm )

25 measured mid-IR spectra for the mixtures of glucose and sucrose. Water absorption was subtracted to enhance the absorption profile of each component.
1.0

0.8

Absorbance

0.6

0.4

0.2

0.0 960

980

1000

1020

1040

1060
-1

1080

1100

1120

Wavenumber(cm )

Extracted pure-component spectra from measured IR spectra of 25. ‘Pure’ and ‘ICA’ represent pure-component absorption spectrum and the ICA-method extracted absorption spectrum respectively.

Glucose
1.2 1.0 0.8 0.6 0.4

Sucrose Pure
0.8

Absorbance

Absorbance

Pure
0.7 0.6 0.5 0.4 960 980 1000 1020 1040 1060 1080 1100 1120
-1

ICA

ICA

0.2 960 980 1000 1020 1040 1060 1080 1100 1120

Wavenumber(cm )

-1

Wavenumber(cm )

Scatter plot for the reference concentrations and ICs from measured mid-IR spectra

4 -8 3

IC2(a.u.)

2

IC1(a.u.)
1 2 3 4 5

-9

-10

1 -11 0

1

2

3

4

5

Sucrose (a.u.)

Glucose(a.u.)

Summary in Spectroscopic analysis

 Measurement geometry or setup – loading factor analysis can provide actual contribution of wavelength in prediction  Dominant absorber such as RBC(hemoglobin) and water in near infrared effect substantially. A proper care is needed.  A new method that does not require no concentration information and calibration process is introduced.

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