Laser Phys. Lett. 9, No. 4, 317–321 (2012) / DOI 10.1002/lapl.201110126 317

Abstract: We presented the rapid diagnostic test for dengue in-fection based on light spectrum of human blood. The transmis-sion spectra of dengue infected whole blood samples have beenrecorded in ultra violet to near infrared range (400 – 800 nm)of about 30 conformed infected patients and compared to nor-mal blood samples. Transmission spectra of dengue infectedblood illustrate a strong band from 400 – 600 nm with promi-nant peaks at 540 and 580 nm, where is in case of normalblood below 600 nm, total absorption has been observed. Theseprominent peaks from 400 – 600 nm are characteristics of cellsdamage and dangue virus antibodies immunoglobulin G (IgG)and immunoglobulin M (IgM) produced against dengue anti-gen. The presented diagnostic method is non invasive, cost ef-fective, easy and fast screening technique for dengue infectedpatients.

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The dengue infected whole blood spectrum 400 – 1000 nmwavelength of different patients

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Transmission spectroscopy of dengue viral infection

S. Firdous, 1,∗ M. Ahmed, 1 A. Rehman, 1 M. Nawaz, 1 S. Anwar, 1 and S. Murtaza 2

1 National Institute of Lasers and Optronics (NILOP), Islamabad, Pakistan2 Department of Pathology, NORI Hospital, Islamabad, Pakistan

Received: 3 November 2011, Revised: 23 November 2011, Accepted: 26 November 2011Published online: 30 January 2012

Key words: blood spectroscopy; viral infection; laser tissue interaction; dengue diagnostic

1. Introduction

Interaction of light with living organisms has been usedfor therapeutic purposes for thousands of years [1,2]. Op-tical spectroscopy is a technique that utilizes the light en-ergy (electromagnetic radiation usually quantified by itswavelength) dependent interaction with a sample. Biolog-ical substances can interact with light energy and producea variety of optical responses, transmission, reflection scat-tering and absorption [3,4]. Infectious diseases have beena major threat to humans since historical times. Infectionwith one or more dengue viruses imperils an estimated 2 –5 billion people living in tropical and subtropical countries[5]. The first record of a case of probable dengue fever isin a Chinese medical encyclopedia (265 – 420 AD), whichreferred to a “water poison” associated with flying insects.When a mosquito carrying dengue virus bites a person, the

virus enters the skin together with the mosquito’s saliva. Itbinds to and enters white blood cells, and reproduces in-side the cells while they move throughout the body. Thewhite blood cells respond by producing a number of sig-naling proteins, such as interferon, responsible for manyof the symptoms, the fever, the flu-like symptoms and thesevere pains [6].

Dengue fever may be diagnosed by microbiologicallaboratory testing. This can be done by virus isolation incell cultures, nucleic acid detection by PCR, viral anti-gen detection or specific antibodies (serology). Virus iso-lation and nucleic acid detection are more accurate thanantigen detection, but these tests are not widely availabledue to their greater cost [7,8]. Diagnostic tests for denguevirus-specific antibodies, immunoglobulin M (IgM), andimmunoglobulin G (IgG) can be useful in confirming a di-agnosis in the later stages of the infection. Both IgG and

∗ Corresponding author: e-mail: shamaraz@gmail.com, shamaraz@nilop.edu.pk

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318 S. Firdous, M. Ahmed, et al.: Transmission spectroscopy of dengue viral infection

Glass slide

Whole blood drop

Objective 10×

MountFiber

Light source

Light spectrometer

Imaging station

Stand

Figure 1 (online color at www.lphys.org) Experimental setup for the whole blood dengue infection diagnostic with light spectroscopy

IgM are produced after 5–7 days. The highest levels ofIgM are detected following a primary infection, but IgMis also produced in secondary and tertiary infections. TheIgM becomes undetectable 30–90 days after a primary in-fection, but earlier following re-infections. IgG, by con-trast, remains detectable for over 60 years and, in the ab-sence of symptoms, is a useful indicator of past infection.In the laboratory test the IgG and the IgM antibodies cancross-react with other flaviviruses, such as yellow fevervirus, which can make the interpretation of the serologydifficult. The disadvantages of this test are the relativelyhigh cost and expertise needed particularly proper qualitycontrol to avoid false positives [9,10].

Whole blood-based optical spectroscopy potentiallyallows the measurement of total changes in whole bloodby means of the interaction of blood cells with light [11].Analysis shows that the primary organism (or pathogen)characteristic of the change in the nature of human, but thischange is now objectively and directly measurable throughthe use of spectral analysis. In the present study opticalspectroscopy using light transmission from tissue has beendemonstrated to diagnose the dengue viral infection. Thepresented results are very effective for initial screening ofdengue infected patients and to minimize the diagnosticcost.

2. Materials and methods

2.1. Samples preparation

The whole blood samples used in this study were collectedfrom the confirmed cases of dengue patients reported tothe NORI and PAEC general hospital, Islamabad alongwith the normal patients for control sample during Au-gust – October 2011. A total of 30 confirmed dengue pa-tients samples were collected and analyzed. Most of these

samples were from the major DEN-2, together with a fewsamples of DEN-3 types of dengue infected patients. Aconfirmed case of dengue virus infection was defined asfebrile illness associated with positive seroconversion orfour-fold increase in dengue-specific IgM or IgG antibodyfrom appropriately timed paired serum.

2.2. Samples analysis

The Avaspec spectrometer (Avantes Inc, Netherlands)wavelength range 400 – 1000 nm with halogen lamp(Avantes Inc, Netherlands) is used as an illuminating lightsource. The data were recorded with the help of Avaspecsoftware. The Halogen lamp spectra were recorded usingglass slide without blood sample. For normal and dengueinfected blood spectrum, 50 μl blood drop is placed onglass slide as shown in Fig. 1. Dengue diagnostic test canbe run on serum or whole blood. The test works best onfresh samples. If testing cannot be done immediately, sam-ples should be stored at 2 – 8◦C and may remain in storagefor up to 3 days. It is recommended that finger prick bloodbe used for quick results.

2.3. Samples precautions

Whole blood samples should be used immediately; allspecimens are handled according to the National Institutesof Health (NIH) recommendations at a biosafety level II[12]. Biological decontamination procedures are followedfor all equipment, containers, surfaces, etc. that come incontact with potentially infectious specimens. All dispos-ables that come in contact with these samples were dis-posed off as infectious waste.

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Laser Phys. Lett. 9, No. 4 (2012) 319

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Figure 2 (online color at www.lphys.org) The glass slide, nor-mal, and dengue infected whole blood spectrum 400 – 1000 nmwavelength

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Figure 3 (online color at www.lphys.org) The dengue infectedwhole blood spectrum 400 – 1000 nm wavelength of different pa-tients

3. Results

Light is absorbed and scattered in human blood throughthe action of a variety of chromospheres, including water,blood cells, various proteins, and hemoglobin. The bloodspectra of light absorption almost all tissue constituents liein the 400 – 1400 nm therapeutic window. Light is verystrongly scattered in tissue from index gradients withinorgans and cells, as well as from discrete structures suchas collagen fibrils, and red blood cells [13–16]. A signifi-cant characteristic of tissue optics is that in almost all tis-sues except for proteins, the scattering coefficient is much

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Figure 4 (online color at www.lphys.org) The whole blood spec-trum 400 – 1000 nm wavelength of glass slide, normal, malaria,and hepatitis c patients

larger than the absorption coefficient at number of wave-lengths. The transmission spectra of glass slide, 50 μlnormal blood, and 50 μl dengue infected samples wererecorded and is shown in Fig. 2, measured with Avaspecspectrometer on illuminating from 400 – 1000 nm of halo-gen lamp light. The normal blood absorbs light completelybelow 600 nm and shows transmission spectra in the rangeof 600 – 900 nm wavelengths.

The dengue infected sample illustrates transmissionspectra from 450 – 600 nm with two prominent peaks at540 and 580 nm, of the blood spectrum as measured withina set of about 30 essentially conformed dengue infectedindividuals, the dengue infected peaks are similar in allthe samples (Fig. 3) ranging from approximately 23 to 70years of age. The sample size may be increased furtherin the future but statistical significance is nevertheless al-ready attained. Such a monumental change in the basic na-ture and character of a fundamental and crucial protein andblood cells within the human body is a manifestation ofsignificant biochemical changes due to dengue infection.The fundamental nature of the protein, i.e., blood, has beenchanged in the case of dengue infection. The antibodiesIgG and IgM are produced against the dengue antigen. Theantibodies IgG activated after 2 – 3 days of dengue virusinfection and remain in blood up to one week. The anti-bodies IgM produced after one weak and remains duringlife. In the present study we have recorded transmissionspectra of other viral infected blood samples, like hepatitisand malaria, beside dengue infection. There is strong ab-sorption in the range of 400 – 600 nm as shown in Fig. 4,but the transmission spectra peaks at 540 and 580 nm wereobserved only in dengue infected samples. The light spec-tra for other infections shows different behavior, the de-

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320 S. Firdous, M. Ahmed, et al.: Transmission spectroscopy of dengue viral infection

tailed study about these infections will be communicatedlater on.

Analysis shows that the primary organism (orpathogen) characteristic of the dengue infection, causesa significant biochemical change in the nature of humanblood in which it resides. The dramatic change in the char-acteristic of the blood has also been presented through vis-ible observation for several years, but this change is nowdirectly measurable through the use of spectral analysis.Biomaterials spectroscopy demonstrates light cellular in-teraction in details for diagnostic and treatment of malig-nant diseases. C.F. Oliveira et al. presented metabolism ofodontoblast-like MDPC-23 cells exposed to laser doses of2, 4, 10, 15, and 25 J/cm2 from a diode laser, and ob-served highest ALP activity at 25 J/cm2, light dose. WhereC. Dressler et.al. explained optical tissue characteristics oftumor cells and their heat-induced changes with control-ling LITT progressions. Other scientists discussed the in-dications and limitations of aesthetic laser medicine, bac-terial, filamentation in Escherichia coli cultures exposed tolow intensity infrared lasers, and healing of the extensivesoft tissues wounds with combined low laser therapy andphotodynamic therapy treatment. In these findings lightabsorbed or scattered in different ways [17–21]. We sug-gest that these two prominent peaks are due to most of celldeath under dengue infection. The spectral methods underdevelopment are anticipated to be of value in the monitor-ing or measurement of change of the condition in a non-invasive manner.

The absorption spectra of human whole blood in thevisible and near-infrared region below 1000 nm are pre-dominantly due to light absorption by blood cells. Opticalproperties of human blood were also studied by A. Rog-gan et al. [22], who presented a set of optical parametersfor blood samples at the 633 nm wavelengths. Their resultsare difficult to compare with our data, as the blood samplesstudied by A. Roggan et al. were washed erythrocyte con-centrates, diluted to the desired hematocrit, and thus theblood samples contained no plasma proteins, leukocytes,or platelets, due to its chemical composition and struc-ture, blood can be considered a diffuse absorber. The majorchromospheres responsible for absorption of visible regionlight in human blood are hemoglobin and bilirubin. Onthe other hand, it is usually assumed that for wavelengthsshorter than 500 nm, high absorption of hemoglobin ef-fectively prevents penetration of light into the tissue. Ac-tually, however, absorption spectrum of hemoglobin hasa minimum at about 400 nm, and even around 500 nmthe absorption of hemoglobin is weak. In dengue infec-tion the broad band transmission spectrum along with twoprominent peaks at 540 and 580 nm may be a substan-tial contribution from scattering through antibodies pro-duced against dengue antigen, since blood derivatives, aswell as tissues, are turbid media, where multiple scatteringregimes are to be expected. The light beam in the bloodsample may be scattered by many cellular organelles andmembranes, or macromolecules, as well as by other ele-ments of the structural matrix. Wavelength dependence of

scattering depends on the size of the particles that scatterlight. When scatterers have dimensions smaller than onetenth the wavelength of the incident light, wavelength de-pendence of scattered light is Rayleigh scattering. Owingto the many-particle and heterogeneous character of blood,we can expect that what we perceive from the blood is acombination of the Mie and Rayleigh scattering. Mathe-matically, an exponential process that accounts for the lossof light intensity from the incident beam may account forboth processes.

In our previous work we have discussed in details thelaser cellular interaction study for photodynamic therapyof normal and malignant samples [23–26]. As a result,scattering of blood constituents and antibodies transmitgreen light and, as white cells and platelets are depleted ofabsorbing and scattering constituents, it will allow deeperpenetration of visible light. Without going deeper into the-oretical considerations, all photons that penetrate a biolog-ical object may interact with it. From the point of viewof biological response, even one percent of incident ra-diant power may be important. In line with expectations,green light penetrated less in both blood derivatives thanred light. Thus, the less penetrated green light may allowfor high light doses.

Future work and papers will focus on the interpretationof the nature of this change, and the development of a spec-tral method to the condition or of more dramatic changesin the blood of the individual. The spectral methods underdevelopment are anticipated to be of value in the monitor-ing or measurement of change of the condition of bloodmalignancy in a non-invasive manner.

4. Conclusions

This study describes a new methodology for opticallyquantifying changes in the blood of dengue infected pa-tients using optical spectroscopy. Knowledge of the opti-cal characteristics of blood tissue in a wide spectral rangeis necessary for the development of various diagnosticand treatment methods in dermatology, virology, and on-cological diseases. In our finding, we studied experimen-tally the optical parameters of the dengue infected bloodsamples of about 30 patients. The experiments were con-ducted in vitro with light spectrophotometer in the range of400 – 1000 nm. Based on the measured transmittance spec-tra, the dengue infected samples shows transmission band400 – 600 nm along with two dominant peaks at 540 and580 nm compared to normal and other infected samples.The results are consistent in all the analyzed infected sam-ples. The presented technique can be used for new lightbased diagnostic apparatus for dengue virus analyses of in-fected patients and further study on Raman excitation andconfocal microscopic imaging of dengue infected bloodsamples is underway.

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Laser Phys. Lett. 9, No. 4 (2012) 321

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