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Diagnosing sickle cell disease and iron deficiency anemia in human blood by Raman spectroscopy


This work proposed the diagnosis of iron deficiency anemia (IDA) and sickle cell disease (SCD) in human blood caused by iron deficiency and hemoglobin S (HbS), which are among the most common anemias, by means of Raman spectroscopy. Whole blood samples from patients diagnosed with IDA and HbS, as well as from normal subjects (HbA), were obtained and submitted to Raman spectroscopy (830 nm, 150 mW, 400–1800 cm−1 spectral range, 4 cm−1 resolution). Difference spectra of IDA–HbA showed spectral features of hemoglobin with less intensity in the IDA, whereas the difference spectra of SCD–HbA showed spectral features of deoxyhemoglobin increased and of oxyhemoglobin decreased in SCD. An exploratory analysis by principal components analysis (PCA) showed that the peaks referred to oxy- and deoxyhemoglobin markedly differentiated SCD and HbA, as well as the increased amount of hemoglobin features in the SCD group, suggesting increased erythropoiesis. The IDA group showed hemoglobin features with lower intensities as well as peaks referred to the iron bonding to the porphyrin ring with reduced intensities when compared to the HbA. Discriminant analysis based on partial least squares (PLS-DA) and PCA (PCA-DA) showed that the IDA and SCD anemias could be discriminated from the HbA spectra with 95.0% and 93.8% of accuracy, for the PLS and PCA respectively, with sensitivity/specificity of 93.8%/95.7% for the PLS-DA model. The iron depletion and the sickling of erythrocytes could be identified by Raman spectroscopy and a spectral model based on PLS accurately discriminated these IDA and SCD samples from the normal HbA.

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  1. 1.

    Kassebaum NJ, Jasrasaria R, Naghavi M, Wulf SK, Johns N, Lozano R, Regan M, Weatherall D, Chou DP, Eisele TP, Flaxman SR, Pullan RL, Brooker SJ, Murray CJ (2014) A systematic analysis of global anemia burden from 1990 to 2010. Blood 123:615–624.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  2. 2.

    World Health Organization, Blood Transfusion Safety (2002) The clinical use of blood in medicine, obstetrics, paediatrics, surgery and anaesthesia, trauma and burns. WHO. Accessed 30 Nov 2018

  3. 3.

    Benoist B, McLean E, Egli I, Cogswell M (2008) Worldwide prevalence of anaemia 1993-2005: WHO Global Database on Anaemia. WHO. Accessed 30 Nov 2018

  4. 4.

    Fernandes AP, Januário JN, Cangussu CB, Macedo DL, Viana MB (2010) Mortality of children with sickle cell disease: a population study. J Pediatr 86(4):279–284.

    Article  Google Scholar 

  5. 5.

    U. S. National Institute of Health, U. S. National Library of Medicine (2019) Sickle cell disease. NIH. Accessed 6 Feb 2019

  6. 6.

    Modell B, Darlison M (2008) Global epidemiology of haemoglobin disorders and derived service indicators. Bull World Health Organ 86(6):480–487.

    Article  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Brazilian Ministry of Health, Pesquisa Nacional de Demografia e Saúde da Criança e da Mulher (2006) Anemia e hipovitaminose A. Brazilian Ministry of Health. Accessed 2 Dec 2018

  8. 8.

    Arduini GA, Rodrigues LP, Trovó de Marqui AB (2017) Mortality by sickle cell disease in Brazil. Rev Bras Hematol Hemoter 39(1):52–56.

    Article  PubMed  Google Scholar 

  9. 9.

    Brazilian Ministry of Health, Secretaria de Atenção à Saúde, Departamento de Atenção Especializada (2012) Doença falciforme: condutas básicas para tratamento. Brazilian Ministry of Health. Accessed 2 Dec 2018

  10. 10.

    Brabin BJ, Hakimi M, Pelletier D (2001) An analysis of anemia and pregnancy-related maternal mortality. J Nutr 131(2):604S–614S.

    CAS  Article  PubMed  Google Scholar 

  11. 11.

    Haas JD, Brownlie T IV (2001) Iron deficiency and reduced work capacity: a critical review of the research to determine a causal relationship. J Nutr 131(2):676S–690S.

    CAS  Article  PubMed  Google Scholar 

  12. 12.

    World Health Organization Regional Office for Europe, UNICEF Regional Office for Central and Eastern Europe the Commonwealth of Independence States and the Baltic States (1999) Prevention and control of iron-deficiency anaemia in women and children. UNICEF/WHO. Accessed 2 Dec 2018

  13. 13.

    Dean J, Schechter AN (1978) Sickle-cell anemia: molecular and cellular bases of therapeutic approaches. N Engl J Med 299(14):752–763.

    CAS  Article  PubMed  Google Scholar 

  14. 14.

    Bryan LJ, Zakai NA (2012) Why is my patient anemic? Hematol Oncol Clin North Am 26(2):205–230.

    Article  PubMed  Google Scholar 

  15. 15.

    Archer NM, Brugnara C (2015) Diagnosis of iron-deficient states. Crit Rev Clin Lab Sci 52(5):256–272.

    CAS  Article  PubMed  Google Scholar 

  16. 16.

    Johnson-Wimbley TD, Graham DY (2011) Diagnosis and management of iron deficiency anemia in the 21st century. Ther Adv Gastroenterol 4(3):177–184.

    Article  Google Scholar 

  17. 17.

    Ali MA, Luxton AW, Walker WH (1978) Serum ferritin concentration and bone marrow iron stores: a prospective study. Can Med Assoc J 118(8):945–946

    CAS  PubMed  PubMed Central  Google Scholar 

  18. 18.

    Burns ER, Goldberg SN, Lawrence C, Wenz B (1990) Clinical utility of serum tests for iron deficiency in hospitalized patients. Am J Clin Pathol 93(2):240–245.

    CAS  Article  PubMed  Google Scholar 

  19. 19.

    Guyatt GH, Patterson C, Ali M, Singer J, Levine M, Turpie I, Meyer R (1990) Diagnosis of iron-deficiency anemia in the elderly. Am J Med 88(3):205–209.

    CAS  Article  PubMed  Google Scholar 

  20. 20.

    Baumann Kurer S, Seifert B, Michel B, Ruegg R, Fehr J (1995) Prediction of iron deficiency in chronic inflammatory rheumatic disease anaemia. Brit J Haematol 91(4):820–826.

    CAS  Article  Google Scholar 

  21. 21.

    Mast AE, Blinder MA, Gronowski AM, Chumley C, Scott MG (1998) Clinical utility of the soluble transferrin receptor and comparison with serum ferritin in several populations. Clin Chem 44(1):45–51

    CAS  Article  Google Scholar 

  22. 22.

    Ogedegbe HO, Csury L, Simmons BH (2004) Anemias: a clinical laboratory perspective. Lab Med 35(3):177–185.

    Article  Google Scholar 

  23. 23.

    Gonçalves MS, Bomfim GC, Maciel E, Cerqueira I, Lyra I, Zanette A, Bomfim G, Adorno EV, Albuquerque AL, Pontes A, Dupuit MF, Fernandes GB, dos Reis MG (2003) βS-Haplotypes in sickle cell anemia patients from Salvador, Bahia, Northeastern Brazil. Braz J Med Biol Res 36(10):1283–1288.

    Article  PubMed  Google Scholar 

  24. 24.

    Hanlon EB, Manoharan R, Koo TW, Shafer KE, Motz JT, Fitzmaurice M, Kramer JR, Itzkan I, Dasari RR, Feld MS (2000) Prospects for in vivo Raman spectroscopy. Phys Med Biol 45(2):R1–R59.

    CAS  Article  PubMed  Google Scholar 

  25. 25.

    de Almeida ML, Saatkamp CJ, Fernandes AB, Pinheiro AL, Silveira L (2016) Estimating the concentration of urea and creatinine in the human serum of normal and dialysis patients through Raman spectroscopy. Lasers Med Sci 31(7):1415–1423.

    Article  PubMed  Google Scholar 

  26. 26.

    Silveira L, Borges RCF, Navarro RS, Giana HE, Zângaro RA, Pacheco MTT, Fernandes AB (2017) Quantifying glucose and lipid components in human serum by Raman spectroscopy and multivariate statistics. Lasers Med Sci 32(4):787–795.

    Article  PubMed  Google Scholar 

  27. 27.

    Bueno Filho AC, Silveira L, Yanai AL, Fernandes AB (2015) Raman spectroscopy for a rapid diagnosis of sickle cell disease in human blood samples: a preliminary study. Lasers Med Sci 30(1):247–253.

    Article  Google Scholar 

  28. 28.

    Atkins CG, Buckley K, Blades MW, Turner RFB (2017) Raman spectroscopy of blood and blood components. Appl Spectrosc 71(5):767–793.

    CAS  Article  PubMed  Google Scholar 

  29. 29.

    Matousek P, Stone N (2013) Recent advances in the development of Raman spectroscopy for deep non-invasive medical diagnosis. J Biophotonics 6(1):7–19.

    CAS  Article  PubMed  Google Scholar 

  30. 30.

    Diem M, Mazur A, Lenau K, Schubert J, Bird B, Miljković M, Krafft C, Popp J (2013) Molecular pathology via IR and Raman spectral imaging. J Biophotonics 6(11-12):855–886.

    CAS  Article  PubMed  Google Scholar 

  31. 31.

    Silveira L, Silveira FL, Bodanese B, Zângaro RA, Pacheco MT (2012) Discriminating model for diagnosis of basal cell carcinoma and melanoma in vitro based on the Raman spectra of selected biochemicals. J Biomed Opt 17(7):077003.

    CAS  Article  PubMed  Google Scholar 

  32. 32.

    Ward KR, Torres Filho I, Barbee RW, Torres L, Tiba MH, Reynolds PS, Pittman RN, Ivatury RR, Terner J (2006) Resonance Raman spectroscopy: a new technology for tissue oxygenation monitoring. Crit Care Med 34(3):792–799.

    CAS  Article  PubMed  Google Scholar 

  33. 33.

    Torres Filho IP, Terner J, Pittman RN, Somera LG 3rd, Ward KR (2005) Hemoglobin oxygen saturation measurements using resonance Raman intravital microscopy. Am J Physiol Heart Circ Physiol 289(1):H488–H495.

    CAS  Article  PubMed  Google Scholar 

  34. 34.

    Eigenvector Research, Eigenvector Documentation Wiki (2017) Advanced preprocessing: sample normalization. Eigenvector Research. Accessed 10 June 2017

  35. 35.

    Chatfield C, Collins AJ (1980) Introduction to multivariate statistics. Chapman & Hall, London

    Google Scholar 

  36. 36.

    Barker M, Rayens W (2003) Partial least squares for discrimination. J Chemom 17(3):166–173.

    CAS  Article  Google Scholar 

  37. 37.

    Nunes CA, Freitas MP, Pinheiro ACM, Bastos SC (2012) Chemoface: a novel free user-friendly interface for chemometrics. J Braz Chem Soc 23(11):2003–2010.

    CAS  Article  Google Scholar 

  38. 38.

    Vanna R, Ronchi P, Lenferink AT, Tresoldi C, Morasso C, Mehn D, Bedoni M, Picciolini S, Terstappen LW, Ciceri F, Otto C, Gramatica F (2015) Label-free imaging and identification of typical cells of acute myeloid leukaemia and myelodysplastic syndrome by Raman microspectroscopy. Analyst 140(4):1054–1064.

    CAS  Article  PubMed  Google Scholar 

  39. 39.

    Lima AMF, Daniel CR, Navarro RS, Bodanese B, Pasqualucci CA, Pacheco MTT, Zângaro RA, Silveira L (2019) Discrimination of non-melanoma skin cancer and keratosis from normal skin tissue in vivo and ex vivo by Raman spectroscopy. Vib Spectrosc 100(1):131–141.

    CAS  Article  Google Scholar 

  40. 40.

    Brunner H, Sussner H (1973) Resonance Raman scattering on haemoglobin. Biochim Biophys Acta 310(1):20–31.

    CAS  Article  PubMed  Google Scholar 

  41. 41.

    Bankapur A, Zachariah E, Chidangil S, Valiathan M, Mathur D (2010) Raman tweezers spectroscopy of live, single red and white blood cells. PLoS One 5(4):e10427.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Wood BR, McNaughton D (2002) Raman excitation wavelength investigation of single red blood cells in vivo. J Raman Spectrosc 33(7):517–523.

    CAS  Article  Google Scholar 

  43. 43.

    De Luca AC, Rusciano G, Ciancia R, Martinelli V, Pesce G, Rotoli B, Selvaggi L, Sasso A (2008) Spectroscopical and mechanical characterization of normal and thalassemic red blood cells by Raman tweezers. Opt Express 16(11):7943–7957.

    Article  PubMed  Google Scholar 

  44. 44.

    Dingari NC, Horowitz GL, Kang JW, Dasari RR, Barman I (2012) Raman spectroscopy provides a powerful diagnostic tool for accurate determination of albumin glycation. PLoS One 7(2):e32406.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  45. 45.

    Perez-Guaita D, de Veij M, Marzec KM, Almohammedi ARD, McNaughton D, Hudson AJ, Wood BR (2017) Resonance Raman and UV-visible microscopy reveals that conditioning red blood cells with repeated doses of sodium dithionite increases haemoglobin oxygen uptake. ChemistrySelect 2(11):3342–3346.

    CAS  Article  Google Scholar 

  46. 46.

    Atkins CG, Schulze HG, Chen D, Devine DV, Blades MW, Turner RFB (2017) Using Raman spectroscopy to assess hemoglobin oxygenation in red blood cell concentrate: an objective proxy for morphological index to gauge the quality of stored blood? Analyst 142(12):2199–2210.

    CAS  Article  PubMed  Google Scholar 

  47. 47.

    Wood BR, Tait B, McNaughton D (2001) Micro-Raman characterisation of the R to T state transition of haemoglobin within a single living erythrocyte. Biochim Biophys Acta 1539(1-2):58–70.

    CAS  Article  PubMed  Google Scholar 

  48. 48.

    Wood BR, Caspers P, Puppels GJ, Pandiancherri S, McNaughton D (2007) Resonance Raman spectroscopy of red blood cells using near-infrared laser excitation. Anal Bioanal Chem 387(5):1691–1703.

    CAS  Article  PubMed  Google Scholar 

  49. 49.

    Rao S, Bálint S, Cossins B, Guallar V, Petrov D (2009) Raman study of mechanically induced oxygenation state transition of red blood cells using optical tweezers. Biophys J 96(1):209–216.

    CAS  Article  PubMed  Google Scholar 

  50. 50.

    Gautam R, Oh JY, Marques MB, Dluhy RA, Patel RP (2018) Characterization of storage-induced red blood cell hemolysis using Raman spectroscopy. Lab Med 49(4):298–310.

    Article  PubMed  PubMed Central  Google Scholar 

  51. 51.

    Talari ACS, Movasaghi Z, Rehman S, Rehman I (2015) Raman spectroscopy of biological tissues. Appl Spectrosc Rev 50(1):46–111.

    CAS  Article  Google Scholar 

  52. 52.

    Jelkmann W (2007) Erythropoietin after a century of research: younger than ever. Eur J Haematol 78(3):183–205.

    CAS  Article  PubMed  Google Scholar 

  53. 53.

    Farrell K, Dent L, Buchowski M, Aguinaga MP (2009) Sickle cell anemia as a disease of oxygen transport: possible implications for the prevention of sickle cell crises. Blood 114(22):4617

    Article  Google Scholar 

  54. 54.

    Chowdhury A, Dasgupta R, Majumder SK (2017) Changes in hemoglobin-oxygen affinity with shape variations of red blood cells. J Biomed Opt 22(10):105006.

    Article  Google Scholar 

  55. 55.

    Brunner H, Mayer A, Sussner H (1972) Resonance Raman scattering on the haem group of oxy-and deoxyhaemoglobin. J Mol Biol 70(1):153–156.

    CAS  Article  PubMed  Google Scholar 

  56. 56.

    Wesełucha-Birczyńska A, Kozicki M, Czepiel J, Łabanowska M, Nowak P, Kowalczyk G, Kurdziel M, Birczyńska M, Biesiada G, Mach T, Garlicki A (2014) Human erythrocytes analyzed by generalized 2D Raman correlation spectroscopy. J Mol Struct 1069:305–312.

    CAS  Article  Google Scholar 

  57. 57.

    Liu R, Mao Z, Matthews DL, Li CS, Chan JW, Satake N (2013) Novel single-cell functional analysis of red blood cells using laser tweezers Raman spectroscopy: application for sickle cell disease. Exp Hematol 41(7):656–661.

    CAS  Article  PubMed  Google Scholar 

  58. 58.

    Yan D, Domes C, Domes R, Frosch T, Popp J, Pletz MW, Frosch T (2016) Fiber enhanced Raman spectroscopic analysis as a novel method for diagnosis and monitoring of diseases related to hyperbilirubinemia and hyperbiliverdinemia. Analyst 141(21):6104–6115.

    CAS  Article  PubMed  Google Scholar 

  59. 59.

    Wood BR, McNaughton D (2002) Micro-Raman characterization of high- and low-spin heme moieties within single living erythrocytes. Biopolymers 67(4-5):259–262.

    CAS  Article  PubMed  Google Scholar 

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L. Silveira Jr. acknowledges FAPESP (São Paulo Research Foundation) for granting the Raman spectrometer (Grant No. 2009/01788-5). L. Silveira Jr. and A. B. Fernandes acknowledge CNPq (National Council for Scientific and Technological Development) for financial support (Grant No. 460014/2014-5). L. Silveira Jr. acknowledges CNPq for the Productivity fellowship (Process No. 306344/2017-3).

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Correspondence to Landulfo Silveira Jr.

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The authors declare that they have no conflict of interest.

Ethical approval

Authors declare compliance with ethical standards when using human blood samples, being the study approved by the Committee for Ethics in Research of Universidade Brasil, Protocol No. 1.578.298 (CAAE No. 53543516.4.0000.5494) following Brazilian regulations for use of human subjects or materials in research (Brazilian Ministry of Health, National Health Council, Resolution CNS No. 466/2012) and have been performed in accordance with the ethical standards as laid down in the 1964 Declaration of Helsinki and its later amendments.

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da Silva, W.R., Silveira, L. & Fernandes, A.B. Diagnosing sickle cell disease and iron deficiency anemia in human blood by Raman spectroscopy. Lasers Med Sci 35, 1065–1074 (2020).

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  • Raman spectroscopy
  • Diagnosis
  • Sickle cell disease
  • Iron deficiency anemia