2. Integrated Cavity Absorption Meter: Design & Validation 1 Introduction 1 Introduction
5.7 Determination of the Transfer Function Equation
A transfer function relates the scattered light from the ICAM to incident light intensity. In the absence of an absorbing sample, such as Mb+, Hb, or RBCs, a small background signal, B, is detected that derives from the presence of milk. The intensity from the ICAM containing the sample of interest, after subtracting B and then dividing by the incident intensity is denoted I*. The transmittance from the ICAM is then related to the absorbance of the sample. For the deoxygenation of oxyhemoglobin or oxygenated RBCs, at a given temperature, the I* quantity is:
𝐼𝐼∗ = 1 − 𝑒𝑒−(𝑘𝑘𝛥𝛥𝑏𝑏𝐶𝐶2[𝛥𝛥𝑏𝑏𝐶𝐶2]+𝑘𝑘𝛥𝛥𝑏𝑏[𝛥𝛥𝑏𝑏]). 5.4 where the k's are absorption coefficients at 620 nm for the indicated species. The apparent fractional saturation of hemoglobin with oxygen in the RBCs was not wavelength
dependent in the region of 620 nm, where I* was measured, in contrast to the 535-580 nm region. Temperature was measured throughout with a calibrated thermistor in the RBC suspension. Nitrogen gas saturated with water vapor was passed over the stirred sample, and the decreasing oxygen concentration data were collected together with the
corresponding temperatures. A very small amount of a dithionite solution was carefully injected at the end of the deoxygenation procedure to ensure complete deoxygenation.
The values of I* were evaluated. We found that the apparent absorbance from the ICAM followed an exponential complement with respect to absorbance per centimeter in the ICAM.
At 20°C, the fractional saturation for oxygen binding by hemoglobin at one atmosphere in the RBC is essentially 1. In terms of the absorption coefficient, kHbO2, within the RBC, the intensity is then:
𝐼𝐼∗= 1 − 𝑒𝑒−(𝑘𝑘𝛥𝛥𝑏𝑏𝐶𝐶2[𝛥𝛥𝑏𝑏𝐶𝐶2]). 5.5 where [HbO2] is within experimental error of HbT. When oxygen is depleted and all hemoglobin is deoxygenated within the RBC, then:
𝐼𝐼∗ = 1 − 𝑒𝑒−(𝑘𝑘𝛥𝛥𝑏𝑏[𝛥𝛥𝑏𝑏]). 5.6 allowing the exponential in equation S1 to be written in terms of fractional saturation, Y, to give:
𝐼𝐼∗= 1 − 𝑒𝑒−𝑘𝑘𝛥𝛥𝑏𝑏𝐶𝐶(𝑌𝑌)−𝑘𝑘𝛥𝛥𝑏𝑏(1−𝑌𝑌)∗𝛥𝛥𝑏𝑏𝑇𝑇. 5.7 Following oxygen removal, Y=0 and the endpoint reading of I* for the
deoxygenated RBC is established, yielding the value for kHb at a given temperature. Y can then be evaluated over the full temperature range for determination of all thermodynamic oxygen binding parameters that characterize oxygen binding for hemoglobin in the RBC.
The ICAM round-bottom flask was held in place via a clamp attached to a camera mount. The camera mount was stable during experimentation. The camera mount and clamp allowed easy removal from the Cary 300 chamber and precision during
replacement.
6. Bibliography
(1) Nencki, M. Über Das Parahämoglobin. Archiv für Experimentelle Pathologie und Pharmakologie 1886, 20 (5–6), 332–346.
(2) Hüfner, G. Über Die Dissociation Des Oxyhämoglobins in Wässeriger Lösung.
Zeitschrift für Physikalische Chemie 1893.
(3) Perutz, M. F.; Rossman, M. G.; Cullis, A. N. N. F.; Muirhead, H.; Will, G.;
North, A. C. T. Structure of Hemoglobin: A Three-Dimensional Fourier Synthesis at 5.5 Å Resolution, Obtained by x-Ray Analysis. Nature 1960, 185 (4711), 416–
422.
(4) Muirhead, H.; Perutz, M. F. Structure of Hemoglobin: A Three-Dimensional Fourier Synthesis of Reduced Human Hemoglobin at 5.5 Å Resolution. Nature 1963, 199 (4894), 633–638.
(5) Chance, M. R.; Parkhurst, L. J.; Powers, L. S.; Chance, B. Movement of Fe with Respect to the Heme Plane in the RT Transition of Carp Hemoglobin. An
Extended x-Ray Absorption Fine Structure Study. Journal of Biological Chemistry 1986, 261 (13), 5689–5692.
(6) Perutz, M. F. The Hemoglobin Molecule. Scientific American 1964, 211 (5), 64–
79.
(7) Perutz, M. F. Science Is Not a Quiet Life: Unravelling the Atomic Mechanism of Haemoglobin; World Scientific, 1997; Vol. 4.
(8) Ashley-Koch, A.; Yang, Q.; Olney, R. S. Sickle Hemoglobin (Hb S) Allele and Sickle Cell Disease: A HuGE Review. American Journal of Epidemiology 2000, 151 (9), 839–845.
(9) Williams, T. N.; Obaro, S. K. Sickle Cell Disease and Malaria Morbidity: A Tale with Two Tails. Trends in Parasitology 2011, 27 (7), 315–320.
(10) Harrison, K. A. Anaemia, Malaria and Sickle Cell Disease. Clinics in Obstetrics and Gynaecology 1982, 9 (3), 445–477.
(11) Konotey-Ahulu, F. I. D. The Sickle Cell Diseases: Clinical Manifestations Including the Sickle Crisis. Archives of Internal Medicine 1974, 133 (4), 611–619.
(12) Platt, O. S.; Brambilla, D. J.; Rosse, W. F.; Milner, P. F.; Castro, O.; Steinberg, M. H.; Klug, P. P. Mortality in Sickle Cell Disease--Life Expectancy and Risk Factors for Early Death. New England Journal of Medicine 1994, 330 (23), 1639–
1644.
(13) Itano, H. A.; Neel, J. v. A New Inherited Abnormality of Human Hemoglobin.
Proceedings of the National Academy of Sciences of the United States of America 1950, 36 (11), 613.
(14) Finch, J. T.; Perutz, M. F.; Bertles, J. F.; Döbler, J. Structure of Sickled
Erythrocytes and of Sickle-Cell Hemoglobin Fibers. Proceedings of the National Academy of Sciences 1973, 70 (3), 718–722.
(15) Gill, S. J.; Benedict, R. C.; Fall, L.; Spokane, R.; Wyman, J. Oxygen Binding to Sickle Cell Hemoglobin. Journal of Molecular Biology 1979, 130 (2), 175–189.
(16) Sunshine, H. R.; Hofrichter, J.; Ferrone, F. A.; Eaton, W. A. Oxygen Binding by Sickle Cell Hemoglobin Polymers. Journal of Molecular Biology 1982, 158 (2), 251–273.
(17) Vichinsky, E. Hemoglobin E Syndromes. Hematology 2007, 2007 (1), 79–83.
(18) Rees, D. C.; Styles, L.; Vichinsky, E. P.; Clegg, J. B.; Weatherall, D. J. The Hemoglobin E Syndromes. Annals of the New York Academy of Sciences 1998, 850 (1), 334–343.
(19) Muncie Jr, H. L.; Campbell, J. S. Alpha and Beta Thalassemia. American Family Physician 2009, 80 (4), 339–344.
(20) Zeng, Y. T.; Huang, S. Z.; Ren, Z. R.; Li, H. J. Identification of Hb D-Punjab Gene: Application of DNA Amplification in the Study of Abnormal Hemoglobins.
American journal of human genetics 1989, 44 (6), 886.
(21) El-Hazmi, M. A. F.; Lehmann, H. Human Haemoglobins and
Haemoglobinopathies in Arabia: Hb O Arab in Saudi Arabia. Acta Haematologica 1980, 63 (5), 268–273.
(22) Chopra, A.; Fisher, C.; Khunger, J. M.; Pati, H. Hemoglobin O Indonesia in India: A Rare Observation. Annals of hematology 2011, 90 (3), 353–354.
(23) Lal, A.; Goldrich, M. L.; Haines, D. A.; Azimi, M.; Singer, S. T.; Vichinsky, E.
P. Heterogeneity of Hemoglobin H Disease in Childhood. New England Journal of Medicine 2011, 364 (8), 710–718.
(24) Efremov, G. D. Hemoglobins Lepore and Anti-Lepore. Hemoglobin 1978, 2 (3), 197–233.
(25) Nagayama, Y.; Yoshida, M.; Kohyama, T.; Matsui, K. Hemoglobin Kansas as a Rare Cause of Cyanosis: A Case Report and Review of the Literature. Internal Medicine 2017, 56 (2), 207–209.
(26) Engelhart, J. F. Commentatio de Vera Materiae Sanguini Purpureum Colorem Impertientis Natura; typis Dieterichianis, 1825.
(27) Bert, P. La Pression Barométrique: Recherches de Physiologie Expérimentale;
G. Masson, 1878.
(28) Bohr, C. Die Saurestoffe Des Genuinen Blutfarbstoffes Und Des Aus Dem Blute Darstellten Hämoglobins. Centralbl. für Physiol 1904, 23.
(29) Barcroft, J.; King, W. O. R. The Effect of Temperature on the Dissociation Curve of Blood. The Journal of Physiology 1909, 39 (5), 374–384.
(30) Haldane, J. S.; Priestley, J. G. Respiration. New Haven. CT: Yale University Press 1922.
(31) van Slyke, D. D.; Neill, J. M. The Determination of Gases in Blood and Other Solutions by Vacuum Extraction and Manometric Measurement. I. Journal of Biological Chemistry 1924, 61 (2), 523–573.
(32) Roughton, F. J.; Severinghaus, J. W. Accurate Determination of O2 Dissociation Curve of Human Blood above 98.7 Percent Saturation with Data on O2 Solubility in Unmodified Human Blood from 0 Degrees to 37 Degrees C. Journal of Applied Physiology 1973, 35 (6), 861–869.
(33) Guidotti, G.; Konigsberg, W.; Craig, L. C. On the Dissociation of Normal Adult Human Hemoglobin. Proceedings of the National Academy of Sciences of the United States of America 1963, 50 (4), 774.
(34) Brissot, P.; Ropert, M.; le Lan, C.; Loréal, O. Non-Transferrin Bound Iron: A Key Role in Iron Overload and Iron Toxicity. Biochimica et Biophysica Acta (BBA)-General Subjects 2012, 1820 (3), 403–410.
(35) Snyder, G. K.; Sheafor, B. A. Red Blood Cells: Centerpiece in the Evolution of the Vertebrate Circulatory System. American Zoologist 1999, 39 (2), 189–198.
(36) Soslau, G. The Role of the Red Blood Cell and Platelet in the Evolution of Mammalian and Avian Endothermy. Journal of Experimental Zoology Part B:
Molecular and Developmental Evolution 2020, 334 (2), 113–127.
(37) Waugh, R.; Evans, E. A. Thermoelasticity of Red Blood Cell Membrane.
Biophysical Journal 1979, 26 (1), 115–131.
(38) Lenormand, G.; Hénon, S.; Richert, A.; Siméon, J.; Gallet, F. Direct
Measurement of the Area Expansion and Shear Moduli of the Human Red Blood Cell Membrane Skeleton. Biophysical Journal 2001, 81 (1), 43–56.
(39) Guest, M. M.; Bond, T. P.; Cooper, R. G.; Derrick, J. R. Red Blood Cells:
Change in Shape in Capillaries. Science 1963, 142 (3597), 1319–1321.
(40) Skalak, R.; Branemark, P. I. Deformation of Red Blood Cells in Capillaries.
Science 1969, 164 (3880), 717–719.
(41) Secomb, T. W.; Skalak, R.; Özkaya, N.; Gross, J. F. Flow of Axisymmetric Red Blood Cells in Narrow Capillaries. Journal of Fluid Mechanics 1986, 163, 405–
423.
(42) Obrist, D.; Weber, B.; Buck, A.; Jenny, P. Red Blood Cell Distribution in
Simplified Capillary Networks. Philosophical Transactions of the Royal Society A:
Mathematical, Physical and Engineering Sciences 2010, 368 (1921), 2897–2918.
(43) Imai, K.; Morimoto, H.; Kotani, M.; Watari, H.; Hirata, W.; Kuroda, M. Studies on the Function of Abnormal Hemoglobins I. An Improved Method for Automatic Measurement of the Oxygen Equilibrium Curve of Hemoglobin. Biochimica et Biophysica Acta (BBA)-Protein Structure 1970, 200 (2), 189–196.
(44) Ackers, G. K.; Halvorson, H. R. The Linkage between Oxygenation and Subunit Dissociation in Human Hemoglobin. Proceedings of the National Academy of Sciences 1974, 71 (11), 4312–4316.
(45) Lederberg, J. Encyclopedia of Microbiology, Four-Volume Set; Academic Press, 2000.
(46) Atha, D. H.; Riggs, A. Tetramer-Dimer Dissociation in Homoglobin and the Bohr Effect. Journal of Biological Chemistry 1976, 251 (18), 5537–5543.
(47) Anderson, N. M.; Sekelj, P. Light-Absorbing and Scattering Properties of Nonhaemolysed Blood. Physics in Medicine & Biology 1967, 12 (2), 173.
(48) Stover, J. C. Optical Scattering: Measurement and Analysis; Society of Photo-Optical Instrumentation Engineers, 2012.
(49) Lambert, J. H. Observationes Variae in Mathesin Puram. Acta Helvetica 1758, 3 (1), 128–168.
(50) Röttgers, R.; Häse, C.; Doerffer, R. Determination of the Particulate Absorption of Microalgae Using a Point‐source Integrating‐cavity Absorption Meter:
Verification with a Photometric Technique, Improvements for Pigment Bleaching, and Correction for Chlorophyll Fluorescence. Limnology and Oceanography:
Methods 2007, 5 (1), 1–12.
(51) Khalizov, A. F.; Xue, H.; Wang, L.; Zheng, J.; Zhang, R. Enhanced Light Absorption and Scattering by Carbon Soot Aerosol Internally Mixed with Sulfuric Acid. The Journal of Physical Chemistry A 2009, 113 (6), 1066–1074.
(52) Walsh, J. W. T. Photometry. Constable, London. 1953.
(53) Elterman, P. Integrating Cavity Spectroscopy. Applied Optics 1970, 9 (9), 2140–
2142.
(54) Fry, E. S.; Kattawar, G. W.; Pope, R. M. Integrating Cavity Absorption Meter.
Applied Optics 1992, 31 (12), 2055–2065.
(55) Jávorfi, T.; Erostyák, J.; Gál, J.; Buzády, A.; Menczel, L.; Garab, G.; Naqvi, K.
R. Quantitative Spectrophotometry Using Integrating Cavities. Journal of Photochemistry and Photobiology B: Biology 2006, 82 (2), 127–131.
(56) Clark JR, L. C.; Wolf, R.; Granger, D.; Taylor, Z. Continuous Recording of Blood Oxygen Tensions by Polarography. Journal of Applied Physiology 1953, 6 (3), 189–193.
(57) Epstein, E. J.; Coulshed, N.; McKendrick, C. S.; Clarke, J.; Kearns, W. E.
Artificial Pacing by Electrode Catheter for Heart Block or Asystole Complicating Acute Myocardial Infarction. British Heart Journal 1966, 28 (4), 546.
(58) Bowen, W. J. Notes on Myoglobin Preparation and Iron Content. Journal of Biological Chemistry 1948, 176 (2), 747–751.
(59) YSI Model 5300 Biological Oxygen Monitor Instruction Manual; YSI Incorporated.
(60) Motulsky, H. Analyzing Data with GraphPad Prism; GraphPad Software Incorporated, 1999.
(61) Gill, S. J.; di Cera, E.; Doyle, M. L.; Bishop, G. A.; Robert, C. H. Oxygen
Binding Constants for Human Hemoglobin Tetramers. Biochemistry 1987, 26 (13), 3995–4002.
(62) Monod, J.; Wyman, J.; Changeux, J.-P. On the Nature of Allosteric Transitions:
A Plausible Model. Journal of Molecular Biology 1965, 12 (1), 88–118.
(63) Millar, S. J.; Moss, B. W.; Stevenson, M. H. Some Observations on the Absorption Spectra of Various Myoglobin Derivatives Found in Meat. Meat Science 1996, 42 (3), 277–288. https://doi.org/https://doi.org/10.1016/0309-1740(94)00045-X.
(64) Parkhurst, L. J.; Gibson, Q. H. The Reaction of Carbon Monoxide with Horse Hemoglobin in Solution, in Erythrocytes, and in Crystals. Journal of Biological Chemistry 1967, 242 (24), 5762–5770.
(65) Schenkman, K. A.; Marble, D. R.; Burns, D. H.; Feigl, E. O. Myoglobin Oxygen Dissociation by Multiwavelength Spectroscopy. Journal of Applied Physiology 1997, 82 (1), 86–92.
(66) Adair, G. S. A Comparison of the Molecular Weights of the Proteins. Biological Reviews 1924, 1 (2), 75–78.
(67) Adair, G. S. The Hemoglobin System VI. The Oxygen Dissociation Curve of Hemoglobin. Journal of Biological Chemistry 1925, 63 (2), 529–545.
(68) Otis, A. B.; Roughton, F. J. W. Improved Gasometric Methods for Estimating Oxygen Content and Percentage Saturation in Haemoglobin Dissociation Curve Determinations. Proceedings of the Royal Society of London. Series B-Biological Sciences 1955, 144 (914), 55–67.
(69) Koshland Jr, D. E.; Nemethy, G.; Filmer, D. Comparison of Experimental Binding Data and Theoretical Models in Proteins Containing Subunits.
Biochemistry 1966, 5 (1), 365–385.
(70) Kiyohiro Imai. Allosteric Effects of Hemoglobin; Cambridge University Press:
Osaka, 1982.
(71) Severinghaus, J. W. Blood Gas Calculator. Journal of Applied Physiology 1966, 21 (3), 1108–1116.
(72) Hartridge, H.; Roughton, F. J. W. The Rate of Distribution of Dissolved Gases between the Red Blood Corpuscle and Its Fluid Environment: Part I. Preliminary EExperiments on the Rate of Uptake of Oxygen and Carbon Monoxide by Sheep’s Corpuscles. The Journal of Physiology 1927, 62 (3), 232–242.
(73) Astrup, P.; Engel, K.; Severinghaus, J. W.; Munson, E. The Influence of Temperature and Ph on the Dissociation Curve of Oxyhemoglobin of Human Blood. Scandinavian Journal of Clinical and Laboratory Investigation 1965, 17 (6), 515–523.
(74) Francis, A. T.; Berry, K.; Thomas, E. C.; Hill, A. H.; Fu, D. In Vitro Quantification of Single Red Blood Cell Oxygen Saturation by Femtosecond Transient Absorption Microscopy. The Journal of Physical Chemistry Letters 2019, 10 (12), 3312–3317.
(75) Vorger, P. Thermodynamic Studies on Oxygen Binding by Human Red Blood Cells. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology 1999, 123 (4), 329–336.
(76) Pezzella, M.; el Hage, K.; Niesen, M. J. M.; Shin, S.; Willard, A. P.; Meuwly, M.; Karplus, M. Water Dynamics around Proteins: T-and R-States of Hemoglobin and Melittin. The Journal of Physical Chemistry B 2020, 124 (30), 6540–6554.
(77) Bucci, E.; Pucciarelli, S.; Angeletti, M. Free Energy Changes and Components Implicit in the MWC Allosteric Model for the Cooperative Oxygen Binding of Hemoglobin. Biochemistry 2013, 52 (24), 4149–4156.
https://doi.org/10.1021/bi400319c.
(78) Salvay, A. G.; Grigera, J. R.; Colombo, M. F. The Role of Hydration on the Mechanism of Allosteric Regulation: In Situ Measurements of the Oxygen-Linked Kinetics of Water Binding to Hemoglobin. Biophysical journal 2003, 84 (1), 564–
570. https://doi.org/10.1016/S0006-3495(03)74876-0.
(79) Beilin, L. J.; Knight, G. J.; Munro-Faure, A. D.; Anderson, J. The Sodium, Potassium, and Water Contents of Red Blood Cells of Healthy Human Adults. The Journal of Clinical Investigation 1966, 45 (11), 1817–1825.
(80) Asakura, T.; Minakata, K.; Adachi, K.; Russell, M. O.; Schwartz, E. Denatured Hemoglobin in Sickle Erythrocytes. The Journal of clinical investigation 1977, 59 (4), 633–640.
(81) Itano, H. A. Solubilities of Naturally Occurring Mixtures of Human
Hemoglobin. Archives of biochemistry and biophysics 1953, 47 (1), 148–159.
(82) Yuile, C. L.; Gold, M. A.; Hinds, E. G. Hemoglobin Precipitation in Renal Tubules: A Study of Its Causes and Effects. The Journal of experimental medicine 1945, 82 (5), 361.
(83) Amiconi, G.; Zolla, L.; Vecchini, P.; Brunori, M.; Antonini, E. The Effect of Macromolecular Polyanions on the Functional Properties of Human Hemoglobin.
European journal of biochemistry 1977, 76 (2), 339–343.
(84) Kondubhotla, S.; Olsen, K. W. Formation of Hemoglobin Octomers Cross-Linking of Hemoglobin at Reactive Cysteine Beta-93 with Bismaleidohexane. In Abstracts of Papers of the American Chemical Society; AMER CHEMICAL SOC PO BOX 57136, WASHINGTON, DC 20037-0136, 1995; Vol. 210, pp 201-PHYS.
(85) Agafonov, R. v.; Negrashov, I. v.; Tkachev, Y. v.; Blakely, S. E.; Titus, M. A.;
Thomas, D. D.; Nesmelov, Y. E. Structural Dynamics of the Myosin Relay Helix by Time-Resolved EPR and FRET. Proc. Natl. Acad. Sci. 2009, 106 (51), 21625–
21630. https://doi.org/10.1073/pnas.0909757106.
(86) Hochreiter, B.; Kunze, M.; Moser, B.; Schmid, J. A. Advanced FRET Normalization Allows Quantitative Analysis of Protein Interactions Including
Stoichiometries and Relative Affinities in Living Cells. Scientific Reports 2019, 9 (1). https://doi.org/10.1038/s41598-019-44650-0.
(87) Parkhurst, L. J.; Larsen, T. M.; Lee, H.-Y. Effects of Wavelength on Fitting Adair Constants for Binding of Oxygen to Human Hemoglobin. In Methods in Enzymology; Elsevier, 1994; Vol. 232, pp 606–632.
(88) Larsen, T. M.; Mueser, T. C.; Parkhurst, L. J. Use of Dual Wavelength Spectrophotometry and Continuous Enzymatic Depletion of Oxygen for Determination of the Oxygen Binding Constants of Hemoglobin. Analytical Biochemistry 1991, 197 (1), 231–246.
(89) Cade, R.; Conte, M.; Zauner, C.; Mars, D.; Peterson, J.; Lunne, D.; Hommen, N.;
Packer, D. Effects of Phosphate Loading on 2, 3-Diphosphoglycerate and Maximal Oxygen Uptake. Medicine and Science in Sports and Exercise 1984, 16 (3), 263–
268.
(90) Bartlett, G. R. Phosphate Compounds in Vertebrate Red Blood Cells. American Zoologist 1980, 20 (1), 103–114.
(91) Lenfant, C.; Torrance, J.; English, E.; Finch, C. A.; Reynafarje, C.; Ramos, J.;
Faura, J. Effect of Altitude on Oxygen Binding by Hemoglobin and on Organic Phosphate Levels. The Journal of clinical investigation 1968, 47 (12), 2652–2656.
(92) Loutit, J. F.; Mollison, P. L.; Young, I. M.; Lucas, E. J. Citric Acid‐sodium Citrate‐glucose Mixtures for Blood Storage. Quarterly Journal of Experimental Physiology and Cognate Medical Sciences: Translation and Integration 1943, 32 (3), 183–202.
(93) Lovelock, J. E.; Bishop, M. W. H. Prevention of Freezing Damage to Living Cells by Dimethyl Sulphoxide. Nature 1959, 183 (4672), 1394–1395.
(94) Pogozhykh, D.; Pakhomova, Y.; Pervushina, O.; Hofmann, N.; Glasmacher, B.;
Zhegunov, G. Exploring the Possibility of Cryopreservation of Feline and Canine Erythrocytes by Rapid Freezing with Penetrating and Non-Penetrating
Cryoprotectants. PLoS One 2017, 12 (1), e0169689.
(95) Brunskill, S. J.; Wilkinson, K. L.; Doree, C.; Trivella, M.; Stanworth, S.
Transfusion of Fresher versus Older Red Blood Cells for All Conditions. Cochrane Database of Systematic Reviews 2015, No. 5.
(96) Valeri, C. R.; Ragno, G.; Pivacek, L. E.; Cassidy, G. P.; Srey, R.; Hansson-Wicher, M.; Leavy, M. E. An Experiment with Glycerol-Frozen Red Blood Cells Stored at–80 C for up to 37 Years. Vox Sanguinis 2000, 79 (3), 168–174.
(97) Valeri, C. R.; Srey, R.; Tilahun, D.; Ragno, G. The in Vitro Quality of Red Blood Cells Frozen with 40 Percent (Wt/Vol) Glycerol at −80° C for 14 Years, Deglycerolized with the Haemonetics ACP 215, and Stored at 4° C in Additive Solution‐1 or Additive Solution‐3 for up to 3 Weeks. Transfusion 2004, 44 (7), 990–995.
(98) Heaton, A.; Keegan, T.; Holme, S. In Vivo Regeneration of Red Cell 2, 3‐
diphosphoglycerate Following Transfusion of DPG‐depleted AS‐1, AS‐3 and CPDA‐1 Red Cells. British Journal of Haematology 1989, 71 (1), 131–136.