Kiinische Wochenschrift

Klin. Wschr. 54, 159-167 (1976)

© by Springer-Verlag t976

Red Cell Aggregation in Blood Flow* II. Effect on A p p a r e n t Viscosity o f B l o o d H. Schmid-Sch6nbein**, G. Gallasch, J.v. Gosen, E. Volger, and H.J. Klose Physiologisches Institut der Universit/it Mfinchen, D-8000 Mfinchen 2

Erytrocytenaggregation und Blutstr&nung II. Effekt auf die scheinbare Blutviskositiit Zusammenfassung. Die scheinbare Viskosit/it des Blutes steigt bei langsamer Scherung in Rotationsviskosimetern durch die reversible Bildungvon Erythrocytenaggregaten sehr stark an. Das Ausmal3 des Viskositgtsanstiegs hgngt jedoch wesentlich von dem H/imatokritwert, daneben vonder Plasmaviskosit~it und schlieBlich von den mikrorheologisehen Eigenschaften der Aggregate ab. Die unabh~ingige Messung der mikrorheologischen Eigenschaften und des Viskositgtsverhaltens erlaubt die detaillierte Analyse des hfimodynamischen Effektes yon Erythrocytenaggregaten unter definierten Fliel3bedingungen in vitro. Die vergleichende Analyse zeigt, dab die konventionelte Viskosimetrie die rheologischen Unterschiede zwischen normaler und pathologisch gesteigerter Aggregation eindeutig untersch/itzt. Basierend auf der detailtierten Analyse unter definierten Bedingungen in vitro werden die biologische Bedeutung von viskometrischen Befunden und die h/imodynamische Relevanz yon Erythrocytenaggregaten abgehandelt. Schliisselw6rter: Blutrheologie, Fahraeus-Effekt, RouleauxKlumpen, Rouleaux-Netzwerke, scheinbare Blutviskositfit, scheinbare relative Blutviskosit~it, Viscosimetrie.

Summary. The apparent viscosity of blood strongly increases at low shear in rotational viscometers, this phenomenon is based on the reversible formation of red cell aggregates. The magnitude of this increase strongly depends on the hematocrit value, on plasma viscosity and lastly on the microrheological properties of the aggregates. The independent measurement of the microrheological behavior and the effects on viscosity allows a detailed analysis of the hemodynamic effects of red cell aggregates under defined flow conditions in vivo. The comparative analysis shows that the conventional viscometry strongly underestimates the rheological differences between normal and pathologically intensified aggregation. Based on detailed analysis under defined flow conditions in vitro, the biological significance of viscometric results and the hemodynamic relevance of red cell aggregates are discussed. Key words: Apparent blood viscosity, blood theology, Fahraeus-effect, relative apparent blood viscosity, rouleaux clumps, rouleanx networks, viscometry. * Presented in parts as an invited lecture during the tst International Congress of Biorheology, Lyon (France) September 1972. The lecture was part of a session dedicated to the memory of the late Professor Aharon Katschalsky. ** Supported by Deutsche Forschungsgemeinsehaft: Present address: Abtlg. Physiologic der RWTH Aachen, 51 Aachen, Melatenerstr. 211-213.

Biological Significance of R e d Cell Aggregation Traditionally, the p h e n o m e n o n o f the red cell aggregation has been studied with regards to its influence u p o n the sedimentation rate o f b l o o d in vitro (i.e. under purely diagnostic aspects) or with regards to its interference with microvascular flow in vivo (i.e. u n d e r pathogenetic aspects). Recent developments o f h e m o r h e o l o g y have put new emphasis on the c o m m o n d e n o m i n a t o r o f both aspects: it has been established b e y o n d reasonable d o u b t that R C A is responsible for the increase in apparent blood viscosity at low rates o f shear, a n d at near stasis. As such a prestatic flow regime can be induced m u c h m o r e readily in rotational than in conventional capillary viscometers, rotational viscometers o f various design have b e c o m e p o p u l a r research instruments during the last 15 years. However, the h y d r o d y n a m i c significance o f such measurements has not always been subject o f critical analysis. I f one tries to attribute the biological r61e o f red cell aggregates to their flow behavior, 1. the h y d r o d y n a m i c (i.e. general rheological) significance o f red cell aggregation must be evaluated and, based u p o n this, 2. the specific circulatory significance o f in vitro values o f blood viscosity must be assessed and weighed against other h e m o d y n a m i c parameters.

What is Being in Rotational Viscometers? K n o w i n g the dependence o f the microrheological behavior o f red b l o o d cells on the incident flow forces, it is n o t difficult to appreciate that their effect on the fluidity o f b l o o d - o r its reciprocal value, the apparent viscosity o f blood-greatly depends on the conditions under which the cells are flowing or under which the apparent viscosity o f b l o o d is being measured, respectively.

160

H. Schmid-Sch6nbein et al. : Red Cell Aggregation in Blood Flow. II

The fluidity of blood increases with decreasing vessel radius and increasing shear stress. The two factors together are known as the Fahraeus-Lindqvist-effect [13] and this effect governs blood flow in all blood vessels smaller than about 300 gm in diameter [21]. At least 99% of all blood vessels are smaller than 300 gm, and at least 99% of the energy produced by the heart is dissipated in these blood vessels. However, the Fahraeus-Lindqvist-effect depends on the physiological deformability of the cells (Devendran and SchmidSch6nbein 1974 [8] and are based on a progressive fall in the hydro-dynamically effective red cell volume fraction (hematocrit), (Barbee and Cokelet) [1]. Due to axial migration, a rapidly flowing core of red blood cells remains unsheared and is surrounded by a lubricating layer containing practically pure plasma. Thus, by far the largest number of blood vessels (and those giving rise to the greatest hydraulic resistance) are being perfused with blood exhibiting a very low apparent viscosity. To the best of our knowledge the viscosity of the blood in these tubes is not greatly different from that of pure plasma - as long as rapid perfusion is maintained [1, 2, 3, 8, 13].

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H. Schmid-Sch6nbein et al. : Red Cell Aggregation in Blood Flow. II

before about 1 min has passed, the rapid process of RCA formation is concluded long before a steady state viscosity reading can be obtained. The time dependent fall in apparent blood viscosity at constant low rates of shear is thus the consequence of aggregate rearrangement and condensation rather than dispersal of RCA [23]. The apparent viscosity of blood at high rates of shear is governed by quite different flow phenomena: the red cells become desaggregated, oriented, aligned in flow and eventually participate in flow in a fluid drop-like manner [22, 23]. By this "streamlining", the hemodynamic effect of a given volume fraction of red cells is reduced, their "effective hematocrit" is lower than that of either aggregated or of non-deformed red cells. This effect is again reflected in the value of the "relative apparent viscosity" of blood taken at high shear stresses. If, as in the present experiments with spontaneously increased plasma viscosity, the adaptation of the fluid drop-like erythrocytes is facilitated, a decrease in relative apparent viscosity occurs. This Surprising finding is not an isolated one: an inverse relationship between plasma viscosity and relative apparent blood viscosity at high shear rates is found regularly (for a detailed discussion see [7, 24] and most recently [32]). This finding stresses that the rheological behavior of the dispersed cells must be taken into consideration even when evaluating the hemodynamics of reversible red cell aggregation. It should be added in paranthesis that in occasional blood samples with a very shear resistant aggregation (TTminhigher than 180 sec-1) relative apparent viscosity at 160 sec -1 was higher than in control blood. In these samples, a shear rate of 160 sec- 1 was presumably not sufficient to deform and Nign the spherical short rouleaux (" flocs") described above (s.p. Fig. 1C in [30]). While the results depicted in this paper can be made plausible by Chien's theory, they illustrate the difficulties encountered in assessing the hemodynamic significance of red cell aggregates in vitro, let alone in vivo [14, 7]). Intravascular "sludging" is assumed to be noxious (for a review see 17) but also quite harmless [21]. The data listed in Table 3 show the microrheology of 4 different blood samples supplied to us from patients which have a sedimentation rate of more than 70 mm/hour (modo Westergreen). All were severely ill and one is fairly safe in assuming that they would have had ".sludging" [14] of the flowing blood in their conjunctival microcirculation. As can be seen from Table 3, the values of apparent blood viscosity of the three acutely ill patients at 8 sec - 1 were within the normal range or below it at 160 sec- 1, the viscosity was slightly elevated (the latter even after

H. Schmid-Sch6nbein et at. : Red Cell Aggregation in Blood Flow. II

165

Table 3. Sedimentation rate, hematocrit value, microrheological and macrorheological behavior of 4 blood samples with extremely augmented red cell aggregation

Sedimentation rate Hct value RCA in stasis t~/2 (sec) ~ RCA in slow flow FSAR ~ Hydrodynamic RCA-dispersion ~Tm~. (sec ~)~ Apparent viscosity at intermediate shear (8 sec-~) ~ Apparent viscosity at high shear rate ~

cholangitis

myocard, inf.

bronchiaI Ca

D.I.C. 35, ~

64, c~

63, ~'

toxinemia of pregnancy 27, ~?

95/132 m.W. 38% 0.95 1.386 460 sec- ~ 5.6 cP 4.2. cP

72/I 10 m.W. 42% 0.75 0.926 49 sec-~ 8.7 cP 3.9 cP

70/95m.W. 35% 0.3 1.306 49 sec- ~ 5.4 cP 3.8 cP

80/110 m.W. 35% 0.9 0.904 90 sec- ~ 5.8 cP 4.0 cP

Normal values see Table 1. b Normal value 63 + 18 sec- 1

correction for hematocrit and plasma viscosity, not shown). Largely undetected by viscometry, the rheology of these patients RCA differed considerably in many respects. In the patient with cholangitis and disseminated intravascular coagulation, the rate and extent of aggregation in stasis was quite normal, while the aggregation in slow flow (TT~, zt T7 and FSAR) are highly pathological, as is the shear resistance (ZTm~.). The myocardial infarction patient had normal shear resistance, rate and extent of RCA in flow and stasis were primarily increased. The bronchial carcinoma patient had again normal rT ~,, but grossly accelerated rate of aggregate reformation (tt/2 = 0.3 s) and strong clump formation (FSAR 1.306!) in slow flow. The patient toxinemia of pregnancy had only an increased shear resistance, but no signs of clump formation (FSAR within the normal range). Table 3 also justifies the measurement of so many different aggregation parameters as there is clear indication from these and other studies [27], that rate of formation, flow behavior in low shear and shear resistance of red cell aggregates are related to different protein molecules which are presently under study in our laboratory [31, 32]. The blood from patients with occlusive vascular disease appears only insignificantly elevated, but again the aggregation parameters are distinctly abnormal. It should be noticed in parenthesis that after therapeutic defibrination (by streptokinase treatment) the viscosity values of these patients were returned to normal, whereas the aggregation parameters were significantly below normal, a change associated with marked improvement of the peripheral circulation in the affected limbs (unpublished observation). The correlation of the microrheological behavior as tested by the present methods, by particle volume analysis (Boss et al., [5, 6]) to the flow behavior in

vivo remains an unsolved problem. Above all, future analysis should take into consideration the paramount significance of the hematocrit level on apparent blood viscosity and on the flow behavior of aggregates. Identification of small aggregates (" doublets ") in the blood from patients with myocardial infarction and risk factors thereof (Boss et al.), greatly improves the diagnostic spectrum beyond the mere measurement of sedimentation rate, viscometry and photometry, it does, however, not allow any prediction about their hemodynamic relevance. The postulates of Knisely [16] about the pathogenetic significance of "blood sludging"

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166

H. Schmid-Sch6nbein et at. : Red Cell Aggregation in Blood Flow. II

remain to be substantiated by future quantitative intravital microscopic studies. The complex rheology of blood makes this an extremely difficult task. The results compiled in the present study can already explain one apparent paradox that has previously shed doubt on Knisely's and Gelin's postulates about the pathogenetic significance of intravascular "sludging". While pronounced aggregation was always seen associated with such diseases as irreversible shock, myocardial infarction or myeloma, it was occasionally seen in subjects with no other symptoms of disease or of circulatory disturbance. The presence of severe intravascular aggregation without signs of hemodynamic impairment might easily be explained by the accompanying anemia (Gelin [14]). While low hematocrit levels might favour the presence of aggregates in flowing blood, it might also keep them from exerting adverse hemodynamic effects in vivo. Much remains to be learned about the microrheology of blood in the living microvasculature. It can be hoped that the combination of the presently available methods with quantitative in vivo analysis will supply a sounder basis for the understanding of intravascular aggregation and its cause-effect relationship to flow retardation and stasis. There can be no doubt that aggregation both in vitro and in vivo and intravascular flow disturbances in the microvessels are frequent pathogenetic phenomena observed in disease. No single method, least of all the measurement of the sedimentation rate, (omitted intentionally from the present report) can elucidate their interrelationship. Even before the ultimate details are understood, the methods now available are helpful for objective monitoring of the effects of dessaggregating therapeutic measures, such as they have been proposed by means of hemodilution [20] anti-adhesive drugs [4] and therapeutic defibrination [12]. All these therapies have been shown to reduce intravascular aggregation, and improve microcirculatory perfusion.

References 1. Barbee, J.H., Cokelet, G.R. : The Fahraeus effect. Microvascular Res. 3, 6-16(1971) 2. Barras, J.P., Maibach, E. : Effect du HR sur la viscosit~ sanguine duns l'insuffisance v~neuse chronique. Abstr. 4th Int. Congr. Phlebol., Lucerne (1971) 3. Berman, H.J., Fuhro, R.L. : Quantitative red cell aggregometry of human and hamster blood. Proc. VIIth Conf. Microcirc. Aberdeen 1972 (1973) 4. Bicher, H. : Blood cell aggregation in thrombotic processes. Thomas, (Springfield) (1972) 5. Boss, N., Chmiel, H., Kachel, V., Ruhenstroth-Bauer, G.: Erythrozytenaggregation bei Nichtrauchern, Rauchern und Herzinfarktpatienten. Blut. 27, 191-195 (1973)

6. Boss, N., Koenig, S., Ruhenstroth-Bauer, G.: Die Erythrozytenaggregation bei Menschen mit Risikofaktoren eines Herzinfarkts. Klin. Wschr. 53, 385-389 (1975) 7. Chien, S. : Shear dependence of effective cell volume as a determinant of blood viscosity. Science 168, 977- 979 (1970) 8. Devendran, T., Kline, K.A., Schmid-Sch6nbein, H. : Capillary viscometry of erythrocyte suspensions in various media. Amer. Soc. Mech. Eng. Publ. 73-WA/Bio 35, 1 - 4 (1973) 9. Dintenfass, L. : Blood microrheology- viscosity factors in blood flow, ischemia and thrombosis. London, (Butterworths) (1971) 10. Dintenfass, L. : A coaxial rhombospheroid viscometer, a further development of the cone in cone viscometer. Biorheology 6, 33-43 (1969) 11. Dintenfass, L. : Principle and diagnostic uses of the blood viscosity factors. Abstr. Vol. XV Congr. Intern. Soc. Hematology, Jerusalem 1974, p. 25 12. Ehrly, A. : Verbesserung der Fliel3eigenschaften des Blutes: Ein neues Prinzip zur medikament6sen Therapie chronisch peripherer arterieller Durchblutungsst6rungen. Suppl. 1 zu VASA (1973) p. 1 - 1 8 13. Fahraeus, R., Lindqvist, T. : The viscosity of blood in narrow tubes. Amer. J. Physiol. 96, 562-570 (1931) 14. Gelin, L.E. : Intravascular aggregation and capillary flow. Acta clair, scand. 113, 463-465 (1957) 15. Gillison, P.J, Dauwalter, C.R., Merrill, E.W.: A rotational viscometer using A.C. torque to balance loop and airbearing. Trans. Soc. Rheol. 7, 319-331 (1963) 16. Knisely, M.H., Bloch~ E.H., Eliot, T.S., Warner, L.: Sludged blood. Science 106, 431-433 (1947) 17. Knisely, M.H.: Intravascular erythrocyte aggregation (blood sludge). In: Handbook of physiology, Sect. 2, Vol. III, W.F. Hamilton and P. Dow, eds., American. Physiological Society, Washington D.C., p. 2249-2292 (1965) 18. Merrill, E.W., Cheng, C., Pelletier, G.A. : Yield stress of normal human blood as a function of endogenous fibrinogen. J. appl. Physiol. 26(1), I (1968) 19. Merrill, E.W. : Rheology of blood. Physiol. Rev. 49, 863 -888 (1969) 20. Messmer, K., Schmid-Sch6nbein, H. (Eds.): Hemodilution, Theoretical basis and clinical application. Karger, Basel (1972) 21. Robertson, H.S., Wolf, S., Wolf, H.G.: Blood "sludge" phenomenon in human subjects. Notes on its significance and on the effects of vasomotor drugs. Amer. J. Med. Sci. 219, 534- 537 (1950) 22. Schmid-Sch6nbein, H., Wells, R. : Fluid drop-like transition of erythrocytes under shear. Science 165, 288 - 291 (1969) 23. Schmid-Sch6nbein, H., Wells, R.E. : Rheological properties of human erythrocytes and their influence upon the "anomalous" viscosity of blood. Ergebn. Physiol. 63, 146-219 (1971) 24. Schmid-Sch6nbein, H., Volger, E., Klose, H.J. : Microrheology and light transmission of blood. II. The photometric quantification of red cetl aggregate formation and dispersion on flow. Pfliiger's Arch. 333, 140-155 (1972) 25. Schmid-Sch6nbein, H. : A simpte device allowing blood viscometry at low rates of shear with the Wells-Brookfield viscometer. Res. exp. Med. 161, 4 9 - 5 7 (1973) 26. Schmid-Sch6nbein, H., Gosen, J. v., Heinich, L., Klose, H.J., Volger, E. : A counter-rotating "rhe0scope" chamber for the study of the microrheology of blood cell aggregation by microscopic observation and microphotometry. Microvasc. Res. 6, 366-376 (1973) 27. Schmid-Sch6nbein, H., Gallasch, G., Volger, E., Klose, H.J. : Microrheology and protein chemistry of pathological red cell aggregation (" blood sludge") studied in vitro. Biorheology 10, 213 -227 (1973) 28. Schmid-Sch6nbein, H.: Einftihrung in die H/imorrheologie:

H. Schmid-Sch6nbein et at. : Red Cell Aggregation in Blood Flow. II Fliegeigenschaflen, Fliegbedingungen, Methodik. Phtebol. Proctol. 2, 205-226 (1973) 29. Schmid-Sch6nbein, H., Ktine, K.A., Heinich, L., Volger, E., Fischer, T. : Microrheology and light transmission of blood III. The veIocity of red cell aggregate formation. Pflfigers Arch. 354, 299--317 (I975) 30. Schmid-Sch6nbein, H., Gallasch, G., Gosen, J. v., Volger, E., Ktose, H.J. : Red cell aggregation in blood flow. I. New methods of quantification. Kiln. Wschr. 4, 149-157 (1976) 31. Volger, E., Schmid-Sch6nbein, H., Merishi, J.N. : Artificial red cell aggregation caused by reduced salinity: production of a Polyalbumin. Bibl. anat. 11,296-302, Karger Basel (1973) 32. Volger, E, Schmid-Sch6nbein, H., Gosen, J, v., Klose, H.J.,

167 Kline, K.A.: Microrheology and light transmission of blood IV. The kinetics of artificial red cell aggregation induced by Dextran. Pfliigers Arch. 354, 319-337 (1975) 33. Zweifach, B.W. : Quantitative studies of microcirculatory structure and function, I. Circular. Res. 34, 843-857 (1974) Professor Dr. H. Schmid-Sch6nbein Abteilung Physiotogie der Medizinischen Fakultfit der RWTH D-5100 Aachen Melatener Strage 211 Federal Republic of Germany