STUDIES ONTHE DESTRUCTIONOF REDBLOODCELLS. XII. FACTORSINFLUENCING THE ROLEOF S HEMOGLOBININ THE PATHO-

LOGIC PHYSIOLOGYOF SICKLE CELL ANEMIA ANDRELATEDDISORDERS1

BY MORTIMERS. GREENBERG,2EDWARDH. KASS, ANDWILLIAM B. CASTLE

(From the Thorndike Memorial Laboratory and Second and Fourth (Harvard) MedicalServices Boston City Hospital, and the Department of Medicine, Harvard Medical

School, Boston, Mass.)

(Submitted for publication January 7, 1957; accepted February 15, 1957)

The deoxygenation of blood from patients withsickle cell anemia leads to transformation of thered blood cells from biconcave discs into thebizarre elongated forms from which the diseasederives its name (1). This change in morphologyof the red cells is associated with increased vis-cosity of the blood proportional to the number ofred cells so altered (2, 3). Much of the abnormalphysiology of sickle cell anemia is attributable tothis increase in the viscosity of deoxygenated blood(2-4) as well as to the greater mechanical fragilityof the red cells that accompanies the sickled state(5-7). Thus, factors which impede the flow ofblood may result in local hypoxia sufficient to in-crease the extent of sickling. The concomitantincrease in viscosity of the blood further decreasesblood flow so that local hypoxia becomes moremarked, thereby increasing the degree of sickling.In this way the "vicious cycle" of sickle cell ane-mia (2, 3) is instituted, resulting in localizedischemia and ultimately thrombosis, with necrosisof tissue. The two chief features of sickle cellanemia are thereby explained, the chronic hemo-lytic anemia on the basis of the increased mechani-cal fragility and diminished life span of the eryth-rocytes in zivo, and the appearance of painfulcrises on the basis that various circumstances maytemporarily so alter the flow of blood in an organthat the "vicious cycle" of erythrostasis, increasedsickling, increased viscosity, etc., occurs and leadsto localized ischemia or infarction (2).

Studies of the properties of hemoglobin solu-tions indicate that the sickling phenomenon is aresultant of the presence of S hemoglobin in the

1This investigation was supported in part by grantsfrom the National Institutes of Health, Public HealthService.

2 Present Address: Lemuel Shattuck Hospital, Boston30, Mass.

red cells of patients with sickle cell anemia (8, 9)and that the sickled cell may be considered to bea hemoglobin "tactoid" covered by a passivelydistorted cell membrane. Harris (8) has demon-strated that concentrations of S hemoglobin ofat least 10 to 12 grams per cent are required fortactoid formation to occur upon deoxygenation ofhemoglobin solutions and that this tactoid forma-tion is associated with increased viscosity of thehemoglobin solutions. Indeed, at concentrationsof S hemoglobin of 20 grams per cent or more,deoxygenation leads to gel formation.

These facts and in particular the contrast withthe asymptomatic sickle cell trait suggest the de-sirability of defining in greater detail some of thequantitative aspects of the relationship between Shemoglobin and viscosity changes in whole bloodand in solutions of hemoglobin in order that thesemay be related to physiologic events occurringduring the course of the disease. The presentstudy was undertaken in order to add to knowl-edge of such relationships.

CLINICAL MATERIAL

Twenty-one Negro patients with various hereditaryhemoglobinopathies involving S hemoglobin were stud-ied. The pertinent clinical and laboratory data are givenin Table I. There were 8 cases of sickle cell anemia(hemoglobin phenotype: S hemoglobin), 9 of sickle celltrait (hemoglobin phenotype: A and S hemoglobins) and3 of sickle cell-hemoglobin C disease (hemoglobin pheno-type: S and C hemoglobins). Finally, in 1 patient 73per cent of the hemoglobin was S hemoglobin, 21 percent was A hemoglobin and 6 per cent was F hemoglobin.These findings are not characteristic of sickle cell traitas it is usually defined (10). Morphologically the redcells were normochromic and microcytic even aftercorrection of pre-existing iron deficiency. This hemato-logic pattern has been called sickle cell-thalassemia dis-ease by Singer, Singer, and Goldberg (11), but inasmuchas the patient's mother was hematologically normal and

833

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the father was not available for study, the genetic back-ground of the abnormality in this patient could not beconfirmed. For convenience, this patient will be desig-nated as having S-A-F disease.

Whole blood will be described according to the con-tained hemoglobin, so that the designation S-S bloodrefers to whole blood taken from patients with sicklecell anemia, A-S to blood from patients with sickle celltrait, and so forth.

METHODS

Red blood cell counts, hematocrits, hemoglobin con-centrations, mean corpuscular indices, icterus indices,reticulocyte counts and other hematologic determinationswere made by standard methods (12). All hematocritdeterminations were made on aliquots of whole bloodafter oxygenation in order to avoid errors introducedby the poor packlng of red cells in the sickled form.The presence of the sickling phenomenon was de-tected by the metabisulfite method (13). The percentageof "irreversibly sickled" red cells (14), or cells whichretain their distorted form in the presence of oxygen,was determined by counting the number of such cells perthousand red cells in smears of capillary blood from theear lobe. The smears were made on cover slips andstained with Wright's stain.

Stroma-free solutions of hemoglobin were prepared bya modification of the method described by Drabkin (15).The modification consisted of using only physiologicsaline as the wash fluid for the red cells, and of usingthe hemoglobin solutions after removal of the stroma bytoluene, without crystallization of the hemoglobin.Methemoglobin concentration was measured by themethod of Evelyn and Malloy (16), in all hemoglobinsolutions, after viscosity measurements had been com-pleted. Because methemoglobins, unlike oxyhemoglobins,do not form tactoids when exposed to low oxygen ten-sions, viscosity measurements are included in this re-port only if the percentage of methemoglobin was lessthan 4 per cent of the total hemoglobin concentration.

Paper electrophoresis of stroma-free hemoglobin solu-tions was carried out in an apparatus modified slightlyfrom that described by Smith and Conley (17). Thepaper strips were scanned with a photoelectric densitom-eter3 and the area under the resultant curves was meas-ured with a polar planimeter in order to calculate therelative amounts of the various hemoglobins present.The mean corpuscular S hemoglobin concentration(MCSHC) of red cells was calculated by multiplyingthe mean corpuscular hemoglobin concentration (MCHC)by the percentage of S hemoglobin as obtained from analy-sis of the paper electrophoretic strips. The methods usedfor determining MCSHCare reproducible within a rangeof 5 to 10 per cent. Fetal hemoglobin was measured bythe method of Singer, Chernoff, and Singer (18). Thepercentage of S hemoglobin in the red cells of patientswith sickle cell anemia was calculated by subtracting thepercentage of fetal hemoglobin present from 100 per cent.

3 Photovolt densitometer, Model 52 C.

The survival time of labeled red cells in the circu-lation and sites of their sequestration were determinedusing radioactive chromium by the methods describedby Jandl, Greenberg, Yonemoto, and Castle (19).

The viscosity of whole blood or of hemoglobin solu-tions was determined according to the methods of Har-ris, Brewster, Ham, and Castle (3) in Ostwald viscosi-meters having bores of about 1 millimeter and flow timesfor water of 25 to 50 seconds at 370 C. Aliquots of oxa-lated bloods were equilibrated with gas mixtures contain-ing known percentages of oxygen and nitrogen in thepresence of 10 per cent carbon dioxide. Unless otherwisestated the viscosity of samples of whole blood was de-termined under the following "standard conditions":hematocrit adjusted to 35 per cent when the sample wasoxygenated, pH 7.2 to 7.4, and temperature 370 C. main-tained in a water bath. The pH of the blood was deter-mined potentiometrically immediately after the flow timehad been measured. In some experiments, the pH ofwhole blood was altered by. adding 0.33 M HCI or 0.09MNaHCO. prior to equilibration with the gas mixture.It was thus possible to vary the pH of the equilibratedblood from 6.8 to 7.5 without affecting greatly either thehematocrit or the mean corpuscular hemoglobin concen-tration.

RESULTS

Relation of hemoglobin concentration to viscosityof hemoglobin solutionsThe viscosities of solutions of S hemoglobin

and of A hemoglobin were determined after com-plete oxygenation and complete reduction, respec-tively. The changes in the relative viscosities thatoccurred in hemoglobin solutions whose concen-

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FIG. 1. THE EFFECT OF HEMOGLOBIN CONCENTRA-TION UPON THE VISCOSITY RELATIVE TO WATER OFSTROMA-FRFm HEMOGLOBIN SOLUTIONS

835

MORTIMERS. GREENBERG,EDWARDH. KASS, AND WILLIAM B. CASTLE

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Note that the viscosity of reduced blood at an hemato-crit of 25 per cent, an hematocrit frequently found tooccur in patients with sickle cell anemia, is about thesame as the viscosity of oxygenated blood at an hemato-crit of 50 per cent. The curve shown here for oxygenatedsickle cell anemia blood is not significantly different fromthe curve for normal blood. In the case of normal bloodthere is no difference between the curve for oxygenatedblood and that for reduced blood.

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trations varied from about 5 to 25 per cent areplotted in Figure 1. The values for reduced Ahemoglobin, oxygenated A hemoglobin and oxy-genated S hemoglobin fell along a common, gradu-ally ascending curve. The values for reduced Shemoglobin also fell along the same curve for con-centrations of less than 10 grams per cent butabove this concentration the viscosity of reducedS hemoglobin rose precipitously to levels abovethose of even the most concentrated of the otherhemoglobin solutions. Although not shown inFigure 1 because of the limitations of the scale,the viscosities relative to water of solutions of re-duced S hemoglobin in concentrations of 15.2 percent and 15.9 per cent were 61.2 and 72.2, re-spectively. At concentrations above 20 gramsper cent, the solutions of reduced S hemoglobinwere complete gels as observed by Harris (8).

Relation of hematocrit to viscosity of sickle cellanemia blood

Blood from a patient with sickle cell anemia wasadjusted to various hematocrit values between 15and 50 per cent by the addition or removal of auto-logous plasma. Aliquots of the blood at each he-matocrit were fully oxygenated or completely re-

SICKLE CELL TRAIT

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duced by equilibration with appropriate gas mix-tures. As shown in Figure 2, the viscosity of theoxygenated S-S blood increased slightly as the he-matocrit increased. These values correspond withthose for normal blood, whether oxygenated or re-duced. There is no increase in the viscosity ofnormal blood upon deoxygenation. However, theviscosity of the reduced S-S blood increased muchmore rapidly than did that of the oxygenated ali-quot especially at hematocrit values above 20 percent. The figure shows that at a hematocrit of 25per cent the viscosity of the completely deoxy-genated S-S blood was approximately that of oxy-genated blood at a hematocrit of 50 per cent.

Effect of pH upon viscosity and the sicklingphenomenonThe effect of change in pH upon the degree of

sickling and the viscosity of S-S and A-S bloodsat several oxygen tensions is shown in Figures3 and 4. There were no significant effects of pHon viscosity or degree of sickling of S-S or A-Soxygenated blood. However, when the effects ofpH were studied at oxygen tensions at which notall of the cells were ordinarily sickled, strikingeffects on the degree of sickling were observed(Figure 4). Thus, at an oxygen tension of 60millimeters of mercury the viscosity and degreeof sickling of S-S blood increased as the pH waslowered, until a maximum was reached at about

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pH 6.8 to 7.0. These changes in pH did notcause significant alterations in mean corpuscularhemoglobin concentration. For example, theMCHCwas 31 per cent at pH 7.0 when the vis-cosity was high, and was 32 per cent at pH 7.3when the viscosity had decreased.

As the pH of A-S blood was lowered to 7.0 atzero oxygen tension the viscosity of the blood ap-proached the maximal values obtained for S-Sblood and almost all the cells were sickled. Thissuggests that A hemoglobin may be incorporatedinto tactoids at low pH values and oxygen tensions.No effect of pH was observed on the viscosity offully reduced A-A blood.

FIG. 5. THE EFFECT OF VARIATIONS IN OXYGENTENSION UPONTHE

VISCOSITY OF WHOLEBLOODOF PATIENTS WITH A VARIETY OF ABNORMALHEMOGLOBINOPATHIESINVOLVING S HEMOGLOBIN

The hematocrit was adjusted to 35 per cent by the addition or removalof autologous serum in all cases, pH was maintained between 7.2 and 7.3in all cases by maintaining a pCO, of 76 millimeters of mercury, and allobservations of viscosity were made on blood at a temperature of 370 C.

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837

MORTIMERS. GREENBERG,EDWARDH. KASS, AND WILLIAM B. CASTLE

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THE VISCOSITY OF FuLLY DEOXYGENATEDBLOOD AT A

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Relation of S hemoglobin concentration within thered cells to the viscosity of whole blood

The alterations in viscosity of oxalated wholeblood that occurred at various oxygen tensions un-

der the standard conditions of hematocrit, pH andtemperature referred to previously, are illustratedin Figure 5. The curves are based on observationsmade on the blood of each of the patients describedin Table I. The viscosity at complete deoxygena-tion was greater for S-S blood than for A-S, or

S-C bloods, but the viscosity of S-A-F blood fell

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within the range of S-S bloods. The oxygen ten-sion that barely permitted sickling was also dis-tinctly greater for S-S bloods than for A-S bloods,and was possibly greater than for the other vari-ants of the S hemoglobinopathies. The viscositycurve of S-C blood fell between the curves thatcharacterize S-S and A-S bloods. That of S-A-Fblood was among those of the less viscous S-Sbloods.

The data in Figure 5 suggest that the mean cor-

puscular S hemoglobin concentration (MCSHC)and the degree of change in viscosity upon deoxy-genation are related phenomena. In Figure 6 isplotted the MCSHCof the red cells of each of thepatients described in Table I in relation to the in-crease in viscosity that occurred when the corre-

sponding blood was deoxygenated completely un-

der the standard conditions of hematocrit, pH,and temperature. The resultant sigmoid curve iscomparable to that in Figure 1, in which the vis-cosity of solutions of various concentrations of Shemoglobin is plotted. Both in the solutions andin the whole blood, and irrespective of the totalMCHC, the first increase in viscosity occurredwhen the MCSHCwas greater than 10 grams per

cent. Solutions of reduced S hemoglobin becamecompletely gelled at concentrations above 20 gramsper cent. Similarly, in whole blood, there was no

further increase in viscosity at MCSHCvaluesabove about 20 grams per cent, presumably be-

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FIG. 7. CHANGESIN THE EFFECT OF DEOXYGENATIONUPON THE VIS-COSITY In Vitro OF BLOOD FROMA PATIENT WITH SICRLE Cuxi TRAIT(A-S BLOOD) AND COEXISTING IRON DEFICIENCY FOLLOWINGTREATMENTOF THE PATIENT Wi FEmous SULFATE

MCSHCis mean corpuscular S hemoglobin concentration.

838

FACTORS IN PATHOPHYSIOLOGICROLE OF S HEMOGLOBIN

cause all the hemoglobin within the red cells wascompletely gelled.

Further confirmation of the role of MCSHCindetermining susceptibility to sickling of red cellscame from observations on the bloods of two pa-tients with sickle cell trait and coexisting irondeficiency. The data from one of these patients arepresented in Figure 7. Initially, the MCHCwas24 grams per cent and the MCSHCwas 10.6grams per cent. At this time there was no in-crease in viscosity and no sickling of red cells uponcomplete deoxygenation of the blood under thestandard conditions. However, paper electro-phoresis and the metabisulfite tests indicated thepresence of S hemoglobin. After iron had beengiven by mouth for 47 days the ratio of S to Ahemoglobin within the red cells did not change,but, as the total MCHCrose, the degree of sicklingand the corresponding changes in viscosity ap-proached values commonly seen in sickle cell trait.An increase of MCSHCto 11.9 grams per centwhen the MCHCwas 27 grams per cent was suffi-cient to permit sickling after complete dexoygena-tion; this effect was still greater when the MCHCreached 30 grams per cent and the MCSHCwas13.2 grams per cent.

DISCUSSION

The importance of changes in the viscosity ofwhole blood in vivo in determining much of thepathologic physiology of the sickle cell syndromes(2, 3, 8, 20, 21) is supported by the present data.The S hemoglobin concentration is evidently thechief determinant in bringing about the changesthat are observed in vitro. Thus, there are evidentsimilarities between the curve of viscosity of wholeblood plotted against the calculated mean cor-puscular S hemoglobin concentration (Figure 6)and that of the viscosity of solutions of S hemo-globin plotted against concentration (Figure 1).Similarly, the point at which deoxygenation firstalters viscosity is related to the concentration ofS hemoglobin, whether in solution or in cells.

When the viscosity curves of the bloods of pa-tients with various S hemoglobinopathies arecompared, a similar relationship is seen betweenthe mean corpuscular S hemoglobin concentra-tions and both the degree of viscosity change upondeoxygenation and the point at which deoxygena-

tion first alters viscosity. The viscosity curves ofthe bloods from patients with various S hemoglo-binopathies have recently been described by Har-ris, Brewster, Ham, and Castle (3) and by Griggsand Harris (20). Our observations are in agree-ment with theirs.

The dependence of the alterations of the viscositycurve upon the concentration of S hemoglobinsuggests relatively little role for the other hemo-globins in the red cells of the patients under study.Evidence that other hemoglobins may react withreduced S hemoglobin has been presented bySinger and Singer (21). In the present observa-tions the interaction of other hemoglobins with Shemoglobin was greatest at pH values lower thanthe usual physiologic range, as shown by the in-creased viscosity of fully reduced A-S blood asthe pH was lowered below 7.4 (Figure 4). Onthe other hand, the viscosity changes that occurredin reduced A-S and S-C bloods at physiologic andhigher pH values fell in the range that would beanticipated from the S hemoglobin concentrationsalone. Thus, the interaction of A and C hemo-globins with S hemoglobin is probably minimalat normal physiologic pH values, but may play arole if intracellular pH is lowered. Conversely,the interaction of reduced hemoglobin molecules isdiminished at pH values above 7.4 and even S-Sinteraction appears to be decreased slightly undersuch circumstances.

Alteration in pH is not the sole determinantof the extent of interaction among hemoglobinmolecules, and it may be anticipated that othermeans for altering such interaction are presentwithin the red cell. Thus, glutathione, which ispresent in increased amounts in the red cells of pa-tients with sickle cell anemia (22) itself causessickling and also reacts with the sulfhydryl groupsof hemoglobin (23). Following treatment of thepatient with adrenal steroids, decreases in theviscosity of S-S blood exposed to reduced oxygentensions under the standard conditions were ob-served and were found to be associated with low-ered blood glutathione levels (22). Similarly,there is evidence that there is an optimal concen-tration of potassium ions for sickling to occur un-der physiologic conditions (24).

The requirement of a mean corpuscular S he-moglobin concentration of approximately 10 gramsper cent before sickling appears in deoxygenated

839

MORTIMERS. GREENBERG,EDWARDH. KASS, AND WILLIAM B. CASTLE

blood under standard conditions indicates thelower limit of sensitivity of the deoxygenation testfor the sickling phenomenon. Thus, the blood ofthe patients with sickle cell trait and iron deficiencyanemia did not sickle after deoxygenation untilthe MCSHChad increased to more than 10 gramsper cent following the administration of iron (Fig-ure 7). However, sickling of the bloods of thepatients with iron deficiency was observed in thepresence of metabisulfite even when the MCSHCwas insufficient to permit sickling upon deoxy-genation. The greater sensitivity of the metabi-sulfite test may be ascribed to the lowered pH atwhich the test is performed and perhaps to thepossibility that strong reducing agents may in-crease the degree of interaction of hemoglobinmolecules beyond that observed with deoxygena-tion alone. We have observed that when theMCSHCis reduced by suspending A-S cells inhypotonic solutions, with resulting dilution of cellcontents by the entry of water, the lower limit ofS hemoglobin concentration in the red cells thatpermits sickling with metabisulfite is about 7grams per cent.

The determination of the limits of sensitivity ofthe usual screening tests for the presence of thesickling phenomenon poses certain problems.Since bloods with MCSHCvalues of less than 7grams per cent do not sickle in the metabisulfitetest and since this concentration of S hemoglobinapproximates one-fourth to one-fifth of the totalhemoglobin in a red cell, it follows that when Shemoglobin accounts for less than 20 to 25 percent of the hemoglobin present in the fully hemo-globinated cell, the cells will not sickle. Hence itis not surprising that surveys of the ratios of S toA hemoglobins in patients with sickle cell traithave not uncovered instances in which less than20 per cent of the hemoglobin was S hemoglobin(10). Yet, there is no apparent reason why indi-viduals may not carry lower concentrations of Shemoglobin in their bloods. Such an eventualitywould remain undetected because of the limitationsof the sickling tests and the paper electrophoreticmethod as usually conducted. For example,Singer and Fisher (25) have shown by carefulelectrophoretic study of the blood of one patientwhose blood did not sickle, that only 5 per centof the total hemoglobin was S hemoglobin(MCSHC, 1.7 grams per cent). It may be an-

ticipated that more sensitive methods will increasethe number of instances in which S hemoglobin inamounts lower than 20 per cent of the total willbe identified in the red cells of patients whose bloodfails to sickle with metabisulfite. Conceivably,the present methods of detecting sickling can bemodified to increase the likelihood of hemoglobininteraction, and hence to increase the sensitivityof such tests.

There is little difference between the viscosityof completely deoxygenated S-S blood at a he-matocrit of 25 per cent and that of fully oxygenatedblood at a hematocrit of 50 per cent (Figure 2).Thus, the physiologic consequences of sicklingin vivo are presumably small except at sites oferythroconcentration. The spleen is such a site(2, 26); it is frequently enlarged, and may be alocus of distress in children with sickle cell anemia(27). The small fibrotic spleen characteristic ofadult patients with sickle cell anemia is undoubt-edly the end result of repeated episodes of spleniccongestion and infarction. Again, patients withsickle cell trait or sickle cell-hemoglobin C disease,who are generally asymptomatic, may developsplenic infarction as a consequence of the hypoxiathat may occur during travel at high altitudes(28). Similar adverse effects are to be anticipatedas a consequence of hypoxia or of increased carbondioxide concentrations in the blood, such as mayoccur during anesthesia.

The auto-splenectomy that occurs in adults withsickle cell anemia may act to the patient's advan-tage. Thus, the red cells of patient Co. Ho., whosespleen was not palpable, had a half-life of 11 daysin the patient herself by the Cr51 method (19).The patient's labeled red cells tended to be se-questered in the liver, a site of low pressure bloodflow, with no apparent concentration in the area ofthe spleen. However, when her labeled red cellswere transfused into a normal recipient with anintact spleen, the half-life of her cells was only 6days. In this recipient radioactivity appearedpredominantly over the area of the spleen as wellas that of the liver (19).

The clinical manifestations of the presence of Shemoglobin are related to the likelihood of sick-ling under physiologic oxygen tensions, which, inturn, appears to be dependent upon a sufficientintracellular S hemoglobin concentration. Thus,anemia, painful crises, and destructive bone

840

FACTORSIN PATHOPHYSIOLOGICROLE OF S HEMOGLOBIN

changes, such as aseptic necrosis of the head of thefemur, occurred in our patients only in those withsickle cell anemia in whom the MCSHCwas 28per cent or higher. The patient with S-A-F dis-ease in whom the MCSHCwas 23 per cent alsohad both anemia and painful crises. No lesions ofthe bones were present but the patient is only 10years old. Such lesions have been reported inother patients with S-A-F disease (29). Smithand Conley (30) have noted aseptic necrosis ofthe head of the femur in patients with sickle cell-hemoglobin C disease, but data permitting quanti-tation of the MCSHCin their patients were notpublished.

Mild anemia, without painful crises or bone le-sions, was noted in our 3 patients with sickle cell-hemoglobin C disease, in whomthe MCSHCwas16 to 18 per cent. In addition, the half-life of thered cells of one of these patients was found to be17 days by the Cr5' method, which is appreciablyshorter than the normal range of 30 to 33 days forthis method. Similarly, one patient with sicklecell trait and an MCSHCof 15 grams per centevidenced a moderate decrease in the red cell sur-vival time, the half-life being 22 days. Otherpatients with sickle cell trait were not anemic un-less a complication was present, such as iron de-ficiency (patients A.D. and B. T.) or uremia (pa-tient A.B.) as noted in Table I.

One patient (C. Ha.) with sickle cell trait andan MCSHCof 14.1 grams per cent was observedduring a severe attack of lobar pneumonia to havedeveloped mild anemia and increased susceptibilityto sickling of her red cells at physiologic oxygentensions capable of initiating sickling of S-S blood.However, after recovery, the susceptibility of herred cells to sickling at such oxygen tensions dimin-ished and their survival time in vivo became nor-mal. The subsequent administration of cortisonefailed to induce an increase in the susceptibility tosickling of her red cells.

The increased viscosity that occurs when redcells have been deoxygenated under standard con-ditions of hematocrit, pH and temperature suppliesa simple method for quantitating the susceptibilityto sickling of red cells. Many uses may be sug-gested for this technique. The viscosity responsesmay provide a means for following the alteredsusceptibility to sickling of red cells during treat-ment, as has been done here in patients with iron

deficiency, or as has been done in studies of theeffects of adrenocortical hormones in patients withsickle cell anemia (22). The viscosity curveshave also been useful in patients with sickle cellanemia who have received transfusions of normalred cells. Under these circumstances many normalred cells are in the circulation and paper electro-phoretic patterns of the hemolyzed blood may showrelatively large amounts of A hemoglobin. How-ever, because the patient's own red cells containa high concentration of S hemoglobin, the viscosityof the blood under the standard conditions beginsto increase at oxygen tensions above 40 millimetersof mercury, a finding that has not been observedin sickle cell trait. On the other hand, becausethe normal transfused cells do not sickle, the maxi-mumviscosity of the blood under the standard con-ditions is not as great as that of the blood beforetransfusion. Finally, the viscosity curves are ofvalue in following patients who develop painfulcrises. During a crisis and for variable periodsof time thereafter, the blood of patients with Shemoglobin syndromes may evidence increasedcapacity to sickle under standard conditions atoxygen tensions intermediate between completeoxygenation and complete reduction (31).

Since this increased susceptibility to sickling ofred cells can be reproduced simply by lowering thepH of the blood, it follows that acidosis may in-crease the degree of sickling in vivo and that alka-losis may reduce the magnitude of such changes.Indeed, it has been possible to induce painful crisesin a patient with sickle cell anemia by administer-ing acidifying agents, and to terminate the crisesso produced, as well as spontaneous crises, by theadministration of alkali (32).

SUMMARY

Twenty-one patients with various hereditaryhemoglobinopathies involving S hemoglobin werestudied with respect to the concentration of thedifferent hemoglobins in their red cells, the clini-cal manifestations of the sickling phenomenon, andthe changes in viscosity of their bloods upon de-oxygenation in vitro under standard conditions ofhematocrit, carbon dioxide tension and tempera-ture.

A general relationship was demonstrated be-tween the mean corpuscular S hemoglobin con-

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centration (MCSHC) and the clinical manifesta-tions of the S hemoglobinopathies. Patients inwhom the MCSHCwas less than 15 grams percent were not anemic and had no painful crises.Those in whom the MCSHCwas 15 to 18 gramsper cent had evidence only of a mild hemolyticanemia. Significant anemia, bony lesions, andpainful crises were limited to patients withMCSHCvalues of more than 20 grams per cent.

The effect of progressive deoxygenation uponthe viscosity of whole blood at standard hemato-crit and pH was also a function of MCSHC. Inthe pH range 7.2 to 7.4, the changes in viscosityupon deoxygenation were similar to those of solu-tions of S hemoglobin and viscosities rose sharplyas the concentrations of S hemoglobin increasedabove 10 grams per cent. Similarly, less completedeoxygenation was necessary to elicit the firstchanges in viscosity at higher than at lower Shemoglobin concentrations.

Viscosity changes in bloods from patients withS hemoglobinopathies were also related to pH.Decreasing the pH of such bloods when partiallydeoxygenated led to increases in viscosity maxi-mal at about pH 6.8 to 7.0. Conversely, raisingthe pH decreased the viscosity of partially deoxy-genated blood. However, decreasing the pH offully reduced S-S blood did not significantly in-crease its viscosity, whereas with fully reducedS-A blood the effect was striking. The evidencesuggests that at pH values of 7.2 to 7.4, the hemo-globin concentrations are the major determinantsof the changes in viscosity upon deoxygenation,but that at lower pH values S hemoglobin inter-acts with other hemoglobins.

The observations are consistent with the previ-ously expressed concepts that the increased vis-cosity of blood that occurs upon deoxygenation is amajor factor in the pathophysiology of the S hemo-globinopathies. From the in vitro data it may beinferred that the changes in viscosity are greatestat sites of erythroconcentration, and are augmentedby lowering the pH and diminished by raising thepH of the blood.

ACKNOWLEDGMENTS

The authors are grateful for the assistance of MissGeneva Daland, Mrs. Rose Blumenthal, Mrs. JoanneNorton, and Miss Mary Emily Miller.

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