Acta med. scand. Vol. 190, pp. 127-131, 1971

MECHANICAL FRAGILITY OF ERYTHROCYTES IN NORMALS AND IN PATIENTS WITH HEART VALVE PROSTHESES

Jon Dale and Erik Myhre

From Medical Department B, University Hospital, Rikshospitalet, Oslo, Norway

Absfruct. Hemolysis following insertion of heart ball valves shows great individual differences. Besides the mechanical properties of the prosthetic valves, individual variations in the mechanical fragility (MF) of the erythro- cytes might be important in this respect. The M F has been studied with the hemoresistometer of Fleisch and Fleisch, which exposes the cells to a standardized trauma by rotation of the blood in a cylindrical chamber. The M F is defined as the amount of heme liberated. A nor- mal material is presented, and the error of the method and the normal variation of the M F are estimated, using two different rotors. The hemoresistometer seems to be a valuable tool for evaluation of the MF, offering sev- eral advantages compared to other methods. Reference has been made to a material of nine consecutive patients with heart valve disease examined before and after prosthetic ball valve implantation. All had a normal M F before the operation, and the mean M F remained normal in spite of moderate intravascular hemolysis de- veloping in all but one patient postoperatively. In four patients with valve prostheses readmitted for hemolytic anemia the plasma heme concentrations were markedly increased, but the MF was normal. The question whether ti lowered M F may be the cause of the severe intravascu- lar hemolysis developed by some patients remains un- settled, but the present study suggests that individual dif- ferences in M F are of minor importance for the develop- ment of severe hemolytic anemia in these patients.

Implantation of prosthetic ball valves in the heart provokes chronic intravascular hemolysis of vary- ing degree in the majority of patients (2, 8, 9, 10, 11, 14, 16, 20); in about 5 % hemolytic anemia develops (8, 9, 14). We studied patients with different types of aortic and mitral ball valve prostheses and found that the degree of hemolysis was largely determined by the type of valve implanted, indicating the direct mechanical trauma on the erythrocytes to be the main cause of hemolysis (8, 9). However, there were great in- dividual differences in red blood cell destruc- tion within patient groups with the same type of

valve. The question therefore arose whether the severity of hemolysis following heart valve re- placement was due to individual differences in mechanical fragility (MF) of the erythrozytes.

Several methods have been used to measure the MF. Rotating tubes containing glass beads or other particles have been most commonly used (3, 7, 15, 17, 18). Andreasen (1) “atomized” suspensions of erythrocytes in order to avoid the influence of the plasma viscosity (12). In order to expose the cells to a standardized mechanical trauma, Fleisch and Fleisch (5) constructed the hemoresistometer, in which heparinized blood is made to rotate in a cylindrical chamber.

In the present study the hemoresistometer has been used to determine the M F in normals, in patients before and after implantation of prosthe- tic ball valves, and in patients with hemolytic anemia following heart valve replacement. The accuracy of the method was also evaluated.

MATERIAL AND METHODS Three groups of subjects were studied. Group I repre- sented a normal material of 20 healthy volunteers and 47 patients with no blood o r circulatory disorder. In group I1 were nine patients with valvular heart disease who were examined before and after replacement of the valves. Seven patients had Starr-Edwards aortic and two Starr-Edwards mitral valves inserted; in all but one (no. 4) the newer types with hollow Stellite (metallic) balls were used. Group I11 consisted of four patients who developed marked intravascular hemolysis with anemia following prosthetic valve implantation. Two pa- tients (nos, 10 and l l) had Starr-Edwards aortic ball valves with hollow metal balls, one (no. 12) a Beall mitral prosthesis and the fourth patient (no. 13) had three valves replaced (Starr-Edwards aortic, Key-Suzuki mitral and a Beall prosthesis in the tricuspid ostium).

The MF was determined by the use of the hemo- resistometer (Fig. 1). Blood sampling was made as non-

Acla med. scand. 190

128 J . Dale and E . Myhre

I-J I ---L 7 2

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Fig. 1. Schematical drawing of the hemoresistometer after Fleisch and Fleisch. Ten millilitres of heparinized blood ((5) is poured into the cylindrical container (5). The rotor (4) is mewed on to the motor spindle, the connect- ing part (3 ) is placed in position in the lop piece (2), and the container fastened by the arm (7) and the screw (8 ) . l’he motor ( I ) is started and turns the rotor at 3 000 rpm for 15 min.

traumatic :IS possible, in most cases during bed rest in the morning. Fifteen to twenty millilitres of blood were drawn into heparinized plastic syringes. Ten millilitres of the heparinized blood were transferred into the cy- lindrical container of the hemoresistometer, the equip- ment was put together and fastened. The motor was started and ran for exactly 15 min at 3 000 rpm. The container wiiq then disconnected, and 2 ml of the blood was transferred into each of two plastic tubes. Two simi- lar tubes Here filled from the remainder of the he- parinized blood in the syringe. The four tubes were then centrifugated at 300 G for 10 min, the plasma was care- fully collected and transferred to four new tubes and centrifugated at 2 000 G for 30 min. The plasma was then analyzed for total heme pigments by the benzidine method of Crosby and Furth (4). The hematocrit was also determined.

The lactic dehydrogenase (LDH) was cstimated in serum according to the method described by Wroblewski and IaDue (19). The upper normal value in our labora- tory is 200 pmol/min/l. The degree of intravascular heinolysis w;is predicted from the serum LDH level as described elsewhere (11).

RESULTS The hemoresistometer was delivered with three different sets of rotors, designated “Hemolysc env. 20 mg%”, “H.e. 35 mg%” and “H.e. 60 mg%”. We used the two rotors that provoked most hemolysis. The M F was defined as the amount of heme pigments (mg/100 ml) released during rotation of the whole blood in the hemo- resistometer. It was calculated as the difference between plasma heme concentration after and before the fragility test. Duplicate determinations were always performed.

Table I shows the values in the normal in- dividuals (group I). The heme concentration in the untreated plasma was somewhat higher than that found by others (4) who have proposed an upper normal limit of 5 mg/100 ml, but it cor- responded well with earlier results from our laboratory (1 3). The hemolysis provoked by the hemoresistometer was very slight; only about 0.2 and 0.5% of the total content of heme in the erythrocytes were detected in plasma after treat- ment of whole blood with the respective rotors. It is interesting to note that the scatter of the individual values was of the same magnitude for both rotors.

The error of the analysis of heme concentra- tion was determined by duplicate estimations (Table 11). As described, two plastic tubes were filled with heparin-blood directly from the syringe and blood rotated in the hemoresistometer was transferred to two other tubes. The difference in heme concentration between each of the tubes in a pair reflected the error in pipetting, cen- trifugation and analysis (Table 111). Obviously the additional error due to pipetting and centri- fugation was small. In calculating thc M F the mean of paired observations was used; this re- duced the error still further.

In order to obtain information about the total error of the method, including blood sampling,

Table I. Plasma heme concentrution and MF of erythrocytes in nornials

Plasma heme pigments (mg/100 ml)

Before trauma After trauma MF No. of

Rotor type subjects Mean S.D. Mean S.D. Mean S.D.

1-i.e. 35 mg”,, 63 6 0 2.4 28.3 8.0 22.3 7.6 H.e. 60 nig”,, 9 5.9 2.5 67.8 7.4 61.9 6.3

Mechanical fragility of erythrocytes 129

treatment of the blood in the hemoresistometer and the heme analysis, blood was drawn from the same individual twice at an interval of about 30 min (Table IV). The spontaneous variation of the M F was evaluated by repeated examination of 17 subjects after 48 h.

The M F varies with the hematocrit values within wide limits (5) and therefore should be corrected to a chosen hematocrit value. This was not done in our material of normals; the hema- tocrit ranged from 38 to 46 with a mean of 42%.

Table 11. Errors of heme analysis. Duplicate deter- minations of plasma samples from different individuals

Plasma heme pigments (mg/100 ml)

After trauma with rotor

Before H.e. H.e. trauma 35 mg% 60 mg%

Mean of all deter-

Mean difference minations 5.9 27.5 68.4

between duplicate determinations 0.75 2.3 4.4

S.D. of differences between duplicate determinations 1.15 3.2 5.4

determinations 9 13 7 No. of duplicate

Table 111. Errors at centrifugation, pipetting and ana- lysis. Differences between equally handled samples in a pair

Plasma heme pigments (mg/100 ml)

After trauma with rotor

Before H.e. H.e. trauma 35 mg% 60 mg%

Mean of all deter-

Mean difference minations 6.0 27.9 67.0

between determina- tions in pairs 1.35 3.5 5.8

S.D. of differences

No. of paired deter-

within pairs 2.0 4.3 8.0

minations 60 55 7

9 - 712998

Table IV. Error of the method and spontaneous variation. Differences in MF between blood samples obtained at intervals of about 30 min and 48 h, respectively. The rotor “H. e. 35 mg%” was used

M F of erythrocytes (blood drawn at interval)

30min 48 h

Mean of all determinations 17.3 22.3 Mean difference between duplicate determinations 3.6 6.5

S.D. of difference between duplicate determinations 4.6 8.0

No. of duplicate deter- minations 7 17

Table V. Plasma heme concentrations (mg/IOO ml) and MF in group 11 before and after ball valve im- plantation A = untreated blood, B = blood treated in the hemoresisto- meter. The rotor “H.e. 35 mg%” was used. The LDH values in serum after operations are listed

Before After operation opera tion

Pat. Prosthe- no. sis A B M F A B M F LDH

Aortic Aortic Aortic Mitral Aortic Mitral Aortic Aortic Aortic

9.6 33.0 23.4 10.5 34.7 24.2 8.1 26.7 18.6 7.2 14.7 7.5 3.8 32.9 29.1 4.0 61.7 57.7 4.6 33.8 29.2 10.3 40.0 29.7 6.9 34.4 27.5 20.6 58.2 37.6 5.7 24.7 19.0 5.5 22.0 16.5 4.1 23.6 19.5 3.1 17.8 14.7 6.2 37.5 31.3 2.8 18.3 15.5 7.4 28.1 20.7 8.7 32.8 24.1

3 30 353 306 210 308 340 470 380 346

Mean 6.3 30.5 24.2 8.1 33.4 25.3 338 S.D. 2.4 4.9 5.0

Table V shows the M F in nine patients with valve disease before and 3-6 weeks after the prosthetic valve implantation (group 11). The hematocrit level was normal in all patients both before and after the operation. Hemolysis was moderate in all cases, the erythrocyte destruc- tion rates were between one and two times the normal as predicted from the serum LDH levels (11). The preoperative M F was within normal limits in all patients, while it was increased in one case (no. 3) after valve replacement. The plasma heme concentration was elevated in an- other operated patient (no. 5>, but the MF was

Acta med. scand. 190

130 J . Dole and E. Myhre

Table VI. Plasma heme concentrations before ( A ) and after treatment ( B ) in the hemoresistometer, and M F corrected to a hematocrit value of 40 in group 111

Heme pig- ments

ml) MF Hb Pat. Prosthe- - correc- (g/tOO Hct LDH no. sis A B ted ml) ( Y o ) (Ujl)

(mg/100

10 Aortic 45.6 57.9 16.4 7.3 30 1 570 I 1 Aortic 65.2 89.6 27.4 9.1 32 1 160 I2 Mitral 39.1 71.0 35.0 10.1 35 920 13 Triple 25.3 38.7 15.8 10.4 34 765

normal. There was no correlation between the M F and the degree of hemolysis postoperatively.

In group 111, operated patients readmitted be- cause of severe hemolysis, the hematocrit val- ues were low. The MF was therefore corrected to a hematocrit value of 40 (Table VI). The plasma heme concentration was markedly elevated in all four patients, while the M F was found to be normal.

DISCUSSION

The great individual differences in hemolysis fol- lowing insertion of heart valve prostheses (8, 9) could be due to individual differences in the red blood cell resistance to mechanical trauma. This factor has not previously been studied in such patients, probably because most routine meth- ods for M F determination are rather rough and difficult to standardize. Because the expected dif- ferences in MF are small, a strictly standardized and sensitive technique is a matter of necessity. The hemoresistometer is claimed to have several advantages compared to the common methods using rotating test tubes containing glass beads ( 5 ) . The trauma is homogenous and is provoked by rapid rotation of the blood instead of crush- ing the erythrocytes by beads. Ideally, only fragile erythrocytes should be destroyed. The hemoresisto- meter provokes a relatively low degree of hemo- lysis; its traumatic effect is more similar to the physiological trauma of the erythrocytes in the circulation. Because the test chamber is built of plexiglass, the contact with air minimal, and the duration of the test short, chemical influence on the erythrocyte membrane is probably negligible.

Acta med. scand. 190

We mainly used the rotor designated “Hemo- lyse env. 35 mg%”, because we wanted a direct comparison of our normal material with the find- ings of Fleisch and Fleisch (5). We found an approximately similar mean M F and a somewhat smaller normal variation (Table I). The error of the various steps of the procedure was analyzed and the method was found to be sensitive and the results reproducible. The differences observed represented very small variations in the degree of hemolysis. We believe that the rotor producing most hemolysis should be preferred. The hemoly- sis provoked by this rotor is far less than by other methods (1, 3, 7, 18) and probably lies within “physiological” limits. Using this rotor one may be able to detect decreased as well as increased MF.

In most other methods (3, 7, 18) the normal variation of the M F (in per cent of the mean value) has been as great or greater than that found with the hemoresistometer, and the ery- throcyte destruction higher. Andreasen (1) used a quite different technique, atomizing a suspen- sion of erythrocytes. He found a much smaller variation of the MF in normals, probably be- cause the influence of plasma viscosity was eliminated.

Preoperatively, patients with valvular heart dis- ease showed a normal MF of the erythrocytes. However, no significant changes occurred in the M F after ball valve insertion. Eight patients de- veloped moderate intravascular hemolysis, but there was no correlation between the preopera- tive M F and the degree of hemolysis postopera- tively. Thus the individual differences in hemoly- sis following prosthetic valve implantation could not be explained by different M F of the erythro- cytes. However, we would need preoperative es- timations of the MF in patients who later de- velop severe hemolysis in order definitely to settle the question of the influence of individual dif- ferences in the MF.

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