Small angle light scattering by erythrocytes

Small Angle Light Scattering by Erythrocytes In an earlier report Livesey and Billmeyer (1) measured the small angle light scattering from samples of human blood. When plotted according to the method of Sloan and Arrington (2), their sample of fresh blood gave scattering maxima at 3.2 °, corresponding to a diameter of 6.5 t~, and at approximately 10% The authors concluded that the maximum at I0 ° was too far removed from the main peak to be a secondary diffraction maximum. They suggested rather, after consulting with this laboratory, that it may have resulted from the thrombocytes (platelets) present in the whole blood sample. The data presented below supports the opposite view, that the maximum observed at I0 ° is not due to platelet scattering but appears rather to be a secondary diffraction maximum. The measurements were carried out with a Phoenix small angle light-scattering photometer as previously described (3), except, the samples were measured in a 10-ram cuvette rather than the standard scattering cell. Whole blood was obtained from CBA/2 mice by decapitation and draining into tubes containing a small quantity of heparin. The scattering measurements were made on aliquots of whole blood added to the cuvette filled with Alsever's solution (4), an isotonic solution of dextrose, citrate and sodium chloride. Suspensions of e r y t h r o c y t e s were o b t a i n e d b y c e n t r i f u g a t i o n of t h e whole blood in a n I n t e r n a t i o n a l clinical centrifuge for 10 min. T h e p a c k e d cells were decanted, washed once w i t h Alsever's solution, a n d resuspended in Alsever's solution. Aliquots of this stock suspension were added to the c u v e t t e as above for t h e s c a t t e r i n g measurements. Figure 1 is a Sloan plot of whole mouse blood a n d s e p a r a t e d e r y t h r o c y t e s . T h e angular s c a t t e r ing d i s t r i b u t i o n of the whole blood is q u a l i t a t i v e l y similar to t h e sample m e a s u r e d b y Livesey and Billmeyer (1). I t has a p r i m a r y s c a t t e r i n g maxim u m at 4 ° a n d a secondary p e a k at 10 °. These cells are s o m e w h a t smaller t h a n t h e h u m a n cells however, a n d t h e angular position of t h e i r p r i m a r y s c a t t e r i n g m a x i m u m is shifted t o w a r d smaller angles. I t should also be n o t e d t h a t a l t h o u g h Livesey a n d B i l l m e y e r s t a t e d t h a t the secondary s c a t t e r i n g p e a k was located at 10 °, a careful examin a t i o n of t h e i r p u b l i s h e d Sloan plot shows its position to be closer to 9.5 °.

I(e)

Q..

ell

t

L

I

2

5 4

5

6 7

8 9

IO I[ 12 13 t4

0 FIG. 1. Sloan plot of isolated mouse e r y t h r o cytes (upper) and whole mouse blood (lower).

T h e second curve in Fig. 1 represents t h e angular s c a t t e r i n g d i s t r i b u t i o n from isolated e r y t h r o cytes. This sample was freed from b o t h t h r o m b o cytes and leucocytes. I t is quite a p p a r e n t t h a t t h e secondary m a x i m u m is still p r e s e n t a n d even more p r o m i n e n t , a l t h o u g h shifted to 11 ° . This corresponds to a slight shift of t h e p r i m a r y m a x i m u m to approximately 4.5 ° . These results d e m o n s t r a t e unequivocally t h a t the secondary m a x i m u m could not be s c a t t e r i n g from t h r o m b o c y t e s . Figure 2 also shows t h e s c a t t e r i n g d i s t r i b u t i o n of the s e p a r a t e d e r y t h r o c y t e s . I n this case t h e int e n s i t y I (0) was normalized to 0 ° s c a t t e r i n g a n d plotted against the actual scattering angle obrained by correcting the instrument angle for refraction. This curve was compared to the Fraunhofer diffraction distribution calculated for a particle 5 tt in diameter. Although the secondary peak of the Sloan plot appears only as a shoulder in the normalized curve, its position does correspond quite well with the first maximum beyond the forward lobe of the theoretical diffraction curve.

We

have concluded

that these results demon-

Journal of Colloid and Interface Science, Vol. 37, No. 1, S e p t e m b e r 1971

249

250

L E T T E R S TO THE EDITORS ANGULAR DEPENDENCELIGHT SCATTERING

strate that the secondary maximum seen in our Sloan plots of mouse erythrocytes and whole blood and in the fresh sample of human blood measured by Livesey and Billmeyer is clearly a secondary diffraction maximum. ACKNOWLEDGMENT The authors thank Dr. Roger Priore of the Roswell Park Memorial Institute Computer Center for the Fraunhofer diffraction computations. REFERENCES 1. LIVESEY, P. J., AND BILLMEYER, F. W., JR. J . Colloid Interface Sci. 30,447 (1969). 2. SLOAN, C. K., paper presented at the 125th National Meeting, Amer. Chem. Soc., Kansas City, Missouri, April 1954. 3. FIEL, R., J. Exp. Cell Res. 59,413 (1970). 4. BUKANTZ,S. C., REIN, C. R., AND KENT, J. F., J. Lab. Clin. Med. 31,394 (1946). ROBERT J. FIEL I-IAROLD M. SCtIEINTATTR

ANGLE(e. RADIUS ~ O.I tN MICRONSJ"

62

0.5

t

~

5

JO

So

50

SPHERE DISK FILAMENT

FIo. 2. Plot of the normalized angular scattering of isolated erythroeytes (points) and a theoretical curve (solid line) for diffraction from a particle 5 ~ in diameter.

Springville Laboratories of Roswell Park Memorial Institute Springville, New York 14141 Received April 12, 1971; accepted May, 1971

Journal of Ooll~dand InlerfaccScience, Vol. 37, 1~o. l, September 1971