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SEM Processing Of Blood Samples

  • Sara E. Miller (a1)

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A long time ago, we ciid some work on RBC's from muscular dystrophy patients, We found that the cells are very sensitive to pH, salt concentration, glass, and other things (maybe including the moon???), Low pH, increased salt, especially NaCl, and sitting in a glass tube or on a glass slide unfixed would cause echinocyte formation.

We got around the problem by drawing the blood and then IMMEDIATELY dropping (drop by drop) blood samples into 1% glutaraldehyde in phosphate buffer, pH 7.4 (not PBS). You can use a Maunsbach formula, where 0.135 U P04 gives 300 milliosmoles, or 0.1 M P04 plus sucrose (try 1.8 % sucrose). Test the osmolarity of the buffer; it should be 300 ± 10 or so. The final osmolarity of the buffered fix will be 400 milliosmoles; 300 from the buffer and 100 from the glutaraldehyde! The fix should be room temperature (not cold) when the blood is added. It can be stored refrigerated.

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References

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Miller, S.E., Roses, A.D. and Appel, S.H., 1975. Erythrocytes in human muscular dystrophy, Science 188:1131.
Roses, A. D., Appet, S. H., Butterfield, D. A., Miller, S. E. and Chesnut, D. B.. 1975. Specificity of biochemical and biophysical tests in Duchenne and myotonic muscular dystrophy, carrier states and congenital myotonia. Trans. Amer. Neurol. Assoc. 100:131134.
Roses, A.O., Roses, M.J., Miller, S.E., Hull, K.J. Jr. and Appel, S.H., 1976, Carrier detection in Duchenne muscular dystrophy, New Engl. J. Med. 294:193198.
Miller, S.E., Roses, A D. and Appel, S. H. 1976. Scanning electron microscopy studies in muscular dystrophy. Arch. Neurol. 33:172174.

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SEM Processing Of Blood Samples

  • Sara E. Miller (a1)

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