ENERGY DISPERSIVE X-RAY MICROANALYSIS OF PLATELETS AND MEGAKARYOCYTES OF SEVERAL MAMMALS USING AIR-DRIED SPREADS AND FRESH FROZEN DRIED SECTIONS

Abstract

Semiquantitative energy dispersive X-ray microanalysis of blood platelets on fresh air-dried blood smears and megakaryocytes on fresh frozen dried ultrathin sections of rat bone' marrow and monkey spleen indicates that the dense bodies of blood platelets are derived from the dense granules of megakaryocytes and that their calcium and magnesium content is determined when they are produced in the megakaryocytes. Dense bodies of human blood platelets contain 5-HT, nucleotides (ATP and ADP) and calcium (1). Electron microscopy (EM) of human, rat, mouse, guinea pig and dog platelets on fresh air-dried spreads of blood disclosed dense bodies, alpha granules, bodies with fine dense particles and peripheral vacuoles in the platelets (2,3,4,5). EM of megakaryocytes in mouse spleen and rat bone marrow on fresh frozen dried ultrathin sections (120 nm) revealed a large cytoplasm containing many dense granules and a larger number of less dense granules and a huge irregular nucleus (4,5). Energy dispersive X-ray microanalysis (EDX) of the dense bodies of human blood platelets revealed high peaks for phosphorus and calcium (2,3,6). Semiquantitative EDX of platelet dense bodies of the rat, mouse, guinea pig, rabbit and dog and dense granules of megakaryocytes of the rat and mouse detected high amounts of phosphorus and magnesium and varying small quantities of calcium (4). Examination of blood platelets and spleen megakaryocytes of a crab-eating macaque and cat blood platelets are included in this study. MATERIALS AND METHOD A male monkey (Macaca irus) (1.5 Kg), five humans (three males and two females, two adults and three children), six albino adult Wistar rats (female), six albino adult DDY mice (male, 25 gO, four adult Hartley guinea pigs (female) and five adult and young tabby cats (two males and three females) were used for the study of blood platelets. Drops of blood from finger and tail tips were placed on collodion-film covered copper grids (200 mesh), immediately blotted with filter paper and air-dried. Wedge biopsy specimens of the monkey spleen were obtained by laparotomy, and bone marrow of six rats (female adult) was removed after decapitation. Fresh frozen dried ultrathin sections (120 nm) were prepared by pressing the specimens on the chuck onto the copper disk surface plated with gold (RF-2,Eikow Eng.) precooled with liquid jnitrogen, and cutting them in a cryochamber (FTS, Sorvall) attached to the ultramicrotome MT2-B (Sorvall). The sections were frozen dried in a glass apparatus within the cryochamber with a continuous flow of dry nitrogen gas from a liquid nitrogen dewar (7). They were further dried in a high vacuum at less than 10^ Torr Article published online by EDP Sciences and available at http://dx.doi.org/10.1051/jphyscol:19842110 C2-486 JOURNAL DE PHYSIQUE f o r more than 48 hr . The specimens were evaporated with carbon about 15 nm thick. A 200 kV e lec t ron microscope (JEM-200CX), equipped with an energy d i spers ive spectrometer (EEDS-11, Ortec) , a scanning transmission attachment (STEM) (JEM-ASID), a low dose system (MDS) and a goniometer s tage was used. The specimens were observed a t an acce le ra t ion vol tage of 200 kV by t h e low dose method and then with the STEM a t an acce le ra t ion vol tage of 80 kV. EDX was performed a t an accelerat ion vol tage of 80 kV, a t a bean current of 95 PA, a t a magnification of 30,000, with condenser aperture of 300 Urn i n diameter with a t i l t i n g of goniometer s tage a t 30 degrees and a t an in tegra ted pulse counting time of 100 sec. The specimen current was about 0.04 nA. Count r a t i o s of t h e elements, Na, Mg, P, S, C 1 , K and Ca were calculated by dividing t h e peak counts of K-alpha X-ray l i n e s of these elements by those of t h e background over t h e energy range of 4.50-5.50 KeV (8) . Dense bodies, alpha granules and regions between t h e granules of t h e p l a t e l e t s of t h e above species and dense granules, l e s s dense granules and regions between t h e granules i n t h e megakaryocyte cytoplasm and regions i n t h e nucleus were examined with a beam spot of about 10 nm i n diameter.