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

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1 ENERGY DISPERSIVE X-RAY MICROANALYSIS OF PLATELETS AND MEGAKARYOCYTES OF SEVERAL MAMMALS USING AIR-DRIED SPREADS AND FRESH FROZEN DRIED SECTIONS K. Takaya To cite this version: K. Takaya. ENERGY DISPERSIVE X-RAY MICROANALYSIS OF PLATELETS AND MEGAKARYOCYTES OF SEVERAL MAMMALS USING AIR-DRIED SPREADS AND FRESH FROZEN DRIED SECTIONS. Journal de Physique Colloques, 1984, 45 (C2), pp.c2-485-c < /jphyscol: >. <jpa > HAL Id: jpa Submitted on 1 Jan 1984 HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

2 JOURNAL DE PHYSIQUE Colloque C2, supplément au n 2, Tome 45, février 1984 page C2-485 ENERGY DISPERSIVE X-RAY MICROANALYSIS OF PLATELETS AND MEGAKARYOCYTES OF SEVERAL MAMMALS USING AIR-DRIED SPREADS AND FRESH FROZEN DRIED SECTIONS K. Takaya Department of Anatomy, Toyama Medical and Pharmaceutical Faculty of Medicine, Toyama, Japan University, Résumé - La microanalyse X semiquantitative de frottis de plaquettes sanguines [obtenues à partir de sang frais séché à l'air libre] et de mégacaryocytes [dans des coupes ultraminces de moelle osseuse de rat et de rate de singe] montre que les corps denses des plaquettes sanguines proviennent de ceux des mêgacaryocytes et montre aussi que leur teneur en Ca et Mg est fonction de celle trouvée dans les mêgacaryocytes. 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 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, 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 and a larger number of less dense 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 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

3 C2-486 JOURNAL DE PHYSIQUE for more than 48 hr. The specimens were evaporated with carbon about 15 nm thick. A 200 kv electron microscope (JEM-200CX), equipped with an energy dispersive spectrometer (EEDS-11, Ortec), a scanning transmission attachment (STEM) (JEM-ASID), a low dose system (MDS) and a goniometer stage was used. The specimens were observed at an acceleration voltage of 200 kv by the low dose method and then with the STEM at an acceleration voltage of 80 kv. EDX was performed at an acceleration voltage of 80 kv, at a bean current of 95 PA, at a magnification of 30,000, with condenser aperture of 300 Urn in diameter with a tilting of goniometer stage at 30 degrees and at an integrated pulse counting time of 100 sec. The specimen current was about 0.04 na. Count ratios of the elements, Na, Mg, P, S, C1, K and Ca were calculated by dividing the peak counts of K-alpha X-ray lines of these elements by those of the background over the energy range of KeV (8). Dense bodies, alpha and regions between the of the platelets of the above species and dense, less dense and regions between the in the megakaryocyte cytoplasm and regions in the nucleus were examined with a beam spot of about 10 nm in diameter. RESULTS EM with the 200 kv electron microscope and a low dose system of monkey blood platelets on fresh air-dried blood smears disclosed dense bodies, alpha, and bodies with fine dense particles as observed in the platelets of other mammals so far examined (Fig. 1). EM of megakaryocytes of the monkey spleen on fresh frozen dried ultrathin sections disclosed a large cytoplasm with a number of dense of varying sizes and a smaller number of less dense and a huge irregular nucleus (Fig. 2) which had already been disclosed in the rat bone marrow and mouse spleen megakaryocytes (4,s). Single or aggregates of structures containing quite similar to those of magakaryocytes in shape, size and density but with no identified nucleus were disclosed, so these were tentatively classified as platelets in monkey spleen. They may, of course, be mixed with parts of the magakaryocyte cytoplasm. Feg. 1 - A blood platelet of a Macaca irus on a fresh air-dried smear as observed by a 200 kv EM at an acceleration voltage of 200 kv with a low dose system (MDS), x. d:dense bodies a:alpha bp:bodies with fine dense particles v :vacuole. Fig. 2 - A megakaryocyte of a Macaca irus spleen on a fresh frozen dried ultrathin section (120 nm) as observed by a 200 kv EM in the same conditions as in Fig. 1, 6000 x. N:nucleus. Arrows indicate dense and arrow heads, less dense. Semiquantitative EDX of platelet dense bodies and megakaryocyte dense of the above species revealed that they commonly contained high amounts of phosphorus, varying high amounts of phosphorus and magnesium in the rat megakaryocyte dense as in rat platelet dense bodies and high amounts of phosphorus and calcium

4 in the monkey magakaryocyte dense as in platelet dense bodies in comparison with very low amounts of these elements in alpha, less dense and regions between the of both cells of the respective species. The difference of phosphorus and calcium content in the dense compared with that in less dense and regions between the in the monkey megakaryocytes was smaller than the difference of these elements in the dense bodies compared with alpha and regions between the of the monkey platelets (Table I), and also smaller than that of phosphorus and magnesium in the dense compared with less dense and regions between the of the rat megakaryocytes (5 and submitted). Table 1. Energy dispersive X-ray microanalysis of monkey blood platelets and spleen megakaryocytes Number Na Mg P S C1 K Ca Monkey blood 19 platelets X*X Dense bodies '191 31'51?' ' ' '199 Alpha ' ' '87 52'63 Regions between '156 17'26 204' ' '128 52'53... Monkey spleen 29 platelets ** ** * Dense '486 29' ' '349 Less dense 9 31'48 35' ' Regions between ?56 272'147 42'51 41' '59... Monkey spleen 4 megakaryocytes Dense 89 27'38 47'51 978' ' ?193 Less dense 26 54' ' t Regions between '38 362tl ' '57 Nucleus 30 51'54 64'56 795' ' t216 12'24 * P<0.001 against reg.btw.grs. * P<0.001 against other two P<O.01 against 1.d.gr. but P<0.001 against reg.btw.grs. *** P<0.001 against al.gr. 1. Count ratio : Element counts/back ground counts x Back ground, over the range of KeV A count ratio of more than 500 for phosphorus and of more than 100 for magnesium and calcium in the individual dense bodies and dense were arbitrarily set to indicate high amounts of these elements in those structures. All the dense bodies of the blood platelets of six species and all the dense of megakaryocytes of two species were classified as containing either phosphorus, phosphorus and magnesium, phosphorus, magnesium and calcium or phosphorus and calcium according to the above criteria. The percentages of the dense bodies of the platelets were summarized in Table 2. The comparison of the percentages of the dense bodies of blood platelets and dense of the megakaryocytes of the rat and monkey is shown in Table 3. Table 2. Phosphorus, calcium and magnesium in platelet dense bodies animal species platelets dense bodies percentage Of dense bodies (%) (number) P PEMg P,MgECa PECa human15) ~acaca irus(1) rat (6) mouse( guinea pig (4) cat (5)

5 C2-488 JOURNAL DE PHYSIQUE Table 3. animal species (number) Phosphorus, calcium and magnesium in platelet dense bodies and megakaryocyte dense dense bodies & percentage of dense bodies & (%) P P&Mg P,Mg&Ca PECa -- Macaca irus(1) platelets( megakaryocytes (6) rats (6) platelets(l7) megakaryocytes megakaryocytes megakaryocytes Table 2 shows that in the platelets of all the species except humans, there are dense bodies with high quantities of phosphorus only. The divalent cation in the dense bodies of human blood platelets was exclusively calcium and most of the dense bodies of the monkey platelets contained calcium but 17 % of the dense bodies contained magnesium in addition to calcium. In the rat, mouse and guinea pig, half of the dense bodies contained phosphorus and magnesium and about a third to fourth of the dense bodies contained calcium as well. 3-5 % of the dense bodies had phosphorus and calcium in the rat and mouse, and in the guinea pig the percentage was higher, at 12 %. Table 3 also indicates quite similar distribution of the dense bodies of blood platelets and dense of megakaryocytes of the rat and monkey regarding their content of phosphorus, magnesium and calcium. Rat megakaryocytes differ from each other in their percentage of dense bodies with calcium,i.e., in megakaryocytes 1-3, the dense bodies with calcium are 20 % but in megakaryocytes 4-5, the dense bodies with calcium are increased up to 55 %. However, the frequency of the dense with calcium in all the dense of the rat megakaryocytes is quite similar to that of the dense bodies with calcium in the rat blood platelets, about 40 %. DISCUSSION The conticuum normalization method of Hall (8) has been widely applied to biological X-ray microanalysis especialy in the studies using thin sections. The count ratios of the elements which are obtained by dividing the element peak counts of the characteristic X-ray lines by the background counts over the energy range far from the elemental X-ray peaks together with several corrections are proportional to the mass fractions of the elements in the analyzed microvolumes. Using the count ratios of the specified elements, it is possible to compare the concentration of the elements in different examined structures. However, studies with a standard section are necessary to compare the concentration of different elements in the same and different microregions which has not been done in this study. The divalent cation in the dense bodies of blood platelets is different from species to species in mammals. It is exclusively calcium in human platelet dense bodies (2, 3,4,5,6,10,11) while in the dog, rabbit, rat,mouse, guinea pig and pig a large amount of magnesiumisalso present in the dense bodies of the platelets. This is confirmed by biochemical methods (9) and by X-ray microanalysis (4,5,6,10,11). Predominance of magnesium in the divalent cation of the dense bodies of blood platelets in the rat, mouse and guinea pig and almost equal frequency of four types of dense bodies on their content of calcium and magnesium in the cat platelet dense bodies were shown in this study. Findings indicating high amounts of calcium in most of the dense bodies of monkey blood platelets and small amounts of magnesium in a small percentage of dense bodies are interesting considering that in most species except this species and human, a large amount of magnesium is present in the dense bodies of the platelets. Comparison of the percentage of the dense bodies of blood platelets of the rat and rnonkey and the dense of the megakaryocytes of the rat bone marrow and monkey spleen clearly indicates that dense bodies of the platelets are derived from

6 megakaryocyte dense. Phosphorus and calcium in the monkey platelet dense bodies and phosphorus and magnesium in the rat platelet dense bodies are already present when they are produced in the megakaryocyte of the respective species. It is also posgible they may be modified to a limited extent after they are discharged into the circulation. References. (1) HOLMSEN H. and WEISS H. J., Ann. Rev. Med. - 30(1979) 119. (2) SKAER R. J., PETERS P. D. and EMMINES J. P., J. Cell Sci. s(1974) 679. (3) TAKAYA K., Arch. Histol. Jap. z(1975) 335. (4) TAKAYA K., KAWAHARA M., YOSHII R. and ASADA M., J. Electr. Microsc. x(l982) 317. (5) TAKAYA K., Seitainokagaku x(1983) 137. (6) DAIMON T., MIZUHIRA V., ON0 M. and UCHIDA K., Histochemistry z(1983) 329. (7) TAKAYA K., Okajimas Folia Anat. Jpn. 58(1982) 797. (8) HALL T. A., J. Microsc. x(1979) 145: (9) KINLOUGH-RATHBONE R. L., CHAHIL A. and MUSTARD J. F., Am. J. Physiol. E(1973) 941. (10) SKAER R. J., PETERS P. D. and EMMINES J. P., J. Cell Sci. z(1976) 441. (11) COSTA J. L., SMITH M. A., TANAKA Y. and CUSHING R. J., Res. Commun. Chem. Path. Pharmacology E(1981) 137.