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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-C2-489.
�10.1051/jphyscol:19842110�. �jpa-00223778�
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 University, Faculty of Medicine, Toyama, Japan
Résumé - La microanalyse X semiquantitative de frottis de plaquettes sangui- nes [obtenues à partir de sang frais séché à l'air libre] et de mégacaryocy- tes [dans des coupes ultraminces de moelle osseuse de rat et de rate de sin- ge] 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 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 ultra- microtome 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
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PHYSIQUEf o r more t h a n 48 h r . The specimens were evaporated with carbon about 15 nm t h i c k . A 200 kV e l e c t r o n microscope (JEM-200CX), equipped w i t h an energy d i s p e r s i v e
spectrometer (EEDS-11, O r t e c ) , a scanning t r a n s m i s s i o n attachment (STEM) (JEM-ASID), a low dose system (MDS) and a goniometer s t a g e was used. The specimens were
observed a t an a c c e l e r a t i o n v o l t a g e of 200 kV by t h e low dose method and t h e n with t h e STEM a t an a c c e l e r a t i o n v o l t a g e of 80 kV. EDX was performed a t an a c c e l e r a t i o n v o l t a g e of 80 kV, a t a bean c u r r e n t of 95 PA, a t a m a g n i f i c a t i o n o f 30,000, with condenser a p e r t u r e of 300 Urn i n diameter w i t h a t i l t i n g of goniometer s t a g e a t 30 degrees and a t an i n t e g r a t e d p u l s e counting time of 100 s e c . The specimen c u r r e n t 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 c a l c u l a t e d by d i v i d i n g t h e peak counts of K-alpha X-ray l i n e s of t h e s e elements by t h o s e of t h e background over t h e energy range of 4.50-5.50 KeV ( 8 ) . Dense b o d i e s , a l p h a g r a n u l e s and r e g i o n s between t h e g r a n u l e s of t h e p l a t e l e t s of t h e above s p e c i e s and dense g r a n u l e s , l e s s dense g r a n u l e s and r e g i o n s between t h e g r a n u l e s i n t h e megakaryocyte cytoplasm and r e g i o n s i n t h e nucleus were examined w i t h a beam s p o t of about 10 nm i n diameter.
RESULTS
EM w i t h t h e 200 kV e l e c t r o n microscope and a low dose system of monkey blood p l a t e l e t s on f r e s h a i r - d r i e d blood smears d i s c l o s e d dense b o d i e s , a l p h a g r a n u l e s , and bodies with f i n e dense p a r t i c l e s a s observed i n t h e p l a t e l e t s of o t h e r mammals s o f a r examined (Fig. 1 ) . EM of megakaryocytes of t h e monkey s p l e e n on f r e s h f r o z e n d r i e d u l t r a t h i n s e c t i o n s d i s c l o s e d a l a r g e cytoplasm with a number of dense g r a n u l e s of v a r y i n g s i z e s and a s m a l l e r number of l e s s dense g r a n u l e s and a huge i r r e g u l a r nucleus (Fig. 2) which had a l r e a d y been d i s c l o s e d i n t h e r a t bone marrow and mouse s p l e e n megakaryocytes (4,s). S i n g l e o r a g g r e g a t e s of s t r u c t u r e s c o n t a i n i n g g r a n u l e s q u i t e s i m i l a r t o t h o s e of magakaryocytes i n shape, s i z e and d e n s i t y b u t w i t h no i d e n t i f i e d nucleus were d i s c l o s e d , s o t h e s e were t e n t a t i v e l y c l a s s i f i e d a s p l a t e l e t s i n monkey s p l e e n . They may, of c o u r s e , be mixed with p a r t s of t h e magakaryocyte cytoplasm.
Feg. 1
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A blood p l a t e l e t of a Macaca i r u s on a f r e s h a i r - d r i e d smear a s observed by a 200 kV EM a t an a c c e l e r a t i o n v o l t a g e of 200 kV with a low dose system (MDS), 14000 x. d:dense bodies a:alpha granules bp:bodies with f i n e dense p a r t i c l e s v :vacuole.Fig. 2
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A megakaryocyte of a Macaca i r u s s p l e e n on a f r e s h f r o z e n d r i e d u l t r a t h i n s e c t i o n (120 nm) a s observed by a 200 kV EM i n t h e same c o n d i t i o n s a s i n Fig. 1, 6000 x . N:nucleus. Arrows i n d i c a t e dense g r a n u l e s and arrow heads, l e s s dense granules.S e m i q u a n t i t a t i v e EDX of p l a t e l e t dense bodies and megakaryocyte dense g r a n u l e s of t h e above s p e c i e s r e v e a l e d t h a t t h e y commonly contained high amounts of phosphorus, v a r y i n g high amounts of phosphorus and magnesium i n t h e r a t megakaryocyte dense g r a n u l e s a s i n r a t p l a t e l e t dense bodies and high amounts of phosphorus and calcium
r e g i o n s between t h e g r a n u l e s o f b o t h c e l l s o f t h e r e s p e c t i v e s p e c i e s . The d i f f e r e n c e o f phosphorus and c a l c i u m c o n t e n t i n t h e d e n s e g r a n u l e s compared w i t h t h a t i n l e s s d e n s e g r a n u l e s and r e g i o n s between t h e g r a n u l e s i n t h e monkey mega- k a r y o c y t e s was s m a l l e r t h a n t h e d i f f e r e n c e o f t h e s e e l e m e n t s i n t h e d e n s e b o d i e s compared w i t h a l p h a g r a n u l e s and r e g i o n s between t h e g r a n u l e s o f t h e monkey p l a t e l e t s ( T a b l e I ) , and a l s o s m a l l e r t h a n t h a t o f phosphorus and magnesium i n t h e d e n s e g r a n u l e s compared w i t h l e s s d e n s e g r a n u l e s and r e g i o n s between t h e g r a n u l e s o f t h e r a t megakaryocytes (5 and s u b m i t t e d ) .
T a b l e 1. Energy d i s p e r s i v e X-ray m i c r o a n a l y s i s o f
monkey b l o o d p l a t e l e t s and s p l e e n megakaryocytes
Number Na Mg P S C1 K Ca
Monkey blood 19
p l a t e l e t s X * X
Dense b o d i e s 124 312'191 31'51 ?'37+467 180'136 443'223 2012137 504'199 Alpha g r a n u l e s 23 188+140 19'38 1492100 213'117 3754197 111'87 52'63
Regions between 27 340'156 17'26 204'111 354'150 6314223 216'128 52'53
g r a n u l e s
. . . Monkey s p l e e n 29
p l a t e l e t s ** **
*
Dense g r a n u l e s 176 76297 35246 819'486 29'48 55261 620'372 376'349
L e s s d e n s e 9 31'48 35'57 4372207 47275 38271 330'235 52244
g r a n u l e s
Regions between 50 59279 31?56 272'147 42'51 41'59 3278180 51'59
g r a n u l e s
. . . Monkey s p l e e n 4
megakaryocytes
Dense g r a n u l e s 89 27'38 47'51 978'273 3282168 157277 571'148 544?193
L e s s d e n s e 26 54'59 55254 525'277 275+151 141297 497t105 157+202
g r a n u l e s
Regions between 22 28233 35'38 3 6 2 t l 2 4 3381182 129'77 538+186 55'57 g r a n u l e s
Nucleus 30 51'54 64'56 795'427 179'212 85288 691t216 12'24
* P<0.001 a g a i n s t r e g . b t w . g r s . * P<0.001 a g a i n s t o t h e r two
P<O.01 a g a i n s t 1 . d . g r . b u t P<0.001 a g a i n s t r e g . b t w . g r s .
***
P<0.001 a g a i n s t al.gr.1. Count r a t i o : Element c o u n t s / b a c k ground c o u n t s x 1000 2. Back ground, o v e r t h e r a n g e o f 4.50-5.50 KeV
A count r a t i o o f more t h a n 500 f o r phosphorus and o f more t h a n 100 f o r magnesium and c a l c i u m i n t h e i n d i v i d u a l d e n s e b o d i e s and d e n s e g r a n u l e s were a r b i t r a r i l y s e t t o i n d i c a t e h i g h amounts o f t h e s e e l e m e n t s i n t h o s e s t r u c t u r e s . A l l t h e d e n s e b o d i e s o f t h e b l o o d p l a t e l e t s o f s i x s p e c i e s and a l l t h e d e n s e g r a n u l e s o f megakaryocytes o f two s p e c i e s were c l a s s i f i e d a s c o n t a i n i n g e i t h e r phosphorus, phosphorus and magnesium, p h o s p h o r u s , magnesium and c a l c i u m o r phosphorus and c a l c i u m a c c o r d i n g t o t h e above c r i t e r i a . The p e r c e n t a g e s o f t h e d e n s e b o d i e s o f t h e p l a t e l e t s were summarized i n T a b l e 2. The comparison o f t h e p e r c e n t a g e s o f t h e d e n s e b o d i e s o f b l o o d p l a t e l e t s and d e n s e g r a n u l e s o f t h e megakaryocytes o f t h e r a t and monkey i s shown i n T a b l e 3.
T a b l e 2. Phosphorus, c a l c i u m and magnesium i n p l a t e l e t d e n s e b o d i e s animal s p e c i e s p l a t e l e t s d e n s e b o d i e s percentage O f dense bodies (%)
(number) P PEMg P,MgECa PECa
human15) 8 4 9 0 0 4 96
~ a c a c a i r u s ( 1 )
-- 3 24 8 0 1 7 7 5
r a t ( 6 ) 17 41 12 51 34 3
mouse(61 . . 1 8 111 22 5 0 2 3 5
g u i n e a p i g (4) 12 96 5 5 1 3 2 12
c a t ( 5 ) 9 64 2 5 22 1 9 34
C2-488 JOURNAL DE PHYSIQUE
Table 3. Phosphorus, calcium and magnesium i n p l a t e l e t dense bodies and megakaryocyte dense g r a n u l e s
animal s p e c i e s dense bodies percentage of dense bodies (number) & g r a n u l e s & g r a n u l e s (%)
P P&Mg P,Mg&Ca PECa Macaca i r u s ( 1 )
--
p l a t e l e t s ( 9 1 69 7 1 2 2 70megakaryocytes (6) 198 11 5 19 65
. . . r a t s (6)
p l a t e l e t s ( l 7 ) 41 1 2 5 1 34 3
megakaryocytes 1-5 84 7 5 0 37 6
megakaryocytes 1-3 30 10 70 2 0 0
megakaryocytes 465 5 4 6 39 46 9
Table 2 shows t h a t i n t h e p l a t e l e t s of a l l t h e s p e c i e s except humans, t h e r e a r e dense bodies with high q u a n t i t i e s of phosphorus only. The d i v a l e n t c a t i o n i n t h e dense bodies of human blood p l a t e l e t s was e x c l u s i v e l y calcium and most o f t h e dense bodies of t h e monkey p l a t e l e t s contained calcium but 17 % of t h e dense bodies contained magnesium i n a d d i t i o n t o calcium. In t h e r a t , mouse and guinea p i g , h a l f of t h e dense bodies contained phosphorus and magnesium and about a t h i r d t o f o u r t h of t h e dense bodies contained calcium a s w e l l . 3-5 % of t h e dense bodies had phosphorus and calcium i n t h e r a t and mouse, and i n t h e guinea p i g t h e percentage was h i g h e r , a t 12 %. Table 3 a l s o i n d i c a t e s q u i t e s i m i l a r d i s t r i b u t i o n of t h e dense bodies of blood p l a t e l e t s and dense g r a n u l e s o f megakaryocytes of t h e r a t and monkey r e g a r d i n g t h e i r c o n t e n t of phosphorus, magnesium and calcium. Rat megakaryocytes d i f f e r from each o t h e r i n t h e i r percentage of dense bodies w i t h c a l c i u m , i . e . , i n megakaryocytes 1-3, t h e dense bodies w i t h calcium a r e 20 % b u t i n megakaryocytes 4-5, t h e dense bodies with calcium a r e i n c r e a s e d up t o 55 %. However, t h e frequency of t h e dense g r a n u l e s w i t h calcium i n a l l t h e dense g r a n u l e s of t h e r a t megakaryocytes i s q u i t e s i m i l a r t o t h a t of t h e dense bodies with calcium i n t h e r a t blood p l a t e l e t s , about 40 %.
DISCUSSION
The conticuum n o r m a l i z a t i o n method of H a l l (8) has been widely a p p l i e d t o b i o l o g i c a l X-ray m i c r o a n a l y s i s e s p e c i a l y i n t h e s t u d i e s u s i n g t h i n s e c t i o n s . The count r a t i o s of t h e elements which a r e obtained by d i v i d i n g t h e element peak counts of t h e c h a r a c t e r i s t i c X-ray l i n e s by t h e background counts over t h e energy range f a r from t h e elemental X-ray peaks t o g e t h e r w i t h s e v e r a l c o r r e c t i o n s a r e p r o p o r t i o n a l t o t h e mass f r a c t i o n s of t h e elements i n t h e analyzed microvolumes. Using t h e count r a t i o s of t h e s p e c i f i e d elements, i t i s p o s s i b l e t o compare t h e c o n c e n t r a t i o n of t h e elements i n d i f f e r e n t examined s t r u c t u r e s . However, s t u d i e s with a s t a n d a r d s e c t i o n a r e n e c e s s a r y t o compare t h e c o n c e n t r a t i o n of d i f f e r e n t elements i n t h e same and d i f f e r e n t microregions which has n o t been done i n t h i s s t u d y .
The d i v a l e n t c a t i o n i n t h e dense bodies o f blood p l a t e l e t s i s d i f f e r e n t from s p e c i e s t o s p e c i e s i n mammals. I t i s e x c l u s i v e l y calcium i n human p l a t e l e t dense bodies ( 2 , 3,4,5,6,10,11) w h i l e i n t h e dog, r a b b i t , rat,mouse, guinea p i g and p i g a l a r g e amount of m a g n e s i u m i s a l s o p r e s e n t i n t h e dense bodies of t h e p l a t e l e t s . This i s confirmed by biochemical methods (9) and by X-ray m i c r o a n a l y s i s (4,5,6,10,11). Predominance of magnesium i n t h e d i v a l e n t c a t i o n of t h e dense bodies of blood p l a t e l e t s i n t h e r a t , mouse and guinea p i g and almost equal frequency of f o u r t y p e s of dense bodies on t h e i r c o n t e n t of calcium and magnesium i n t h e c a t p l a t e l e t dense bodies were shown i n t h i s s t u d y . Findings i n d i c a t i n g high amounts of calcium i n most of t h e dense bodies of monkey blood p l a t e l e t s and small amounts of magnesium i n a small percentage of dense bodies a r e i n t e r e s t i n g c o n s i d e r i n g t h a t i n most s p e c i e s except t h i s s p e c i e s and human, a l a r g e amount of magnesium i s p r e s e n t i n t h e dense bodies of t h e
p l a t e l e t s .
Comparison of t h e percentage o f t h e dense bodies of blood p l a t e l e t s of t h e r a t and rnonkey and t h e dense g r a n u l e s of t h e megakaryocytes of t h e r a t bone marrow and monkey s p l e e n c l e a r l y i n d i c a t e s t h a t dense bodies of t h e p l a t e l e t s a r e d e r i v e d from
p r e s e n t when t h e y a r e produced i n t h e megakaryocyte o f t h e r e s p e c t i v e s p e c i e s . I t i s a l s o p o s g i b l e t h e y may b e m o d i f i e d t o a l i m i t e d e x t e n t a f t e r t h e y a r e d i s c h a r g e d i n t o t h e c i r c u l a t i o n .
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