Embed Size (px)
Citation preview
J. clin. Path. (1958), 11, 535.
THE EFFECT OF REDUCED OXYGEN TENSION ONPLATELET FORMATION IN VITRO
BY
R. J. V. PULVERTAFTFrom the John Burford Carlill Pathological Laboratories, Westmninster Medical School, London
It is now generally accepted that platelets areformed in the bone marrow from megakaryocytes.This suggestion was first clearly made by Wright(1906), who based his case on embryological andmicroscopical arguments as well as on considera-tions of comparative biology. The question was,however, settled when Thiery and Bessis in 1956produced their beautiful film showing conclusivelythat megakaryocytes throw out long processeswhich become fragmented to form platelets. Thiswas confirmed by Izak, Nelken, and Gurevitch in1957.Examination in this laboratory of some hun-
dreds of bone-marrow cultures from human beingsand animals by tissue culture methods did not,however, in any case disclose activity in mega-karyocytes (Pulvertaft and Humble, 1956). Ontwo occasions only they were seen to rotate; onone spidery processes were seen reminiscent ofthe film by Thidry and Bessis; in all other casesthey remained spherical until, after a lapse of a
few days, they disintegrated.Nearly all the specimens examined in this series
were, however, human; Thiery and Bessis workedwith the rat. More important is the fact thatthey worked with isolated megakaryocytes sealedbetween coverslip and microscope slide, whereasthe specimens personally examined were mountedon agar in a special chamber.
It was found that when bone-marrow cultureswere submitted to low oxygen tensions great acti-vity was shown by megakaryocytes, includingplatelet formation.
TechniqueThe culture chamber used was similar to a blood-
counting chamber, having a central pillar recessed by2 mm., surrounded by a ditch which was normallyhalf-filled witfi culture medium. The central pillarwas coated with 2% agar, and the medium used was
10% human serum, 0.1% " difco " yeast extract, and90% Hanks' balanced salt solution. Streptomycinand penicillin were added, 100 units per ml.The marrow was collected into heparinized human
serum, usually from the same patient. When this is
poured into a dish and allowed to settle, small frag-ments are usually seen. These consist of fat andblood vessels with bone marrow, and the smallestpossible fragments are placed on the agar. .A cover-slip is applied and sealed with beeswax. After half-filling the ditch with medium through a lateralaperture, later sealed, the preparation is incubated,coverslip downwards.Agar repels cells, and glass attracts them; the
marrow cells are therefore flattened on to the glassin a manner not seen unless agar is used. Motile cellsmove peripherally, and in 18 hours megakaryocytesare clearly seen as dense spheres. They congregatealong blood vessels and are usually not obvious untilmigration is established (Figs. 1, 2, 3).
After 18 hours the culture medium is withdrawn.and the slide is placed in a desiccator with the lateralaperture unsealed. The desiccator is then evacuatedwith a mercury pump, and the air is replaced by amixture of 95% nitrogen and 5% C02. Meanwhileculture medium is placed in shallow dishes, and alldissolved gas is removed by a mercury pump in adesiccator. When bubbling has ceased, 95°' nitrogenand 5% C02 is introduced, and the desiccator is sealedfor 18 hours, during which time the medium issaturated with this gas mixture. Five per cent. C02is of course essential in order to maintain pH at 7.4.The chamber is then filled with this fluid, making surethat no bubble of air remains. It is sealed withbeeswax.The chamber is mounted on a phase-contrast
microscope in an incubator box. Cinematographicrecords are taken by time-lapse.
Results
During the first five hours the cells normallymotile continue to move, but the megakaryocytesremain spherical. After five hours motility ofmegakaryocytes is observed.
It must be appreciated that these cells are invarious stages of maturity, and that only fullymature cells can form platelets. These can berecognized from their peculiar mottled texture,identical with the appearance of coherent plate-lets collected from the buffy coat of heparinizedblood.
copyright. on N
ovember 12, 2021 by guest. P
rotected byhttp://jcp.bm
j.com/
J Clin P
athol: first published as 10.1136/jcp.11.6.535 on 1 Novem
ber 1958. Dow
nloaded from
R. J. V. PULVERTAFT
OAx~*~
~ ~ ~ W ~~~~~~~~ouseboremarrW.
536
copyright. on N
ovember 12, 2021 by guest. P
rotected byhttp://jcp.bm
j.com/
J Clin P
athol: first published as 10.1136/jcp.11.6.535 on 1 Novem
ber 1958. Dow
nloaded from
EFFECT OF REDUCED OXYGEN TENSION ON PLATELETS IN VITRO
around, so that they cover a wide circular area,extending over many microscopical fields of aI1/12 lens. Such cells never develop further (Figs.4 and 5).They may also become sausage-shaped, and
then become motile, progressing and elongatingsimultaneously (Fig. 6). Since they are at firstspheres they make only point contact with thecoverslip, and when this serpentine development
* sets in they are not adherent to the glass or agar,but progress in the fluid. Serpentine developmentmay proceed to platelet formation.
Finally, true platelet formation develops by theextrusion of thick processes which elongate pro-
p ~~~~~~~~~~~gressivelyand which eventually become frag-mented. This process alone represents platelet
1<'t j formation, and only megakaryocytes which havethe peculiar mottled appearance characteristic ofmaturity develop in this way (Figs. 7 and 8). Suchcells in the early stages of platelet formation,
AP.A5 . 9 while still spherical, are seen to be studded withsmall projections which make contact with thecoverslip.The very tenuous threads which are developed
_61> frequently are seen to expand and to containnuclear fragments. The threads are readily
FIG. 2.-Megakaryocytes in medium saturated with air. x 560. broken up by motile cells, and it must be remem-
bered that these "in vitro" experiments areunlike conditions in living marrow in that there isno circulation of blood ; in life doubtless thesethreads are broken up by currents very quickly.In the slide preparations the threads often persistfor 24 hours.Those who do not examine living cells on a
warm stage are apt to forget that the sphericalforms seen in sections and stained films are notrepresentative of living and active cells. Thesphere is a defence mechanism, a form adoptedwhen cells are chilled or otherwise insulted, and
FIG. 3.-Spherical megakaryocyte. Appearance in medium saturatedwith air. x 840.
Four types of motility are observed. The first isrotation, in either direction, at a rate of one fullrotation in three to four minutes This continuies ._for many hours, and rotating cells have not beenobserved to develop in any way. Although thenuclei of cells often rotate, I have never seen anyother human cell rotating in nitrogen, in air, orin oxygen.
Secondly, megakaryocytes are seen to spreadover the surface of the coverslip. This they doby sending out pseudopodia symmetrically all FIo. 4.-Spreading megakaryocyte, in nitrogenated medium. x 840.
537
I
copyright. on N
ovember 12, 2021 by guest. P
rotected byhttp://jcp.bm
j.com/
J Clin P
athol: first published as 10.1136/jcp.11.6.535 on 1 Novem
ber 1958. Dow
nloaded from
FIG. 5.-Spreading megakaryocyte, in nitrogenated medium. Sequence from film. x 840.
copyright. on N
ovember 12, 2021 by guest. P
rotected byhttp://jcp.bm
j.com/
J Clin P
athol: first published as 10.1136/jcp.11.6.535 on 1 Novem
ber 1958. Dow
nloaded from
FIG. 6.-Elongating and progressing megakaryocyte, in nitrogenated medium. Sequence from film. x 390.
copyright. on N
ovember 12, 2021 by guest. P
rotected byhttp://jcp.bm
j.com/
J Clin P
athol: first published as 10.1136/jcp.11.6.535 on 1 Novem
ber 1958. Dow
nloaded from
FIG. 7.-Platelet formation in nitrogenated medium. Sequence from film. x 840.
copyright. on N
ovember 12, 2021 by guest. P
rotected byhttp://jcp.bm
j.com/
J Clin P
athol: first published as 10.1136/jcp.11.6.535 on 1 Novem
ber 1958. Dow
nloaded from
FIG. 8.-Platelet formation in nitrogenated medium. Sequence from film. x 390.
copyright. on N
ovember 12, 2021 by guest. P
rotected byhttp://jcp.bm
j.com/
J Clin P
athol: first published as 10.1136/jcp.11.6.535 on 1 Novem
ber 1958. Dow
nloaded from
R. J. V. PULVERTAFT
this is perhaps true of megakaryocytes as well asof polymorphs. We do not know from observa-tion what the shape of a living megakaryocytemay be. They tend to collect around bloodvessels, and therefore probably do not wander far.However, the number of controls in the presentexperiments is exceptionally large; about 2,500bone-marrow preparations have been examined,in fluid saturated with air and 5% CO2, withoutnoting motility or any other activity in those cells,except the rotation twice observed. Almost in-variably activity is seen in nitrogen after a lapseof approximately five hours.
It is not possible to say precisely how muchoxygen is present in the preparations showingactivity. The nitrogen used contained a maximumresiduum of 0.1% oxygen, and not all the oxygencould have been removed from the agar cap bythe vacuum.The stimulus to megakaryocyte activity, includ-
ing platelet formation, does, however, appear to bea sudden fall in oxygen tension, and this fits inwith the known fact that platelets are increasedfollowing haemorrhage and in persons living athigh altitudes. It is plausible to suggest that thereaction is a defence mechanism, considering themany ways in which platelets control haemor-rhage.The other forms of activity may be related to
speed of megakaryocyte maturation. The largerthe surface area of a cell the greater is its meta-
bolic activity; oxygen deficiency may thusaccelerate the development of these cells to thepeculiar form which Bessis (1954) describes asthrombocytogenic. However, such maturationwas not observed in vitro.
SummaryIn the course of the examination of 2,500
slide cell cultures of human bone marrow,megakaryocytes remained spherical in all cases, andthe only activity noted was rotation in two cases.When the oxygenated culture medium was re-placed by one saturated with nitrogen and 5%cO2, they showed activity after the lapse of fivehours. The activity noted was (1) rotation, (2)elongation and progression, (3) wide spreadingover a plane surface, (4) platelet formation.
All the bone-marrow samples and continuousadvice were provided by Dr. J. G. Humble, con-sultant haematologist to Westminster Hospital, andtechnical help by Mr. J. A. Haynes, chief technician.Extracts from films were prepared by Miss A.Readman.
All the expenses were covered by a grant from theBritish Empire Cancer Campaign.
REFERENCESBessis, M. (1954). Traite de Cytologie Sanguine. Masson et Cie, Paris.Izak, G., Nelken, D., and Gurevitch, J. (1957). Blood, 12, 507.Pulvertaft, R. J. V., and Humble, J. G. (1956). Rev. Hemat., 11, 349.Thi6ry, J. P., and Bessis, M. (1956). Ibid., 11, 162.Wright, J. H. (1906). Boston med. surg. J., 154, 643.
542
copyright. on N
ovember 12, 2021 by guest. P
rotected byhttp://jcp.bm
j.com/
J Clin P
athol: first published as 10.1136/jcp.11.6.535 on 1 Novem
ber 1958. Dow
nloaded from
