Red Cell Structure and Its Breakdown

132 Pages·1955·6.839 MB·German

by  Eric Ponder (auth.)

PROTOPLASMATOLOGIA
HANDBUCH
DER PROTOPLASMAFORSCHUNG
HERAUSGEGEBEN VON
L. V. HEILBRUNN F. WEBER
UND
PHILADELPHIA GRAZ
MITHERAUSGEBER
W. H. ARISZ·GRONINGEN . H. BAUER·WILHELMSHAVEN • J. BRACHET·
BRUXELLES . H. G. CALLAN· ST. ANDREWS . R. COLLANDER· HELSINK[ .
K. DAN·TOKYO . E. FAURE·FREMIET·PARIS . A. FREY·WYSSLING-ZORICH·
1. GEITLER-WIEN . K. HÖFLER-WIEN . M. H. JACOBS-PHILADELPHIA .
D. MAZIA-BERKELEY . A. MONROY·PALERMO . J. RUNNSTRÖM-STOCKHOLM·
W. J. SCHMIDT -GIESSEN . S. STRUGGER -MONSTER
BAND X
PATHOLOGIE DES PROTOPLASMAS
2
RED CELL STRUCTURE AND ITS BREAKDOWN
SPRINGER-VERLAG WIEN GMBH
1955
RED CELL STRUCTURE
AND ITS BREAKDOWN
BY
ERle PONDER
MINEOLA, N. Y.
WITH 58 FIGURES
SPRINGER-VERLAG WIEN GMBH
1955
ALLE RECHTE, INSBESONDERE DAS DER ÜBERSETZUNG
IN FREMDE SPRACHEN, VORBEHALTEN.
OHNE AUSDRÜCKLICHE GENEHMIGUNG DES VERLAGES IST ES AUCH NICHT GESTATTET,
DIESES BUCH ODER TEILE DARAUS AUF PHOTOMECHANISCHEM WEGE (PHOTOKOPIE, MIKROKOPIE)
ZU VERVIELFÄLTIGEN.
ISBN 978-3-662-23122-7 ISBN 978-3-662-25095-2 (eBook)
DOI 10.1007/978-3-662-25095-2
Protoplasmatologia
X. Pathologie des Protoplasmas
2. Red Cell Structure and Its Breakdown
Red Cell Structure and Its Breakdown
By
ERIC PONDER
Tbe Nassau Hospital, Mineola, N.Y.
Witb 58 Figures
Table 01 Contents
Page
Preface .................. . 2
I. Tbe Structure of tbe Mammalian Red Cell 3
1. Dimensions and numbers of red cells . 3
2. Red cell sbape ........... . 7
3. Structure and tbe disk-spbere transformations 8
4. Scatter of properties of red cells about their means 13
5. Polarisation optics ............ . i4
6. Leptoscopic measurements . . . . . . . . . . 22
7. Evidence provided by tbe electron microscope 25
8. X-ray scattering ............. . 38
9. Quantities and nature of lipids and proteins . 40
10. Volume of gbosts, rt:sidual Rb, and a concept of structure 43
11. Tbe variety of type3 of ghost . . . . . . . . . 49
12. Contraction of gbosts . . . . . . . . . . . . . 53
13. Miscellaneous observations bearing on structure 55
II. Osmotic Hemolysis . . . . . . . 59
14. Prolytic ion exchange3. . . . . . . . 59
15. Tbc van 't Hoff-Mariotte law .... 61
16. Tbe critical volume and its variations 63
17. Modification of tbe van 't Hoff-Mariotte law: tbe meaning of tbe
constant R . . . . . . . . . . . . . . . . . . . . 66
18. Modification of tbe van 't Hoff-Mariotte law: departures from linearity
and tbe rigidity of gbosts . . . . . . . . . . . . . . . . . . . 69
19. The dual mechanism of bemolysis, or colloid-osmotic bemolysis . .. 70
III. The Kinetics of Hemolysis . . . . . . . . . . . . . . . . . . . . . .. 73
20. The tbeory of S. C. BROOKS and tbe distribution of red cell resistances
to a lysin . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 73
21. YULE'S tbeory of chance occupancy of reactive sites . . . . . . . ., 79
22. Effects of varying the number of red cells; tbree different explanations 82
23. Progressive reactions .a nd explanations for them . 86
24. Atypical time-dilution curves . . . . . . . . . . . . . . . . . . .. 90
Protov1asmatologla x, 2
2 X, 2: E. PONDER, Red Cell Structure and Its Breakdown
IV. Fragmentation, Erythrophagocytosis, and the Effects of Tissue Lysins 95
25. Fragmentation and myelin form formation 95
26. Properties of red cells fragmented by heat . . 91
21. The kinetics of fragmentation by heat . . . . 101
28. Fragmelltation by urea and related substances 104
29. Memallical fragmentatioll in ammonium sulfate solutions 106
30. Contact hemolysis and mechallical fragility 108
31. Erythrophagocytosis 109
32. Tissue hemolysins 111
Referellces ......... 116
Preface
In writing this chapter for "Protoplasmatologia," I have tried to
confine the material to a consideratioll of the problems of the red cell and
its breakdowll as they appear at the moment. The problems of 1955 are
very different from those which presented themselves tell years ago, as
will be realized by anybody who cOlllpares this chapter with my lIlono
graph, "Helllolysis and Related Phellolllena," 1948. In these ten years,
the situation has <hallged because of six new departures: the observation
of fine structure, made with the electron microscope,. the realization that
there are many varieties of ghosts which have properties of their own, the
illcreasing amoullt of evidence that some of the simplifying hypotheses
regardillg the osmotic behaviour of the red cell have broken down, the
observation of the hitherto neglected fragmentation phenomena, the
realization that many Iytic reactiollS cannot be described by the equations
for simple <hemical reactions, and, fillally, the appreciation of the fact that
the mammalian red cell has a complex metabolism and that this metabolism
is concerned with processes such as active ion transport.
This <hapter is concerned with all these matters except the last. By
dividing it into sections and by illustrating thern freely, I have tried to
rnake its contents readable to students of general physiology who do not
iutend to become specialists in what has now becorne a very cornplex sub
ject. I have further restricted the material by treating red cell destruc
tion from the point of view of the cell rather than frorn the point of view
of the organism. In the third section, it has becorne necessary to use the
language of rnathernatics, but in the other seetions I have tried to use
mathernatical express ions and arguments based upon thern as little as
possible.
The references are not exhaustive. They are principally references
to work whi<h has been done within the last ten years, often with the
addition of one key reference added to guide the student to work done
earlier.
My colleagues have been very generous in sending me original photo
graphs of material whi<h often has not been published elsewhere: I have
to thank the editors and copyright owners of the following journals. for
permission to reproduce material: in the case of' Fig. 2, the Journal of
The Structure of the Mammalian Red Cell 3
Laboratory and Clinical Medicine; in the case of Figs. 4 and 40, the Pro
ceedings of the Society for Experimental Biology and Medicine; in the case
of Figs. 6, 7, 11, 36, 51, and 54, the Journal of Experimental Biology; in
the case of Figs. 12 and 21, the Journal of Cellular and Comparative Physio
logy; in the case of Figs. 18, 25, 26, 27, 28, 29, 30, 31, 35, and 43, the Revue
d'Hematologie; in the case of Figs. 37, 55, and 56, Acta Hematologica; in
the case of Figs. 38, 39, and 57, Blood, The Journal of Hematology; in the
case of Figs. 44, 46, 53, and 58, the Journal of General Physiology; and in
the case of Fig. 45, the Journal of Immunology. Fig. 41 is reproduced from
International Clinics (J. B. Lippincott & Co.). Fig. 15 is reproduced from
the Cold Spring Harbor Symposia, and Figs. 8, 9, and 10 from Trends in
Physiology and Biochemistry (Academic Press).
A number of the line drawings have been made by Miss RUTH J. MANDLE
BAUM of the Rockefeller Institute, Illustration Division, and it is a pleasure
to thank her for the time and trouble which she has taken with them.
The four seetions of this chapter eontain a good deal of unpublished
work. Most of this has been done at the Nassau Hospital, but some of it
at l'Institut Pasteur and at the Centre National de Transfusion Sanguine,
in the laboratories of Dr. D. G. DERVICHIAN and Dr. MARCEL BESSIS, respect
ively. It all represents work supported by a grant from the Eli Lilly
Company, Special Grants Committee.
This Chapter is dedieated to my friend MARCEL BESSIS. E. P.
J. The Structure 01 the Mammalian Red Cell
1. Dimensions and Numbers "01 Red Cells
Hemolysis is a proeess in which at least some of the Hb of the red cell
is lost and in which the eell is replaced by a ghost (stroma, hemolytie
residue, ete.). Just as the red eell has the properties of size, shape, Hb con
tent per unit volume, ete., so ghosts have eorresponding properties. It
cannot be over-emphasised, however, that ghosts are more varied in their
physieal and chemical properties than red cells are, so that it is possible
to prepare many kinds of ghost from a single kind of red eell by using
different methods (Section 11). Mueh eonfusion and many misunderstand
ings have arisen from a failure to realise this, and in the past it has been
almost customary for investigators to deseribe the properties of what they
eall "the ghost," unaware that the descriptions apply only to the kind of
ghost prepared by the method adopted. To give an example, the thiclmess
(50 A) of the ghost "membrane" found by HILLIER and HOFFMAN (1953)
eannot profitably be compared with the thickness (300 to 800 A) found by
BERNHARD (1952) or by BESSIS and BRICKA (500 to 1000 A; 1949), because the
ghosts were prepared by different methods. HILLIER and HOFFMAN'S ghosts
are "clean" or "attenuated" ghosts, washed until nothing but the material
most resistant to washing remains; in other preparations, the washiug and
the removal of Hb and other eomplexed material is less complete. If Olle
chooses, oue may restriet the term "ghost" to the structure which contains
1*
4 X, 2: E. PONDER, Red Cell Structure and Its Breakdown
the minimum quantity of material, and so can avoid the difficulties
associated with the idea that the red cell is bounded by more than a
paucimolecular "membrane" surrounding Hb in solution, but by doing this
one excludes ghosts whim are prepared by a variety of other methods,
and whim have interesting properties of their own.
The red cell has volume V, measured by a variety of methods, some of
whim are absolute and some of whim are relative, but aH of wh im (exGept
the photographie [PONDER and MILLAR 1924) and diffractometric [P~JPER
1947, Cox and PONDER 1941) methods) depend on an enumeration of N, the
number of cells per unit volume. It has area A, related to the volume by
a function whim describes itsshape. The cell also has a content of Rb per
unit volume, this being on the average
A.163..u2 the same as the mean corpusculllr Rb con
V. 61",,3 centration.
The volume V of the mammalian red
cell varies from 87 ± 5 pS (man and higher
~ A.J10",,2 apes) to 20 ± 5 p3 (goats), and is inclosed
~ V. 57",3 in a surface area whim va ries from about
170 p2 to about 30 p2. In the nucleated
red cells of birds, the volume is usually
A. 67#2 between 120 and 200 p3, in reptiles be-
v. 30#3 tween 170 and 450 #3, in amphibia be-
tween 670 p3 (frog) and 13,800 #3 (Am
sFhigee. p1 . aBR seede nc elolsn oef dmgaen a, nthde drraabw bnit , taon sdc athlee. phiuma), and in fishes between 108 #3
(flounder) and 1,500 #3 (dogfish). Tables 42,
43 and 44 of Standard Values in Blood (ALBRITTON 1951) contain as mum
detailed information as is available. There is a tendency for the red cell
count to be low when the cell volume is large, so that the volume concen
e
tration of the cells tends to be roughly eonstant. Even in the mammals
the shape is variable, not only from mammal to mammal but from cell to
cell, and from what is known about the correlation between eell diameter
and cell thiclmess in the mammals, or between ceHlength and eell breadth
in the lower vertebrates, it is eertain that the red cells of a given animal
are not all built on the same plan (PONDER 1930, 1934).
Many of the properties of the red cell depend on its shape. In the cas e
of mammalian erythroeytes, this is determined by measuring a number of
the dimensions of the eell as seen and photographed in its biconeave cross
section (Fig. 1). When the measurements have been made, the cross
sectional view of the eell is drawn to seale and its volume and surface area
are eomputed from the drawing. These measurements and eomputations
are not simple. The shape of the eell regulates the rate of diffusion of
gases into its interior; it also determines, at least in part, the osmotie
fragility of the eell, the extent to wh im it breaks into fragments when
heated, and (to a lesser degree) its memanieal fragility. Further, the shape
determines the surfaee area exposed to the action of hemolysins, although
this is not a matter of great importance sinee the area of the diseoidal red
eell is only about 1.7 times that of the same eell in its spherieal form.
The Structure of the Mammalian Red Cell 5
When we speak of the surface exposed to the action of a hemolysill,
moreover, we must remember that, on a molecular scale, the surface is
better represented as aseries of hills and valleys than as smooth. The
roughness of the surface may even depend on the ionic strength of the
medium surrounding the cell (FURCHGOTT and PONDER 1941).
If more restricted information is sufficient, the diameter of the in
dividual cell can be measured by photography, which is applicable either
to cells in plasma or in dried films. The thidmess of the individual red
cell ean be estimated by an adaptation of the method of shadowing (LARSEN
1952); this method is applicable to cells in dried films only. The mean
diameter can be found by diffraction, although there is some doubt regard
ing the constant to be used in the calculation; this seems to have different
Fig. 2. (1) Unstalned human red cells as seen with the ordinary mlcroscope. (2) A slmilar preparatlon seen
with phase contrast.
(From a paper by TOMPKINS 1954.)
va lues according to wh ether the cells are in plasma or dried (PONDER 1933).
Mean thidmess is conventionally found by dividing the mean cell volume
by 1l r2, where r is the radius of the cell as seen on the flat.
The introduction of phase contrast microscopy has made the measure
ment of red cell diameter easier, if not more exact. Using phase contrast,
TOMKINS (1953) has obtained 8.6 ft as the mean diameter of the human red
cell in plasma; this value is essentially the same as that found by PONDER,
MILLAR, SASLOW and others working in the period between 1925 and 1930.
LARSEN (1948) has published results obtained with a modificatiQIl of Price
Jones' method; he believes that there is no shrinkage when red cells dry,
and that the average mean diameter of the human red cell is about 7.7 p.
His data show internal inconsistancies, not apparent at first sight because
of a tendency to place emphasis on statistical treatment. Photographs
made with the phase microscope leave no doubt as to the shrinkage (from
0.5 to 1.0 p) on drying. It should be remarked that the shrinkage on drying
may be masked if the wet cells, instead of being flat disks, are crenated so
that their shape tends towards that of a sphere; cells rendered spherical
with lecithin, for example, increase in diameter when dried in films.
Within the past ten years, there have been a number of improvements
in the methods for measuring red cell volume. The colorimetric methods
wh ich use Evans Blue have been extended by the use of non-penetratillg
6 X, 2: E. PONDER, Red eeB Structure and Its Breakdown
radioactive isotopes in plaee of the dye; This is sometimes a convenience
e
when is required for blood volume stndies, although the dye and the
isotope do not always give the same result (WISH, FURTH, and STOREY 1950).
The effects of the rate and duration of spinning on the results given by the
hematocrit method, which are principally effects on the quantity of plasma
trapped in the red cell colunm, have been worked out much more thoroughly
than heretofore by several investigators (MOLLISON and CHAPLIN 1952,
MAIZELS 1952, VAZQUEZ, NEWERLY, YALLOW, and BERsoN 1952). Their con
clusions confirm, in the main, the classical ones of MILLAR and show, among
other things, that the "best" rate and duration of spinniug is 55 minutes
at 3000 r. p. m. in a centrifuge with a 15 cm. radius; this corresponds to
1500 G. The conventional 3000 r. p. m. for 30 minutes gives about 5 p. c. of
trapped plasma (MAIZELS 1946; LESSON and REEVE 1951). The conductivity
methods (FRICKE and CURTIS 1935, PONDER 1935) are a littIe bettel' than they
were ten years ago, principally when applied to ghosts, because phase
microseopy gives one a bettel' idea of shape and therefore of the proper
value for the form factor X. The priucipal improvements in the diffracto
metric method have been technical on es associated with bettel' densitometry.
Virtually no progress has been made with the problems relating to the
illumination function as it is affected by scattel' of the sizes in the red ceU
population, but it has come to be realised that, when the population is not
uniform, the simple expression relating the diameter of the cell, whether
e
as a disk 01' as a sphere, to the diffraction angle
= e
d k . i. cosec
is exact only at the first minimum. The optics of the diffractometer should
accordingly be such as to make the first minimum fall nicely on the plate,
and the shortening of the focal length of the camera lens (PONDER 1951 a)
was a step in thc wrong direction. The only change in the photographic
methods has been the introduction of phase microscopy, which allows the
photographic methods to be extended to ghosts. With phase contrast, many
ghosts are as clearly seen as are red cells with direct light and critical
illumination.
The hematocrit method, the conductivity methods, and the dye 01'
e,
non-penetr{lting isotope methods all give the volume concentration of
the red cells in the sampIe, and give V, the mean volume of the red cell,
only when N, the number of cells per unit vohllne of the sampie is known.
The determination of N is tedious if oue counts a sufficiently large lllimber
of cells (about 8000) to reduce the coefficient of variation of N to ± 1 p. c.,
although this degree of aecuracy ean he obtained by a careful worker
nsing calibrated apparatlls. If a llluch smalleI' number of eells (several
hundreds. the number to whim the studies of BERKsoN. MAGATH. and HURN
1940, re fe 1') , one obtains a level of preeision too 10w for anything exeept
clinieal work. Thc eounting of several thollsands of eells without tedium.
and, what is more important, withollt the observcr beeoming so fatigued
that thc precision of his eountillg falls off badly, is therefore a problem of
real importallce. Scveral attempts have heen made to solve it hy seanning