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Measurement of capillary hematocrit by photometric techniques
MICROVASCULAR
RESEARCH
(1973)
$351-356
Measurement of Capillary Hematocrit Photometric Techniques
by
PAUL C. JOHNSON, DAVID L. HUDNALL, AND JOE H. DIAL Department of Physiology, University of Arizona GolIege of Medicine, Tucson, Arizona M7274 Received June 15,1972 The quantitation of capillary hematocrit was approached by two photometric techniques. The first involved measurement of the average red cell velocity while at the same time counting the number of red cells which pass per unit time (red cell flux). The red cell flux (cell&c) divided by the red cell velocity (mm/set) provided a reading termed hematocrit index (cells/mm). This index represents the average number of red cells per unit length of the flow column passing the photo sensors.This method provides a continuous read-out of hematocrit and is generally applicable to low hematocrit vessels. However it is not appropriate in situations where rouleaux might occur or where two or more red cells might pass the sensors simultaneously. The second method involves measurement of capillary opacity which is a measure of the average light absorption by the blood column in the capillary. This average value is compared with the change produced when a red cell passesthe sensor and is expressed as a fraction of the RBC opacity. This method is an off-line technique involving manual analysis but can be used in situations where capillary hematocrit is high and where the hematocrit index technique cannot be employed.
Capillary hematocrit is commonly thought to be influenced to some extent by the volume of flow of blood through the circulation with higher flows producing a greater degree of plasma skimming. To investigate the validity of this hypothesis we devised two different methods for measuring the hematocrit in individual capillaries of mesentery. 1. HEMATOCRIT
INDEX
Studies were performed on the mesentery of the anesthetized cat. An isolated intestinal loop was prepared as described in another paper in this symposium (Gore, 1973).The preparation was mounted on the stageof a Leitz Panphot microscope and perfused from the donor animal through a length of polyethylene tubing. A capillary was selectedfor study and its image projected onto a screenwhich contained the dual slits for red cell velocity measurement as shown in Fig. 1. As red cells passed through the capillary they produced successivepulsesin the two photomultiplier tubes. These signals formed the basis of the velocity measurementas described in the preceding paper (Wayland, 1973). In the present application the output of one of the photo tubes was also employed to activate the trigger circuit of a frequency meter. Any signal greater than 1 V activated the trigger circuit which produced a short pulse. Individual red cells produced signals of 3-6 V, well above the threshold value. The output of the Copyright All rights
0 1973 by Academic Press, Inc. of reproduction m any form reserved.
351
352
JOHNSON, HUDNALL,
AND DIAL
trigger circuit was fed into a 4-bit counter and the latter into a D-A converter which provided an analog output proportional to the cell count per second (red cell flux). Simultaneously the velocity of the same red cells was determined. An analog multiplier circuit computed the product of red cell flux times the reciprocal of velocity to obtain hematocrit index (cells/mm). This index represents at any time the average number of red cells per unit path length of the capillary at the site of measurement. Temporal variations in capillary hematocrit can be readily determined with this technique. The rapidity of the system response will depend upon the time constants of the system. In practice, some time averaging is necessary since the measurements were made between two slits only 5 pm apart. In this short path hematocrit literally varies from O-100 % depending upon whether a red cell or plasma overlies
I
I
,
I
, Velocity
Hemotocrit Index
P-M Tubes
(mm/set)
(cells/mm)
Flux (celldsec)
iill’ \4q
;;I
Clock
1
8 Light Source
FIG. 1. Schematic diagram of system for measurement of hematocrit index in mesenteric capillaries. The velocity measurement is obtained by the dual slit method. Further details of the technique are given in the text. From Am. J. Physiol. (1971) 221, 105, reproduced by permission.
the counting slit. The time interval of the flux measurement could be varied from 37-300 msec. The longer time interval was used with low flux, low hematocrit capillaries. Additional time averaging was introduced by the hematocrit readout circuit which has a time constant of 0.1 sec. We found it useful to regard the time constant of the system in terms of an equivalent length of the flow path. With a velocity of 1 mm/set the corresponding “length constant” is about 200 pm. The system described was used to study variations in capillary hematocrit when total flow through the intestine was changed (Johnson, Blaschke, Burton, and Dial, 1971). In Fig. 2 is shown an experiment in which red cell velocity and hematocrit index were recorded simultaneously from a single capillary of the cat mesentery. Blood pressure was recorded from the arterial circuit perfusing the mesenteric preparation. Arterial pressure to the preparation was reduced from 90 mm Hg to 60 mm Hg for about 2 min by application of a screw clamp to the perfusion system. During the period of hypotension hematocrit gradually fell and returned to the control level after
353
CAPILLARY HEMATOCRIT MEASUREMENT
restoration of pressure. Note that there is no immediate change in hematocrit when pressure and flow are restored. This indicates that capillary hematocrit is not affected by red cell velocityper se.Following reactive hyperemia this capillary showed a secondary drop in flow and an associated large decreasein hematocrit. In the later portion of the recovery period, phasic variation in red cell velocity are accompanied by similar changesin hematocrit. The slow changein hematocrit with reduction in arterial pressure does not correspond to the time constant of the measuring systemwhich is much faster. Rather, it appears to reflect autoregulatory adjustments upstream from the capillary under study. Red cell velocity in capillaries may possessa pulsatile component synchronous with the arterial pulse. However, the systemdescribedhere would have insufficient frequency responseto pick up and record accurately associatedchangesin hematocrit 1.51
I
RED CELL VELOCITY (mm/set) 0 133 HEMATOCRIT INDEX (cells/mm) OL :z.nJ
BLOOD PRESSURE (mm Hg) 0 0
50
IO0
150 TIME
200
250
300
(seconds)
FIG. 2. Simultaneous recording of hematocrit index and red cell velocity in a mesenteric capillary. This capillary showed a modest reduction in hematocrit when arterial pressure to the mesentery was reduced. Note also the phasic variations in hematocrit concurrent with similar changes in velocity during the recovery period. From Am. J. Physiol. (1971) 221,105, reproduced by permission.
of the samefrequency. The system does appear to be capable of responding to slower changescommonly found in the capillary network such asthe vasomotion seenin Fig. 2 during the later stagesof the recovery period. The system described here possessescertain important limitations. First, it requires that each red cell passingthe slits produce a pulse which crossesthe 1 V threshold level. This limits the technique to the very smallestvesselsin the microcirculation in which the red cells will travel single file. Even in these vessels,the technique is only applicable in circumstances where the red cells travel singly rather than as pairs or as rouleaux. Generally this obviates its usein high hematocrit capillaries where the distance between adjacent red cells is so short that the two cells might appear as one to the photo tube. Moreover the question as to whether a capillary is suitable for the type of measurement usedhere can be determined only by careful examination of the capillary. We have found that capillaries with low hematocrit are reasonably stable; average hematocrit index does not change drastically except in association with vasomotion. In addition, the technique provides no measure of volume hematocrit in the capillary, though it could 13
354
JOHNSON,
HUDNALL,
AND
DIAL
be used to obtain such if the capillary diameter were measured. Also, it obviously provides no measureof hematocrit when there is no flow. Techniques for measuring red cell flux have been described previously (Muller, 1966; Harris, 1968; Greenwald, 1969).The limitations described above for hematocrit index determinations would apply also to their measurements. Within the limits described above, the technique appears to provide a rapid and reliable continuous measure of capillary hematocrit. 2. CAPILLARY
OPACITY
The technique described above provides information on capillary hematocrit in capillaries which meet certain criteria, the most important being the restriction to capillaries having low hematocrits. Sincethis limitation is not desirable in somecircumstances,we also devised a method to assessthe hematocrit of capillaries over a greater range. This technique was used specifically to compare the relative hematocrit in adjacent capillaries in an assessmentof plasma skimming (Johnson, 1971). The tissue Photomultiplier
Signals Opacity
Site A
--
Site B
-
0
Min.
-
0.6 Avg.
-
1.0 Max.
Opacity
FIG. 3. Schematic diagram of the principle of the opacity measurement technique. Shown is the phototube output from a capillary with closely spaced red cells (Site A) and output from a capillary with fewer red cells (Site B). The deflection produced by passage of a red cell across the phototube slit window is taken as maximum opacity. The average opacity is considered to be a function of red cell spacing since each red cell is considered to produce the same deflection. From Am. J. Physiol. (1971) 221,99, reproduced by permission.
preparation used in this study is similar to that described above. The image of a capillary is projected onto the slits of a velocity measuring system. The output of one phototube is recorded on FM magnetic tape for later determination of capillary opacity by manual analysis. The principle of the opacity measurementis shown in Fig. 3. The opacity of the capillary overlying the slit will vary during flow from a minimal value when only plasma is over the slit to a maximal value when the window is occupied by a red cell. The opacity of the capillary is expressedas a fraction of the deflection produced by a red cell. If the capillary hematocrit is low asindicated at site B in Fig. 3, the average photomultiplier signal will be a small fraction of that produced by a red cell. On the
CAPILLARY
HEMATOCRIT
MEASUREMENT
355
other hand,,if the capillary hematocrit is high as at site A, the averagephotomultiplier signal will approach the peak value obtained by the red cell. The principle of this technique was previously described by Muller (1966). In our studies the taped signals were replayed at one-fourth the original tape speedonto two channels of a strip chart recorder (Beckman-Offner Type R). One channel was adjusted for high frequency response to obtain the minimal and maximal values for the opacity measurement. The second channel output was set at identical gain but time averaging (time constant approximately 1 set) was introduced to obtain the average opacity reading. Several measurements of maximum, minimum, and average opacity were made for each capillary. The opacity of the capillary as measured by this method would not be expected to bear a precise relationship to the volume hematocrit although it should reflect 0.70 0.60 0.50 : 0 g
0.40 0.30
t “‘0
( IO
/ 20
1 30
I 40
I 50
I 60
I 70
RED CELLS / mm FIG. 4. Plot of capillary opacity versus red cell spacing in four capillaries. The regression line shown
was calculated for opacity on red cell spacing baaed on data from all four capillaries. The correlation coefficient, r, is 0.96 and P i 0.01. The SE of estimate for opacity is 0.03. From Am. J. Physiol. (1971) 221,99, reproduced by permission.
changes and differences in the latter. Several factors would introduce non-linearity into the opacity-hematocrit relationship, perhaps the most important of these is the non-linear relation between light transmission and red cell opacity. This problem could be minimized in one of two ways. First, it would be possible to use monochromatic light in a spectral region where hemoglobin absorption is low. In this instance the time integral of light transmission would represent the average hemoglobin concentration in the flowing stream. However, in our study we used the unfiltered mercury arc and so a good deal of the light absorbed was from the 4070 A peak in the hemoglobin absorption spectrum which coincides with a region of strong emission in the mercury arc spectrum. For precise studies it would also be desirable to use a wave length which is not affected by the degreeof oxygenation of the red cell. The other approach which could be used is to limit the measurement to a spectral region such as the 4070 A peak, providing essentially complete absorption of the light by the red cell. In that instance each red cell would produce an identical signal (provided geometry were uniform) and the opacity reading would represent the fraction of the total time that the slit window was occupied by a red cell. In fact red cell
356
JOHNSON,
HLJDNALL,
AND
DIAL
geometry in the capillary is limited to perhaps a few forms. To determine whether the opacity measurement bears a direct relationship to red cell spacing we measured opacity simultaneously with the hematocrit index described in the preceding section. The results are shown in Fig. 4. These findings indicate that opacity measured with the visible mercury spectrum does indeed appear to behave as a linear function of red cell spacing although the intercept is not zero. It should be noted that the comparisons between opacity and red cell spacing in Fig. 4 were limited to the range O-60 cells/mm. Obviously the relationship becomes non-linear beyond this range since linear extrapolation would indicate an opacity of 1.00 with a red cell spacing of 100 cells/mm. The relation between opacity and cell spacing was virtually identical in the four capillaries tested. It may be valid therefore to use opacity measurements as a basis for comparing hematocrit between capillaries. It is important to note that this opacity value is not an absolute measurement of light transmission by the tissue. Therefore it would not be influenced by differences in opacity of tissue structures surrounding different capillaries. However, comparison between vessels would not be valid if their internal diameters were different. For example the separation between red cells will be 35 % greater in a 6 pm capillary as compared with a 7 pm capillary of the same hematocrit, assuming that red cells travel essentially single file in both vessels and mean plasma velocity is the same. The difference in spacing comes about because of the larger plasma sleeve outside the red cell column in the larger vessel. While our findings indicate a reasonably good relation between opacity and cell spacing, further work on this technique seems advisable. In particular the effect of limiting the spectrum may be an area deserving further study. It may be possible to extend this technique to somewhat larger vessels (of constant diameter) if a region of moderate absorption is used. An additional factor which needs to be considered is that much of the light loss is due to reflection by the red cell membrane rather than absorption by the hemoglobin. ACKNOWLEDGMENTS Supported by NIH grant AM 12065 and a grant-in-aid from The American Heart Association. The technical assistance of Mrs. Susan Neighbors is gratefully acknowledged. REFERENCES GORE, R. W. (1973). Microvas. Res. 5,368-375. GREENWALD, E. K. (1969). Microuas. Rex 1,410. HARRIS, P. D., RANDALL, J. E., AND NICOLL, P. A. (1968). J. Appl. Physiol. 24,722. JOHNSON, P. C. (1971). Am. J. Physiol. 221,99. JOHNSON, P. C., BLASCHKE, J., BURTON, K. S., AND DIAL, J. H. (1971). Am. J. Physiol. 221,105. MULLER, H. (1966). Meth. Med. Res. 11,207. WAYLAND, H. (1973). Microvas. Res. 5,336-350.
RESEARCH
(1973)
$351-356
Measurement of Capillary Hematocrit Photometric Techniques
by
PAUL C. JOHNSON, DAVID L. HUDNALL, AND JOE H. DIAL Department of Physiology, University of Arizona GolIege of Medicine, Tucson, Arizona M7274 Received June 15,1972 The quantitation of capillary hematocrit was approached by two photometric techniques. The first involved measurement of the average red cell velocity while at the same time counting the number of red cells which pass per unit time (red cell flux). The red cell flux (cell&c) divided by the red cell velocity (mm/set) provided a reading termed hematocrit index (cells/mm). This index represents the average number of red cells per unit length of the flow column passing the photo sensors.This method provides a continuous read-out of hematocrit and is generally applicable to low hematocrit vessels. However it is not appropriate in situations where rouleaux might occur or where two or more red cells might pass the sensors simultaneously. The second method involves measurement of capillary opacity which is a measure of the average light absorption by the blood column in the capillary. This average value is compared with the change produced when a red cell passesthe sensor and is expressed as a fraction of the RBC opacity. This method is an off-line technique involving manual analysis but can be used in situations where capillary hematocrit is high and where the hematocrit index technique cannot be employed.
Capillary hematocrit is commonly thought to be influenced to some extent by the volume of flow of blood through the circulation with higher flows producing a greater degree of plasma skimming. To investigate the validity of this hypothesis we devised two different methods for measuring the hematocrit in individual capillaries of mesentery. 1. HEMATOCRIT
INDEX
Studies were performed on the mesentery of the anesthetized cat. An isolated intestinal loop was prepared as described in another paper in this symposium (Gore, 1973).The preparation was mounted on the stageof a Leitz Panphot microscope and perfused from the donor animal through a length of polyethylene tubing. A capillary was selectedfor study and its image projected onto a screenwhich contained the dual slits for red cell velocity measurement as shown in Fig. 1. As red cells passed through the capillary they produced successivepulsesin the two photomultiplier tubes. These signals formed the basis of the velocity measurementas described in the preceding paper (Wayland, 1973). In the present application the output of one of the photo tubes was also employed to activate the trigger circuit of a frequency meter. Any signal greater than 1 V activated the trigger circuit which produced a short pulse. Individual red cells produced signals of 3-6 V, well above the threshold value. The output of the Copyright All rights
0 1973 by Academic Press, Inc. of reproduction m any form reserved.
351
352
JOHNSON, HUDNALL,
AND DIAL
trigger circuit was fed into a 4-bit counter and the latter into a D-A converter which provided an analog output proportional to the cell count per second (red cell flux). Simultaneously the velocity of the same red cells was determined. An analog multiplier circuit computed the product of red cell flux times the reciprocal of velocity to obtain hematocrit index (cells/mm). This index represents at any time the average number of red cells per unit path length of the capillary at the site of measurement. Temporal variations in capillary hematocrit can be readily determined with this technique. The rapidity of the system response will depend upon the time constants of the system. In practice, some time averaging is necessary since the measurements were made between two slits only 5 pm apart. In this short path hematocrit literally varies from O-100 % depending upon whether a red cell or plasma overlies
I
I
,
I
, Velocity
Hemotocrit Index
P-M Tubes
(mm/set)
(cells/mm)
Flux (celldsec)
iill’ \4q
;;I
Clock
1
8 Light Source
FIG. 1. Schematic diagram of system for measurement of hematocrit index in mesenteric capillaries. The velocity measurement is obtained by the dual slit method. Further details of the technique are given in the text. From Am. J. Physiol. (1971) 221, 105, reproduced by permission.
the counting slit. The time interval of the flux measurement could be varied from 37-300 msec. The longer time interval was used with low flux, low hematocrit capillaries. Additional time averaging was introduced by the hematocrit readout circuit which has a time constant of 0.1 sec. We found it useful to regard the time constant of the system in terms of an equivalent length of the flow path. With a velocity of 1 mm/set the corresponding “length constant” is about 200 pm. The system described was used to study variations in capillary hematocrit when total flow through the intestine was changed (Johnson, Blaschke, Burton, and Dial, 1971). In Fig. 2 is shown an experiment in which red cell velocity and hematocrit index were recorded simultaneously from a single capillary of the cat mesentery. Blood pressure was recorded from the arterial circuit perfusing the mesenteric preparation. Arterial pressure to the preparation was reduced from 90 mm Hg to 60 mm Hg for about 2 min by application of a screw clamp to the perfusion system. During the period of hypotension hematocrit gradually fell and returned to the control level after
353
CAPILLARY HEMATOCRIT MEASUREMENT
restoration of pressure. Note that there is no immediate change in hematocrit when pressure and flow are restored. This indicates that capillary hematocrit is not affected by red cell velocityper se.Following reactive hyperemia this capillary showed a secondary drop in flow and an associated large decreasein hematocrit. In the later portion of the recovery period, phasic variation in red cell velocity are accompanied by similar changesin hematocrit. The slow changein hematocrit with reduction in arterial pressure does not correspond to the time constant of the measuring systemwhich is much faster. Rather, it appears to reflect autoregulatory adjustments upstream from the capillary under study. Red cell velocity in capillaries may possessa pulsatile component synchronous with the arterial pulse. However, the systemdescribedhere would have insufficient frequency responseto pick up and record accurately associatedchangesin hematocrit 1.51
I
RED CELL VELOCITY (mm/set) 0 133 HEMATOCRIT INDEX (cells/mm) OL :z.nJ
BLOOD PRESSURE (mm Hg) 0 0
50
IO0
150 TIME
200
250
300
(seconds)
FIG. 2. Simultaneous recording of hematocrit index and red cell velocity in a mesenteric capillary. This capillary showed a modest reduction in hematocrit when arterial pressure to the mesentery was reduced. Note also the phasic variations in hematocrit concurrent with similar changes in velocity during the recovery period. From Am. J. Physiol. (1971) 221,105, reproduced by permission.
of the samefrequency. The system does appear to be capable of responding to slower changescommonly found in the capillary network such asthe vasomotion seenin Fig. 2 during the later stagesof the recovery period. The system described here possessescertain important limitations. First, it requires that each red cell passingthe slits produce a pulse which crossesthe 1 V threshold level. This limits the technique to the very smallestvesselsin the microcirculation in which the red cells will travel single file. Even in these vessels,the technique is only applicable in circumstances where the red cells travel singly rather than as pairs or as rouleaux. Generally this obviates its usein high hematocrit capillaries where the distance between adjacent red cells is so short that the two cells might appear as one to the photo tube. Moreover the question as to whether a capillary is suitable for the type of measurement usedhere can be determined only by careful examination of the capillary. We have found that capillaries with low hematocrit are reasonably stable; average hematocrit index does not change drastically except in association with vasomotion. In addition, the technique provides no measure of volume hematocrit in the capillary, though it could 13
354
JOHNSON,
HUDNALL,
AND
DIAL
be used to obtain such if the capillary diameter were measured. Also, it obviously provides no measureof hematocrit when there is no flow. Techniques for measuring red cell flux have been described previously (Muller, 1966; Harris, 1968; Greenwald, 1969).The limitations described above for hematocrit index determinations would apply also to their measurements. Within the limits described above, the technique appears to provide a rapid and reliable continuous measure of capillary hematocrit. 2. CAPILLARY
OPACITY
The technique described above provides information on capillary hematocrit in capillaries which meet certain criteria, the most important being the restriction to capillaries having low hematocrits. Sincethis limitation is not desirable in somecircumstances,we also devised a method to assessthe hematocrit of capillaries over a greater range. This technique was used specifically to compare the relative hematocrit in adjacent capillaries in an assessmentof plasma skimming (Johnson, 1971). The tissue Photomultiplier
Signals Opacity
Site A
--
Site B
-
0
Min.
-
0.6 Avg.
-
1.0 Max.
Opacity
FIG. 3. Schematic diagram of the principle of the opacity measurement technique. Shown is the phototube output from a capillary with closely spaced red cells (Site A) and output from a capillary with fewer red cells (Site B). The deflection produced by passage of a red cell across the phototube slit window is taken as maximum opacity. The average opacity is considered to be a function of red cell spacing since each red cell is considered to produce the same deflection. From Am. J. Physiol. (1971) 221,99, reproduced by permission.
preparation used in this study is similar to that described above. The image of a capillary is projected onto the slits of a velocity measuring system. The output of one phototube is recorded on FM magnetic tape for later determination of capillary opacity by manual analysis. The principle of the opacity measurementis shown in Fig. 3. The opacity of the capillary overlying the slit will vary during flow from a minimal value when only plasma is over the slit to a maximal value when the window is occupied by a red cell. The opacity of the capillary is expressedas a fraction of the deflection produced by a red cell. If the capillary hematocrit is low asindicated at site B in Fig. 3, the average photomultiplier signal will be a small fraction of that produced by a red cell. On the
CAPILLARY
HEMATOCRIT
MEASUREMENT
355
other hand,,if the capillary hematocrit is high as at site A, the averagephotomultiplier signal will approach the peak value obtained by the red cell. The principle of this technique was previously described by Muller (1966). In our studies the taped signals were replayed at one-fourth the original tape speedonto two channels of a strip chart recorder (Beckman-Offner Type R). One channel was adjusted for high frequency response to obtain the minimal and maximal values for the opacity measurement. The second channel output was set at identical gain but time averaging (time constant approximately 1 set) was introduced to obtain the average opacity reading. Several measurements of maximum, minimum, and average opacity were made for each capillary. The opacity of the capillary as measured by this method would not be expected to bear a precise relationship to the volume hematocrit although it should reflect 0.70 0.60 0.50 : 0 g
0.40 0.30
t “‘0
( IO
/ 20
1 30
I 40
I 50
I 60
I 70
RED CELLS / mm FIG. 4. Plot of capillary opacity versus red cell spacing in four capillaries. The regression line shown
was calculated for opacity on red cell spacing baaed on data from all four capillaries. The correlation coefficient, r, is 0.96 and P i 0.01. The SE of estimate for opacity is 0.03. From Am. J. Physiol. (1971) 221,99, reproduced by permission.
changes and differences in the latter. Several factors would introduce non-linearity into the opacity-hematocrit relationship, perhaps the most important of these is the non-linear relation between light transmission and red cell opacity. This problem could be minimized in one of two ways. First, it would be possible to use monochromatic light in a spectral region where hemoglobin absorption is low. In this instance the time integral of light transmission would represent the average hemoglobin concentration in the flowing stream. However, in our study we used the unfiltered mercury arc and so a good deal of the light absorbed was from the 4070 A peak in the hemoglobin absorption spectrum which coincides with a region of strong emission in the mercury arc spectrum. For precise studies it would also be desirable to use a wave length which is not affected by the degreeof oxygenation of the red cell. The other approach which could be used is to limit the measurement to a spectral region such as the 4070 A peak, providing essentially complete absorption of the light by the red cell. In that instance each red cell would produce an identical signal (provided geometry were uniform) and the opacity reading would represent the fraction of the total time that the slit window was occupied by a red cell. In fact red cell
356
JOHNSON,
HLJDNALL,
AND
DIAL
geometry in the capillary is limited to perhaps a few forms. To determine whether the opacity measurement bears a direct relationship to red cell spacing we measured opacity simultaneously with the hematocrit index described in the preceding section. The results are shown in Fig. 4. These findings indicate that opacity measured with the visible mercury spectrum does indeed appear to behave as a linear function of red cell spacing although the intercept is not zero. It should be noted that the comparisons between opacity and red cell spacing in Fig. 4 were limited to the range O-60 cells/mm. Obviously the relationship becomes non-linear beyond this range since linear extrapolation would indicate an opacity of 1.00 with a red cell spacing of 100 cells/mm. The relation between opacity and cell spacing was virtually identical in the four capillaries tested. It may be valid therefore to use opacity measurements as a basis for comparing hematocrit between capillaries. It is important to note that this opacity value is not an absolute measurement of light transmission by the tissue. Therefore it would not be influenced by differences in opacity of tissue structures surrounding different capillaries. However, comparison between vessels would not be valid if their internal diameters were different. For example the separation between red cells will be 35 % greater in a 6 pm capillary as compared with a 7 pm capillary of the same hematocrit, assuming that red cells travel essentially single file in both vessels and mean plasma velocity is the same. The difference in spacing comes about because of the larger plasma sleeve outside the red cell column in the larger vessel. While our findings indicate a reasonably good relation between opacity and cell spacing, further work on this technique seems advisable. In particular the effect of limiting the spectrum may be an area deserving further study. It may be possible to extend this technique to somewhat larger vessels (of constant diameter) if a region of moderate absorption is used. An additional factor which needs to be considered is that much of the light loss is due to reflection by the red cell membrane rather than absorption by the hemoglobin. ACKNOWLEDGMENTS Supported by NIH grant AM 12065 and a grant-in-aid from The American Heart Association. The technical assistance of Mrs. Susan Neighbors is gratefully acknowledged. REFERENCES GORE, R. W. (1973). Microvas. Res. 5,368-375. GREENWALD, E. K. (1969). Microuas. Rex 1,410. HARRIS, P. D., RANDALL, J. E., AND NICOLL, P. A. (1968). J. Appl. Physiol. 24,722. JOHNSON, P. C. (1971). Am. J. Physiol. 221,99. JOHNSON, P. C., BLASCHKE, J., BURTON, K. S., AND DIAL, J. H. (1971). Am. J. Physiol. 221,105. MULLER, H. (1966). Meth. Med. Res. 11,207. WAYLAND, H. (1973). Microvas. Res. 5,336-350.