(Received for publication, August 3, 1994; and in revised form, November 18, 1994)
From the
We have used differential polarization imaging microscopy to measure the amount and orientation of aligned sickle hemoglobin polymer in quickly deoxygenated sickle red blood cells. Images of the angular orientation of the aligned polymer at each point in the cell allowed for determination of the inclination of individual domains, providing detailed information regarding the polymerization and elongation of sickle hemoglobin polymers ex vivo. We found that the number of aligned polymer domains increased with increasing mean cell hemoglobin concentration. Sickle and holly leaf-shaped cells contained single or few domains of aligned polymer, while more compact cells such as irreversibly sickled cells contained many domains. A new class of cells was discovered by examination of images of the angular orientation of aligned polymer, which contained a single central nucleation site, with growth of polymer occurring outward in all directions in a spherulite-like domain.
The fundamental abnormality of sickle hemoglobin (HbS) ()responsible for the severe clinical disease sickle cell
anemia is its low solubility when deoxygenated(1) . The
substitution of valine for glutamic acid as the sixth amino acid of
mutant
-globin chains (2) results in changes
in the structure of the deoxy form of the HbS molecule, which
diminishes its solubility. As sickle erythrocytes give up oxygen,
poorly soluble deoxy HbS within these cells rapidly aggregates and
polymerizes(3, 4) . Deoxygenated HbS polymer within
red cells results in their deformation and
rigidity(5, 6) . Those cells with the greatest degree
of deformation (sickling) contain a large amount of aligned Hb polymer (7) . Although the exact event that initiates microvascular
occlusion remains controversial, the presence in the circulation of
cells containing polymerized Hb contributes to this process and,
thereby, to the resultant episodic painful crises and the chronic and
acute organ damage(8, 9, 10) . Methods of
quantifying HbS polymer that employ centrifugation(11) ,
nuclear magnetic resonance(12, 13, 14) , and
laser photolysis(15, 16, 17, 18, 19, 20, 21) do not
spatially distinguish the angular distribution of intracellular HbS.
Optical methods that rely on birefringence to measure hemoglobin
polymer are subject to artifactual variations related to the quantity
and alignment of polymer(22, 23) .
Hemoglobin preferentially absorbs light polarized parallel to the plane of the heme(24) . In sickle hemoglobin polymers, the average orientation of the hemes is perpendicular to the polymer long axis(25, 26) . Light polarized perpendicular to the polymer axis will be absorbed more strongly than light polarized parallel to it. Utilizing this phenomenon, imaging of the differential absorption of two light beams polarized at right angles to each other provides information about the quantity and orientation of aligned sickle hemoglobin polymer (AHP) in sickle cells. We have developed the technique of differential polarization imaging microscopy to measure the amount and distribution of AHP at each point within individual sickle erythrocytes(7, 27, 28, 29) . (This method does underestimate the AHP that is aligned parallel to the optical axis of the microscope because the linear dichroism of AHP aligned in this way is axially symmetric.) These measurements allow the calculation of the ratio of AHP to total Hb at each point, thereby minimizing artifacts induced by light scattering, thus providing a more accurate measurement of the fraction of AHP than birefringence dependent methods, which are influenced by light scattering. A thorough mathematical treatment of this subject has previously been presented by Kim et al.(30, 31, 32) The AHP images obtained in our lab are in qualitative agreement with the findings of Beach et al.(33) using substantially different instrumentation.
It must also be pointed out that our method measures the quantity of aligned hemoglobin polymer, where polymer randomly distributed in a given area would not be detected. In solution studies, the initial rise in light scattering associated with the onset of polymerization is followed by a later rise in birefringence(34, 35) . This indicates that the initial polymer in the given area is partially randomly arranged and that individual polymer fibers within that area subsequently coalesce and become more aligned. We allowed a 30-min period following deoxygenation prior to fixation to assure that as many polymer fibers became aligned as possible.
Our goal is to determine how various physical parameters affect the amount and distribution of AHP within individual deoxygenated sickle red blood cells (SRBC). Using this technique, we demonstrated previously that the amount of AHP within deoxygenated sickle cells may have had little relationship to the external cellular morphology but was related to the number of identifiable individual domains of AHP within the cell(27) . A specific example of the lack of correlation between amount of AHP and external cellular morphology is the homokentrocyte, a rare type of deoxygenated SRBC that has a normal biconcave disc morphology but a large amount of AHP arranged concentrically within(27) . We also showed that the speed of deoxygenation has only a minor effect on the number of AHP domains in SRBC(7) .
Since the previous report(7) , we have extended our morphological classification system, which is based on AHP arrangement within the cell and other unique visual characteristics, to include new, more detailed classes. We also present images that show not only the amount of AHP but also its orientation at each point within the cell. We utilized this classification system to assess the effect mean cell hemoglobin concentration (MCHC) had on the nucleation and distribution of AHP in intact cells after deoxygenation. Cell populations with increasing average MCHC, which were prepared by fractionating SRBC by density on discontinuous Stractan density gradients, showed a concomitant increase in the average number of AHP domains/cell after deoxygenation.
After obtaining informed consent, blood samples from two subjects from the Northern California Comprehensive Sickle Cell Center at San Francisco General Hospital were drawn by venipuncture into heparin. Both subjects were homozygous for sickle cell anemia and had fetal hemoglobin levels below 6%. All cell manipulations were performed within 6 h of venipuncture, and fixed cells were imaged within 2 days. Cells were attached to microscope slides with poly-L-lysine (DP = 66) prior to imaging to restrict the motion of the fixed RBC during the course of the measurement.
The array measures transmitted intensities of two orthogonally polarized beams, arbitrarily defined as parallel (par) and perpendicular (perp). The absorption (A) of these beams can be related to the differences in transmitted intensities divided by their sum as shown below (for small differential signals) and described previously(27, 29) .
Combining this measurement with data obtained with each polarization rotated by 45° allowed for the unambiguous determination of the relative amount, distribution, and orientation of AHP in the cells. The images presented here consist of an absorption image, a differential absorption image, and an angle image. These are proportional to total Hb, AHP, and the orientation of the AHP, respectively.
Figure 1: Images of Hb, AHP, and the angular orientation of AHP in sickled red blood cells. The lowerright-hand image represents absorption, with gray scale intensity proportional to total Hb. The lowerleft-hand image is the same field of cells with gray scale intensity representing the amount of AHP. The gray scale coding for absorbance of Hb varies from 0 (black) to 0.5 (white) and for the absorbance of AHP from 0 (black) to 0.05 (white) for most images. In the colored image (topleft), color represents the angular orientation of AHP at each point within the cell and represents the same field of cells. Cell 5 is labeled in all three images to illustrate the relationship between them. The color wheel in the upper right is used to determine the relationship between color and angle. The angular orientation of a color within the color wheel represents the orientation of the AHP for any pixels in the angle image with that color displayed. For example, green areas in the angle image represent AHP oriented with its long axis left to right and red areas represent AHP oriented top to bottom. This image contains examples of several classes of cells: 1 is a single-domain cell, 2 is a three-domain cell, 3 is a multiple domain cell, 4 is a zero-domain cell, 5 and 6 are central constriction cells, and 7 is a spherulite cell. The angle image of the spherulite cell clearly shows that each AHP domain is arranged radially from a central point within the cell and may have resulted from a single nucleation event.
Figure 2: Images of Hb, AHP, and the angular orientation of AHP in sickled red blood cells. As explained in Fig. 1, the lowerright-hand image represents absorption, and the lowerleft-hand image is the same field of cells with gray scale intensity representing the amount of AHP. The colored image (topleft) displays the angular orientation of AHP at each point within the cell and represents the same field of cells. Cell 1 is a classic, sickle shaped, single-domain cell, and cells 2 and 3 are multiple domain cells. Of note is the continuous AHP domain in cell 1 where, by utilizing the angle image, the AHP can be seen to bend gently with the curvature of the cell.
Figure 4: Images representing three of the RBC densities studied including MCHC of 31.2 (A), 38.7 (B), and 42+ g/dl (C). The cells were separated by density on discontinuous Stractan gradients. The right-hand images, which represent Hb level, reveal an increase in hemoglobin concentration from density A to C as indicated by the increase in the average gray level in the cells (angle images have been omitted for clarity). Cells in A had the largest amount of AHP/cell and contained only a few domains. The number of domains of AHP increased in B and C as hemoglobin concentration increased. This AHP image in C is displayed at a 2-fold higher intensity relative to the others to ensure visibility. As a result, the large number of domains may be difficult to discern as reproduced here.
The color portion of the image (topleft) in Fig. 1and Fig. 2represents the angle of orientation of the AHP at each point within the cells. These images are of the same field of cells as the Hb and AHP images. The colorwheel in the upperright can be used to determine the relationship between color and angle of alignment. The angular orientation of a color within the color wheel represents the orientation of the AHP for pixels in the angle image with that color displayed. For example, green areas in the angle image represent AHP oriented with its long axis left to right and red areas represent AHP oriented top to bottom. This allows the determination of the two-dimensional orientation of the AHP within individual AHP domains. Only pixels with a higher level of AHP than that measured in control (nonsickle) cells have a color displayed. This ensures that spurious angles representing edge polarization or other artifacts are not represented.
Depictions of the arrangement of intracellular AHP for each class of cells are shown in Fig. 3. The lines within the cell represent AHP segments. Experimentally, the intracellular AHP arrangement of each cell imaged was deduced from visual inspection of images.
Figure 3: Depictions of the arrangement of hemoglobin polymers within sickled RBC. The intracellular AHP arrangements for the various morphological classes shown here in cartoon form have been deduced from inspection of images of the cells. The AHP and angle images provided unambiguous determination of AHP distribution and orientation for well separated AHP domains. The lines within the cell represent AHP. Distributions are shown for a classic sickled shaped single-domain cell (A), a single nucleation site central constriction cell (B), a single nucleation site spherulite cell (C), a three-domain cell (D), and a multiple domain cell (E).
Two subsets of the one-three-domain class, central constriction cells and spherulite cells, appeared to contain only a single nucleation site and exhibited unusual AHP distributions.
Fig. 4includes representative images from three of the RBC densities studied with MCHC of 31.2, 38.7, and 42+ g/dl. The top pair of images (31.2 g/dl, A) exhibited the lowest hemoglobin concentration as indicated by the low gray scale intensity in the absorbance (right-hand) image and a relatively large amount of AHP represented by the high gray scale intensity on the left. In the middle images (B), the hemoglobin concentration was higher (38.7 g/dl), and the number of domains had increased significantly. Fig. 4C, with an MCHC of 42+ g/dl, exhibited the highest Hb absorbance and a large number of domains. This AHP image of Fig. 4C is displayed at a 2-fold higher intensity relative to the others, and the large number of domains may be difficult to discern as reproduced here.
The percentage of AHP decreased with increasing cell density when cells from all classes were averaged (Table 1). This is somewhat counterintuitive, because with more hemoglobin available for polymerization, a higher level of AHP would be expected. We believe that there was an increase in the total amount of AHP with increasing cell density but that it was masked by the crisscrossing of AHP domains, reducing the total linear dichroism signal. This was supported by the presence of the highest %AHP in single-domain cells and the lowest in myriad domain cells, regardless of cell fraction involved (Table 1). It was also possible that the alignment that occurred following the initial polymerization phase(34, 35) , which is responsible for the sizable dichroic signals we measure, was not able to proceed fully in the higher density cells. This was supported by the decrease in the %AHP within each class as MCHC increased (Table 1). As the number of domains reached the extreme present in myriad domain cells, the measured signal was nearly extinguished. %AHP was therefore inversely representative of the number of domains (except in the zero domain case). Histograms of the percentage of AHP/cell for each MCHC clearly indicated a shift toward lower percentages as MCHC was increased (Fig. 5).
Figure 5: Histograms representing the distribution of the percentage of AHP per cell at 31.2 g/dl (A), 34.5 g/dl (B), 38.7 g/dl (C), and 42+ g/dl (D). The average percentage of AHP/cell decreased as density increased. Cells with %AHP greater than 6% are very scarce in both C and D. The overlapping of the AHP domains, or the inability of the AHP to align in higher MCHC cells, was responsible for the reduction in the linear dichroism signal at higher MCHC. This would have limited the number of multiple or myriad domain cells with a large percentage of AHP. Cells were separated by density on discontinuous Stractan gradients.
Another influence on the length of AHP fibers and the size of domains is the shear stress to which AHP is subjected during deoxygenation(40, 41) . We have not yet examined this variable.
Higher MCHC resulted in changes in morphological presentation. As the average MCHC increased, the percentage of one-three-domain cells decreased (Fig. 6A). Conversely, the percentage of multiple domain cells over the same range in MCHC increased (Fig. 6B). For the most dense fraction, which contained the largest percentage of irreversibly sickled cells, there was a smaller percentage of multiple domain cells compared with the second densest fraction and a larger number of myriad domain cells. This supports the evidence that the number of polymer domains increases with increasing MCHC as predicted by supersaturation behavior(16, 17, 42) .
Figure 6:
The fraction of total cells in the
one-three-domain (A) and multiple () and myriad
(&cjs2113;) domain classes (B) as a function of MCHC. Higher
cell densities resulted in an increase in the number of multiple and
myriad domain cells, and a concomitant decrease in the number of
one-three-domain cells. Cells were separated by density on
discontinuous Stractan gradients and morphological classification was
accomplished by visual inspection of
images.
Intracellular AHP domains probably play an important role in the flow of sickled RBC through the vasculature(43) . To better understand the factors regulating domain formation, we measured the number of AHP domains in fixed, sickled RBC populations with various intracellular HbS concentrations. In these experiments, SRBC were separated into narrow density fractions and then deoxygenated in about 4.5 s to approximate conditions in the human circulation. The number of AHP domains, the amounts of Hb and AHP, and the orientation of AHP were determined for each cell. The number and arrangement of AHP in the different density cells provided support that our morphological classification scheme was based on nucleation and growth phenomena(7, 37, 43) , as predicted by current theories(16, 17, 18, 42) .
The average number of domains present in fully deoxygenated sickled SRBC was found to rise in concert with cellular hemoglobin concentration, while the percentage of Hb existing as AHP decreased. The large number of domains seen in higher density cells implied that overlapping of the AHP domains, or the inability of the AHP to align in higher MCHC cells, was responsible for the reduction in the linear dichroism signal. This would have limited the number of multiple or myriad domain cells with a large percentage of aligned HbS. This reversal in expected distribution was previously calculated and found to fit the experimental distribution(28) . The supersaturation theory for polymer nucleation, which describes the fraction of Hb that is insoluble compared with the total amount of Hb, predicts an increase in domain number with increasing MCHC(16, 17, 18, 42) . This theory describes two distinct polymerization phases: homogeneous nucleation, where a small number of Hb molecules form a stable cluster that undergoes subsequent elongation, and heterogeneous nucleation, where nucleation occurs on the surface of existing polymer fibers. Heterogeneous nucleation proceeds more rapidly than homogeneous nucleation and can therefore lead to explosive polymer growth. The dumbbell-shaped domains of central constriction cells and the radial domains of spherulite cells may illustrate this process. It appears that the central constriction point may have been a single homogeneous nucleation site and that the domains of polymer grew by heterogeneous nucleation. Simulations by Dou and Ferrone (38) have shown that this type of 2-fold symmetry predominates initially with radial or spherulite symmetry dominating at later times. Of the two, central constriction cells predominated here, probably because the cellular Hb was exhausted before full spherulite formation could occur. The striking example of a spherulite cell (cell 7 of Fig. 1) shows that symmetry had begun to approach circular, but again the cellular Hb pool appeared to have become exhausted before a full radial domain was formed. Rare examples of spherulite cells were seen where the radial domain was fully formed (data not shown).
Single domain cells exhibited the highest measured %AHP in these studies partly because a single continuous domain generates a large signal with no loss of signal from overlap with other domains. The average %AHP for the 2 densest fractions were similar, but the fraction of myriad domain cells was higher in the most dense fraction. This suggested that cells with MCHC above 38.7 g/dl contained a very large number of AHP domains. The difficulty in determining %AHP accurately for the denser fractions was exemplified by the small differences in the %AHP between myriad-domain cells (those that contain greater than 10 AHP domains) and zero-domain cells (Table 1). For this reason, at higher MCHC, the only observed effect of increasing MCHC (from 38.7 to 42+ g/dl) was an increase in the number of myriad domain cells. This morphological shift was indicative of an increase in the number of AHP domains but was not significantly represented by a large change in the %AHP. The plateau in the percentages of multiple + myriad domain cells at high MCHC could also be representative of a saturation in the number of nucleii formed due to limitations in oxygen diffusion out of the cells(48) .
Upon closer inspection of cell 2 in Fig. 2, it is possible to imagine that the bottom and the two rightmost pairs of domains were each individual central constrictions with the leftmost quartet either two central constrictions or a near-spherulite domain. All of these apparent domains could then have resulted from only three individual nucleation sites instead of many more. For this reason, our assignment of the number of domains in cells with many domains may be an overestimation of the number of nucleation sites.
As MCHC increases, the supersaturation ratio rises as well (up to 70% with fast deoxygenation(44) ), and this increases the probability of homogeneous nucleation, generating more individual domains of polymer. In accordance with this theory, we saw an increase in the number of domains with increasing MCHC. A rigorous comparison between MCHC and the number of AHP domains would require a knowledge of the exact number of domains/cell, which is not possible with this technique. The persistence of preformed nucleation sites at full oxygenation (7) would also complicate such a calculation.
Sickle (Fig. 3A) and holly leaf (Fig. 3B) shaped cells were found to contain mostly single domains of AHP and are known to be highly undeformable in the deoxygenated state(43, 45) . These rigid cells may be important in vascular occlusion due to their inability to traverse the narrow confines of the microcirculation. It has been shown that reversibly sickled cells and other poorly deformable sickle cells are important to the rheologic impairment that results in vascular occlusions(43) . Flow studies ex vivo have shown that vaso-occlusion is initiated by adherence of low density sickle cells to vascular endothelium and propagated by the trapping of poorly deformable cells behind these niduses(46) . It is important to remember that such cells often result from RBC with low intracellular Hb concentrations and thus long delay times would be predicted. These cells would not be expected to sickle during the time spent in the microcirculation(47) . Uncertainties about full reoxygenation in the lungs and the possibility of preformed nucleation sites existing upon the cells reentry into the microcirculation complicate such predictions.