Abstract
Purpose:
To investigate foveal avascular zone (FAZ) changes in the superficial (SCP) and deep (DCP) capillary plexuses in beta-thalassemia major (BTM) patients, as shown in optical coherence tomography angiography.
Methods:
Nonrandomized, comparative case series of 54 eyes of 27 BTM patients and 46 eyes of 23 healthy controls, utilizing an automated FAZ detection algorithm. Measurements included FAZ area and FAZ shape descriptors (convexity, circularity, and contour temperature). Results were compared between the two groups, and correlated to iron load and chelation therapy parameters.
Results:
SCP and DCP FAZ area were not significantly different between the control and BTM groups (P = 0.778 and P = 0.408, respectively). The same was true regarding SCP FAZ convexity (P = 0.946), circularity (P = 0.838), and contour temperature (P = 0.907). In contrast, a statistically significant difference was detected between controls and BTM group regarding DCP FAZ convexity (P = 0.013), circularity (P = 0.010), and contour temperature (P = 0.014). Desferrioxamine dosage was strongly correlated to the DCP area (r = 0.650, P = 0.05) and liver magnetic resonance imaging/T2-star to DCP circularity (r = −0.492, P = 0.038). Correlations were also revealed between urine Fe excretion and DCP convexity (r = 0.531, P = 0.019), circularity (r = 0.661, P = 0.002), and contour temperature (r = −0.591, P = 0.008).
Conclusions:
Retinal capillary plexuses and especially DCP seem to present unique morphologic changes in BTM patients, not in the FAZ area, but in specific shape descriptors, indicating minor but detectable FAZ changes. These changes correlate well with iron load and chelation therapy parameters. Their clinical importance and pathophysiologic implications remain to be elucidated through further studies.
Beta-thalassemia (BT) is a severe genetic autosomal recessive hemoglobinopathy leading to defective β-chain production, imbalance in α/β-globin chain synthesis, ineffective erythropoiesis, and anemia.
1–3 Homozygous beta-thalassemia major (BTM) requires regular lifelong red blood bell (RBC) transfusions. Without transfusions, BTM leads to death before the age of three years old.
3 Heterozygous patients present with milder symptoms, at an older age, and do not require transfusions. Every year, more than 42,000 newborns are affected by BT worldwide.
4 Areas with high prevalence include the Mediterranean Basin, Central Asia, Transcaucasia, the Indian subcontinent and the Far East. BT is not uncommon in people of African descent.
2
Transfusion therapy, along with iron chelation (the gradual accumulation of iron due to multiple transfusions can lead to organ failure), is the cornerstone of BTM treatment, preventing death and decreasing mortality.
5–7 The whole spectrum of BTM ocular complications includes those due to iron overload,
2,8 but also those related to chelation therapy.
9 The chelation agent more extensively used clinically worldwide is desferrioxamine. Other agents include the orally administered deferiprone and deferasirox.
2
The majority of BTM retinal lesions fall into the category of pseudoxanthoma elasticum-like (PXE-like) lesions, a term introduced by Aessopos et al.,
10 to differentiate them from true pseudoxanthoma elasticum (PXE).
11 PXE-like retinal lesions include angioid streaks, peau d'orange, and peudopapillitis (optic disc drusen).
12,13
Retinal venous tortuosity (RVT) is considered the primary non-PXE-like retinal abnormality, found in 4% to 24% of BTM patients, and attributable to chronic anemia.
14–16 Although RVT is well documented, the possibility of microvascular anomalies in BTM has not yet been explored.
The purpose of this pilot study was to investigate the presence of macular microvascular alterations in BTM patients. Foveal avascular zone (FAZ) is the most sensitive index of insult to the retinal capillary network, providing early and accurate data regarding possible pathologic processes. Under this notion, we utilized optical coherence tomography angiography (OCTA)
17,18 to investigate probable changes in both the superficial (SCP) and deep (DCP) capillary plexuses.
Estimated fractal dimension by the standard box count algorithm. We opted to use the Shape Filter default box sizes 2, 3, 4, 6, 8, 12, 16, 32, 64 as the goodness of fit index; also calculated by Shape Filter, in all cases was ≥0.995.
We also calculated two other possibly useful indexes: the ratio (feret diameter) / (minimum feret diameter), a measure of the elongation of FAZ and the ratio area / (maximum inscribed circle area), a measure of the irregularity of the FAZ outline.
We separately compared area and shape descriptor parameters for both SCP and DCP FAZ. The level of statistical significance according to Hochberg's step-up correction for multiple comparisons was set at 0.016.
In SCP there was no significant difference between the control and BTM groups regarding the FAZ area (μm2) (303,425 ± 118,733 vs. 312,224 ± 120,450, P = 0.778), the area of the FAZ convex hull (μm2) (338,138 ± 135,507 vs. 344,970 ± 133,560, P = 0.763), and the FAZ perimeter (μm) (2301 ± 544 vs. 2302 ± 527, P = 0.884).
Also, the perimeter of SCP FAZ convex hullμ (μm) did not differ significantly between the control and the BTM groups (2087 ± 384 vs. 2095 ± 390, P = 0.99). Elongation indices such as the SCP FAZ feret diameter (μm) (738 ± 125 vs. 735 ± 138, P = 0.661), the minimum feret diameter of FAZ (μm) (584 ± 125 vs. 582 ± 110, P = 0.961), and the ratio FAZ feret diameter/minimum FAZ feret diameter (1.278 ± 0.116 vs. 1.267 ± 0.104, P = 0.861) were not different between the control and BTM groups.
The same was true regarding the SCP maximum inscribed circle diameter (μm) (472 ± 111 vs. 479 ± 116, P = 0.661), the FAZ area/maximum inscribed circle area (1.87 ± 0.91 vs. 3.84 ± 11.60, P = 0.984), and the area/perimeter index (128.40 ± 22.10 vs. 130.38 ± 23.35, P = 0.763).
No statistically significant difference was detected between controls and BTM patients regarding SCP FAZ convexity (0.918 ± 0.050 vs. 0.919 ± 0.047,
P = 0.946), solidity (0.901 ± 0.034 vs. 0.905 ± 0.032,
P = 0.676), circularity (0.720 ± 0.108 vs. 0.728 ± 0.099,
P = 0.838), FAZ contour temperature (0.211 ± 0.043 vs. 0.210 ± 0.039,
P = 0.907), and FAZ fractal dimension (1.725 ± 0.040 vs. 1.722 ± 0.050,
P = 0.800). All the above are summarized in
Table 2 and
Figure 3.
Table 2 Foveal Avascular Zone Shape Descriptors—Superficial Capillary Plexus
Table 2 Foveal Avascular Zone Shape Descriptors—Superficial Capillary Plexus
Regarding DCP, the same parameters as with SCP were measured and compared between the control and BTM group. In DCP there was no significant difference between the control and BTM groups regarding the FAZ area (μm2) (564,368 ± 180,227 vs. 508,743 ± 127,250, P = 0.408) and the area of the FAZ convex hull (μm2) (652,366 ± 211,692 vs. 576,295 ± 141,311, P = 0.298).
Also, the DCP FAZ perimeter (μm) (3668 ± 733 vs. 3289 ± 474, P = 0.089) and the perimeter of FAZ convex hull (μm) (2905 ± 433 vs. 2726 ± 331, P = 0.224) were not different between the two groups. As in SCP above, the FAZ feret diameter (μm), the minimum feret diameter of FAZ, and the ratio FAZ feret diameter/minimum FAZ feret diameter were all within the same range between the DCP control and BTM groups: 1029 ± 142 vs. 957 ± 105 (P = 0.113), 814 ± 139 vs. 767 ± 102 (P = 0.376), and 1.274 ± 0.096 vs. 1.253 ± 0.065 (P = 0.592), respectively.
Other indices tested for the DCP included the maximum inscribed circle diameter (μm) (580 ± 183 vs. 619 ± 109, P = 0.606), the FAZ area/maximum inscribed circle area (6.91 ± 22.00 vs. 1.75 ± 0.62 P = 0.113), and the area/perimeter index (151.28 ± 21.37 vs. 151.46 ± 19.00, P = 0.763), all failing to reveal any statistically significant difference. The same was true for shape descriptors, solidity (0.869 ± 0.024 vs. 0.883 ± 0.021, P = 0.059), and FAZ fractal dimension (1.728 ± 0.048 vs. 1.731 ± 0.046, P = 0.992).
Interestingly, a statistically significant difference was detected between controls and BTM patients regarding DCP FAZ convexity (0.804 ± 0.044 vs. 0.836 ± 0.037,
P = 0.013), circularity (0.535 ± 0.068 vs. 0.588 ± 0.065,
P = 0.010), and DCP FAZ contour temperature (0.298 ± 0.029 vs. 0.276 ± 0.025,
P = 0.014). The results are summarized in
Table 3 and
Figure 4.
Table 3 Foveal Avascular Zone Shape Descriptors—Deep Capillary Plexus
Table 3 Foveal Avascular Zone Shape Descriptors—Deep Capillary Plexus
We subsequently investigated if iron load and chelation therapy indices were related to the FAZ area and/or FAZ shape parameters in the BTM patients. FAZ area correlation to DSF dosage was moderate for the SCP (
r = 0.541,
P = 0.025), and strong for the DCP (
r = 0.650,
P = 0.05). There was also a moderately strong negative correlation between liver MRI/T2* and DCP circularity (
r = −0.492,
P = 0.038). As lower liver MRI/T2* values represent increased iron load in the liver, the correlation indicates a positive correlation between liver iron load and DCP circularity. The strongest correlations as a whole were revealed between UIE and DCP convexity (
r = 0.531, moderate correlation,
P = 0.019), DCP circularity (
r = 0.661, strong correlation,
P = 0.002), and DCP contour temperature (
r = −0.591, strong correlation,
P = 0.008). Spearman's correlation results for all comparisons are presented in detail in
Table 4.
Table 4 Spearman's Regression Analysis of the Correlation Between Shape Descriptors and Iron Load—Chelation Therapy Parameters
Table 4 Spearman's Regression Analysis of the Correlation Between Shape Descriptors and Iron Load—Chelation Therapy Parameters
Although the impact of BTM on the retina has been extensively investigated, possible microvascular changes in BTM patients have never been evaluated. Although significant FAZ size variability has been reported,
34,35 FAZ outline was the parameter of choice in this pilot study, as it is obtained in all OCTA machines, can be readily evaluated for artifacts, and does not carry the burden of the various vascularity indices that vary widely across different manufacturers.
36–38
SCP and DCP are fundamentally different. DCP represents an area of slow circulation and relatively low intravascular hydrostatic pressure, more susceptible to ischemia and oxidative stress. FAZ alterations seem to be evident in the DCP, as it represents the most remote and vulnerable part of the retinal capillary plexus. DCP capillaries form spider-like “vortices,” revealing an entirely different structure than that of SCP.
39,40 DCP capillaries are perfused through other small-diameter vessels, either directly from SCP arterioles, or indirectly through anastomotic branches from the SCP itself.
41 DCP FAZ has proven to be a sensitive biomarker of early retinal vascular insult in a variety of diseases.
42–44
Using shape descriptors, we demonstrated a more circular, convex, and less complex (with less multilobular contour) DCP FAZ in BTM patients than in normal controls.
This seems to be paradoxical, as the notion is that the more circular, the more normal FAZ appearance is. Increased circularity could be an equivalent to RVT, which is not uncommon in BTM patients. RVT in BTM patients increases with age and is attributed to the mild chronic anemia between transfusions, resulting in tissue hypoxia. RVT is also well documented in chronically anemic patients (inverse relationship to hematocrit).
RVT can be evaluated as a function of venous length. For a tortuous vein the distance between two points in its course is by definition longer than that of a less tortuous path between the same points. If a similar effect could be expected in FAZ, it would be expressed as an arcuate or multilobular deformation of the FAZ defining vessels. This deformation could be expressed only as a centrifugal bending of FAZ capillaries leading to an overall increase in circularity. A centripetal deformation is rather unlikely, as the retinal tissue structure in the foveola is arranged in a centrifugal manner, and is incompatible with a centripetal or even randomly tortuous deformation.
Both venous tortuosity and DCP changes could be attributed to the hyperdynamic circulation due to chronic anemia. BTM patients usually have an increased cardiac index, heart rate and stroke volume, lower systolic blood pressure, and a blunted blood pressure temporal variability, all consistent with a markedly decreased systemic vascular resistance.
45,46 DCP changes could be related to unique compensatory/autoregulatory responses to these systemic hemodynamic alterations. DCP's autoregulatory response seems to be independent of that of the SCP, further supporting the notion that DCP is a discrete, sensitive, and accurate index of vascular changes.
47
The observed DCP changes could also be viewed in the context of local vascular remodeling,
48 including capillary dropout, arteriolar–venular anastomoses, and sea fan neovascularization. These changes are thought to be due to repeated episodes of vascular closure and reopening,
14,49 or increased oxidative stress and ischemia in poorly perfused areas, especially between transfusions.
In order to correlate our findings with the known causative agents of BTM retinal abnormalities, namely, the iron accumulation and chelation therapy, we further investigated a possible correlation between FAZ shape descriptors and parameters related to the iron load. We found a moderate negative correlation (r = −0.492) between DCP circularity and liver MRI/T2*, suggesting increased circularity with increasing liver iron load (as lower MRI/T2* values are associated with increased iron load).
There was also a moderate positive correlation between mean values of UIE during the last three years and DCP convexity (r = 0.531), and a strong positive correlation to DCP circularity (r = 0.661) and DCP contour temperature (r = 0.591). A moderate positive correlation (r = 0.541) was revealed between DSF mean dosage (mg/month) and SCP area, and a strong correlation to the DCP area (r = 0.650).
Urinary iron excretion values depend on both iron load (total body iron) and DSF dosage. The higher the iron load and/or DSF dosage, the more elevated the mean iron levels in the urine. Thus, it seems probable that both iron overload and DSF dosage contribute to the observed DCP changes.
Iron accumulation results in endothelial dysfunction due to oxidative stress, by catalyzing the conversion of oxygen into highly reactive free radicals (superoxide-driven Haber-Weiss reaction). Superoxide alone is capable of producing damaging effects in mitochondria.
50 The highly reactive hydroxyl radical is produced in the presence of ferrous iron (Fe
2+) by the decomposition of hydrogen peroxide (Fenton reaction).
51,52
Experimental evidence suggests that iron has the potential to reduce endothelium-derived nitric oxide bioactivity either directly (decreased nitric oxide synthase activity) or indirectly (increased membrane lipid peroxidation generating lipid peroxyl radicals).
53,54 Microcirculatory hemodynamics in the retina seem to be affected by the nitric oxide synthase/nitric oxide pathway.
55,56 Nitric oxide level is well documented to be reduced in BTM patients.
57–59
Human vascular endothelial cells incubated with thalassemic serum present functional disturbances including increased levels of soluble adhesion molecules, reduced mitotic potential, and morphologic changes reminiscent of apoptosis.
60,61 Subsequent in vivo studies confirmed the presence of endothelial dysfunction in BTM patients.
62,63
Interestingly, no correlation was found between DCP and serum ferritin levels. It is, though, known that ferritin levels do not always reflect correctly the iron load, and are not always good predictors of iron load–related complications.
64,65 Ferritin trends, rather than absolute values, provide useful information. Other measurements such as non-transferrin bound iron and labile plasma iron, are alternatives to ferritin serum markers but are not routinely available or completely validated.
65,66
Another possible explanation for the DCP alterations includes changes in RBC deformability, an essential feature of RBCs enabling them to pass through the smallest capillaries, as retinal capillary diameter is smaller than that of the RBCs (6–8 μm). Deformability depends on the integrity of cytoskeletal proteins (spectrin, ankyrin, band 3), intracellular ion concentrations, intracellular water (viscosity), and membrane surface-to-volume ratio,
67 factors that may be altered in BTM patients. Of particular interest is the increase in intracellular Ca
2+ in BTM patients, which has been documented to induce RBC stiffness.
68,69 RBC stiffness could probably act as an internal splint, the column of RBCs pushing like a ram and blunting the angles of FAZ outline.
DSF is capable of reducing iron levels, alleviating the detrimental effects of iron accumulation. On the other hand, a higher mean DSF dosage is mandated by high iron levels. It is possible that in our cases, the correlation between DSF dosage and SCP and DCP area may in part be indirect and related to elevated primary iron levels.
Regarding the possibility that DSF treatment may influence DCP, one should mention the many and well-documented DSF retinal side effects, including night blindness, impaired color vision, loss of visual field, reduced visual acuity, and retinal pigment epithelium (RPE) degeneration. DSF toxicity is attributed to the ion chelation (iron, copper, aluminum) on RPE, resulting in dysfunction or due to defective vasoregulation.
9,70,71
DSF may also directly influence SCP and DCP by upregulating the expression of angiogenic factors, such as vascular endothelial growth factor (VEGF).
72,73 VEGF has been associated with capillary enlargement and efficient response to vasodilators as is nitric oxide.
74–76 In vitro studies revealed mRNA levels of nitric oxide synthase to be profoundly increased by DSF.
77 An enlargement of the SCP and DCP capillaries could lead to outward “bending” (inward bending is prevented by the retinal structure at the foveola). It is possible that an initial phase of oxidative stress–induced capillary damage is followed by a second phase of DSF-induced capillary deformation.
Our study has several limitations, including the relatively small number of patients and the lack of analysis of the SCP and DCP vascular plexus itself, rather than SCP and DCP FAZ. Nevertheless, we believe that given the nature of a pilot study we should concentrate on robust and uniform measures such as those regarding FAZ, rather than more complex and not uniformly defined quantities such as vascular density or vascular patterns.
Another limitation is that the axial length of the eye was not used for scale correction of the FAZ size. This, however, is in part balanced by the minor refractive error in both BTM and control groups. Notably, scale-independent shape descriptors, such as ratios (feret diameter/minimum feret diameter, area/maximum inscribed circle area, area/perimeter), solidity, fractal dimension, circularity, convexity, and contour temperature are not influenced at all.
Regarding the advantages of our study, one should mention the novelty of the approach to the FAZ characteristics by the utilization of shape descriptors that have not been commonly used in other studies, and the automated detection of the FAZ. Also, our study is, to the best of our knowledge, the first study of FAZ in BTM. Further studies should be conducted to elucidate the complex relationship between SCP, DCP and the iron load and chelation therapy.
In conclusion, retinal capillary plexuses and especially DCP seem to present unique morphologic changes in BTM patients. These are not changes regarding the SCP and DCP FAZ area, but specific FAZ shape descriptors, indicating minor but detectable changes in the FAZ of patients with BTM. Moreover, the aforementioned changes correlate well with iron load and chelation therapy parameters of BTM patients. The clinical importance and pathophysiologic implications of these changes remain to be elucidated through further studies.