Sickle cell anemia (SCA) is a genetic disorder that affects millions of people worldwide, for which there is no cure despite substantial understanding of its underlying pathogenesis [1, 2]. Anemia caused by ineffective erythropoiesis and hemolysis is a contributing factor, but vascular complications and abnormal blood flow dynamics account for much of SCA morbidity and mortality. However, there are few real-time in vivo studies on the microcirculation in SCA patients, except for the work by Lipowsky et al.  on intravital microscopy of nailfold capillary hemodynamics in SCA.
We have previously reported three real-time in vivo studies on the microcirculation of the bulbar conjunctiva in SCA patients using computer-assisted intravital microscopy (CAIM) [4–6]. The microvascular bed of the bulbar conjunctiva offers a readily accessible site for noninvasive measurements from which it is possible to extrapolate the in vivo condition of the microvasculature within soft tissues, and to quantify changes in microvascular condition of critical end organs over time. Using our imaging studies of the bulbar conjunctiva in SCA patients, we have characterized and quantified the morphometric and dynamic microvascular abnormalities (vasculopathy) of the disease , demonstrated that abnormal microvascular blood flow dynamics correlate with intracranial blood flow velocity in the Circle of Willis measured by transcranial Doppler ultrasonography , and evaluated the efficacy of the drug Poloxamer 188 (RheothRx® and Flocor™) on vasoocclusion . Thus, microvascular characteristics from image analysis of the bulbar conjunctiva can serve as a reliable surrogate biomarker of the severity of microvascular pathology and the efficacy of interventions designed to treat and ameliorate complications resulting from SCA-associated vasculopathy.
These real-time in vivo studies using CAIM have included both adult [4, 6] and pediatric SCA patients [5, 6]. However, in pediatric patients, these studies have focused primarily on the measurements of vessel diameter and blood flow velocity, and assessments of vasculopathy have not been reported. Moreover, there have been no direct comparisons of microvascular abnormalities and severity of vasculopathy between pediatric and adult SCA patients. Accordingly, the goal of this study was to characterize and compare real-time measurements on the degree of in vivo vasculopathy in pediatric and adult SCA patients, and to test the hypothesis that the severity of vasculopathy increases with age as a natural course of the disease.
Fourteen pediatric and eight adult SCA patients participated in the study. Mean ages of the two groups were significantly different (13.6 ± 4.4 years vs. 36.8 ± 11.9 years, P < 0.001). Conjunctival microvasculature was compared between the pediatric and adults patients, and contrasted with that of healthy, non-SCA control subjects analyzed in previous studies [4, 7, 8]. Figure 1A shows a typical image of the conjunctival microvasculature in a non-SCA subject frame captured from a videotape sequence from an unrelated study [7, 9]. There is an orderly presence of anastomosing networks of capillaries, arterioles, and venules without the presence of ischemic (avascular) zones (Fig. 1A). The normal A:V ratio is typically ∼1:2, and the arterioles and venules exhibit an even distribution without the presence of dilations, narrowing, distension, microaneurysm, sacculated (beaded) vessels, broken/damaged vessels, or hemosiderin deposits. Normal conjunctival blood flow, though variable in red cell velocity, is smooth and nonintermittent. Blood sludging, tortuous vessels, and boxcar blood flow (trickled flow) patterns are typically not observed.
The conjunctival microcirculation in the pediatric and adult SCA patients uniquely differs from those found in non-SCA control subjects [See Supporting Information]. There is a lower amount of vascularity (diminished presence of conjunctival vessels) and abnormal vascular distribution in most patients in both age groups, giving the bulbar conjunctiva a “blanched” avascular appearance. The prevalence of specific microvascular abnormalities in both patient groups is summarized in Tables I and II [See Supporting Information] and some of the abnormalities are shown in Figure 1B–D. SCA patients from both age groups exhibit, to varying degrees, the same morphometric and dynamic abnormalities, including abnormal vessel diameter, abnormal vessel distribution, abnormal vessel morphometry (shape), sludged flow, vessel tortuosity, abnormal A:V ratio, boxcar blood flow, hemosiderin deposits, and abnormal flow (red cell) velocity. These microvascular abnormalities are rarely found in the bulbar conjunctiva of healthy non-SCA subjects [4, 7, 8]. The severity of vasculopathy, as indicated by the severity index (SI), was significantly lower in the pediatric patients than in the adult patients (4.2 ± 1.8 vs. 6.6 ± 2.4, P = 0.028). For comparison, the mean SI values for both the pediatric and adult SCA patients were significantly higher than the mean SI value determined for a previous cohort of healthy non-SCA subjects (n = 10; SI = 0.31 ± 0.72; P < 0.05) . In comparing the prevalence of microvascular abnormalities between pediatric and adult SCA groups, the following significant differences were observed:
Abnormal vessel morphometry was observed in three out of eight adult patients (38%) but was not observed in any of the pediatric patients. The odds ratio (OR) [95% confidence interval (CI)] for the difference in prevalence was ∞ (1.2, ∞) (P = 0.036).
Vessel tortuosity was observed in seven out of eight adult patients (88%) compared with only three out of 14 pediatric patients (21%). The OR (95% CI) for the difference in prevalence was 25.7 (1.7, 1258) (P = 0.006).
In addition, several microvascular abnormalities were highly prevalent in both the pediatric and adult patients. Ten out of 14 pediatric patients (71%) and seven out of eight adult patients (88%) had vessel sludging. Ten out of 14 pediatric patients (71%) and eight out of eight adult patients (100%) had an abnormal A:V ratio. Eight out of 14 pediatric patients (57%) and seven out of eight adult patients (88%) had an abnormal vessel distribution. Eleven out of 14 pediatric patients (79%) and six out of eight adult patients (75%) exhibited boxcar flow patterns.
CAIM is a real-time technology that can be used to noninvasively videotape, analyze and quantify real-time microvascular abnormalities in vascular diseases. The technique has been used successfully in our laboratory to assess microvascular abnormalities in type-1 and type-2 diabetes, Alzheimer's disease, and SCA [4–12]. The in vivo microvascular bed of the bulbar conjunctiva (conjunctival microcirculation) is particularly amenable to the use of CAIM because it is noninvasively and easily accessible, and yields images of excellent quality and clarity. Results from some of the studies on the identification and quantification of microvascular abnormalities in the conjunctival microcirculation [4, 11] have been used as a basis for subsequent translational research and interventional efficacy studies [6, 10].
This study was designed to extend our knowledge base on real-time vasculopathy in pediatric and adult SCA patients. Our overall goal is to understand the ontogeny of vasculopathy based on the hypothesis that, as a genetic disorder, SCA microvascular complications and vasculopathy begin to develop after birth and continue to progress into adulthood as part of the natural course of the disease. Results from this study support this hypothesis: the severity of microvascular abnormalities in the pediatric patients was significantly lower than that observed in the adult patients. Secondary analyses of specific microvascular abnormalities revealed that the observed difference in severity was primarily due to a lower prevalence of abnormal vessel morphometry and vessel tortuosity in the pediatric patients compared with the adults. These findings suggest that these two specific abnormalities develop at a slower rate than other microvascular abnormalities.
The primary limitation of this study is that it is cross-sectional. The observed difference in the severity of vasculopathy between the pediatric and adult patients could be attributable to advances in management of the disease that were not available to the adult patients during their childhood. A longitudinal study in which the microvasculature of SCA patients is evaluated at regular intervals from childhood to adulthood would be required to definitively test the hypothesis that the severity of vasculopathy progresses with age. If confirmed, the results of this study suggest that the pediatric years represent a window of opportunity during which effective treatment and management modalities may slow or ameliorate complications of SCA caused by vasculopathy that arises as a natural progression of the disease from childhood to adulthood. Specific abnormalities, e.g., abnormal vessel morphometry and vessel tortuosity, may serve as landmark biomarkers to evaluate the efficacy of treatment and disease management modalities over time. Moreover, the high prevalence of other abnormalities in both pediatric and adult patients, including vessel sludging, abnormal A:V ratio, abnormal vessel distribution, and boxcar flow patterns—indicative of rapid development of vasculopathy in childhood—suggests an urgency to identify better interventions and treatments that ameliorate or slow the progression of microvascular abnormalities and can be used to treat pediatric SCA patients more aggressively.
Conjunctival vessels have unique shapes and forms (Fig. 1A–D) and can be easily reidentified for follow-up studies using CAIM—each individual vessel can serve as its own baseline (reference) control and then relocalized and reassessed in longitudinal studies [6, 10]. This makes the conjunctival microcirculation an ideal arena and CAIM an excellent noninvasive real-time technology for longitudinal studies of SCA disease progression and evaluations of the efficacy of medications and other treatment or management modalities. At this time, CAIM is not yet widely used as a research tool. However, two identical CAIM systems have been built recently and are functional in other laboratories. A blinded interventional collaborative study to compare independently obtained real-time in vivo vasculopathy data is in progress. These studies will eventually allow for independent confirmation of our results at other institutions and will validate the utility of CAIM as a clinical tool to objectively and noninvasively study vasculopathy in SCA and other vascular diseases.