NTP pattern of avian embryonic red cells: role of RNA degradation and AMP deaminase/5'-nucleotidase activity

During terminal erythroid differentiation, degradation of RNA is a potential source for nucleotide triphosphates (NTPs) that act as allosteric effectors of hemoglobin. In this investigation, we assessed the developmental profile of RNA and purine/pyrimidine trinucleotides in circulating embryonic chick red blood cells (RBC). Extensive changes of the NTP pattern are observed which differ significantly from what is observed for adult RBC. The biochemical mechanisms have not been identified yet. Therefore, we studied the role of AMP deaminase and IMP/GMP 5'-nucleotidase, which are key enzymes for the regulation of the purine nucleotide pool. Finally, we tested the effect of major NTPs on the oxygen affinity of embryonic/adult hemoglobin. The results are as follows. 1) Together with ATP, UTP and CTP serve as allosteric effectors of hemoglobin. 2) Degradation of erythroid RNA is apparently a major source for NTPs. 3) Developmental changes of nucleotide content depend on the activities of key enzymes (AMP deaminase, IMP/GMP 5'-nucleotidase, and pyrimidine 5'-nucleotidase). 4) Oxygen-dependent hormonal regulation of AMP deaminase adjusts the red cell ATP concentration and therefore the hemoglobin oxygen affinity.     

Nitric oxide effect on the hemoglobin-oxygen affinity.

The biological roles of nitric oxide (NO)-hemoglobin (Hb) derivatives are obscure. It is proposed that NO can function as an allosteric regulator of hemoglobin oxygen-binding properties. We aimed to estimate the effects of NO donors and NO-synthase substrate (L-arginine) on hemoglobin-oxygen affinity (HOA) in experiments in vitro with the various ratios between NO formed and Hb and various oxygen pressures. HOA index (p50), blood pH, plasma and red blood cell (RBC) concentrations of nitrite/nitrate and methemoglobin amounts were measured after the experiments. In our experiments, blood incubation with NO donors (glyceryltrinitrate, molsidomine, sodium nitroprusside, S-nitrosocysteine) or NO-synthase substrate (L-arginine) did not change HOA even at NO:Hb ratio of 1:1. At the same time our results showed that oxygenated blood incubation with S-nitrosocysteine induced an oxyhemoglobin dissociation curve shift leftwards. This indicates a leading role of met-Hb in a modification of Hb oxygen-binding properties. However other NO-modified forms of hemoglobin (S-nitroso- and nitrosylhemoglobin) also may be involved in the regulation of HOA. The results obtained indicate that nitric oxide can be the allosteric effector of hemoglobin, increasing or decreasing its oxygen affinity - possibly, through the generation of different NO-Hb derivatives.     

Study of the trajectory of erythrocyte movement in microvessels using a method of automatic image analysis

Many physiological and pathological processes in the circulation are related to changes of blood rheological properties. Since blood represents a specific suspension of cells in plasma the mentioned changes are to a great degree dependent on behavior and interaction of red blood cells (RBC). For investigation of blood flow structure in microvessels we developed an algorithm and worked out a program for automatic treatment of RBC movement on the basis of automatic image analysis system "Leitz-TAS" (Ernst Leitz, FRG). The program was based on computation of coordinates of blood cell centers. Further we calculated the following values: the vector of displacements (S), its projection on both axes (dx, dy), their relationship (dy/dx), the relationship of RBC radial coordinate to vessel radius (y/R), and the velocity of RBC movements (V). Proceeding from these data we obtained such parameters, as e.g., the velocity profile, the radial displacements of red cells, etc. by which we could judge on the blood flow regime, the flow structure and changes of blood rheological properties.     

Decreased Hematocrit-To-Viscosity Ratio and Increased Lactate Dehydrogenase Level in Patients with Sickle Cell Anemia and Recurrent Leg Ulcers

Leg ulcer is a disabling complication in patients with sickle cell anemia (SCA) but the exact pathophysiological mechanisms are unknown. The aim of this study was to identify the hematological and hemorheological alterations associated with recurrent leg ulcers. Sixty-two SCA patients who never experienced leg ulcers (ULC-) and 13 SCA patients with a positive history of recurrent leg ulcers (ULC+) - but with no leg ulcers at the time of the study – were recruited. All patients were in steady state condition. Blood was sampled to perform hematological, biochemical (hemolytic markers) and hemorheological analyses (blood viscosity, red blood cell deformability and aggregation properties). The hematocrit-to-viscosity ratio (HVR), which reflects the red blood cell oxygen transport efficiency, was calculated for each subject. Patients from the ULC+ group were older than patients from the ULC- group. Anemia (red blood cell count, hematocrit and hemoglobin levels) was more pronounced in the ULC+ group. Lactate dehydrogenase level was higher in the ULC+ group than in the ULC- group. Neither blood viscosity, nor RBC aggregation properties differed between the two groups. HVR was lower and RBC deformability tended to be reduced in the ULC+ group. Our study confirmed increased hemolytic rate and anemia in SCA patients with leg ulcers recurrence. Furthermore, our data suggest that although systemic blood viscosity is not a major factor involved in the pathophysiology of this complication, decreased red blood cell oxygen transport efficiency (i.e., low hematocrit/viscosity ratio) may play a role.     

Purification of hemoglobin from red blood cells using tangential flow filtration and immobilized metal ion affinity chromatography

Two methods for purifying hemoglobin (Hb) from red blood cells (RBCs) are compared. In the first method, red blood cell lysate is clarified with a 50 nm tangential flow filter and hemoglobin is purified using immobilized metal ion affinity chromatography (IMAC). In the second method, RBC lysate is processed with 50 nm, 500 kDa, and 50-100 kDa tangential flow filters, then hemoglobin is purified with IMAC. Our results show that the hemoglobins from both processes produce identical Hb products that are ultrapure and retain their biophysical properties (except for chicken hemoglobin, which shows erratic oxygen binding behavior after purification). Therefore, the most efficient method for Hb purification appears to be clarification with a 50 nm tangential flow filter, followed by purification with IMAC, and sample concentration/polishing on a 10-50 kDa tangential flow filter.     

The nicotinamide adenine dinucleotides as allosteric effectors of human hemoglobin

The oxygen binding properties of human hemoglobin are appreciably altered by the nicotinamide dinucleotides NADH, NADP+, and NADPH. These cofactors are important in the control of many metabolic pathways and in providing reductive potential for a number of enzymatic reactions, including in vivo reduction of methemoglobin. Specific binding of these cofactors to hemoglobin and their potential for acting as allosteric modifiers of hemoglobin function have not been previously recognized. Detailed oxygen binding studies utilizing a thin-layer method suggest that the nicotinamide dinucleotides bind with high affinity to the deoxyhemoglobin tetramer at the beta chain anion-binding site and stabilize the low affinity "T-state" conformation. Stripped Hb A in 0.05 M N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer, pH 6.5, at 20 degrees C is half-saturated at a pO2 of 1.6 mm Hg. In the presence of 0.5 mM NADH, NADP+, or NADPH, the P50 is raised to 3.8, 7.1, and 12.5 mm Hg, respectively. The Bohr factor for stripped Hb A in 0.05 M HEPES buffer is sensitive to these effectors and is raised from 0.25 to about 0.65 by the addition of NADPH. The data suggest that routine use of these effectors in studies of human hemoglobin variants or the allosteric mechanism of Hb A be considered carefully. The relatively low intraerythrocytic levels of the nicotinamide dinucleotides in relation to hemoglobin dictate that these cofactors cannot significantly affect in vivo oxygen delivery. However, the converse is theoretically possible. The binding of the cofactors to hemoglobin and the preferential binding of their reduced forms may affect cofactor-dependent metabolic processes in red blood cells.     

Gas exchange properties of goat hemoglobins A and C

Hypoxic or anemic goats with the A hemoglobin genotype switch to the production of hemoglobin C, resulting in a reduced blood oxygen affinity. However, the physiologic consequences of this switch are not clear. We therefore studied the gas exchange properties of the two hemoglobin types. We found that purified hemoglobins A and C have very similar oxygen affinities and H+ Bohr effects, but in the presence of CO2, the affinity of hemoglobin C is substantially less than that of hemoglobin A. That this is not a nonspecific ionic effect is suggested by identical effects of NaCl on O2 binding to the two proteins and by a 2-fold higher capacity of hemoglobin C to bind CO2. The data can be explained by a class of CO2 binding sites in the beta C chain whose affinity is much higher than that of either of the primary sites or of those in Hb A. Our results suggest that in hemoglobin C-containing red cells CO2 acts as a potent allosteric effector, analogous to the role played by 2,3-diphosphoglycerate in human red blood cells. Goat hemoglobin C may have advantages over hemoglobins A or B in O2 transport under hypoxic conditions or in anemia.     

Pulmonary vascular effects of red blood cells containing S-nitrosated hemoglobin

The role of S-nitrosated hemoglobin (SNO-Hb) in the regulation of blood flow is a central and controversial question in cardiopulmonary physiology. In the present study, we investigate whether intact human red blood cells (RBCs) synthesized to contain high SNO-Hb levels are able to export nitric oxide bioactivity and vasodilate the pulmonary circulation, and whether SNO-Hb dependent vasodilation occurs secondary to an intrinsic oxygen-linked, allosteric function of Hb. RBCs containing supraphysiological concentrations (100-1,000x normal) of SNO-Hb (SNO-RBCs) were synthesized and added to isolated, perfused rat lungs during anoxic or normoxic ventilation, and during normoxic ventilation with pulmonary hypertension induced by the thromboxane mimetic U-46619. SNO-RBCs produced dose-dependent pulmonary vasodilation compared with control RBCs during conditions of both normoxic (U-46619) and hypoxic pulmonary vasoconstriction. These effects were associated with a simultaneous, rapid, and temperature-dependent loss of SNO from Hb. Both vasodilatory effects and the rate of SNO-Hb degradation were independent of oxygen tension and Hb oxygen saturation. Furthermore, these effects were not affected by inhibition of the RBC membrane band 3 protein (anion exchanger-1), a putative membrane facilitator of NO export from RBCs. Whereas these data support observations by multiple groups that synthesized SNO-Hb can vasodilate, this effect is not under intrinsic oxygen-dependent allosteric control, nor likely to be relevant in the pulmonary circulation at normal physiological concentrations.     

The importance of a single amino acid substitution in reduced red blood cell carbonic anhydrase function of early-diverging fish

In most vertebrates, red blood cell carbonic anhydrase (RBC CA) plays a critical role in carbon dioxide (CO₂) transport and excretion across epithelial tissues. Many early-diverging fishes (e.g., hagfish and chondrichthyans) are unique in possessing plasma-accessible membrane-bound CA-IV in the gills, allowing some CO₂ excretion to occur without involvement from the RBCs. However, implications of this on RBC CA function are unclear. Through homology cloning techniques, we identified the putative protein sequences for RBC CA from nine early-diverging species. In all cases, these sequences contained a modification of the proton shuttle residue His-64, and activity measurements from three early-diverging fish demonstrated significantly reduced CA activity. Site-directed mutagenesis was used to restore the His-64 proton shuttle, which significantly increased RBC CA activity, clearly illustrating the functional significance of His-64 in fish red blood cell CA activity. Bayesian analyses of 55 vertebrate cytoplasmic CA isozymes suggested that independent evolutionary events led to the modification of His-64 and thus reduced CA activity in hagfish and chondrichthyans. Additionally, in early-diverging fish that possess branchial CA-IV, there is an absence of His-64 in RBC CAs and the absence of the Root effect [where a reduction in pH reduces hemoglobin’s capacity to bind with oxygen (O₂)]. Taken together, these data indicate that low-activity RBC CA may be present in all fish with branchial CA-IV, and that the high-activity RBC CA seen in most teleosts may have evolved in conjunction with enhanced hemoglobin pH sensitivity.

     

Blood rheology of Weddell seals and bowhead whales

Red blood cell (RBC) aggregation and blood viscosity are important determinants of in vivo blood flow dynamics and, in marine mammals, these parameters may impact diving physiology by altering blood oxygen delivery during the diving response. Weddell seals are superb divers and exhibit age-related patterns in blood oxygen chemistry and diving ability. By contrast, bowhead whales are not long duration divers, and little is known of their blood properties relative to diving. The present study was designed to compare rheological characteristics of blood from Weddell seal pups, Weddell seal adults, and from adult bowhead whales: blood viscosity and RBC aggregation in plasma and in polymer solutions (i.e., RBC "aggregability") were measured. Salient findings included: (1) significant 4- to 8-fold greater aggregation in blood from adult seals compared with pups and human subjects; (2) 2-to 8-fold greater aggregation in bowhead whale blood compared with human blood; (3) compared to human red cells, enhanced RBC aggregability of RBC from adult seals and whales as determined by their greater aggregation in polymer solutions; (4) increasing RBC aggregation and aggregability of seal pup blood over a seven day period following birth; (5) significantly greater blood viscosity for adult seals compared with pups at both native and standardized hematocrits. These results indicate that, for both species, hemorheological parameters differ markedly from those of humans, and suggest progressive changes with seal age; the physiological implications of these differences have yet to be fully defined.     

Effects of superoxide anions on red cell deformability and membrane proteins.

The effect of superoxide anions (O2-) on red blood cells (RBC) deformability and membrane proteins was investigated using hypoxanthine-xanthine oxidase system. Exposure of RBC to O2- caused a marked decrease in RBC deformability with a concomitant increase in cell volume and shape changes. The RBC exposed to O2- also displayed pronounced degradation of membrane proteins such as band 3 protein and spectrin; new bands of low molecular weight products appeared as the original membrane proteins tended to diminish, without the appearance of high molecular weight products. Since the membrane proteins are involved in processes regulating membrane properties such as permeability and viscoelasticity, the decreased deformability induced by O2- may be attributable to changes in membrane proteins. Interestingly, resealed ghosts exposed to O2- did not show any significant change in membrane proteins, which suggests the existence of further generation of O2- and subsequent production of other active oxygen species mediated by O2(-)-initiated autoxidation of hemoglobin in intact RBC. Furthermore, electrophoretic analysis suggested that active oxygens increased the endogenous proteolytic susceptibility of RBC. In conclusion, a close linkage was suggested between RBC deformability and the membrane proteins.     

Role of hemoglobin oxygenation in the modulation of red blood cell mechanical properties by nitric oxide

It has been previously demonstrated that both externally generated and internally synthesized nitric oxide (NO) can affect red blood cell (RBC) deformability. Further studies have shown that the RBC has active NO synthesizing mechanisms and that these mechanisms may play role in maintaining normal RBC mechanical properties. However, hemoglobin within the RBC is known to be a potent scavenger of NO; oxy-hemoglobin scavenges NO faster than deoxy-hemoglobin via the dioxygenation reaction to nitrate. The present study aimed at investigating the role of hemoglobin oxygenation in the modulation of RBC rheologic behavior by NO. Human blood was obtained from healthy volunteers, anticoagulated with sodium heparin (15 IU/mL), and the hematocrit was adjusted to 0.4 L/L by adding or removing autologous plasma. Several two mL aliquots of blood were equilibrated at room temperature (22 ± 2 °C) with moisturized air or 100% nitrogen by a membrane gas exchanger, The NO donor sodium nitroprusside (SNP), at a concentration range of 10^?7–10^?4 M, was added to the equilibrated aliquots which were maintained under the same conditions for an additional 60 min. The effect of the non-specific NOS inhibitor l-NAME was also tested at a concentration of 10^?3 M. RBC deformability was measured using an ektacytometer with an environment corresponding to that used for the prior incubation (i.e., oxygenated or deoxygenated). Our results indicate an improvement of RBC deformability with the NO donor SNP that was much more pronounced in the deoxygenated aliquots. SNP also had a more pronounced effect on RBC aggregation for deoxygenated RBC. Conversely, l-NAME had no effect on deoxygenated blood but resulted in impaired deformability, with no change in aggregation for oxygenated blood. These findings can be explained by a differential behavior of hemoglobin under oxygenated and deoxygenated conditions; the influence of oxygen partial pressure on NOS activity may also play a role. It is therefore critical to consider the oxygenation state of intracellular hemoglobin while studying the role of NO as a regulator of RBC mechanical properties.     

Mechanism of oxidative damage to fish red blood cells by ozone

The present study was conducted to elucidate the adverse effects of ozone exposure on rainbow trout (Oncorhynchus mykiss) red blood cells (RBCs). We evaluated whether hemoglobin (Hb) or Hb-derived free iron could participate in the RBC damage using an in vitro ozone exposure system. Ozone exposure induced hemolysis, formation of methemoglobin, and RBC membrane lipid peroxidation. This RBC damage was not suppressed by the addition of a specific iron chelator (deferoxamine mesilate) to the medium but was suppressed by carbon monoxide (CO) treatment before ozone exposure. Generation of hydrogen peroxide (H2O2) in RBC was observed upon ozone exposure but was significantly suppressed by CO treatment before ozone exposure. Thus the Hb status (i.e., Hb redox condition) and H2O2 generation in RBC should play important roles in mediating RBC damage by ozone exposure. In other words, neither ozone nor its derivative directly attacked from the outside of the cell, but ozone that penetrated through the membrane derived the reactive oxygen species from Hb inside of the cell.     

RBC aggregation: laboratory data and models

The reversible aggregation of red blood cells (RBC) into linear and three-dimensional structures continues to be of basic science and clinical interest: RBC aggregation affects low shear blood viscosity and microvascular flow dynamics, and can be markedly enhanced in several clinical states. Until fairly recently, most research efforts were focused on relations between suspending medium composition (i.e., protein levels, polymer type and concentration) and aggregate formation. However, there is now an increasing amount of experimental evidence indicating that RBC cellular properties can markedly affect aggregation, with the term "RBC aggregability" coined to describe the cell's intrinsic tendency to aggregate. Variations of aggregability can be large, with some changes of aggregation substantially greater than those resulting from pathologic states. The present review provides a brief overview of this topic, and includes such areas as donor-to-donor variations, polymer-plasma correlations, effects of RBC age, effects of enzymatic treatment, and current developments related to the mechanisms involved in RBC aggregation.     

Influence of cell-specific factors on red blood cell aggregation

The reversible aggregation of red blood cells (RBC) into linear and three-dimensional structures continues to be of basic science and clinical interest: RBC aggregation affects low shear blood viscosity and microvascular flow dynamics, and can be markedly enhanced in several clinical states. Until fairly recently, most research efforts were focused on relations between suspending medium composition (i.e., protein levels, polymer type and concentration) and aggregate formation. However, there is now an increasing amount of experimental evidence indicating that RBC cellular properties can markedly affect aggregation, with the term "RBC aggregability" coined to describe the cell's intrinsic tendency to aggregate. Variations of aggregability can be large, with some changes of aggregation substantially greater than those resulting from pathologic states. The present review provides a brief overview of this topic, and includes such areas as donor-to-donor variations, polymer-plasma correlations, effects of RBC age, effects of enzymatic treatment, and current developments related to the mechanisms involved in RBC aggregation.     

Rapid Rather than Gradual Weight Reduction Impairs Hemorheological Parameters of Taekwondo Athletes through Reduction in RBC-NOS Activation

Purpose

Rapid weight reduction is part of the pre-competition routine and has been shown to negatively affect psychological and physiological performance of Taekwondo (TKD) athletes. This is caused by a reduction of the body water and an electrolyte imbalance. So far, it is unknown whether weight reduction also affects hemorheological properties and hemorheology-influencing nitric oxide (NO) signaling, important for oxygen supply to the muscles and organs.

Methods

For this purpose, ten male TKD athletes reduced their body weight by 5% within four days (rapid weight reduction, RWR). After a recovery phase, athletes reduced body weight by 5% within four weeks (gradual weight reduction, GWR). Each intervention was preceded by two baseline measurements and followed by a simulated competition. Basal blood parameters (red blood cell (RBC) count, hemoglobin concentration, hematocrit, mean corpuscular volume, mean cellular hemoglobin and mean cellular hemoglobin concentration), RBC-NO synthase activation, RBC nitrite as marker for NO synthesis, RBC deformability and aggregation parameters were determined on a total of eight investigation days.

Results

Basal blood parameters were not affected by the two interventions. In contrast to GWR, RWR decreased activation of RBC-NO synthase, RBC nitrite, respective NO concentration and RBC deformability. Additionally, RWR increased RBC aggregation and disaggregation threshold.

Conclusion

The results point out that a rapid weight reduction negatively affects hemorheological parameters and NO signaling in RBC which might limit performance capacity. Thus, GWR should be preferred to achieve the desired weight prior to a competition to avoid these negative effects.     

A quantitative framework for the design of acellular hemoglobins as blood substitutes: implications of dynamic flow conditions

The delivery of oxygen to tissue by cell-free carriers eliminates intraluminal barriers associated with red blood cells. This is important in arterioles, since arteriolar tone controls capillary perfusion. We describe a mathematical model for O(2) transport by hemoglobin solutions and red blood cells flowing through arteriolar-sized tubes to optimize values of p50, Hill number, hemoglobin molecular diffusivity and concentration. Oxygen release is evaluated by including an extra-luminal resistance term to reflect tissue oxygen consumption. For low consumption (i.e., high resistance to O(2) release) a hemoglobin solution with p50=15 mmHg, n=1, D(HBO2)=3 x 10(-7) cm(2)/s delivers O(2) at a rate similar to that of red blood cells. For high consumption, the p50 must be decreased to 5 mmHg. The model predicts that regardless of size, hemoglobin solutions with higher p50 will present excess O(2) to arteriolar walls. Oversupply of O(2) to arteriolar walls may cause constriction and paradoxically reduced capillary perfusion.     

Relationship between the major phosphorylated metabolic intermediates and oxygen affinity of whole blood in the loggerhead (Caretta caretta) and the Green Sea Turtle (Chelonia mydas) during development.

(1) 2,3-Diphosphoglyceric acid (2,3-DPG) is present in the erythrocytes (RBC) of the 68-day loggerhead turtle embryo and 44-day green sea turtle embryo at levels of 7.4 and 5.5 μmoles/ml of RBC, representing the major organic phosphate during the latter period of embryonic development. (2) Inositol pentaphosphate (IPP) is absent in the red blood cells of the embryos of both the loggerhead and green sea turtle. (3) Near equimolar amounts of 2,3-DPG and IPP are present in the erythrocytes of the adult loggerhead and green sea turtle. The total concentration of these two organic phosphates is approximately 0.75 μmoles/ml of RBC in the adult of both species. (4) There is a switch from embryonic to adult hemoglobin during development of these two species of turtles; the two embryonic bands have identical electrophoretic mobilities, whereas the two adult bands migrate differently on cellulose acetate at pH 8.6. (5) The whole blood oxygen affinity of the adult loggerhead and green sea turtle is 60.3 and 32.6 Torr, respectively. (6) The stripped adult hemoglobins in these two species of turtles show no change in oxygen affinity upon addition of 2,3-DPG, ATP, or IPP. (7) It therefore appears unlikely that whole blood oxygen affinity is controlled by organic phosphate modulation of hemoglobin function in these species of turtles.     

HDP-a novel heme detoxification protein from the malaria parasite

When malaria parasites infect host red blood cells (RBC) and proteolyze hemoglobin, a unique, albeit poorly understood parasite-specific mechanism, detoxifies released heme into hemozoin (Hz). Here, we report the identification and characterization of a novel Plasmodium Heme Detoxification Protein (HDP) that is extremely potent in converting heme into Hz. HDP is functionally conserved across Plasmodium genus and its gene locus could not be disrupted. Once expressed, the parasite utilizes a circuitous "Outbound-Inbound" trafficking route by initially secreting HDP into the cytosol of infected RBC. A subsequent endocytosis of host cytosol (and hemoglobin) delivers HDP to the food vacuole (FV), the site of Hz formation. As Hz formation is critical for survival, involvement of HDP in this process suggests that it could be a malaria drug target.     

In vivo photoacoustic and photothermal cytometry for monitoring multiple blood rheology parameters

Alterations of blood rheology (hemorheology) are important for the early diagnosis, prognosis, and prevention of many diseases, including myocardial infarction, stroke, sickle cell anemia, thromboembolism, trauma, inflammation, and malignancy. However, real-time in vivo assessment of multiple hemorheological parameters over long periods of time has not been reported. Here, we review the capabilities of label-free photoacoustic (PA) and photothermal (PT) flow cytometry for dynamic monitoring of hemorhelogical parameters in vivo which we refer to as photoacoustic and photothermal blood rheology. Using phenomenological models, we analyze correlations between both PT and PA signal characteristics in the dynamic modes and following determinants of blood rheology: red blood cell (RBC) aggregation, deformability, shape (e.g., as in sickle cells), intracellular hemoglobin distribution, individual cell velocity, hematocrit, and likely shear rate. We present ex vivo and in vivo experimental verifications involving high-speed PT imaging of RBCs, identification of sickle cells in a mouse model of human sickle cell disease and in vivo monitoring of complex hemorheological changes (e.g., RBC deformability, hematocrit and RBC aggregation). The multi-parameter platform that integrates PT, PA, and conventional optical techniques has potential for translation to clinical applications using safe, portable, laser-based medical devices for point-of-care screening of disease progression and therapy efficiency.