Identifying Erythrocyte Injury in Toxicology Studies

Abstract

The hematological impacts of a drug can affect erythropoiesis at the level of the bone marrow, or decrease the life span of the RBC (red blood cell). The most common and recognizable clinical manifestation of either type of drug-induced erythropoietic injury is a decrease in RBC mass, or what is clinically referred to as an anemia. A decrease in RBC production can generally be separated from increased destruction (hemolysis) by evaluation of the hemogram for evidence of regeneration. In most healthy mammalian species, hemolysis will result in a regenerative response characterized by an increase in circulating reticulocytes. Hemorrhage as an alternative cause of a regenerative anemia can generally be excluded by careful clinical evaluation of the animal. Subsequently, the investigation of a drug-induced regenerative anemia should involve a very thorough evaluation of RBC morphology for evidence of immune-mediated destruction, RBC oxidative injury, and fragmentation that can help to identify the underlying pathological mechanism(s) involved.

A hemolytic anemia, either intravascular or extravascular, is often easily recognizable in toxicology studies, as the pertinent RBC (red blood cell) parameters (HCT [hematocrit], Hgb (hemoglobin), RBC count) are negatively impacted. The recognition of an intravascular component relies on parameters that are often related to the rate and or severity of the hemolysis, including hemoglobinemia, hemoglobinuria, and serum haptoglobin or bilirubin concentrations. Low levels of hemolysis can be hidden from detection by hemoglobin scavenging mechanisms. These scavenging mechanisms serve to protect the vasculature from the damaging impact of predominantly iron. However, other injurious substances reside within the RBC and localized vascular inflammation remains a possibility. A hemolytic anemia might also manifest as increased iron deposition in tissues or observation of hemosiderophages, which can be recognized on histopathology with the use of special stains such as Perls’ stain or Prussian blue.

There are classic morphologies associated with hemolysis, spherocytes, and ghost cells, for example, whereas other RBC changes are typically considered clinically benign in terms of hemolysis. Several detailed studies have been published documenting how drug-induced hemolytic anemias can be mechanistically evaluated in both these situations. One example is a report documenting a hemolytic anemia due to acanthocytosis and spherocytosis in rats given a CXCR3 receptor antagonist. ¹ The core hematological parameters indicated an anemia marked by a robust reticulocytotic, leading the authors to perform a detailed evaluation of RBC morphology, ex vivo fragility, and in vitro hemolysis. ¹ Spherocytosis is well-recognized as an indicator of hemolysis and acanthocytosis has been documented as a morphology associated with hemolytic anemias in Beagles fed a high-cholesterol diet and in several rodent models that alter lipid metabolism.^2-4 Thus, the morphologies observed in this report are consistent with a process of hemolysis. Conversely, in a study evaluating miltefosine, the RBC alteration that manifested in a dose-responsive manner was a profound echinocytosis.^5,6 Aside from snake envenomation and alternative sources of phospholipases, echinocyte formation is not a morphology typically associated with hemolysis. In addition, the report documented a shift in the hemolytic curves determining the concentration of the drug that causes hemolysis of 50% of the RBC (CH50). Blood with a lower HCT hemolyzed far more severely (lower CH50) than a normal HCT blood.^5,6 Early safety studies would not identify this type of impact, as healthy animals are evaluated. However, this type of titrational study could be done in vitro at early stages if the compound was anticipated to interact with cell membranes (e.g., is amphipathic in nature). The magnitude of hemolysis caused by a cationic cellulose nanoparticle was more severe at lower concentrations, likely due to the distribution of the particle in the RBC membrane. ⁷ This is a recognized phenomenon, and the investigators were actively exploring the concern. These findings highlight the need to think prospectively about the physical nature of the substance being evaluated and the nature of the RBC membrane.

We have documented lipid-induced hemolysis in response to a parenteral lipid emulsion used in small animals for nutritional support. ⁸ Similar to the HCT/hemolysis trend reported in the miltefosine report, we showed hemolysis was more severe in blood collected from dogs with documented inflammation. We hypothesize that oxidative injury to the RBC was a predisposing factor. This type of effect might only become evident when a drug reaches an ill patient population. However, detailed analysis of RBC structure (e.g., electron microscopy) and function (osmotic fragility testing, kinetic testing, evaluation of lipid composition or fluidity) may increase the sensitivity of the evaluation and put low-level injuries on the radar.

Overt hemolytic anemias can be mechanistically dissected by a thorough evaluation of RBC morphology, whereas recognition of low levels of hemolysis may be more occult. In addition, changes in cell morphology can indicate insertion of the drug into the RBC membrane, aberrations in lipid metabolism, or altered organ function. In these cases, a careful inspection of RBC indices, morphology, and enumerating reticulocytes can point toward RBC changes that may be injurious in the presence, or absence, of a decrease in RBC mass that we know can elicit fatigue, shortness of breath, and hypoxic tissue injury that are impactful on day-to-day activities. To truly evaluate the health of the RBC lineage, we should consider the relative rate of production and destruction. If destruction is accelerated, but the bone marrow responds with accelerated and/or increased erythropoietic drive, the animal may preserve enough RBC mass to avoid consequences associated with hypoxia. This does not mean there are no biological consequence to the hemolysis. There are two main pathologies we should consider when using decreased RBC mass as a threshold for further evaluation of the erythroid lineage. One, a prolonged increase in erythropoietic drive is hypothesized to elicit pathologic changes in the bone marrow. Second, an intravascular hemolysis releases pro-inflammatory substances that can damage the vasculature and potentially increase the risk of thrombosis.^9-11

One example of a drug known to bear an increased risk of thrombosis is the chemotherapeutic agent paclitaxel. Drug-induced changes to RBCs likely contribute to this risk. Paclitaxel causes increased exposure of phosphatidyl serine on exposed RBCs. Importantly, this exposure was associated with the expected increase in thrombin generation. ¹² Cancer itself can be prothrombotic and cancer-associated thrombosis is an area of robust investigation. The potential contribution of the RBC membrane to this process warrants consideration. In addition, the release of prothrombotic substances normally sequestered within the RBC membrane (mature RBCs lack organelles) including nucleosides, nitric oxide scavengers, and iron could tip the scales of coagulation in the wrong direction in these high-risk patients administered a drug with even a low degree of hemolysis. The threshold of acceptable hemolysis is more complex than adequate oxygen-carrying capacity. Clearly defining a hemolytic threshold that is acceptable is complicated by the need to separate anemia from the potential oxidative injury and inflammatory impacts of hemolysis. The literature supports a 2% threshold as nonhemolytic, but the rationale and data behind that threshold do not appear readily apparent.^7,13

There are several methods that can be used to explore the structure and function of the RBC if an injury is suspected or overt. Accelerated damage to the RBC, particularly oxidative damage, can result in an increase in the proportion of denser RBCs as detected by density gradient sedimentation. If oxidative injury is suspected based on morphology and/or increased reticulocyte counts, evaluation options can include ratios of co-factors (e.g., [NADH/NAD++ NADH]), quantification of specific enzymes (e.g., glutathione reductase), or measurement of known damaging metabolites (e.g., lipoperoxides). ¹⁴ Injection of biotinylated RBCs can be used to quantify RBC turnover. ⁴ Digital methods are emerging that allow investigators to score a high number of RBCs (e.g., blood smear evaluations using digital methods and artificial intelligence algorithms), or use flow cytometric nonsphered approach to evaluate morphology in an automated system. There are reports using the CellaVision system to evaluate RBC morphology, but one must verify the proper classification of the morphology. ¹⁵ These newer methods could be considered on an as-needed basis, but would likely require additional validation in the pertinent veterinary specie(s).

With every challenge comes opportunity. The development of therapeutic nanoparticles, lipid-encapsulated drugs, and drugs that use the RBC as a delivery device may lead to more RBC pathologies identified in safety studies that require mechanistic investigation. Patient population needs, such as the need for long-term administration of drugs or use of therapeutics in anemic patients, may drive a reconsideration of how findings in early safety studies might inform parameters including in subsequent clinical trials.