Transplant-associated thrombotic microangiopathy (TA-TMA) and acute graft-versa-host disease (aGVHD) are typically manifesting within the first 100 days post-transplant. These conditions represent critical factors influencing the success of HSCT, with aGVHD being the most prevalent, but TA-TMA poses a greater threat due to its rapid progression and high mortality rate (1–7). Diagnosis is usually based on the criteria stipulated by either Europe (European Bone Marrow Transplant International Working Group) (8) or United States (Blood and Marrow Transplantation Clinical Trial Network) (9), but the uniformity of these standards remains to be studied. Grade 2–4 aGVHD was found to be an adjusted risk factor for TA-TMA (10) and lead to an increased risk of death in TA-TMA (11), but further research is needed to understand the overlap of disease mechanisms and the underlying relationships. aGVHD usually occurs in the acute phase after allo-HSCT, when TA-TMA is most commonly observed (5). Both TA-TMA and grade III-IV aGVHD exhibit numerous overlapping clinical symptoms, making it more difficult to distinguish between these two diseases. To date, there have been no reports on distinguishing TA-TMA from aGVHD, an accurate diagnosis and early start of etiological treatment that alter the natural history of these serious conditions may best improve patient outcomes.

TA-TMA is a microangiopathic hemolytic anemia and platelet consumption resulting from complement-mediated endothelial injury following allo-HSCT, resulting in microvascular thrombosis and fibrin deposition in the microcirculation (16). aGVHD, another more common and early complication after allo-HSCT, is induced by the direct endothelial injury mediated by host-reactive, donor-derived cytotoxic T cells (5). TA-TMA and grade 2–4 aGVHD are recognized risk factors for mortality after allo-HSCT (5, 17, 18), the clinical differentiation of the two represents a diagnostic challenge for transplant clinicians. All TA-TMAs in this study occurred during the process of GVHD. In our study, the cumulative mortality rate of TA-TMA was 90.9%, while the cumulative mortality rate of grade III-IV aGVHD was also as high as 72.0%, both of which are serious complications after allo-HSCT. Most studies have shown that the occurrence time of TA-TMA is between 32 days and 303 days after HSCT (5, 13, 17–25). In our study, the median onset time of TA-TMA was the 94th day after allo-HSCT, which was later than that of aGVHD, indicating that the occurrence of TA-TMA is more insidious, and may have a slow onset but a dangerous development.

The impact of various transplantation factors on TA-TMA has been reported by multiple institutions, including aGVHD, sex, prior autologous transplant, GVHD prophylaxis, donor type, source of stem cells (25, 26). And factors related to the risk of aGVHD are mainly reported as age and donor type (27–29). Our results showed no obvious tendency in age and gender between TA-TMA and aGVHD, nor were there any notable differences in the transplant-related factors examined in this study. To date, no literature has concurrently compared and analyzed these factors in both TA-TMA and aGVHD, precluding reference to differing outcomes. It is therefore worthy to incorporate a broader range of transplant-related indicators in future multi-center and long-term studies.

In 2012, the International Council for Standardization in Hematology (ICSH) proposed that more than 1% of morphologically identified schistocytes on the blood film are considered suspicious for TMA (30). Report for 2021 suggested that the detection of more than 1% schistocytes on PB smears in adults and full-term neonates remained a robust cytomorphological threshold that favors a diagnosis of TMA (31). In our study, the proportion of fragmented red blood cells in TA-TMA was more than 1%, which was consistent with the literature reports. The proportion of fragmented red blood cells in TA-TMA was significantly higher compared to those in aGVHD, and the decrease in the proportion of these fragmented cells can reduce the risk of TA-TMA. The increased proportion of fragmented red blood cells elevated the risk of death of TA-TMA by 80%, which can independently predict TA-TMA, highlighting its close association with the occurrence, development and prognosis of TA-TMA. On the other hand, it is worth noting that although all TA-TMA patients in this study consistently exhibited fragmented red blood cells in peripheral blood, a lack such cells does not exclude a priori the diagnosis of TMA, it had been reported that the absence of fragmented red blood cells in patient with TA-TMA had led to a delay diagnosis and treatment (32), emphasizing the importance of fragmented red blood cells combined with specific laboratory tests and comprehensive analysis.

Not only that, the injury to vascular endothelial cells in TA-TMA patients may lead to microangiopathic hemolytic anemia and thrombocytopenia, as well as thrombosis in the microcirculation (33, 34). These adverse effects may subsequently cause ischemic damage to end-organs, particularly the kidneys, as well as the lungs, heart, and gastrointestinal tract. In contrast, the initial phase of aGVHD is characterized by tissue damage mediated through the regulation and release of inflammatory cytokines, primarily affecting three areas: the skin, gastrointestinal tract, and liver (35). Based on the different pathogenesis and the analysis results of this study, we identified five symptoms that are more likely to co-occur with TA-TMA: hypertension, renal injury, cardiac insufficiency, pulmonary infection, and CMV-emia. Additionally, three significantly elevated indicators in TA-TMA patients were identified: proportion of fragmented red blood cells in peripheral blood, serum LDH levels, and serum BUN levels. Two significantly reduced indicators were also noted: red blood cell counts and hemoglobin levels. For grade III-IV aGVHD, TBIL levels were higher and associated with a higher risk of death, and increased serum TBIL was a unique marker of hepatic involvement (35), suggesting that patients with grades III-IV aGVHD are more likely to liver involvement. Although TA-TMA was associated with a higher incidence of hemorrhagic cystitis and urinary occult blood and a reduction in platelet and ALB levels, these indicators were similarly observed in our grade III-IV aGVHD patients, therefore, hemorrhagic cystitis, urinary occult blood, platelets counts, and ALB levels are more significant only when differentiating between TA-TMA and grade I-II aGVHD cases.

Existing studies have reported varying evidence of the prognostic impact of LDH and renal injury in TA-TMA. High baseline LDH level was associated with definite and probable TMA, definite and probable TMA were each independently associated with long-term kidney dysfunction (19), elevated LDH levels, proteinuria on routine urinalysis, and hypertension were the earliest markers of TMA (13). HSCT-TMA was associated with renal dysfunction, renal failure, renal replacement therapy and hypertension (36). Our findings determined that LDH elevation and renal injury were more likely significantly associated with an increased risk of TA-TMA. On this basis, we also noticed that elevated LDH, and renal injury predicted higher mortality for TA-TMA, representing one of the novel findings of this study. This finding is consistent with recent literature indicating that the ASTCT/CIBMTR/EBMT/APBMT consensus risk stratification for pediatric TA-TMA identifies LDH >2 times ULN is one of the most important predictors of nonrelapse mortality of TA-TMA, allowing risk stratification even in the absence of available sC5b-9 testing (37). Although proteinuria is closely related to TA-TMA, our study did not reveal a significant difference in the incidence of proteinuria between TA-TMA and aGVHD, so no further analysis was conducted, considering that proteinuria frequently occurs when aGVHD involves the kidneys. These data suggest that more transplant recipients should be included in our subsequent studies and clinical trials to verify the discriminatory and prognostic value of proteinuria between these two diseases.

A pre-emptive strategy has successfully decreased the incidence of CMV disease after allo-HSCT, however, it is difficult to completely prevent that (38). Positive CMV serostatus is also associated with probable TMA (19), which may act as an independent risk factor for TA-TMA and directly damage microvascular endothelial cells, causing TMA (13, 14). Kraft S’s research showed that CMV reactivation/end-organ disease was a risk factor for development of TA-TMA (5), only a limited number of studies suggested that the risk factors for TA-TMA were not associated with CMV-emia (28). In this study, no significant difference was observed in the CMV positivity rate between TA-TMA and aGVHD prior to transplantation. However, at the time of diagnosis, the CMV positivity rate of TA-TMA was significantly higher than that of aGVHD. Compared to grade I-II and III-IV aGVHD, post-transplant CMV viremia may increase the risk of TA-TMA development and death, although the result was not statistically significant after adjusting for risk variables. Regardless, post-transplant CMV viremia should not be ignored, while considering aGVHD after transplantation, clinicians should be more vigilant for the development of TA-TMA.

Hemoglobin levels, platelet counts, ALB levels were identified as protective factors for the occurrence, development, and prognosis of TA-TMA in our study. Severe anemia and severe thrombocytopenia had been recognized as significant prognostic indicators in a large-scale external validation trial of the TA-TMA prognostic model (39), and our findings are in agreement with the existing literature reports. Furthermore, we found that the levels of hemoglobin and platelets in TA-TMA were significantly lower than those in aGVHD, which has certain clinical value for the differential diagnosis between TA-TMA and aGVHD, and increased hemoglobin and platelet levels can reduce the risk of death for TA-TMA and grade III-IV aGVHD, serving as protective factors against the death for TA-TMA and grade III-IV aGVHD. Notably, platelet levels retained independent prognostic significance for TA-TMA even after adjusting for various risk variables. Although platelets are often at low levels due to the insufficiency of megakaryocyte engraftment in the early stages after allo-HSCT, the onset of TA-TMA typically occurs later than that of aGVHD, so we speculate that it is not difficult to exclude the interference of low platelet levels in the differential diagnosis of TA-TMA and aGVHD at this time, despite the current inability to fully evaluate this hypothesis. Furthermore early post-allo-HSCT studies are warranted to validate the applicability of this finding in contemporary patient cohorts. In addition, ALB has been identified as an independent prognostic factor following allo-HSCT in leukemia patients, capable of predicting long-term outcomes (40). In this study, ALB levels in TA-TMA and grade III-IV aGVHD were significantly lower compared to those in grade I-II aGVHD, and was significantly associated with a reduced risk of mortality in TA-TMA, acting as an independent predictor of favorable prognosis for TA-TMA. Patients with TA-TMA may consume ALB through urinary albumin loss and inflammation. The underlying mechanism is primarily associated with kidney lesions and inflammation resulting from microvascular endothelial injury, while ALB depletion in aGVHD is mainly attributed to protein loss caused by gastrointestinal injury, so in clinical practice, dynamic monitoring of ALB levels should be integrated with other biomarkers for a comprehensive evaluation. Despite limitations of sample size and retrospective design, our exploratory analysis revealed a non-significant trend toward improved survival with complement inhibitor in TA-TMA. Recent studies have demonstrated that complement inhibitors can markedly enhance survival outcomes in both adult and pediatric patients with TA-TMA (3, 41), suggesting a potential survival benefit for high-risk individuals. Prospective studies are urgently needed to definitively establish the efficacy of targeting the complement pathway in high-risk TA-TMA.

To our knowledge, this study provides one of the first comprehensive analyses aimed at differentiating TA-TMA from aGVHD following transplantation. Our findings suggest that fragmented red blood cells, renal injury, and serum LDH levels may serve as independent diagnostic indicators for distinguishing TA-TMA from aGVHD. Furthermore, hemoglobin levels appear to have diagnostic utility in differentiating TA-TMA from severe (grade III-IV) aGVHD. In terms of prognosis, our data provide preliminary evidence that fragmented red blood cells, LDH, and renal injury are associated with an increased risk of mortality, whereas platelet counts and ALB levels may be potential protective factors in TA-TMA patients.

There are some limitations in this study, due to the limited sample size and the large range of screened variables, there is no further analysis of all potentially significant and important indicators. An important aspect of our study design was the comparative analysis of TA-TMA and aGVHD as mutually exclusive groups, despite the established fact that aGVHD is a significant risk factor for TA-TMA development. Our rationale for this approach was pragmatic and aimed at addressing a specific clinical question: at the moment of initial clinical deterioration, which parameters can most effectively help the clinician distinguish whether the primary driver is aGVHD or TA-TMA? We sought to delineate the relatively “pure” clinical profiles of each entity at onset. We acknowledge that this design is a simplification and does not capture the complexity of cases where both entities manifest simultaneously or sequentially. Future dynamic studies should build upon to unravel their complex interplay. In addition, the small sample size and single-center design limit the statistical power and generalizability of our findings. The prognostic outcomes of TA-TMA in this study, particularly the independent prognostic factors identified through multivariate analysis-including schistocytes, lactate dehydrogenase, renal injury, platelets, and albumin should be regarded as exploratory preliminary results. These findings require validation in larger sample sizes and should not be considered definitive independent prognostic factors. The primary value of this study lies in its detailed descriptions and the provision of valuable real-world insights, which offer robust hypotheses for future research.

Based on our findings, we recommend a rapid TA-TMA diagnostic approach using key lab parameters (Hb, fragmented RBCs, LDH, renal injury), alongside monitoring platelets and albumin for risk stratification and renal protection. Future studies should investigate the protective roles of these factors, assess early interventions, and validate the survival benefit of complement blockade in high-risk patients.