Validation of a SCID mouse model for transfusion by concurrent comparison of circulation kinetics of human platelets, stored under various temperature conditions, between human volunteers and mice.

BACKGROUND: Initial evaluation of new platelet (PLT) products for transfusion includes a clinical study to determine in vivo recovery and survival of autologous radiolabeled PLTs in healthy volunteers. These studies are expensive and do not always produce the desired results. A validated animal model of human PLTs in vivo survival and recovery used pre-clinically could reduce the risk of failing to advance product development. STUDY DESIGN AND METHODS: An immunodeficient (SCID) mouse model to evaluate recovery of human PLTs was compared to a radiolabeling study in human volunteers. Autologous apheresis PLTs stored for 7 days at room temperature (RT), thermo-cycled (TC), and cold temperature (CT) were radiolabeled and infused into healthy humans (n = 16). The same PLTs, non-radiolabeled, were also infused into mice (n = 160) on the same day. Blood samples from humans and mice were collected to generate clearance curves of PLTs in circulation. Flow cytometry was used to detect human PLTs in mouse blood. RESULTS: Human and mouse PLTs were cleared with one phase exponential clearance. Relative differences for initial recovery and AUC, expressed as ratio of test and control PLTs, were similar in humans and mice. The initial recovery ratio of TC/RT was 0.73 ± 0.07 in humans and 0.67 ± 0.14 in mice. The ratio for CT/TC was 0.53 ± 0.06 in humans and 0.75 ± 0.18 in mice. CONCLUSION: The SCID mouse model can provide information on relative differences of initial in vivo recovery and AUC between control and alternatively stored/processed human PLTs that is predictive of performance in healthy human volunteers.

Temperature cycling during platelet cold storage improves in vivo recovery and survival in healthy volunteers.

BACKGROUND: Room temperature (RT) storage of platelets (PLTs) can support bacterial proliferation in contaminated units, which can lead to transfusion-transmitted septic reactions. Cold temperature storage of PLTs could reduce bacterial proliferation but cold exposure produces activation-like changes in PLTs and leads to their rapid clearance from circulation. Cold-induced changes are reversible by warming and periodic rewarming during cold storage (temperature cycling [TC]) has been proposed to alleviate cold-induced reduction in PLT circulation. STUDY DESIGN AND METHODS: A clinical trial in healthy human volunteers was designed to compare in vivo recovery, survival, and area under the curve (AUC) of radiolabeled autologous apheresis PLTs stored for 7 days at RT or under TC or cold conditions. Paired comparisons of RT versus TC and TC versus cold PLTs were conducted. RESULTS: Room temperature PLTs had in vivo recovery of 55.7 ± 13.9%, survival of 161.3 ± 28.8 hours, and AUC of 5031.2 ± 1643.3. TC PLTs had recovery of 42.6 ± 16.4%, survival of 48.1 ± 14.4% hours, and AUC of 1331.3 ± 910.2 (n = 12, p < 0.05). In a separate paired comparison, cold PLTs had recovery of 23.1 ± 8.8%, survival of 33.7 ± 14.7 hours, and AUC of 540.2 ± 229.6 while TC PLTs had recovery of 36.5 ± 12.9%, survival of 49.0 ± 17.3 hours, and AUC of 1164.3 ± 622.2 (n = 4, AUC had p < 0.05). CONCLUSION: TC storage for 7 days produced PLTs with better in vivo circulation kinetics than cold storage but is not equivalent to RT storage.

Macrophage Depletion Mitigates Platelet Aggregate Formation in Splenic Marginal Zone and Alleviates LPS-Associated Thrombocytopenia in Rats.

Sepsis is often accompanied with thrombocytopenia partly due to platelet sequestration in the lung and liver. The spleen can store up to one-third of circulating platelets and can also significantly affect platelet transfusion outcomes by accumulating platelets. However, in sepsis, it is not clear whether there are platelet changes in the spleen which could contribute to sepsis-associated thrombocytopenia and also influence platelet transfusion outcomes. By using confocal microscopy, we examined endogenous rat platelets and infused human platelets in the spleen of severe combined immune deficient Rag2 KO rats which were injected intraperitoneally with lipopolysaccharide (LPS). LPS-injected Rag2 KO rats developed sepsis as indicated by increased TNFa, IL-6, IL-1b, and IL-10 levels and thrombocytopenia. Large platelet aggregates were observed in the spleen with majority located in the marginal zone and closely associated with CD169+ macrophages. Depletion of macrophages by clodrosome resulted in reduction of LPS-induced cytokine generation and alleviated LPS-induced thrombocytopenia. Macrophage depletion also remarkedly diminished large platelet aggregate formation in the splenic marginal zone but had less effect on those in red pulp. Infusion of human platelets into LPS-injected rats failed to raise platelet counts in the peripheral blood. In LPS-injected rat spleen, human platelets interacted with aggregated rat platelets in the marginal zone. In contrast, human platelets infused into control rats were located outside of splenic marginal zone. This study provides morphological evidence of platelet aggregates in the splenic marginal zone in sepsis which can interact with infused platelets and thus can contribute to platelet infusion refractoriness in sepsis. It indicates that macrophages play an important role in LPS-associated thrombocytopenia. It also suggests that CD169+ macrophages support platelet aggregate formation in the splenic marginal zone.