Improved Cryopreservation of Human Induced Pluripotent Stem Cell (iPSC) and iPSC-derived Neurons Using Ice-Recrystallization Inhibitors.

Human induced pluripotent stem cells (iPSCs) and iPSC-derived neurons (iPSC-Ns) represent a differentiated modality toward developing novel cell-based therapies for regenerative medicine. However, the successful application of iPSC-Ns in cell-replacement therapies relies on effective cryopreservation. In this study, we investigated the role of ice recrystallization inhibitors (IRIs) as novel cryoprotectants for iPSCs and terminally differentiated iPSC-Ns. We found that one class of IRIs, N-aryl-D-aldonamides (specifically 2FA), increased iPSC post-thaw viability and recovery with no adverse effect on iPSC pluripotency. While 2FA supplementation did not significantly improve iPSC-N cell post-thaw viability, we observed that 2FA cryopreserved iPSC-Ns re-established robust neuronal network activity and synaptic function much earlier compared to CS10 cryopreserved controls. The 2FA cryopreserved iPSC-Ns retained expression of key neuronal specific and terminally differentiated markers and displayed functional electrophysiological and neuropharmacological responses following treatment with neuroactive agonists and antagonists. We demonstrate how optimizing cryopreservation media formulations with IRIs represents a promising strategy to improve functional cryopreservation of iPSCs and post-mitotic iPSC-Ns, the latter of which have been challenging to achieve. Developing IRI enabling technologies to support an effective cryopreservation and an efficiently managed cryo-chain is fundamental to support the delivery of successful iPSC-derived therapies to the clinic.

An economic and robust TMT labeling approach for high throughput proteomic and metaproteomic analysis.

Multiplexed quantitative proteomics using tandem mass tag (TMT) is increasingly used in -omic study of complex samples. While TMT-based proteomics has the advantages of the higher quantitative accuracy, fewer missing values, and reduced instrument analysis time, it is limited by the additional reagent cost. In addition, current TMT labeling workflows involve repeated small volume pipetting of reagents in volatile solvents, which may increase the sample-to-sample variations and is not readily suitable for high throughput applications. In this study, we demonstrated that the TMT labeling procedures could be streamlined by using pre-aliquoted dry TMT reagents in a 96 well plate or 12-tube strip. As little as 50 µg dry TMT per channel was used to label 6-12 µg peptides, yielding high TMT labeling efficiency (∼99%) in both microbiome and mammalian cell line samples. We applied this workflow to analyze 97 samples in a study to evaluate whether ice recrystallization inhibitors improve the cultivability and activity of frozen microbiota. The results demonstrated tight sample clustering corresponding to groups and consistent microbiome responses to prebiotic treatments. This study supports the use of TMT reagents that are pre-aliquoted, dried, and stored for robust quantitative proteomics and metaproteomics in high throughput applications.

In vivo methods for imaging blood-brain barrier function and dysfunction.

The blood-brain barrier (BBB) is the interface between the central nervous system and systemic circulation. It tightly regulates what enters and is removed from the brain parenchyma and is fundamental in maintaining brain homeostasis. Increasingly, the BBB is recognised as having a significant role in numerous neurological disorders, ranging from acute disorders (traumatic brain injury, stroke, seizures) to chronic neurodegeneration (Alzheimer's disease, vascular dementia, small vessel disease). Numerous approaches have been developed to study the BBB in vitro, in vivo, and ex vivo. The complex multicellular structure and effects of disease are difficult to recreate accurately in vitro, and functional aspects of the BBB cannot be easily studied ex vivo. As such, the value of in vivo methods to study the intact BBB cannot be overstated. This review discusses the structure and function of the BBB and how these are affected in diseases. It then discusses in depth several established and novel methods for imaging the BBB in vivo, with a focus on MRI, nuclear imaging, and high-resolution intravital fluorescence microscopy.

Inhibition of ice recrystallization during cryopreservation of cord blood grafts improves platelet engraftment.

BACKGROUND: Platelet engraftment following cord blood (CB) transplantation remains a significant hurdle to this day. The uncontrolled growth of ice, a process referred to as ice recrystallization, is one of several mechanisms that lead to cell loss and decreased potency during freezing and thawing. We hypothesized that reducing cell damage induced by ice recrystallization in CB units (CBUs) would reduce losses of stem and progenitor cells and therefore improve engraftment. We previously demonstrated that the ice recrystallization inhibitor (IRI) N-(2-fluorophenyl)-D-gluconamide (IRI 2) increases the postthaw recovery of CB progenitors. Herein, we set out to ascertain whether IRI 2 can enhance platelet and bone marrow engraftment activity of hematopoietic stem cells (HSCs) in cryopreserved CBUs using a serial transplantation model. STUDY DESIGN AND METHODS: CBUs were processed following standard volume/red blood cell reduction procedure and portions frozen with dimethyl sulfoxide (DMSO) supplemented or not with IRI 2. Thawed CB samples were serially transplanted into immunodeficient mice. RESULTS: Our results show that supplementation of DMSO with IRI 2 had several beneficial effects. Specifically, higher levels of human platelets were observed in the peripheral blood (p < 0.05; n = 4) upon transplant of CBUs preserved with the IRIs. In addition, human BM chimerism and the number of human CFU progenitors in the bone marrow were superior in IRI 2 recipients compared to DMSO recipients. Moreover, IRI 2 had no negative impact on the multilineage differentiation and self-renewal activities of HSCs. DISCUSSION: Taken together, these results demonstrate that supplementation of a hematopoietic graft with IRI can improve the postthaw engraftment activities of HSCs.

Trehalose-Based Polyethers for Cryopreservation and Three-Dimensional Cell Scaffolds.

The capability to slow ice growth and recrystallization is compulsory in the cryopreservation of cells and tissues to avoid injuries associated with the physical and chemical responses of freezing and thawing. Cryoprotective agents (CPAs) have been used to restrain cryoinjury and improve cell survival, but some of these compounds pose greater risks for the clinical application of cryopreserved cells due to their inherent toxicity. Trehalose is known for its unique physicochemical properties and its interaction with the phospholipids of the plasma membrane, which can reduce cell osmotic stress and stabilized the cryopreserved cells. Nonetheless, there has been a shortage of relevant studies on the synthesis of trehalose-based CPAs. We hereby report the synthesis and evaluation of a trehalose-based polymer and hydrogel and its use as a cryoprotectant and three-dimensional (3D) cell scaffold for cell encapsulation and organoid production. In vitro cytotoxicity studies with the trehalose-based polymers (poly(Tre-ECH)) demonstrated biocompatibility up to 100 mg/mL. High post-thaw cell membrane integrity and post-thaw cell plating efficiencies were achieved after 24 h of incubation with skin fibroblast, HeLa (cervical), and PC3 (prostate) cancer cell lines under both controlled-rate and ultrarapid freezing protocols. Differential scanning calorimetry and a splat cooling assay for the determination of ice recrystallization inhibition activity corroborated the unique properties of these trehalose-based polyethers as cryoprotectants. Furthermore, the ability to form hydrogels as 3D cell scaffolds encourages the use of these novel polymers in the development of cell organoids and cryopreservation platforms.

Characterizing the ability of an ice recrystallization inhibitor to improve platelet cryopreservation.

Improving aspects of platelet cryopreservation would help ease logistical challenges and potentially expand the utility of frozen platelets. Current cryopreservation procedures damage platelets, which may be caused by ice recrystallization. We hypothesized that the addition of a small molecule ice recrystallization inhibitor (IRI) to platelets prior to freezing may reduce cryopreservation-induced damage and/or improve the logistics of freezing and storage. Platelets were frozen using standard conditions of 5-6% dimethyl sulfoxide (Me2SO) or with supplementation of an IRI, N-(2-fluorophenyl)-d-gluconamide (2FA), prior to storage at -80 °C. Alternatively, platelets were frozen with 5-6% Me2SO at -30 °C or with 3% Me2SO at -80 °C with or without 2FA supplementation. Supplementation of platelets with 2FA improved platelet recovery following storage under standard conditions (p = 0.0017) and with 3% Me2SO (p = 0.0461) but not at -30 °C (p = 0.0835). 2FA supplementation was protective for GPVI expression under standard conditions (p = 0.0011) and with 3% Me2SO (p = 0.0042). Markers of platelet activation, such as phosphatidylserine externalization and microparticle release, were increased following storage at -30 °C or with 3% Me2SO, and 2FA showed no protective effect. Platelet function remained similar regardless of 2FA, although functionality was reduced following storage at -30 °C or with 3% Me2SO compared to standard cryopreserved platelets. While the addition of 2FA to platelets provided a small level of protection for some quality parameters, it was unable to prevent alterations to the majority of in vitro parameters. Therefore, it is unlikely that ice recrystallization is the major cause of cryopreservation-induced damage.

Modulating Intracellular Ice Growth with Cell-Permeating Small-Molecule Ice Recrystallization Inhibitors.

Ice formation remains central to our understanding of the effects of low temperatures on the biological response of cells and tissues. The formation of ice inside of cells and the net increase in crystal size due to recrystallization during thawing is associated with a loss of cell viability during cryopreservation. Because small-molecule ice recrystallization inhibitors (IRIs) can control the growth of extracellular ice, we sought to investigate the ability of two aryl-glycoside-based IRIs to permeate into cells and control intracellular ice recrystallization. An interrupted graded freezing technique was used to evaluate the IRI permeation into human red blood cells (RBCs) and mitigate cell damage during freezing and thawing. The effect of IRIs on the intracellular growth of ice crystals in human umbilical vein endothelial cells (HUVECs) was visualized in real time under different thawing conditions using fluorescence cryomicroscopy. Adding an aryl glycoside to 15% glycerol significantly increased post-thaw RBC integrity by up to 55% during slow cooling compared with the 15%-glycerol-only control group. The characteristics of the cryobiological behavior of the RBCs subjected to the interrupted graded freezing suggest that the aryl-glycoside-based IRI is internalized into the RBCs. HUVECs treated with the IRIs were shown to retain a large number of small ice crystals during warming to high subzero temperatures and demonstrated a significant inhibition of intracellular ice recrystallization. Under slow thawing conditions, the aryl glycoside IRI p-bromophenyl-ß-d-glucoside was shown to be most effective at inhibiting intracellular ice recrystallization. We demonstrate that IRIs are capable of internalizing into cells, altering the cryobiological response of cells to slow and rapid freezing and controlling intracellular ice recrystallization during freezing. We conclude that IRIs have tremendous potential as cryoprotectants for the preservation of cells and tissues at high subzero temperatures.

Presence of a basic secretory protein in xylem sap and shoots of poplar in winter and its physicochemical activities against winter environmental conditions.

XSP25, previously shown to be the most abundant hydrophilic protein in xylem sap of Populus nigra in winter, belongs to a secretory protein family in which the arrangement of basic and acidic amino acids is conserved between dicotyledonous and monocotyledonous species. Its gene expression was observed at the same level in roots and shoots under long-day conditions, but highly induced under short-day conditions and at low temperatures in roots, especially in endodermis and xylem parenchyma in the root hair region of Populus trichocarpa, and its protein level was high in dormant buds, but not in roots or branches. Addition of recombinant PtXSP25 protein mitigated the denaturation of lactate dehydrogenase by drying, but showed only a slight effect on that caused by freeze-thaw cycling. Recombinant PtXSP25 protein also showed ice recrystallization inhibition activity to reduce the size of ice crystals, but had no antifreezing activity. We suggest that PtXSP25 protein produced in shoots and/or in roots under short-day conditions and at non-freezing low temperatures followed by translocation via xylem sap to shoot apoplast may protect the integrity of the plasma membrane and cell wall functions from freezing and drying damage in winter environmental conditions.

Characterization of ice recrystallization inhibition activity in the novel freeze-responsive protein Fr10 from freeze-tolerant wood frogs, Rana sylvatica.

Fr10 is a secreted freeze-responsive protein found in the wood frog (Rana sylvatica). This protein has gained notable research attention for its highly dynamic expression in response to seasonal freezing stress, while its over-expression has been documented to enhance freeze tolerance in cold-susceptible cultured cells. This study further characterizes the properties of this novel protein with regards to thermal stability and ice recrystallization inhibition (i.e. IRI) activity. Thermal stability was assessed using differential scanning fluorimetry, with an experimental Tm value of 50.8 ±â€¯0.1 °C. Potential IRI activity of Fr10 was evaluated using a recently developed nanoparticle-based colorimetric assay, where Fr10 displayed the ability to prevent freeze-induced aggregation of gold nanoparticles. Based upon this assay, Fr10 protein appeared to have a low level of IRI activity and it was therefore predicted that one of Fr10's biological functions may be to inhibit ice crystal growth via recrystallization. A SPLAT cooling assay was then employed to directly characterize the IRI properties of Fr10 and provide further insight into this hypothesis. In the presence of 30 µM of Fr10, a 40% reduction in the mean grain size of ice crystals relative to the control samples was observed, thus introducing the possibility of Fr10 to inhibit ice recrystallization. Collectively, the results from this study provide new insight into the potential of further exploring the potential of this vertebrate freeze-responsive protein in cryoprotection.

1D Self-Assembly and Ice Recrystallization Inhibition Activity of Antifreeze Glycopeptide-Functionalized Perylene Bisimides.

Antifreeze glycoproteins (AFGPs) are polymeric natural products that have drawn considerable interest in diverse research fields owing to their potent ice recrystallization inhibition (IRI) activity. Self-assembled materials have emerged as a promising class of biomimetic ice growth inhibitor, yet the development of AFGP-based supramolecular materials that emulate the aggregative behavior of AFGPs have not yet been reported. This work reports the first example of the 1D self-assembly and IRI activity of AFGP-functionalized perylene bisimides (AFGP-PBIs). Glycopeptide-functionalized PBIs underwent 1D self-assembly in water and showed modest IRI activity, which could be tuned through substitution of the PBI core. This work presents essential proof-of-principle for the development of novel IRIs as potential supramolecular cryoprotectants and glycoprotein mimics.

MRI findings of serous atrophy of bone marrow and associated complications.

OBJECTIVES: To report the MRI appearance of serous atrophy of bone marrow (SABM) and analyse clinical findings and complications of SABM. METHODS: A retrospective search of MRI examinations of SABM was performed. Symptoms, underlying conditions, MRI findings, delay in diagnosis and associated complications were recorded. RESULTS: We identified 30 patients (15 male, 15 female; mean age: 46 ± 21 years) with MRI findings of SABM. Underlying conditions included anorexia nervosa (n = 10), cachexia from malignant (n = 5) and non-malignant (n = 7) causes, massive weight loss after bariatric surgery (n = 1), biliary atresia (n = 1), AIDS (n = 3), endocrine disorders (n = 2) and scurvy (n = 1). MRI showed mildly hypointense signal on T1- weighted and hyperintense signal on fat-suppressed fluid-sensitive images of affected bone marrow in all cases and similar signal abnormalities of the adjacent subcutaneous fat in 29/30 cases. Seven patients underwent repeat MRI due to initial misinterpretation of bone marrow signal as technical error. Superimposed fractures of the hips and lower extremities were common (n = 14). CONCLUSIONS: SABM occurs most commonly in anorexia nervosa and cachexia. MRI findings of SABM are often misinterpreted as technical error requiring unnecessary repeat imaging. SABM is frequently associated with fractures of the lower extremities. KEY POINTS: • SABM occurs in several underlying conditions, most commonly anorexia nervosa and cachexia. • Abnormal marrow signal is often misinterpreted as technical error requiring unnecessary repeat imaging. • SABM is frequently associated with stress fractures. • Fractures in SABM can be obscured by marrow signal abnormality on MRI.

Modulation of antifreeze activity and the effect upon post-thaw HepG2 cell viability after cryopreservation.

Most antifreeze proteins (AFPs) exhibit two types of "antifreeze activity" - thermal hysteresis (TH) and ice recrystallization inhibition (IRI) activity. The mechanism of TH activity has been studied in depth and is the result of an adsorption of AFPs to the surface of ice with an ice-binding face (IBF). In contrast, the mechanism of ice recrystallization and its inhibition is considerably less understood. In this paper, we examine several different antifreeze proteins, glycoproteins and mutants of the Lolium perenne AFP (LpAFP) to understand how IRI activity is modulated independently of TH activity. This study also examines the ability of the various AF(G)Ps to protect HepG2 cells from cryoinjury. Post-thaw cell viabilities are correlated to TH, IRI activity as well as dynamic ice shaping ability and single ice crystal growth progressions. While these results demonstrate that AF(G)Ps are ineffective as cryoprotectants, they emphasize how ice crystal habit and most importantly, ice growth progression affect HepG2 cell survival during cryopreservation.

The role of pubic symphyseal CT arthrography in the imaging of athletic pubalgia.

OBJECTIVE: The purpose of this article is to describe the results of pubic symphyseal CT arthrography compared with MRI in patients with suspected athletic pubalgia. MATERIALS AND METHODS: In this study, two musculoskeletal radiologists retrospectively searched our department's PACS to identify patients who had undergone CT-guided injection with concurrent pubic symphyseal CT arthrography for evaluation and treatment of groin pain, sports hernia, or athletic pubalgia over a 5.5-year period (January 1, 2007-July 1, 2012). The MR and CT arthrography images and reports, clinical findings at presentation, pain response to injection, and operative findings were reviewed using the electronic medical record. RESULTS: Twelve patients underwent CT-guided injection and pubic symphyseal CT arthrography at our institution during the 5.5-year study period. Nine of the 12 patients had undergone MRI before the procedure. In two of the three patients who had not undergone MRI, CT arthrography revealed secondary clefts. Three of four patients who had secondary clefts on MRI had contrast extravasation reproducing the cleft at CT. Three patients had MRI findings suggestive of athletic pubalgia without MRI evidence of a secondary cleft; in all three of these patients, CT arthrography showed a secondary cleft. In four patients, CT arthrography revealed tendon tears at the adductor origin that were not apparent on MRI. All 12 patients reported decreased groin pain after injection. CONCLUSION: Pubic symphyseal CT arthrography is a useful technique for the diagnosis and short-term pain relief of athletic pubalgia. It can be used to identify secondary clefts and to detect tendon tears that can potentially be overlooked on MRI.

It's a Trap! Aldolase-Prescribed C4 Deoxyradiofluorination Affords Intracellular Trapping and the Tracing of Fructose Metabolism by PET.

Fructose metabolism has been implicated in various diseases, including metabolic disorders, neurodegenerative disorders, cardiac disorders, and cancer. However, the limited availability of a quantitative imaging radiotracer has hindered its exploration in pathology and diagnostic imaging. Methods: We adopted a molecular design strategy based on the catalytic mechanism of aldolase, a key enzyme in fructolysis. We successfully synthesized a radiodeoxyfluorinated fructose analog, [18F]4-fluoro-4-deoxyfructose ([18F]4-FDF), in high molar activity. Results: Through heavy isotope tracing by mass spectrometry, we demonstrated that C4-deoxyfluorination of fructose led to effective trapping as fluorodeoxysorbitol and fluorodeoxyfructose-1-phosphate in vitro, unlike C1- and C6-fluorinated analogs that resulted in fluorolactate accumulation. This observation was consistent in vivo, where [18F]6-fluoro-6-deoxyfructose displayed substantial bone uptake due to metabolic processing whereas [18F]4-FDF did not. Importantly, [18F]4-FDF exhibited low uptake in healthy brain and heart tissues, known for their high glycolytic activity and background levels of [18F]FDG uptake. [18F]4-FDF PET/CT allowed for sensitive mapping of neuro- and cardioinflammatory responses to systemic lipopolysaccharide administration. Conclusion: Our study highlights the significance of aldolase-guided C4 radiodeoxyfluorination of fructose in enabling effective radiotracer trapping, overcoming limitations of C1 and C6 radioanalogs toward a clinically viable tool for imaging fructolysis in highly glycolytic tissues.

Synthesis of peptides and glycopeptides with polyproline II helical topology as potential antifreeze molecules.

A library of peptides and glycopeptides containing (4R)-hydroxy-L-proline (Hyp) residues were designed with a view to providing stable polyproline II (PPII) helical molecules with antifreeze activity. A library of dodecapeptides containing contiguous Hyp residues or an Ala-Hyp-Ala tripeptide repeat sequence were synthesized with and without α-O-linked N-acetylgalactosamine and α-O-linked galactose-ß-(1→3)-N-acetylgalactosamine appended to the peptide backbone. All (glyco)peptides possessed PPII helical secondary structure with some showing significant thermal stability. The majority of the (glyco)peptides did not exhibit thermal hysteresis (TH) activity and were not capable of modifying the morphology of ice crystals. However, an unglycosylated Ala-Hyp-Ala repeat peptide did show significant TH and ice crystal re-shaping activity suggesting that it was capable of binding to the surface of ice. All (glyco)peptides synthesized displayed some ice recrystallization inhibition (IRI) activity with unglycosylated peptides containing the Ala-Hyp-Ala motif exhibiting the most potent inhibitory activity. Interestingly, although glycosylation is critical to the activity of native antifreeze glycoproteins (AFGPs) that possess an Ala-Thr-Ala tripeptide repeat, this same structural modification is detrimental to the antifreeze activity of the Ala-Hyp-Ala repeat peptides studied here.

Isolated fractures of the trapezoid as a sports injury.

The aim of this work is to describe the radiographic findings of isolated trapezoid fractures and determine the utility of these findings in guiding treatment. A second aim is to heighten awareness of an uncommon sports-related injury that is often radiographically occult because of the lack of primary or overt secondary radiographic findings. A retrospective review of radiology reports at our institution from 2007 to 2010 was performed to identify isolated trapezoid fractures. Two musculoskeletal radiologists and one orthopedic hand surgeon reviewed the patient presentations, images, treatments, and outcomes of the patients' injuries. This project had institutional review board approval. We describe three patients who presented with isolated sports-related trapezoid fractures. Each patient was successfully treated with activity modification, cast immobilization, and/or surgery based on their specific radiographic findings. Isolated sports-related trapezoid fractures are rare injuries. Only one prior case report in the English literature exists. Treatment success in patients with trapezoid fractures depends upon the degree of activity modification, splint protection, and especially fragment displacement. We report the largest series to date of isolated trapezoid fractures, all of which resulted from sports participation, and we analyze the success of diagnostic and treatment interventions.

Probing the Mechanism of Action of Small-Molecule Ice Recrystallization Inhibitors Using Proton Nuclear Magnetic Resonance Relaxation.

N-2-Fluorophenyl-d-gluconamide (2FA) improves the recovery and function of cryopreserved biological materials by inhibiting ice recrystallization. However, as for many small-molecule ice recrystallization inhibitors, the mechanism of action of 2FA is not well-understood. In this study, the IC50 of 2FA for ice recrystallization was determined to be 3.5 mM (95% CI [3.41-3.52]). 1H transverse and longitudinal relaxations were then characterized by NMR at 2FA concentrations from 0 to 10 mM and at temperatures between -15 °C and +30 °C. Corresponding activation energy of water molecule motion (EAH2O) was calculated, showing that at each concentration 2FA did not affect EAH2O in the solid state, whereas in the liquid state EAH2O was significantly higher with 2FA than for pure water. Therefore, 2FA is excluded from the ice lattice upon freezing and concentrated in the interstitial liquid phase. This restricts the migration of water molecules between ice crystals via the liquid phase, inhibiting ice recrystallization.

Inhibiting ice recrystallization and optimization of cell viability after cryopreservation.

The ice recrystallization inhibition activity of various mono- and disaccharides has been correlated with their ability to cryopreserve human cell lines at various concentrations. Cell viabilities after cryopreservation were compared with control experiments where cells were cryopreserved with dimethylsulfoxide (DMSO). The most potent inhibitors of ice recrystallization were 220 mM solutions of disaccharides; however, the best cell viability was obtained when a 200 mM d-galactose solution was utilized. This solution was minimally cytotoxic at physiological temperature and effectively preserved cells during freeze-thaw. In fact, this carbohydrate was just as effective as a 5% DMSO solution. Further studies indicated that the cryoprotective benefit of d-galactose was a result of its internalization and its ability to mitigate osmotic stress, prevent intracellular ice formation and/or inhibit ice recrystallization. This study supports the hypothesis that the ability of a cryoprotectant to inhibit ice recrystallization is an important property to enhance cell viability post-freeze-thaw. This cryoprotective benefit is observed in three different human cell lines. Furthermore, we demonstrated that the ability of a potential cryoprotectant to inhibit ice recrystallation may be used as a predictor of its ability to preserve cells at subzero temperatures.

Increased apoptosis in cryopreserved autologous hematopoietic progenitor cells collected by apheresis and delayed neutrophil recovery after transplantation: a nested case-control study.

BACKGROUND AIMS: Delayed neutrophil recovery following autologous hematopoietic stem cell transplantation (aHSCT) increases transplant-related morbidity. Apoptosis induced by cryopreservation and thawing of hematopoietic progenitor cells collected by apheresis (HPC-A) was investigated in this nested case-control study as a factor associated with delayed neutrophil recovery following aHSCT. METHODS: Among patients with lymphoma who underwent aHSCT between 2000 and 2007 (n = 326), 13 cases of primary delayed neutrophil recovery and 22 age- and sex-matched controls were identified. Apoptosis and viability were measured using multiparameter flow cytometry, and colony-forming capacity was determined using semi-solid methylcellulose assays. RESULTS: HPC-A grafts from cases and controls had similar percentages of viable mononuclear cells (MNC) and CD34+ progenitor cells, as determined by standard 7AAD dye exclusion methods measured before and after cryopreservation. Patients with delayed neutrophil recovery received increased numbers of apoptotic MNC (P = 0.02) but similar numbers of apoptotic CD34+ cells per kilogram measured after thawing. Apoptosis was more pronounced in MNC compared with CD34+ cells after thawing, and apoptosis was negligible in freshly collected HPC-A products. Patients with delayed neutrophil recovery had fewer total colony-forming unites (CFU) and CFU-granulocyte-macrophages (GM) per 10(5) viable post-thaw MNC compared with controls (P < 0.05). CONCLUSIONS: Increased numbers of apoptotic MNC in thawed HPC-A products are associated with delayed neutrophil recovery after aHSCT. Studies that address factors contributing to increased apoptosis are needed, and measuring apoptosis in thawed HPC-A may have a role in the assessment of graft adequacy.