Validation of Serum Calprotectin Relative to Other Biomarkers of Infection in Febrile Infants Presenting to the Emergency Department.

Antimicrobial stewardship involves a delicate balance between the risk of undertreating individuals and the potential societal burden of overprescribing antimicrobials. This balance is especially crucial in neonatal care. In this observational study, the usefulness of biomarkers of infectious diseases (calprotectin, procalcitonin (PCT), C-reactive protein (CRP), and white blood cells (WBCs) were evaluated in 141 febrile infants aged 28-90 days presenting to an emergency department. Since our focus was on the usefulness of serum calprotectin, this biomarker was not part of clinical decision-making. A significant difference was observed in the levels of all biomarkers, related to final discharge diagnosis and disposition status. The difference in levels related to antibiotic prescription was significant for all biomarkers but WBCs. The performance of calprotectin in the detection of bacterial infections (AUC (95% CI): 0.804 (0.691, 0.916)) was comparable to the performance of both PCT (0.901 (0.823, 0.980)) and CRP (0.859 (0.764, 0.953)) and superior to the WBC count (0.684 (0.544, 0.823)). Procalcitonin and CRP demonstrated a statistically significantly higher specificity relative to calprotectin. In this cohort, antibiotic use did not always correlate to a definite diagnosis of confirmed bacterial infection. The sample size was limited due to associated challenges with recruiting febrile infants. Hence, there is a need for adequate diagnostic tools to help discriminate between various kinds of infections. This study suggests serum calprotectin, procalcitonin, and CRP may serve as valuable biomarkers to differentiate between types of infection, in addition to clinical input and decision-making.

Analysis of Calprotectin as an Early Marker of Infections Is Economically Advantageous in Intensive Care-Treated Patients.

Calprotectin is released from neutrophil granulocytes upon activation. Several studies have indicated that plasma calprotectin is an early determinant of bacterial infections, which may serve as a diagnostic tool facilitating decision making on antibiotic treatment. The study objective was to explore the health and economic implications of calprotectin as a predictive tool to initiate antimicrobial therapy in a cohort of critically ill patients. Thus, data obtained from a previously published study on calprotectin as a hypothetical early biomarker of bacterial infections in critically ill patients were evaluated regarding the potential cost-effective impact of early analysis of calprotectin on an earlier start of antibiotic treatment. Under the assumption that calprotectin is used predictively and comparators (white blood cells, procalcitonin, and C-reactive protein) are used diagnostically, a cost-effective impact of EUR 11,000-12,000 per patient would be obtained. If calprotectin would be used predictively and comparators would be used predictively for 50% of patients, it is hypothesized that cost-effectiveness would be between EUR 6000 and 7000 per patient, based on reduced stay in the ICU and general ward, respectively. Furthermore, predictive use of calprotectin seems to reduce both mortality and the length of hospital stay. This health economic analysis on the predictive use of plasma calprotectin, which facilitates clinical decision making in cases of suspected sepsis, indicates that such determination has a cost-saving and life-saving impact on the healthcare system.

Early kinetics of calprotectin in plasma following inguinal hernia surgery.

Calprotectin is one of the most abundant proteins of neutrophil granulocytes. It is released upon neutrophil activation and is considered a sensitive and clinically useful marker for neutrophil-mediated inflammation, including bacterial infections. However, early kinetics of calprotectin activation following inflammatory activation has hitherto been unknown. The aim of the present study was to determine the early phase of the kinetics of calprotectin, in comparison with the inflammatory markers CRP, IL-6, TNF-α, and procalcitonin, in plasma following a standardized temporary mild inflammatory response, using uncomplicated inguinal hernia surgery as a model. The study cohort consisted of 17 adult patients (15 male and 2 female) undergoing elective surgery for hernia. Values of calprotectin increased significantly at 2 h following surgery, and continued to increase to reach the highest level at 24-36 h after surgery, values still not exceeding upper normal reference level. This contrasts to IL-6 and CRP, for which an elevation was found first later, 4 h and 24-36 h post-surgery, respectively, for IL-6, and CRP. No significant increase was seen for TNF-α, or procalcitonin. The data demonstrate a very rapid and significant but modest increase in calprotectin following induction of mild inflammation, supporting that calprotectin can be useful for early detection of inflammatory response.

Circulating levels of calprotectin, a signature of neutrophil activation in prediction of severe respiratory failure in COVID-19 patients: a multicenter, prospective study (CalCov study).

OBJECTIVE: Severe COVID-19 is characterized by a dysregulated immune response in which neutrophils play a critical role. Calprotectin reflects neutrophil activation and is involved in the self-amplifying thrombo-inflammatory storm in severe COVID-19. We aimed to evaluate the role of calprotectin in early prediction of severity in COVID-19 patients. METHODS: This was a multicenter prospective observational study enrolling consecutive adult COVID-19 patients. On arrival to emergency department, blood samples were collected for laboratory tests, including serum calprotectin. The primary outcome was severe respiratory failure requiring invasive mechanical ventilation and the secondary outcome was need for Intensive Care Unit (ICU) admission. RESULTS: Study population included 395 patients, 57 (14.4%) required invasive mechanical ventilation and 100 (25.3%) were admitted to ICU. Median serum calprotectin levels were significantly higher in intubated (3.73 mg/L vs. 2.63 mg/L; p < 0.001) and ICU patients (3.48 mg/L vs. 2.60 mg/L; p = 0.001). Calprotectin showed a significant accuracy to predict the need for invasive mechanical ventilation (ROC AUC 0.723) and ICU admission (ROC AUC 0.650). In multivariate analysis, serum calprotectin was an independent predictor of invasive mechanical ventilation (OR 1.161) and ICU admission (OR 1.068). CONCLUSION: Serum calprotectin can be used as an early predictor of severity in COVID-19 patients.

Calprotectin, a new biomarker for diagnosis of acute respiratory infections.

Respiratory tract infections require early diagnosis and adequate treatment. With the antibiotic overuse and increment in antibiotic resistance there is an increased need to accurately distinguish between bacterial and viral infections. We investigated the diagnostic performance of calprotectin in respiratory tract infections and compared it with the performance of heparin binding protein (HBP) and procalcitonin (PCT). Biomarkers were analyzed in patients with viral respiratory infections and patients with bacterial pneumonia, mycoplasma pneumonia and streptococcal tonsillitis (n = 135). Results were compared with values obtained from 144 healthy controls. All biomarkers were elevated in bacterial and viral infections compared to healthy controls. Calprotectin was significantly increased in patients with bacterial infections; bacterial pneumonia, mycoplasma pneumonia and streptococcal tonsillitis compared with viral infections. PCT was significantly elevated in patients with bacterial pneumonia compared to viral infections but not in streptococcal tonsillitis or mycoplasma caused infections. HBP was not able to distinguish between bacterial and viral causes of infections. The overall clinical performance of calprotectin in the distinction between bacterial and viral respiratory infections, including mycoplasma was greater than performance of PCT and HBP. Rapid determination of calprotectin may improve the management of respiratory tract infections and allow more precise diagnosis and selective use of antibiotics.

Calprotectin is superior to procalcitonin as a sepsis marker and predictor of 30-day mortality in intensive care patients.

Sepsis is the most frequent cause of death in the intensive care unit (ICU). A rapid and correct diagnosis and initiation of therapy is crucial for improving patient outcomes. The aim of this study was to compare the performance of calprotectin with the more widely used sepsis biomarker procalcitonin (PCT) in ICU patients. The performance of calprotectin and PCT as sepsis and prognostic markers for 30-d mortality was compared in a prospective, observational study in an eight-bed ICU. We investigated concentrations of the biomarkers in plasma collected at admission from all ICU patients admitted during a year (2012-2013, n = 271) together with simplified acute physiology 3 scores (SAPS3) and sequential organ failure assessment (SOFA) scores. The receiver operating characteristic (ROC) analysis showed a higher area under the curve (AUC) value for calprotectin (0.79) than for PCT (0.49) when used as a sepsis marker. The calprotectin concentrations at admission were higher in non-survivors than in survivors at day 30. In our study, calprotectin was superior to PCT for distinguishing between ICU patients with sepsis and non-sepsis patients. Calprotectin also had higher predictive ability regarding 30-d mortality.

Reference Intervals for Fecal Calprotectin in Adults Using Two Different Extraction Methods in the Uppsala-SCAPIS Cohort.

BACKGROUND: Fecal calprotectin measurement is generally recommended to exclude inflammatory bowel disease (IBD) in patients with suspected IBD. A problem with the fecal calprotectin assays so far has been the rather long test-turnaround times. Recently a particle enhanced turbidimetric immunoassay (PETIA) for fecal calprotectin with assay times of approximately 10 minutes has been introduced on the European market. The aim of this study was to define reference intervals for adults with this new fecal calprotectin PETIA using two different extraction methods. METHODS: Samples were collected from 382 healthy individuals from the Swedish CArdioPulmonary bioImage Study (SCAPIS) Uppsala cohort in the age range 50 - 65 years. 202 samples were processed with CALEX® Cap extraction device (BÜHLMANN, Schönenbuch, Switzerland) and 180 samples were extracted using weighed samples. The extracted samples were analyzed on a Mindray BS-380 using the fCal Turbo PETIA reagent (BÜHLMANN). RESULTS: The calculated reference values for the Calex device were < 199 µg/g for the whole cohort, < 184 µg/g for females, and < 215 µg/g for males, while the corresponding values for weighed samples were < 153 µg/g for the whole cohort, < 141 µg/g for females, and < 215 µg/g for males. There were no significant statistical differences for calprotectin levels in males and females. CONCLUSIONS: The CALEX device yielded slightly higher calprotectin values. As there were no significant gender differences, the study indicates gender independent reference intervals of < 199 µg/g feces for the CALEX device and < 153 µg/g feces for weighed samples in patients in the 50 - 65 year age range.

Turbidimetric Determination of Fecal Calprotectin Using Two Table Top Chemistry Analyzers: Mindray BS-200E and Cobas® c111.

BACKGROUND: Fecal calprotectin assays are widely used in diagnosis and monitoring of inflammatory bowel disease (IBD) in patients with suspected IBD. The most frequently used technique is ELISA and microtiter plates. Turbidimetric assays for analysis of fecal calprotectin can significantly reduce turnaround time. Many laboratories may be reluctant to run fecal samples on their large chemistry analyzers. The aim of this study was to evaluate fecal calprotectin particle enhanced turbidimetric immunoassay (PETIA) on smaller chemistry analyzers that could be dedicated for fecal samples. METHODS: The BÜHLMANN fCAL® turbo assay was validated on two table top chemistry analyzers, Mindray BS-200E and cobas® c111. RESULTS: The assay was linear in the range between 20 and 1,900 µg/g with a limit of quantification around 20 µg/g on both instruments. The total coefficient of variation was < 7% in the range between 50 and 1,300 µg/g on both instruments. No antigen excess hook effect was observed up to 18,000 µg/g on the Mindray BS-200E and up to 20,000 µg/g on cobas® c111. The BÜHLMANN fCAL® turbo assay showed a high correlation with the BÜHLMANN fCAL® ELISA. CONCLUSIONS: Running the BÜHLMANN fCAL® turbo on Mindray BS-200E or cobas® c111 chemistry analyzers can provide rapid test results without exposing large routine chemistry analyzers to stool samples.

Analysis of HbA1c on an automated multicapillary zone electrophoresis system.

Hemoglobin A1c (HbA1c) is a frequently requested laboratory test and there is thus a need for high throughput instruments for this assay. We evaluated a new automated multicapillary zone electrophoresis instrument (Capillarys 3 Tera, Sebia, Lisses, France) for analysis of HbA1c in venous samples. Routine requested HbA1c samples were analyzed immunologically on a Roche c6000 instrument (n = 142) and then with the Capillarys 3 Tera instrument. The Capillarys 3 Tera instrument performed approximately 70 HbA1c tests/hour. There was a strong linear correlation between Capillarys 3 Tera and Roche Tina-Quant HbA1c Gen 3 assay (y = 1.003x - 0.3246 R2 = .996). The total CV for the 12 capillaries varied between 0.8 and 2.2% and there was a good agreement between duplicate samples (R2 = .997). In conclusion, the Capillarys 3 Tera instrument has a high assay capacity for HbA1c. It has a good precision and agreement with the Roche Tina-Quant HbA1c method and is well suited for high volume testing of HbA1c.

A novel turbidimetric immunoassay for fecal calprotectin optimized for routine chemistry analyzers.

BACKGROUND: Fecal calprotectin assays are widely used to exclude inflammatory bowel disease (IBD) in patients with suspected IBD. A problem with the fecal calprotectin assays is the rather long test-turnaround times. A particle enhanced turbidimetric immunoassays (PETIA) for fecal calprotectin would reduce test-turnaround times and would permit more laboratories to perform the measurements. The aim of this study was to evaluate a new feces calprotectin PETIA. METHOD: Using routine fecal samples the feces calprotectin PETIA was validated on two chemistry analyzers, Mindray BS-380 and Cobas 501. RESULTS: The assay is linear in the range 11-2000 µg/g, with a limit of quantitation of approximately 10 µg/g. No antigen excess hook effect was observed up to 10 000-15 000 µg/g depending on the instrument used. The turbidimetric method showed a good agreement with the Bühlmann ELISA. The total coefficient of variation was 3%-8% in the 50-100 µg/g range. CONCLUSION: The fecal calprotectin PETIA, fCal Turbo, is well suited for rapid analysis of fecal calprotectin on Mindray BS-380 or Cobas 501 clinical chemistry analyzers. The test results are commutable with Bühlmann fecal MRP8/14 ELISA.

The Roche Immunoturbidimetric Albumin Method on Cobas c 501 Gives Higher Values Than the Abbott and Roche BCP Methods When Analyzing Patient Plasma Samples.

BACKGROUND: Serum/plasma albumin is an important and widely used laboratory marker and it is important that we measure albumin correctly without bias. We had indications that the immunoturbidimetric method on Cobas c 501 and the bromocresol purple (BCP) method on Architect 16000 differed, so we decided to study these methods more closely. METHOD: A total of 1,951 patient requests with albumin measured with both the Architect BCP and Cobas immunoturbidimetric methods were extracted from the laboratory system. A comparison with fresh plasma samples was also performed that included immunoturbidimetric and BCP methods on Cobas c 501 and analysis of the international protein calibrator ERM-DA470k/IFCC. RESULTS: The median difference between the Abbott BCP and Roche immunoturbidimetric methods was 3.3 g/l and the Roche method overestimated ERM-DA470k/IFCC by 2.2 g/l. The Roche immunoturbidimetric method gave higher values than the Roche BCP method: y = 1.111x - 0.739, R² = 0.971. CONCLUSION: The Roche immunoturbidimetric albumin method gives clearly higher values than the Abbott and Roche BCP methods when analyzing fresh patient samples. The differences between the two methods were similar at normal and low albumin levels.

Safety and tolerability of a modified filter-type device for leukocytapheresis using ACD-A as anticoagulant in patients with mild to moderately active ulcerative colitis. Results of a pilot study.

Cellsorba™ is a medical device for leukocytapheresis (LCAP) treatment of ulcerative colitis (UC). Cellsorba™ EX Global type has been developed from Cellsorba E for intended use with ACD-A as anticoagulant. We evaluated safety and efficacy of the modified Cellsorba using ACD-A in a pilot trial comprising patients with active UC, despite receiving 5-ASA. A total of 10 LCAP treatments/patients were administered. Safety assessment focused on clinical signs and symptoms, hematological variables, as well as levels of bradykinin and IL-6. Efficacy was determined using the Mayo clinical/endoscopic scoring index as well histological assessment of biopsies. Additional aim was to evaluate the impact of apheresis system lines and filter on selected regulatory molecules. All six subjects completed the trial without any serious adverse events. WBC, platelet counts, and levels of bradykinin and IL-6 were not significantly affected. The median Mayo score decreased from 8.0 to 3.5 at week 8 (and to 2 at week 16 for the responders). Four patients were responders, of whom two patients went into remission. Median histological scores decreased from 3.5 to 2.0 in these four patients. Concentration of LL-37 increased within the apheresis system lines. LCAP with Cellsorba EX using ACD-A as anticoagulant was found to be a safe and well-tolerated procedure in patients with active UC. The positive impact on efficacy parameters merits further evaluation in a controlled fashion.

Chemical biology suggests a role for calcium signaling in mediating sustained JNK activation during apoptosis.

Calcium (Ca(2+)) is used as a signaling molecule to regulate many cellular processes. Calcium signaling generally involves transient elevations of the concentration of free Ca(2+) in the cytosol. More pronounced and sustained elevations of intracellular Ca(2+) concentrations are observed during apoptosis (programmed cell death). These Ca(2+) elevations have been shown to lead to the activation of proteases (calpains) and to changes in protein phosphorylation. Recent evidence, using chemical biology, has raised the possibility that calcium signaling is involved in sustained JNK activation during late phases of apoptosis. For at least some stimuli, calcium release leads to activation of calmodulin kinase II (CaMKII), apoptosis signaling kinase 1 (ASK1) and JNK. Calcium signaling may help to orchestrate the apoptotic response during the execution phase.

Induction of the lysosomal apoptosis pathway by inhibitors of the ubiquitin-proteasome system.

The lysosomal apoptosis pathway is a potentially interesting therapeutic target. Since apoptosis involving the lysosomal pathway has been described to involve cathepsins, we screened a drug library for agents that induce cathepsin-dependent apoptosis. Using pharmacological inhibitors and siRNA, we identified 2 structurally related agents (NSC687852 and NSC638646) that induced cathepsin D-dependent caspase-cleavage activity in human breast cancer cells. Both agents were found to induce the mitochondrial apoptosis pathway. NSC687852 and NSC638646 were found to inhibit the activity of ubiquitin isopeptidases and to induce the accumulation of high-molecular-mass ubiquitins in cells. We show that 3 other inhibitors of the proteasome degradation pathway induce lysosomal membrane permeabilization (LMP) and that cathepsin-D siRNA inhibits apoptosis induced by these agents. We conclude that a screen for cathepsin-dependent apoptosis-inducing agents resulted in the identification of ubiquitin isopeptidase inhibitors and that proteasome inhibitors with different mechanisms of action induce LMP and cathepsin D-dependent apoptosis.

Charting calcium-regulated apoptosis pathways using chemical biology: role of calmodulin kinase II.

BACKGROUND: Intracellular free calcium ([Ca2+]i) is a key element in apoptotic signaling and a number of calcium-dependent apoptosis pathways have been described. We here used a chemical biology strategy to elucidate the relative importance of such different pathways. RESULTS: A set of 40 agents ("bioprobes") that induce apoptosis was first identified by screening of a chemical library. Using p53, AP-1, NFAT and NF-kappaB reporter cell lines, these bioprobes were verified to induce different patterns of signaling. Experiments using the calcium chelator BAPTA-AM showed that Ca2+ was involved in induction of apoptosis by the majority of the bioprobes and that Ca2+ was in general required several hours into the apoptosis process. Further studies showed that the calmodulin pathway was an important mediator of the apoptotic response. Inhibition of calmodulin kinase II (CaMKII) resulted in more effective inhibition of apoptosis compared to inhibition of calpain, calcineurin/PP2B or DAP kinase. We used one of the bioprobes, the plant alkaloid helenalin, to study the role of CaMKII in apoptosis. Helenalin induced CaMKII, ASK1 and Jun-N-terminal kinase (JNK) activity, and inhibition of these kinases inhibited apoptosis. CONCLUSION: Our study shows that calcium signaling is generally not an early event during the apoptosis process and suggests that a CaMKII/ASK1 signaling mechanism is important for sustained JNK activation and apoptosis by some types of stimuli.

Mechanisms of action of DNA-damaging anticancer drugs in treatment of carcinomas: is acute apoptosis an "off-target" effect?

DNA damage induces apoptosis of cells of hematological origin. Apoptosis is also widely believed to be the major antiproliferative mechanism of DNA damaging anticancer drugs in other cell types, and a large number of laboratories have studied drug-induced acute apoptosis (within 24 hours) of carcinoma cells. It is, however, often overlooked that induction of apoptosis of carcinoma cells generally requires drug concentrations that are at least one order of magnitude higher than those required for loss of clonogenicity. This is true for different DNA damaging drugs such as cisplatin, doxorubicin and camptothecin. We here discuss apoptosis induction by DNA damaging agents using cisplatin as an example. Recent studies have shown that cisplatin induces caspase activation in enucleated cells (cytoplasts lacking a cell nucleus). Cisplatin-induced apoptosis in both cells and cytoplasts is associated with rapid induction of cellular reactive oxygen species and increases in [Ca(2+)](i). Cisplatin has also been reported to induce clustering of Fas/CD95 in the plasma membrane. Available data suggest that the primary responses to cisplatin-induced DNA damage are induction of long-term growth arrest ("premature cell senescence") and mitotic catastrophe, whereas acute apoptosis may be due to "off-target effects" not necessarily involving DNA damage.

Cytokeratin-18 is a useful serum biomarker for early determination of response of breast carcinomas to chemotherapy.

PURPOSE: With a widening arsenal of cancer therapies available, it is important to develop therapy-specific predictive markers and methods to rapidly assess treatment efficacy. We here evaluated the use of cytokeratin-18 (CK18) as a serum biomarker for monitoring chemotherapy-induced cell death in breast cancer. EXPERIMENTAL DESIGN: Different molecular forms of CK18 (caspase cleaved and total) were assessed by specific ELISA assays. Drug-induced release of CK18 was examined from breast carcinoma cells and tissue. CK18 protein composition was examined in serum. CK18 levels were determined in serum from 61 breast cancer patients during docetaxel or cyclophosphamide/epirubicin/5-fluorouracil (CEF) therapy. RESULTS: Caspase-cleaved CK18 molecules were released from monolayer cultures and tumor organ cultures to the extracellular compartment. CK18 was present in complexes with other cytokeratins in serum. Such CK18 protein complexes are remarkably stable, leading to favorable performance of CK18 biomarker assays for clinical investigations. Docetaxel induced increased levels of caspase-cleaved CK18 in serum from breast cancer patients, indicating apoptosis. CEF therapy led to increases predominantly in uncleaved CK18, indicating induction of necrotic cell death in many tumors. The increase in total CK18 at 24 h of the first treatment cycle correlated to the clinical response to CEF therapy (P < 0.0001). CONCLUSIONS: Induction of necrotic cell death may explain the clinical efficacy of anthracycline-based therapy for breast carcinomas with defective apoptosis pathways. We suggest that CK18 biomarkers are useful for early prediction of the response to CEF therapy in breast cancer and may be useful biomarkers for clinical trials.

Acute apoptosis by cisplatin requires induction of reactive oxygen species but is not associated with damage to nuclear DNA.

Cisplatin is a broad-spectrum anticancer drug that is also widely used in experimental studies on DNA damage-induced apoptosis. Induction of apoptosis within 24-48 hr requires cisplatin concentrations that are at least one order of magnitude higher than the IC(50). Here, we show that such high, apoptosis-inducing cisplatin concentrations induce cellular superoxide formation and that apoptosis is inhibited by superoxide scavengers. The same concentration limit and the requirement for superoxide are also true for induction of caspase activation in enucleated cells (cytoplasts), showing that cisplatin-induced apoptosis occurs independently of nuclear DNA damage. In contrast, lower cisplatin concentrations, which do not induce acute apoptosis, are sufficient for induction of DNA damage signaling. We propose that the antiproliferative effects of cisplatin at IC(50) doses involve premature senescence and secondary, nonstress-induced apoptosis. The higher doses currently used in in vitro studies lead to acute, stress-induced apoptosis that involves induction of superoxide but is largely DNA damage-independent.

Preferential cell death of CD8+ effector memory (CCR7-CD45RA-) T cells by hydrogen peroxide-induced oxidative stress.

T cells are used in many cell-based cancer treatments. However, oxidative stress that is induced during various chronic inflammatory conditions, such as cancer, can impair the immune system and have detrimental effects on T cell function. In this study, we have investigated the sensitivity of different human T cell subsets to H(2)O(2)-induced oxidative stress. We showed that central memory (CD45RA(-)CCR7(+)) and effector memory (CD45RA(-)CCR7(-)) T cells are more sensitive to H(2)O(2) as compared with naive (CD45RA(+)CCR7(+)) T cells. Furthermore, the study showed that CD8(+) effector memory T cells are more sensitive to low levels of H(2)O(2) (5 microM) compared with other types of T cells investigated. H(2)O(2)-exposed CD45RO(+) T cells showed mitochondrial depolarization prior to caspase 3 activity. Moreover, the pan-caspase inhibitor z-Val-Ala-Asp(OMe)-fluoromethylketone rescued cells from death. These experiments suggest that H(2)O(2)-induced cell death of CD45RO(+) T cells acts via the mitochondrial pathway and that caspase involvement is needed. This study suggests that oxidative stress in cancer patients can be disadvantageous for T cell-based adoptive cell transfer therapies, since effector memory T cells are the primary phenotype of the cells administered.