The number of HLA confirmatory tests during unrelated donor search as a driver for the evaluation of back-up haploidentical donor(s).

INTRODUCTION: the identification of a suitable donor in an appropriate timing represents a crucial step in the preparation of allogeneic stem cell transplantation (HSCT). At our Institution, for patients lacking an HLA-identical sibling, a haploidentical donor is considered in the absence of a 10/10-matched or a one-locus HLA-mismatched unrelated donor (UD), but the optimal timing of work-up of potential familiar haploidentical donor(s) by the Apheresis Team is actually unknown. PATIENTS & METHODS: we analyzed here n = 167 UD searches launched at our Hospital between July 2013 and July 2018 and looked for any correlation between the number of HLA confirmatory tests received and the final type of donor selected for HSCT, in an attempt to identify those situations where prompt evaluation of haploidentical donor(s) is warranted. RESULTS: a total of n = 117 transplants were performed and haploidentical HSCTs were n = 16 (14 %). In n = 93 cases (56 %) the number of HLA confirmatory tests received were two; they were one, zero and three for n = 52, n = 14 and n = 8 patients, respectively. Only 5 % of haploidentical donors were used when two confirmation test samples were received whereas this percentage rises to 17 % when only one sample reached the HLA lab. When no confirmation tests were available, haploidentical transplant occurred in 100 % of cases. CONCLUSIONS: besides the situations with no HLA confirmation tests, the evaluation of any haploidentical donor(s) should be promptly started also when only one HLA confirmatory test is received, in order to optimise the potential work-up process and avoid delay in transplantation.

Response of steroid-refractory chronic graft-versus-host disease to extracorporeal photopheresis correlates with the dose of CD3+ lymphocytes harvested during early treatment cycles.

BACKGROUND: Extracorporeal photopheresis (ECP) is a recognized second-line treatment for steroid-refractory chronic graft-versus-host disease (cGVHD). Treatment course is usually long, expensive, and demanding for patients, so predictors for response are needed. We carried out a retrospective study on cGVHD patients treated at our institution with the aim to identify a possible correlation between apheretic yields composition and probability of response. STUDY DESIGN: Patients treated for at least 6 months were eligible for the study. Flow cytometry data, including absolute counts of lymphocytes and their subpopulations in ECP products from cGVHD patients, were collected. For each cell population 1) the median dose per procedure harvested during the first 3 months of treatment and 2) the cumulative dose collected in the same period were compared with clinical response. RESULTS: A total of 726 ECP procedures were performed in 15 patients. Overall response, defined as either a complete response (CR) or a partial response according to National Institutes of Health criteria, was obtained in 10 of 15 patients (66.7%), and CR, in eight of 15 (53.3%). According to Cox regression analysis, the probability of achieving an overall response is significantly correlated with the median number of CD3+, CD3+CD4+, and CD3+CD8+ lymphocytes collected during the early treatment phase (first 3 months). CONCLUSION: Our data suggest that CD3+ cell evaluation in ECP during the early phase of treatment course could predict response and help identify patients who deserve further treatment.

Identification of hematopoietic progenitor cell donor characteristics predicting successful mobilization: results of an Italian multicenter study.

BACKGROUND: Peripheral blood (PB) hematopoietic progenitor cells (HPC) collected by apheresis are the first-choice source for allogeneic stem cell transplantation. The target HPC dose is usually considered to be 4 × 10(6) CD34+ cells/kg of the recipient, but higher doses are required in reduced-intensity conditioning and haploidentical transplants. Thus, prolonged stimulation and repeated collections or failure to reach HPC target may occur, increasing risks for donors and recipients. We carried out a retrospective multicenter study on healthy donors, to identify donor variables which may correlate with HPC mobilization. STUDY DESIGN AND METHODS: HPC allogeneic donations from sibling and unrelated donors performed in two centers from 1995 to 2012 were analyzed. We defined a mobilization cutoff of 50 × 10(6) CD34+ cells/L and tested somatic variables, blood counts, and granulocyte-colony-stimulating factor (G-CSF) dose and molecular form. RESULTS: A total of 360 donors were analyzed (male, 201; female, 159; sibling, 348; unrelated, 12; median [range] age, 44.8 [13-80] years). Median peak CD34+ in PB was 54.4 × 10(6) /L (range, 5 × 10(6) -299 × 10(6) ). By multivariate analysis, we identified the following variables to correlate with good mobilization: 1) male sex (p<0.0005); 2) younger age (p=0.007); 3) higher baseline (premobilization) white blood cell (WBC) count (p<0.0005); 4) higher G-CSF dosage (p<0.0005); and 5) use of lenograstim rather than filgrastim (p<0.002). CONCLUSION: In healthy donors it is possible to predict successful HPC mobilization by donor sex, age, WBC count, and G-CSF form and dose. Furthermore, based on these data, it may be possible, at least in parental setting, to modulate G-CSF dosage on the basis of donor characteristics.

Polymorphism of the HLA system and weak antibody response to BNT162b2 mRNA vaccine.

The polymorphism of the HLA system has been extensively studied in COVID-19 infection, however there are no data about the role of HLA on vaccine response. We report here the HLA-A, -B, -C, and DRB1 allelic frequencies of n = 111 individuals after BNT162b2 mRNA vaccine, selected on the basis of lower antibody levels (<5% percentile) after the second dose among a total of n = 2569 vaccinees, and compare them with the frequencies of a reference population. We found that differences in the frequencies of the alleles HLA-A*03:01, A*33:03, B*58:01 and at least one haplotype (HLA-A*24:02~C*07:01~B*18:01~DRB1*11:04) are associated with a weaker antibody response after vaccination, together with the age of vaccinees. Our results might suggest a role played by some HLA alleles or haplotypes in antibody production after the BNT162b2 mRNA vaccine, giving insights into the tracking of potentially susceptible individuals across populations. Further studies are needed to better define our exploratory findings and dissect the role of HLA polymorphism on response to anti-COVID-19 vaccines.

Flow cytometry and cytomorphology evaluation of hematologic malignancy in cerebrospinal fluids: comparison with retrospective clinical outcome.

An independent clinical assessment was compared with flow cytometry (FCM) and cytomorphology results obtained on 227 cerebrospinal fluids investigated for hematologic malignancy, in a retrospective longitudinal study with a median observation time of 11 months. A combined method assessment (CMA), defining "positive" a sample if at least one method gave "positive" results, was also tested. Eleven out of 55 screening samples and 53 out of 166 follow-up samples resulted positive at clinical evaluation. FCM and CM were concordant with positive clinical assessment in 68.5% and 51.5% of cases, respectively. According to CMA, 10.5% of samples (resulting false negative by either FCM or cytomorphology) were rescued as true positive. FCM retained significantly higher accuracy than cytomorphology (p=0.0065) and 100% sensitivity when at least 220 leukocytes were acquired. CMA accuracy was higher than FCM accuracy and significantly higher than cytomorphology accuracy in the analysis of all samples (p<0.0001), samples from mature B/T cell neoplasms (p=0.0021), and samples drawn after intrathecal treatment (p=0.0001). When acquiring ≤220 leukocytes, FCM accuracy was poor, and combining cytomorphology added statistically significant diagnostic advantage (p=0.0043). Although FCM is the best diagnostic tool for evaluating CSF, morphology seems helpful especially when clinically positive follow-up samples are nearly acellular.

Correlation analysis between virtual and Complement-Dependent-Cytotoxicity crossmatch in a monocenter retrospective series of 118 allografted patients.

PURPOSE OF THE STUDY: The detection of patients' anti-HLA antibodies before allogeneic hematopoietic stem cell transplantation (HSCT) may affect post-transplant outcome, due to a potential detrimental impact on engraftment or toxicity-related issues. Crossmatch (XM) techniques provide support to physicians during the pre-transplant phase but the role of Complement-Dependent Cytotoxicity XM (CDC-XM) is not well-defined when performed routinely and in parallel with the virtual XM. PATIENTS AND METHODS: We report here our experience with both virtual and CDC-XM tests on n = 118 patients undergoing search for a donor other than HLA-identical sibling from July 2013 to June 2018 at our Institution. When anti-HLA antibodies (Abs) were present, they were classified as donor-specific Abs (DSA) or non-DSA. RESULTS: On the n = 118 patients, n = 35 (29.7 %) had a positive virtual XM test (of which one of more DSA were found in n = 8; 6.8 %) and n = 5 had a positive CDC-XM test. These latter, positive for HLA class II only, were interpreted as false-positive results due to prior administration of anti-CD20 to the patients, all affected by lymphoma; none of them had a positive virtual XM for anti-HLA Abs of class II. Importantly, all these patients successfully engrafted, further supporting the lack of significant impact of CDC-XM positive results in this series. CONCLUSIONS: According to our data on more than a hundred patients, routinely performed CDC-XM does not seem to add significant information with respect to virtual XM. We cannot exclude the usefulness of CDC-XM in specific situations, although a positive CDC-XM result was an unfrequent event.

Clinical, magnetic resonance imaging, and genetic study of 5 Italian families with cerebral cavernous malformation.

BACKGROUND: Cerebral cavernous malformations (CCMs) are congenital vascular anomalies of the central nervous system that can result in seizures, hemorrhage, recurrent headaches, and focal neurologic deficits. These CCMs can occur as sporadic or autosomal dominant conditions, although with incomplete penetrance and variable clinical expression. Three CCM loci have been identified, on chromosomes 7q21-22 (CCM1; Online Mendelian Inheritance in Man [OMIM] 116860), 7p13-15 (CCM2; OMIM 603284), and 3q25.2-27 (CCM3; OMIM 603285), and 3 genes have been cloned, KRIT1 on CCM1, MGC4607 on CCM2, and PDCD10 on CCM3. Mutations in KRIT1 account for more than 40% of CCMs. OBJECTIVE: To describe the results of a comprehensive evaluation of 5 Italian families affected with CCM. DESIGN: Clinical, magnetic resonance imaging, and KRIT1 gene analysis. SETTING: University academic teaching hospitals. PATIENTS: Fifteen patients with CCM diagnosed according to defined criteria and 45 at-risk, symptom-free relatives. RESULTS: Three novel and 2 described mutations were found in KRIT1. The families included 33 KRIT1 mutation carriers, 57.6% of whom had no symptoms. Magnetic resonance imaging revealed CCM lesions in 82.3% of symptom-free mutation carriers. CONCLUSIONS: The data confirm both incomplete clinical and neuroimaging penetrance in families with the KRIT1 mutation. This consideration is important in genetic counseling. Moreover, the data emphasize both the importance of magnetic resonance imaging in the diagnosis of CCM and the potential for DNA-based diagnosis to identify subjects at risk.

A novel type of familial transthyretin amyloidosis, ATTR Asn124Ser, with co-localization of kappa light chains.

A 68-year-old Italian woman who had a clinical history of thyroidectomy in 2002 presented with slowly progressing renal insufficiency and non-nephrotic proteinurea in 2004. A renal biopsy showed the occurrence of amyloid; the thyroid biopsy previously taken also revealed amyloid infiltration. Other amyloid-containing tissues included bone marrow and heart. The plasma cell level in the bone marrow was found to be less than 5% and both serum and urine samples were positive for a monoclonal kappa light chain band. DNA analysis unexpectedly revealed the presence of a novel transthyretin (TTR) mutation, ATTR Asn124Ser. Histologically, amyloid deposits in the thyroid had a homogeneous appearance with moderate Congophilia. In immunohistochemistry, a kappa light chain antiserum showed positive immunoreactivity with amyloid deposits in the thyroid. Furthermore, a TTR antiserum, anti-TTR50-127, also recognized a number of amyloid deposits stained positive with the kappa light chain antiserum. Overall, the kappa light chain antiserum reacted with most of the amyloid deposits in the thyroid, whereas TTR immunoreactivity was scarcer, with a scattered appearance. In contrast, only the anti-TTR50-127 antiserum labeled amyloid in the kidney, albeit not all deposits. In this study, we report a patient having a novel TTR variant, ATTR Asn124Ser, with co-localization of kappa light chains in the amyloid deposits in the thyroid tissue.

SOD1 mutations in amyotrophic lateral sclerosis. Results from a multicenter Italian study.

Amyotrophic Lateral Sclerosis (ALS), the most common form among motoneuron diseases, is characterized by a progressive neurodegenerative process involving motor neurons in the motor cortex, brain stem and spinal cord. Sporadic (SALS) accounts for the majority of patients but in about 10% of ALS cases the disease is inherited (FALS), usually as an autosomal dominant trait. In the present study we show the results of a referred based multicenter study on the distribution of SOD1 gene mutations in the largest cohort of Italian ALS patients described so far. Two hundred and sixty-four patients (39 FALS and 225 SALS) of Italian origin were studied. In 7 out of 39 FALS patients we found the following SOD1 gene mutations: i) a new G12R missense mutation in exon 1, found in a patient with a slowly progressive disease course; ii) the G41S mutation, in four unrelated patients with rapidly progressive course complicated with cognitive decline in two of them; iii) the L114F mutation, in a patient with a slowly progressive phenotype; iv) the D90A mutation, in a heterozygous patient with atypical phenotype. In addition, in one SALS patient a previously reported synonymous variant S59S was identified. In 17 (3 FALS and 14 SALS) out of 264 patients (6.4 %) the polymorphism A-->C at position 34 of intron 3 (IVS3: + 34 A-->C) was found, and in one FALS patient a novel variant IVS3 + 62 T-->C was identified. The frequency of SOD1 gene mutations (17.9 %) in FALS cases was comparable with that found in other surveys with a similar sample size of ALS cases. No SOD1 gene mutations have been identified in SALS cases. Within FALS cases, The most frequent mutation was the G41S identified in four FALS.

Frequency of butyrylcholinesterase gene mutations in individuals with abnormal inhibition numbers: an Italian-population study.

OBJECTIVES: More than 30 genetic variants of serum cholinesterase (butyrylcholinesterase, BChE) have been described. Some of them (the atypical and the fluoride-resistant variants) are well known because carriers are prone to develop prolonged apnea following the administration of the muscle relaxant succinylcholine. Genotype characterization is therefore important in order to prevent such episodes. Genetic studies have so far focused on selected individuals or families rather than on the random population. METHODS: From a large group of healthy blood donors (n = 2609), we selected all the 58 individuals with low serum cholinesterase activity: among them 28 subjects had abnormal dibucaine and fluoride inhibition numbers. Twenty-five mutations in the coding region of the human cholinesterase gene were analyzed. RESULTS: All individuals with abnormal inhibition numbers were homozygotes or double heterozygotes in several mutations. Asp70Gly (Atypical variant) and Ala539Thr (K variant) were the most frequently observed amino acid substitutions. The majority of subjects with low BChE activity but normal dibucaine and fluoride number presented only the K form. We analyzed 106 randomly chosen subjects for K and atypical variants. Carriers of these alleles were at risk of low BChE activity (OR = 9.55, 95%CI, 5.61-16.26 and OR = 30.33, 95%CI, 7.05-130.52 respectively). CONCLUSIONS: Data obtained from this study help to better define the etiology of low BChE activity and the role of the rather common K allele. It is the first time that such a large population has been screened for so many mutations. BChE is also implicated in detoxifying cocaine; therefore genetic analysis could be useful in cases of cocaine toxicity in Italian subjects.

Flow cytometry vs cytomorphology for the detection of hematologic malignancy in body cavity fluids.

Flow cytometry and cytomorphology results on 92 body cavity fluids [61 effusions and 31 bronchoalveolar lavage fluids (BALF)] from hematologic malignancy were compared with retrospective clinical outcome. We observed double true positive/negative results in 67 cases (73%), and double false negative results in 2 cases (2%). Immunophenotyping accounted for true positive/negative results in 22 out of 23 mismatched cases (25%), and retained significantly higher accuracy than that of cytomorphology especially in effusions and differentiated lymphoma. In BALF analysis, immunophenotyping and cytomorphology sensitivity was 75% and 0%, respectively. Flow cytometry retains the highest accuracy in detecting neoplastic cells in body cavity fluids.

Assay using succinyldithiocholine as substrate: the method of choice for the measurement of cholinesterase catalytic activity in serum to diagnose succinyldicholine sensitivity.

No comparative information is available concerning the ability of various cholinesterase (ChE) methods to identify succinyldicholine-sensitive patients, purely on the basis of the enzyme activity recorded in serum. Here, we evaluated six different methods for the measurement of ChE activity; 131 subjects were subdivided according to ChE phenotype and, therefore, to succinyldicholine sensitivity. ChE phenotype was determined by measuring dibucaine and fluoride numbers. DNA analysis was also performed to confirm correlation between the phenotype classification used in the study and the ChE genotype. The tested methods were significantly different in their ability to discriminate between the subjects with and without succinyldicholine-sensitive phenotypes. The succinyldithiocholine/5,5'-dithio-bis(2-nitrobenzoate) (DTNB) method showed the highest accuracy (area under the receiver operating characteristic (ROC) curve 0.97) followed by the propionylthiocholine/DTNB method (area under the ROC curve 0.94). On the other hand, the two methods using butyrylthiocholine as substrate and that employing benzoylcholine showed limited clinical utility in discriminating subjects at risk of prolonged apnea (area under the ROC curve < or = 0.9). Using the succinyldithiocholine method, a value < or = 23 U/l was approximately five times as likely to occur in a sensitive individual as in a normal one.