Vascular access for autologous peripheral blood stem cells collection by large volume leukapheresis: Single center experience.

INTRODUCTION: Peripheral blood stem cell (PBSC) harvesting requires reliable and safe vascular access. In our institution, a change of practice was implemented and the central venous catheter (CVC) placement for all autologous PBSC collections was abandoned in favor of a careful evaluation of peripheral venous access (PVA) for each individual patient. The aim of this prospective study was to evaluate the rate of patients with adequate peripheral veins for autologous PBSC collection and compare patient characteristics, collection efficacy, and complication rate between patients with PVA and CVC. METHOD: Peripheral veins were assessed by the apheresis nurse team in all patients referred between January 2020 and July 2021 to autologous PBSC collection. Only in case of difficult venous access, CVC was inserted. Large volume leukapheresis (LVL) procedures, which processed ≥3 total blood volumes, were performed. RESULTS: In 65 (57%) patients PVA was used, while 49 (43%) patients required placement of short-term CVC. Peripheral venous access was successfully used significantly more often in males (69.8%) (P = 0.010), and patients with multiple myeloma (71.0%) than in patients with non-Hodgkin's lymphoma (35.9%) and Hodgkin's lymphoma patients (33.3%) (P < 0.001). There was a significant difference in the type of prior administered chemotherapy; in the patients who received cytostatics free chemotherapy, PVA was used more often (75.0%) (P = 0.007). In terms of the efficacy and safety of LVLs, there were no differences between procedures performed using PVA and CVCs. CONCLUSION: Peripheral venous access is feasible for autologous PBSC collection in more than a half of patients, in particular in those with multiple myeloma. Changes in the treatment of multiple myeloma, using new proteasome inhibitors-based and immunomodulatory agents that do not adversely affect peripheral veins, have enabled the use of PVA even at the high blood flow rates required by LVL. Peripheral venous access is not associated with safety issues or with a lesser collection efficiency, and it is cost-effective as well. Each patient referred to autologous PBSC collection needs to be evaluated individually by the experienced apheresis team for the most appropriate venous access.

Intra- and intergenotype characterization of D6 measles virus genotype.

Determination of inter- and intragenotype stability and variability are the basic tools for the molecular epidemiology and evolutionary investigation of measles virus (MV). We made a comparison between complete genome sequences of four MVs (two wt MV strains-WA.USA/17.98 and 97-45881, and two SSPE MV strains-MVs/Zagreb.CRO/47.02/and MVs/Zagreb.CRO/08.03/), all belonging to genotype D6. Results of analyses clearly confirm that MV genome continuously changes within the viruses of the same or different genotypes by accumulation of mutations in different parts of the genome. Only a small number of these accumulated mutations induce amino acid substitutions and thus possibly introduce new biological characteristics or a new genotype over a long time period. This study clearly reveals a long untranslated region between M and F genes as the most variable region of the MV genome and detects the presence of unique residues on the level of the entire genome as a new important parameter in the investigation of molecular evolution of MVs.

Genetic characterization of L-Zagreb mumps vaccine strain.

Eleven mumps vaccine strains, all containing live attenuated virus, have been used throughout the world. Although L-Zagreb mumps vaccine has been licensed since 1972, only its partial nucleotide sequence was previously determined (accession numbers , and ). Therefore, we sequenced the entire genome of L-Zagreb vaccine strain (Institute of Immunology Inc., Zagreb, Croatia). In order to investigate the genetic stability of the vaccine, sequences of both L-Zagreb master seed and currently produced vaccine batch were determined and no difference between them was observed. A phylogenetic analysis based on SH gene sequence has shown that L-Zagreb strain does not belong to any of established mumps genotypes and that it is most similar to old, laboratory preserved European strains (1950s-1970s). L-Zagreb nucleotide and deduced protein sequences were compared with other mumps virus sequences obtained from the GenBank. Emphasis was put on functionally important protein regions and known antigenic epitopes. The extensive comparisons of nucleotide and deduced protein sequences between L-Zagreb vaccine strain and other previously determined mumps virus sequences have shown that while the functional regions of HN, V, and L proteins are well conserved among various mumps strains, there can be a substantial amino acid difference in antigenic epitopes of all proteins and in functional regions of F protein. No molecular pattern was identified that can be used as a distinction marker between virulent and attenuated strains.

Screening of serologically negative plasma pools for hepatitis C virus by nucleic acid amplification testing in Croatia, 2001-2003.

A residual risk of HCV infection by different blood products exists due to blood donations collected during the serological window period in the early stages of infection. The aim of this study is nucleic acid amplification technique (NAT)-based screening of the anti-HCV negative plasma pools obtained from various Croatian transfusion centres between 2001 and 2003 for HCV RNA. During this period 2647 anti-HCV negative plasma pools were tested by NAT and 12 (0.45%) HCV RNA positive pools were detected. In comparison to the results of our previous study [Forcic D, Zgorelec R, Branovic K, Kosutic-Gulija T, Santak M, Mazuran R. Incidence of hepatitis C virus RNA in anti-HCV negative plasma pools in Croatia. Transfus Apher Sci 2001;24:269-78], a remarkable decrease in the number of positive plasma pools (from 2.1% to 0.45%) was demonstrated.

Detection and characterization of measles virus strains in cases of subacute sclerosing panencephalitis in Croatia.

Two cases of subacute sclerosing panencephalitis (SSPE), diagnosed in Croatia in 2002, were investigated. The coding regions of the matrix (M), hemagglutinin (H) and nucleoprotein (N) genes of measles virus were sequenced following direct RT-PCR amplification of viral RNA extracted from brain tissue. Phylogenetic analysis of the sequences of H and N genes, showed that both strains belonged to genotype D6. No vaccine strain was detected although both patients had been previously immunized. The comparison of analyzed sequences of two SSPE causative viruses with corresponding sequences of D6 genotype and with each other revealed a number of mutations in N and H gene sequences. In comparison to the Edmonston reference strain, the M gene of the SSPE viruses showed the characteristic biased hypermutation and a premature termination codon in one of the patients.

Genetic characterization of a mumps virus isolate during passaging in the amniotic cavity of embryonated chicken eggs.

The aim of this study was the molecular characterization of a historical mumps isolate (an alleged individual sample). After RNA extraction and cDNA synthesis, selective nested PCR amplification with specific primers, automated DNA sequencing and RFLP analyses were performed. The relative ratios of the detected virus sequences were determined by GeneScan electrophoresis. Phylogenetic tree based on the 316 nucleotide region of the SH gene of the mumps virus was generated by the neighbor-joining method. Results obtained by the described molecular approach show: (a) there are two mumps virus variants, A and B, detected in the fourth passage of wild type virus in the amniotic cavity of embryonated chicken eggs (ECE); (b) variants A and B belong to different genotypes; (c) variants A and B differ in the HN and NP genes which code for amino acid sequences comprising immunogenic epitopes; (d) variant B contains one or more minor variants. We discuss whether the observed differences between the two variants are a consequence of natural heterogeneity or of laboratory contamination in the early passages.

The first genetic characterization of a D4 measles virus strain derived from a patient with subacute sclerosing panencephalitis.

Measles virus (MV) strains derived from patients with subacute sclerosing panencephalitis (SSPE), SSPE strains, possess numerous mutations when compared to viruses belonging to the same genotype and circulating in similar time period. Although many SSPE strains have been extensively characterized, none of them belongs to D4 genotype which currently predominates in Europe where it has caused a number of recent outbreaks/epidemics. We sequenced an MV derived from a patient with long-term SSPE; the virus was named MVs/Zagreb.CRO/30.06[D4] (SSPE). Initial genetic analysis showed that it belongs to D4 genotype. The sequences of genes encoding matrix and fusion proteins indicate premature protein terminations. Putative hemagglutin (H) protein is lengthened for 20 amino acids, which is the longest H protein elongation so far found in SSPE viruses. Nucleotides 1421 A, 1422 G, 1507 C and 1542 C in nucleoprotein gene open reading frame seem to be specific for this D4 strain, differentiating it from other D4 non-SSPE strains. Besides, a unique mutation at position 543 of H protein was found, histidine instead of tyrosine. As persistent MV infections are initially established by "normal" wild-type MV strains, the presented comparative analyses describe alterations that could be involved in the maintenance of persistent infection, disease development and progression.

A comparison of complete untranslated regions of measles virus genomes derived from wild-type viruses and SSPE brain tissues.

We compared complete untranslated regions (UTRs) of two subacute sclerosing panencephalitis (SSPE) measles virus (MV) strains and two wild-type (wt) MV strains, all belonging to the same genotype (D6). In comparison to wt MVs of the same genotype, base changes were identified in the two SSPE measles virus strains at 27 and 33 noncoding positions, respectively. Majority of these residues are unique for each of the SSPE virus sequences in comparison to all other reported measles virus strain sequences. The location of some of these changes indicates that they may modify cis-acting regulatory sequences including gene-end signal of the P gene, H/L gene junction and Kozak consensus element of the L gene. Further, within the long UTR between M and F genes, deletions and insertions were identified. Thus, our study could be significant for additional investigation using reverse genetics and recombinant viruses, of possible influence of mutations in UTRs on establishment and maintenance of chronic progressive CNS disease caused by MV persistence.

Restriction enzyme cleavage of fluorescently labeled DNA fragments--analysis of the method and its usage in examination of digestion completeness.

Restriction enzymes have proven to be among the most valuable tools in molecular biology. In this work, we demonstrate that the cleavage of fluorescently labeled, PCR-amplified DNA can be used as a simple and highly sensitive technique for detection of sequences present in a percentage as low as 0.6% in a DNA pool. Due to the fact that fluorescent labeling of DNA fragments enables such sensitive detection and quantification of restriction enzyme cleavage, the method was further exploited in monitoring of the enzymatic digestion completeness and in determination of factors that influence restriction enzyme effectiveness. We analyzed the activity of six restriction endonucleases; the percentage of uncleaved DNA fragments predominantly ranged between 2.0 and 2.5 and the highest value was 8.00%. We conclude that, since the enzymatic digestion completeness may not always be assured, each assay based on restriction enzyme cleavage that is intended to be used in investigations of heterogeneity in a DNA pool should be constructed so that the presence of cleaved sequences is the indication of pool nonuniformity. When the presence of uncleaved sequences indicates pool heterogeneity, the results could be misleading due to possible incompleteness of enzymatic cleavage.

Determination of the coding and non-coding nucleotide sequences of genuine Edmonston-Zagreb master seed and current working seed lot.

To confirm the genetic stability of the Edmonston-Zagreb vaccine strain, we determined and compared the nucleotide sequences of genuine Edmonston-Zagreb master seed (EZ D22) and current working seed lot (EZ D24 2/99). Sequence analysis and comparison of the two sequences confirmed that these two sequences are the same at the molecular level. The obtained sequences were also compared to reference strains, i.e. Edmonston wild-type (Edmonston Wt) AF266288 and Edmonston-Zagreb (EZ) AF266290 vaccine strain. The sequence of EZ D22 differed from the Edmonston Wt in 32 nucleotides. EZ D22 differed from EZ AF266290 in six nucleotides. Coding substitution at position 441 and two silent substitutions at positions 11999 and 14612 in the L gene are unique to EZ D22. The differences found between EZ from different sources can be a good reason for periodical sequence analysis of the same strain in the hands of different manufactures.