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Biomedical subjects

D F Stroncek

Publications and source records attributed to D F Stroncek.

At least 19 recordsLinked to original sources
PubMed2001

Evaluation of solutions for the storage of granulocyte colony-stimulating factor-mobilized granulocyte concentrates.

BACKGROUND: High cell counts in granulocyte colony-stimulating factor (G-CSF)-mobilized granulocytes are detrimental to concentrate storage. An eightfold dilution with autologous plasma improves storage, but this method is impractical. The purpose of this study was to identify an infusible solution that could be used in place of autologous plasma to dilute and store granulocytes. MATERIALS AND METHODS: Granulocytes collected from donors given dexamethasone (8 mg per os) and/or G-CSF (5 micrograms/kg subcutaneously [SQ]) were diluted eightfold in the following cell culture media: X-Vivo 10, Dulbecco's modified Eagle's minimal essential medium (DMEM) or Iscoves modified Dulbecco's medium (IMDM); or in the following infusible solutions: Plasma-Lyte A; Normosol R; lactated ringers, supplemented with 1% human serum albumin and 50 mM histidine (LRAH); or Plasma-Lyte A supplemented with 50 mM histidine buffer or 25 mM HEPES buffer plus 1% human serum albumin. The granulocytes were stored for 48 h at room temperature. White blood cell (WBC) counts, WBC viability and pH were measured after approximately 2 h, 24 h and 48 h of storage. RESULTS: Cell counts, viability and pH were maintained after 2 h, 24 h and 48 h in cells stored in the three cell culture media. The pH fell slightly after 48 h to 6.86 +/- 0.10 in granulocyte concentrates diluted in LRAH, but fell to a greater extent after 24 h and 48 h, to 6.36 +/- 0.23 (48-h value) in granulocyte concentrates diluted in Plasma-Lyte A and to 6.40 +/- 0.19 (48-h value) in granulocyte concentrates diluted in Normosol R. The cell counts of concentrates diluted in LRAH were stable for 48 h, but fell in granulocyte concentrates stored in Plasma-Lyte A and Normosol R. Plasma-Lyte A supplemented with histidine maintained the pH of diluted granulocyte concentrates better than Plasma-Lyte A supplemented with HEPES; 6.91 +/- 0.10 and 6.65 +/- 0.11, respectively, after 24 h. Cell counts were maintained best in granulocyte concentrates diluted in Plasma-Lyte A supplemented with albumin and one or both of the buffers. CONCLUSIONS: Culture media were best for granulocyte storage, but they are not approved for in vivo use. Infusible solutions are not buffered adequately and lack sufficient protein, but infusible solutions, such as lactated Ringer's solution or Plasma-Lyte A supplemented with buffers and albumin, hold promise as effective and licensable solutions for granulocyte storage.

Buffers↗
PubMed2001

RBC autoantibodies in autoimmune lymphoproliferative syndrome.

BACKGROUND: Patients with autoimmune lymphoproliferative syndrome (ALPS) have an autosomal dominant genetic defect that affects lymphocyte apoptosis and is associated with chronic nonmalignant lymphadenopathy, splenomegaly, and autoimmunity, particularly affecting RBCs, WBCs, and platelets. STUDY DESIGN AND METHODS: DATs were performed on 34 consecutive patients with ALPS and 37 of their clinically unaffected relatives. The effects of age, sex, race, and immunoglobulin levels on the incidence of autoantibodies and clinical hemolysis were assessed. RESULTS: The DAT was positive in 21 (62%) of ALPS patients but in only 1 (3%) of their relatives (p = 0.001). The DAT reacted because of IgG alone in 43 percent, complement alone in 5 percent, and IgG plus complement in 19 percent; 33 percent of the patients' cells had a positive reaction with polyspecific reagent only. All 10 ALPS patients with a history of hemolytic anemia had a positive DAT. Sixty percent of them had only IgG on their cells, 30 percent had IgG and complement, and 10 percent reacted only with polyspecific reagent. Of the 11 patients with a positive DAT and no history of hemolytic anemia, IgG alone was present in 27 percent, complement alone in 9%, and IgG plus complement in 9 percent; 55 percent had positive DATs only with polyspecific reagent. Among ALPS patients, those with a positive DAT had greater quantities of cells with increased alpha and ss T-cell receptors that phenotyped as CD4-CD8- and higher IgG levels. CONCLUSIONS: The DAT results in ALPS patients are most similar to those found in warm autoimmune hemolytic anemia. The DAT is useful to distinguish affected and unaffected persons within an ALPS family.

Adolescent↗
PubMed2001

The expression of NA antigens in people with unusual Fcgamma receptor III genotypes.

BACKGROUND: The Fcgamma receptor IIIb (FcgammaRIIIB) genes that encode neutrophil-specific antigens NA1 and NA2 differ at 5 nucleotides (nts); in 4, the result is an amino acid (AA) difference between the two alleles. The role of each of these differences in antigen expression is not known. Persons with FcgammaRIIIB genes that differ from NA1-FcgammaRIIIB and NA2-FcgammaRIIIB by 1 nt have been described. This study compared NA1 and NA2 expression on granulocytes in persons with variant FcgammaRIIIB genes and in healthy blood donors. STUDY DESIGN AND METHODS: Reactions of NA1- and NA2-specific MoAbs and alloantibodies with granulocytes were assessed by flow cytometry in 74 healthy blood donors and 6 persons with known variant FcgammaRIIIB genes. The granulocytes were tested with 1 NA1-specific MoAb, 1 NA2-specific MoAb, 4 NA1-specific alloantibodies, and 4 NA2-specific alloantibodies. RESULTS: Analysis of granulocytes from persons with variant NA genotypes found that single-base substitutions in FcgammaRIIIB at 141 and at 349 are important in NA1 expression and those at 227 and 277 are important in NA2 expression. Among blood donors, neither age, sex, nor race affected the expression of NA1 or NA2. The NA2-specific MoAb reacted more intensely with granulocytes from NA2-double-dose cells than with those from NA-single-dose cells, but this was not true for the NA2-specific alloantibodies. There was no difference in the reactions of the NA1-specific MoAbs and alloantibodies with donor samples of known NA1-double-dose or NA-single-dose cells. The intensity of reactions of both the NA1- and NA2-specific MoAbs and alloantibodies were strongly correlated on double-dose cells but not on single-dose cells. In fact, granulocytes from 7 healthy blood donors, phenotyped as NA-single-dose with the MoAbs, were phenotyped as NA2-double-dose with the alloantibodies. Variations in FcgammaRIIIB are common in blacks, but 5 of the 6 donors were white. These results suggest that FcgammaRIIIB variations may be common in both whites and blacks. CONCLUSIONS: NA2 expression is affected by polymorphisms in FcgammaRIIIB 227 and FcgammaRIIIB 277, both of which are involved in an FcgammaRIIIb N-glycosylation site. Polymorphisms in FcgammaRIIIB at 141 and 349 appear more important to NA1 expression.

Adolescent↗
PubMed2000

Detection of HLA antibodies by using flow cytometry and latex beads coated with HLA antigens.

BACKGROUND: Detection of HLA class I antibodies in sera is needed in various clinical situations. The standard method for detecting HLA class I antibodies is the complement-dependent lymphocytotoxicity (CDC) assay, but solid-phase assays are now available. STUDY DESIGN AND METHODS: This study assessed the ability of a flow cytometric assay using latex beads coated with HLA class I antigens to detect HLA class I-specific antibodies. The CDC assay was compared with the pooled-bead assay for the detection of HLA class I antibodies. Thirty-one randomly selected serum samples previously tested by CDC assay were tested with pooled beads and analyzed by flow cytometry. Twenty-seven additional serum samples, chosen by clinical criteria and CDC assay results, were tested against the pooled beads. Next, samples from six patients from whom three or more serum samples were drawn on consecutive days were tested with both methods. Finally, serum samples that were proved positive by both methods were tested with selected beads coated with antigens from a single person. RESULTS: Among the randomly selected serum samples, there was 90-percent agreement between the two assays. There was 96-percent agreement between the two assays of the 27 samples that were selected by clinical criteria and CDC assay results. Testing the sera with individual beads suggested that the HLA class I antibodies react with beads expressing the corresponding HLA antigen and beads expressing antigens in the same cross-reactive group. CONCLUSION: The pooled-bead assay can be used as an alternative method for detecting HLA class I antibodies. However, if the specificity of the HLA class I antibody is required, another assay must be used.

Antibodies↗
PubMed2000

Storage of G-CSF-mobilized granulocyte concentrates.

BACKGROUND: Current standards limit granulocyte storage to 24 hours. Since G-CSF inhibits granulocyte apoptosis, it may be possible to store G-CSF-mobilized granulocytes for longer periods while maintaining cell viability and function. However, G-CSF mobilization increases the yield of granulocytes several times, and the resulting higher cell concentrations may diminish viability during storage and significant levels of pyrogenic cytokines may be produced. STUDY DESIGN: Ten granulocyte donors were given dexamethasone (8 mg PO), G-CSF (5 microg/kg SQ), or both and on the next day granulocyte concentrates were collected using a blood cell separator. Component cell counts, cell viablilities, pH, and IL-1beta, IL-6, IL-8 and TNF levels were measured at 2 to 4 (2), 20 to 28 (24), and 44 to 52 hours (48 hours). RESULTS: Significantly more granulocytes were collected when donors were given G-CSF (4.2 +/- 2.3 x 10(10)) or G-CSF plus dexamethasone (6.4 +/- 2.5 x 10(10)) compared with that collected with dexamethasone alone (2.2 +/- 1.2 x 10(10)); p = 0.03 and p = 0.002, respectively. Storage had little effect on WBC count. Slight but significant increases in IL-1beta and IL-8 occurred after 24 and 48 hours as compared to the levels at 2 hours' storage. Levels of IL-6 and TNF did not change. The pH dropped significantly with time in granulocytes mobilized with each regimen. Granulocytes mobilized with G-CSF plus dexamethasone were acidic immediately after collection, and pH was below 6.0 after 24 hours. To assess the effect of cell concentrations on pH, serial dilutions were performed on 13 granulocyte concentrates in autologous plasma prior to storage. The pH remained above 7.0 only when dexamethasone-mobilized granulocytes were diluted 1-in-8 and when the G-CSF plus dexamethasone-mobilized granulocytes were diluted 1-in-16. CONCLUSIONS: To optimize storage pH, mobilized granulocyte concentrates require a 1-in-8 to 1-in-16 dilution, which is operationally impractical. Clinical-grade granulocyte preservative solutions are needed to maintain pH during storage.

Apoptosis↗
PubMed1999

Combined ultrafiltration-transduction in a hollow-fiber bioreactor facilitates retrovirus-mediated gene transfer into peripheral blood lymphocytes from patients with mucopolysaccharidosis type II.

The process of growing and transducing large quantities of human primary peripheral blood lymphocytes (PBLs) with high gene transfer efficiency continues to be one of the major challenges for clinical and experimental gene therapy. Toward developing a clinical trial of lymphocyte gene therapy for mucopolysaccharidosis type II (i.e., Hunter syndrome), we investigated a novel method that exploited the innate capability of a hollow-fiber bioreactor system to filter large quantities of vector supernatant and facilitate transduction. An aliquot (5 x 10(7)) of PBL apheresis product was precultured in a gas-permeable culture bag or a bioreactor, and then transduced with a retroviral vector L2SN containing the iduronate-2-sulfatase (IDS) and neomycin resistance genes. We observed that the total number of PBLs could be expanded up to 187-fold, yielding up to 10(10) cells at the end of a 7-day culture period. The multiplicity of infection could be increased (up to 20-fold) by ultrafiltrating a large volume of vector supernatant through the semipermeable membrane of this system. A high level of transduction efficiency (up to 57%) was achieved, resulting in IDS enzyme activity as high as 1250 U/mg/hr in transduced PBL(MPS) 15 days after transduction. This level was markedly increased from that of nontransduced cells (<3 U/mg/hr) and was even greater than that of normal PBLs (mean, 809; n = 10). After 12 days of G418 selection, PBL(MPS) transductants exhibited a proviral IDS enzyme level approximately threefold higher than that in normal PBLs. These results indicated that the hollow-fiber bioreactor could be used to culture and transduce human primary PBLs in clinically useful quantities with relatively high gene transfer efficiency and transgene expression.

Bioreactors↗
PubMed1999

Collection of two peripheral blood stem cell concentrates from healthy donors.

When peripheral blood stem cell (PBSC) concentrates are used for allogeneic transplants, two or more apheresis procedures must often be performed. To determine how many cells could be collected from healthy people by two back-to-back apheresis procedures and what effect these collections would have on donors, we gave 19 healthy people 5 micrograms kg-1 day-1 and 21 people 10 micrograms kg-1 day-1 of granulocyte colony stimulating factor, filgrastim, for 5 days. We then collected two PBSC concentrates, one on day 5 and one on day 6. A third group of six people was given filgrastim 10 micrograms kg-1 day-1 for 5 days but had no PBSC concentrates collected. PBSC concentrate cell counts and donor cell counts, symptoms, and blood chemistries were assessed for up to 1 year. On day 5, three times more CD34+ cells were collected from donors given 10 micrograms kg-1 day-1 than those given 5 micrograms kg-1 day-1 (P = 0.009) but on day 6 the quantity of cells collected was the same (P = 0.23). The total number of CD34+ cells collected was two times greater in donors given the higher dose of filgrastim (median = 579 x 10(6); range = 174-1639 x 10(6) compared to 237 x 10(6); 103-1670 x 10(6); P = 0.061). Platelet counts fell after each PBSC concentrate collection, but there were no differences between the two groups of donors in platelet counts measured immediately after each collection. The platelet counts also fell in people who did not donate PBSC concentrates. The lowest counts in all three groups of people also occurred on day 10. In PBSC donors given 10 micrograms kg-1 day-1 of filgrastim the absolute neutrophil count (ANC) fell below premobilization counts on day 14. In donors given 5 micrograms kg-1 day-1 the ANC fell below premobilization counts on days 21, 28 and 49, CD34+ cell counts were significantly lower than premobilization counts on days 14 and 28 in donors given 10 micrograms kg-1 day-1 of filgrastim and on day 14 in those given 5 micrograms kg-1 day-1. No decrease in neutrophil or CD34+ cell counts occurred after filgrastim was given in the people who did not donate PBSC concentrates. The incidence of symptoms was similar in both groups of PBSC concentrate donors, except that those given 10 micrograms kg-1 day-1 were more than twice as likely to experience myalgias as those receiving the lower dose (P = 0.029). Several blood chemistries changed. Levels of alkaline phosphatase, LDH, SGPT, SGOT, uric acid and sodium increased. Levels of bilirubin, total protein, potassium, calcium and chloride decreased. In conclusion, twice as many CD34+ cells were collected from donors given 10 micrograms kg-1 day-1 of filgrastim. Platelet, neutrophil and CD34+ cell counts fell after the PBSC concentrate collections. The fall in platelet counts was due to both the collection and the administration of filgrastim. The falls in neutrophil and CD34+ cell counts were due to the loss of haematopoietic progenitor cells in the PBSC concentrates. Allogeneic PBSC concentrate donors should be given 10 micrograms kg-1 day-1 of filgrastim, and if possible only one component should be collected in order to avoid thrombocytopenia.

Adult↗
PubMed1999

Granulocyte storage and antigen stability.

BACKGROUND: Current methods for the detection of granulocyte antibodies require panels of freshly isolated cells. This makes these assays time-consuming, costly, and technically difficult. STUDY DESIGN AND METHODS: The immunofluorescence method of detecting the binding of antibodies to granulocytes was modified for use with a flow cytometer, and methods were tested to store granulocytes for use in that assay. Granulocytes were stored at 4 degrees C for 7 days under three conditions: 1 -percent formaldehyde-fixed cells were stored in Hanks' balanced salt solution (HBSS); untreated cells were stored in tissue culture medium (RPMI-1640); and cells were fixed and stored with a commercial white cell-storage solution (Cyto-Chex Reagent). Antigen stability was evaluated by using monoclonal antibodies (MoAbs) and alloantibodies. Serologic studies were done by an indirect immunofluorescence assay and assessed by flow cytometric analysis. RESULTS: On Day 2, only 2 to 7 percent of granulocytes stored in RPMI-1640 remained. On Day 7, 67 to 76 percent of granulocytes fixed in formaldehyde and stored in HBSS remained, and 47 to 87 percent of granulocytes stored in a white cell-storage solution remained. All antigens were detectable by the MoAbs and alloantisera on Day 7. However, nonspecific staining by the fluorescein isothiocyanate (FITC)-conjugated secondary antibody hindered interpretation of test results on Day 4. Non-specific staining occurred over time and was associated with increased cell permeability during storage. Two sources of nonspecific staining were identified. The first source was the FITC-conjugated secondary antibody; it was eliminated by switching to a phycoerythrin conjugate. The second source was factors in human serum; it was resolved by examining only viable, impermeable cells identified by using 7-aminoactinomycin-D. CONCLUSION: Granulocytes and their antigens can be preserved for at least 7 days, but evaluation of antibody reactions was possible for only 4 days as a result of non-specific staining due to enhanced membrane permeability of dying cells.

Animals↗
PubMed1999

Biotinylation modifies red cell antigens.

BACKGROUND: Chemical biotinylation of red cell membranes may be useful for several clinical applications, including red cell survival studies. STUDY DESIGN AND METHODS: To examine the possible effects of biotinylation on red cell antigens, standard hemagglutination assays were performed on matched sets of control and biotinylated red cells. The red cells were biotinylated at a final concentration of 2.0 pg of sulfo-N-hydroxysuccinimide-biotin per cell, and antigen-negative cells were directly compared to antigen-positive cells when possible. The hemagglutination assays were graded in a blinded fashion. Forty-one red cell antigens from 21 of the 23 established blood group systems were tested. RESULTS: Hemagglutination based upon antibody binding to A, A1, M, N, S, s, P1, D, C, E, c, e, C(w), Lu(b), K, k, Kp(b), Le(a), Le(b), Fy(a), Fy(b), Jk(a), Jk(b), Di(a), Wr(a), Wr(b), Yt(a), Xg(a), Sc1, Do(b), Co(a), Ch, H, Ge2, Cr(a), Kn(a), I, and P was not affected by biotinylation. Unexpectedly, the hemagglutination of Di(b+) and LW(a+) red cells was blocked after biotinylation. Conversely, MH04 monoclonal anti-A agglutinated red cells expressing B only after biotinylation. BIRMA-1 monoclonal anti-A and polyclonal anti-A from sera did not agglutinate the biotinylated B red cells. CONCLUSION: Biotinylation of human red cells specifically modified their antigenicity, as measured by standard hemagglutination assays.

Biotinylation↗
PubMed1999

Evaluation of the gel system for ABO grouping and D typing.

BACKGROUND: The gel agglutination assay has been approved by the Food and Drug Administration as an alternative to the tube assay for the detection of red cell antibodies. It has also been approved recently by the Food and Drug Administration for ABO blood grouping and D typing. STUDY DESIGN AND METHODS: Tube and gel agglutination assays were compared for ABO grouping and D typing of 100 donor and 100 patient specimens. ABO grouping of 14 specimens of known ABO groups and D typing of 10 specimens with weak D were also compared. When antigen typing or isohemagglutinin results differed, gel testing was repeated by the use of modified incubation times, reagent or specimen volumes, and red cell concentrations. RESULTS: ABO grouping and D typing in all patient and donor specimens concurred. B isohemagglutinins were not detected in seven group A specimens. Six of seven discrepancies were resolved when gel tests were incubated at room temperature with increased serum or plasma volume. Weak D was detected in all 10 specimens tested by both assays. When weak A and/or B were tested with monoclonal antibody reagents, the correct phenotypes were identified in 9 specimens by gel assay and in 10 by tube assay. Using human antisera, 6 specimens were correctly phenotyped by gel assay and 7 by tube assay. CONCLUSION: The gel assay performed as well as the tube assay in detection of A, B, and D, but the tube assay was slightly better at detecting B isohemagglutinins. The gel assay can be used in place of the tube assay for ABO blood grouping and D typing.

ABO Blood-Group System↗
PubMed1999

Retroviral transduction and expansion of peripheral blood lymphocytes for the treatment of mucopolysaccharidosis type II, Hunter's syndrome.

BACKGROUND: Gene therapy using autologous peripheral blood lymphocytes (PBLs) has been used to produce adenosine deaminase with which to treat patients with severe combined immunodeficiency. Patients with mucopolysaccharidosis type II (MPS II) lack iduronate-2-sulfatase (IDS), and serial PBL gene therapy may benefit these patients. STUDY DESIGN AND METHODS: The purpose of these studies was to develop a method to transduce PBLs from a patient with MPS II by using a retroviral vector, LS2N, containing the IDS gene. PBLs were collected by apheresis and cryopreserved in aliquots for the performance of multiple transductions and expansions. The PBLs were expanded in number and then transduced in a hollow-fiber bioreactor (HFBR). Additional culture allowed for further expansion. RESULTS: Fresh PBLs (6.2 x 10(7)) from a patient with MPS II were transduced with L2SN and expanded in an HFBR with an extracapillary space of 11 mL. After 10 days of culture, 4.1 x 10(9) cells were harvested. Cryopreserved MPS II PBLs could not be reliably expanded if they were placed in the HFBR immediately after being thawed; however, cells were successfully transduced and expanded in the HFBR if they were first cultured in a bag. To increase the cell yield, PBLs were expanded in a 60-mL HFBR after transduction and expansion in an 11-mL HFBR. In four separate experiments, 2 x 10(8) cryopreserved PBL were cultured for 3 days in a bag and transferred to an 11-mL HFBR, where they were transduced daily with L2SN for 3 days and then expanded for 4 additional days. Cells were then transferred into a 60-mL HFBR and expanded for an additional 7 days. In the four experiments, 5.5 x 10(9), 7.4 x 10(9), 1.12 x 10(9), and 19.4 x 1(9) cells were produced. The vector was detected in the harvested cells, but the proportion of cells transduced was less than 2.5 percent, the lowest standard used in the assay. In two of the experiments, cells harvested from the HFBR were used in a gene therapy clinical trial. CONCLUSION: Autologous cryopreserved PBLs can be transduced and expanded to produce >1 x 10(10) cells. This procedure is being used for a Phase I/II clinical trial of lymphocyte gene therapy.

Cells, Cultured↗
PubMed1999

Changes in serum osteocalcin and bone-specific alkaline phosphatase are associated with bone pain in donors receiving granulocyte-colony-stimulating factor for peripheral blood stem and progenitor cell collection.

BACKGROUND: Granulocyte-colony-stimulating factor (G-CSF) has been used to increase the number of CD34+ peripheral blood stem and progenitor cells collected by apheresis for use in autologous or allogeneic progenitor cell transplantation. The most frequent side effect of G-CSF treatment is bone pain, which occurs in over 80 percent of healthy progenitor cell donors. STUDY DESIGN AND METHODS: The possible mechanism of bone pain was investigated by measuring serum levels of osteocalcin (OC), bone-specific alkaline phosphatase (BAP), acid phosphatase (ACP), and tartrate-resistant acid phosphatase (TRAP) in seven healthy progenitor cell donors treated with human recombinant G-CSF administered subcutaneously for 5 consecutive days. RESULTS: All seven patients experienced bone pain during the treatment period. Serum levels of OC, BAP, ACP, and TRAP were measured in blood samples drawn on Days 0, 4, 5, 6, and 14. Levels of BAP were increased (p<0.05) over baseline on Days 4, 5, and 6, while those of OC decreased on Days 4, 5, and 6 (p<0.05). No significant changes occurred in ACP or TRAP levels. OC and BAP are considered markers of bone formation (osteoblast activity), and they correlate in many patients with metabolic bone disorders. The pattern of increased BAP and decreased OC has been reported in patients with osteolytic bone metastases. CONCLUSION: G-CSF treatment in healthy stem and progenitor cell donors may affect osteoblastic activity, and this activity may be associated with bone pain.

Alkaline Phosphatase↗
PubMed1998

Therapeutic apheresis for babesiosis.

Infection with the tick-borne protozoa Babesia is becoming more common. Babesiosis is usually successfully treated with antibiotics but, in some cases, apheresis may also be indicated. We report two patients with babesiosis and hemolysis treated by apheresis and antibiotics. One case had traditional indications for red blood cell (RBC) exchange, and a second patient was treated with RBC exchange, and plasmapheresis for hemolysis, probably secondary to Babesia parasitemia. Case 1 involved a 44-year-old man with chronic relapsing pancreatitis who had become infected with Babesia from a unit of RBCs transfused during surgery. At 5 weeks after surgery, fever and severe hemolysis developed, along with a hemoglobin of 69 g/L; 30% of his RBCs were found to be infected with Babesia. This patient had several postoperative complications; the babesiosis was treated with clindamycin, quinine, and three RBC exchanges. Parasitemia fell to less then 1% of RBCs, but the patient died of pancreatitis. Case 2 was a 47-year-old man with a renal transplant who had been receiving immunosuppressive therapy for 8 years. He had a history of tick bites, fever, and hemolytic anemia. Analysis of a peripheral blood smear detected Babesia. He was initially treated with antibiotic therapy and two RBC exchanges. Hemolysis improved transiently but worsening parasitemia developed later, as well as an IgG RBC autoantibody. He was then treated by plasmapheresis and RBC exchange. Although his condition improved, he had a third hemolytic episode, which was treated with plasmapheresis and RBC exchange before the parasitemia and autoimmune hemolytic anemia disappeared. In conclusion, immunosuppressed or severely ill people who become infected with Babesia may benefit from RBC exchange or plasmapheresis, or both.

Adult↗
PubMed1998

The expression of the NB1 antigen on myeloid precursors and neutrophils from children and umbilical cords.

The neutrophil-specific antigen NB1 is expressed by neutrophils from 97% of healthy adults. However, membrane expression of this molecule is unique in that it is found on only a subpopulation of neutrophils present in NB1-positive adults. We have investigated the ontogeny of NB1 antigen expression by haematopoietic progenitor cells to determine the stage and pattern of antigen expression during granulocytic cell differentiation. In addition, we examined whether the ontogeny and frequency of granulocytic cells expressing the NB1 antigen might vary in subjects according to age. A monoclonal antibody (MoAb) specific for NB1 (1B5) and flow cytometry was used to assess the frequency and characteristics of the NB1-positive cells found in umbilical cord blood (n = 11), children (n = 37), healthy adults (n = 46) and patients with chronic myelogenous leukaemia (n = 8). We also used flow cytometry to isolate NB1-positive and NB1-negative bone marrow and peripheral blood cells from various tissue sources. The separated subpopulations were then analysed by Wright stain and light microscopy. The size of the NB1-positive neutrophil subpopulation in 46 healthy adults (56 +/- 19%) was identical to that found for neutrophils from 36 children ranging in age from 8 months to 18 years (56 +/- 11%). In contrast, expression of the NB1 antigen by the neutrophils present in umbilical cord blood (91 +/- 3%, n = 11) was significantly greater than that in adults (P < 0.002) or children (P < 0.002). We also examined the size of the NB1-positive subpopulation among neutrophils from eight patients with chronic myelogenous leukaemia (CML). The NB1-positive subset in CML subjects (29.5 +/- 22.4%) was significantly less that in healthy adults (P < 0.02) or children (P < 0.02). Marrow cells from eight adults were similarly separated and analysed. We found that 69 +/- 17% of segmented and band forms of neutrophils, 70 +/- 2% of metamyelocytes and 61 +/- 23% of myelocytes were NB1-positive. In fetal bone marrow, 86 +/- 9% of the segmented and band forms, 82 +/- 10% of the metamyelocytes and 3 +/- 4% of myelocytes were NB1-positive. In conclusion, neutrophil-specific antigen NB1 is first expressed at the myelocyte stage of myeloid differentiation. In adult bone marrow, the percentages of myelocytes, metamyelocytes and segmented or band cells that expressed this antigen were similar and comparable in magnitude to the frequency of NB1-positive neutrophils found in the circulation. Although the size of the NB1-positive neutrophil subpopulation was the same in healthy adults and children, it was significantly increased in umbilical cord blood, and in fetal marrow cells.

Adolescent↗
PubMed1998

Expression of neutrophil antigens after 10 days of granulocyte-colony-stimulating factor.

BACKGROUND: Granulocyte-colony-stimulating factor (G-CSF) is becoming the standard agent for mobilizing granulocytes. Most granulocyte donors are given a single dose of G-CSF, but in some cases they are given G-CSF for several days, and multiple granulocyte concentrates are collected. The administration of a single dose of G-CSF induces several changes in the expression of neutrophil antigens, but the effects of multiple daily doses of G-CSF are not known. STUDY DESIGN AND METHODS: Seven healthy people received 5 microg per kg of G-CSF for 10 days. Their expression of several neutrophil antigens before, during, and after the administration of G-CSF was analyzed through the use of flow cytometry. RESULTS: The expression of L-selectin (CD62L), Fcgamma receptor (FcgammaR) III (FcgammaRIII, CD16), and the leukocyte function antigen (CD11a) decreased throughout the course of G-CSF administration, while the expression of FgammaR I (FcgammaRI, CD64) and lipopolysaccharide-binding protein receptor (CD14) increased. The expression of FcgammaR II (FcgammaRII, CD32) also increased, but not until the fourth day of G-CSF administration. The expression of amino peptidase N (CD13), C3bi receptor (CD11b), and the neutrophil beta2 integrin unit (CD18) did not change during the administration of G-CSF, but that of both CD13 and CD18 increased 3 days after the last dose. The expression of neutrophil-specific antigen NB1 initially increased, returned to pre-G-CSF levels after 4 days, and then increased again after 10 days of G-CSF administration. CONCLUSION: Changes in the expression of several neutrophil antigens occurred throughout a 10-day course of G-CSF Most of the changes occurred after one dose, but additional changes occurred later in the 10-day course and after its completion. These changes may affect the function of G-CSF-mobilized granulocytes.

Adult↗
PubMed1997

Comparison of two blood cell separators in collecting peripheral blood stem cell components.

To ensure that a sufficient number of CD34+ cells are collected for an allogeneic blood progenitor cell transplant, the most effective blood cell separator should be used to collect peripheral blood stem cell (PBSC) components. We compared the effectiveness of two blood cell separators. We gave 29 healthy people 7.5 or 10 micrograms kg-1 of granulocyte colony stimulating factor (G-CSF) daily for 5 days and collected one PBSC component with either a Fenwal CS3000 (n = 15) or a Cobe Spectra (n = 14) blood cell separator. The volume of blood processed was the same for each machine (8.4 +/- 1.0 L; range = 4.9-9.4 L for the CS3000 and 8.9 +/- 1.0 L; range 6.7-10.9 L; P = 0.71). The components collected with the CS3000 contained more mononuclear cells (39.6 +/- 21.9 x 10(9) compared with 26.9 +/- 5.6 x 10(9), P = 0.02) and fewer neutrophils (1.38 +/- 1.88 x 10(9) compared with 5.53 +/- 8.71 x 10(9), P = 0.001). The total number of CD34+ cells collected with the two instruments was the same (470 +/- 353 x 10(6) for the CS3000 and 419 +/- 351 x 10(6) for the Spectra; P = 0.64) as was the number of CD34+ cells collected per litre of whole blood processed (55.9 +/- 42.0 x 10(6) L-1 compared with 45.9 +/- 37.9 x 10(6) L-1; P = 0.59). The mononuclear cell collection efficiency was greater for the CS3000 (82.4 +/- 54.9% compared with 53.3 +/- 14.1; P = 0.04) but the CD34+ cell collection efficiencies were the same (87.4 +/- 61.1% for the CS3000 compared with 56.3 +/- 23.5% for the Spectra; P = 0.07). In conclusion, both blood cell separators collected components which contained large numbers of CD34+ cells, but those collected with the CS3000 contained fewer neutrophils and the CS3000 was more efficient at collecting mononuclear cells.

Adult↗
PubMed1997

The kinetics of G-CSF mobilization of CD34+ cells in healthy people.

When healthy people are given granulocyte colony stimulating factor (G-CSF) for 10 days the number of CD34+ cells in the peripheral blood begins to increase on the fourth day, reaches a maximum on the sixth day and then decreases. In this study, we further define the time and variability of peak mobilization of CD34+ cells. Twenty-two healthy people were given G-CSF (7.5 or 10 micrograms kg-1 day-1) subcutaneously each morning for 5 days and peripheral blood CD34+ cell counts were analysed immediately prior to the fourth (day 4) and fifth (day 5) G-CSF injection and 24 h after the fifth injection (Day 6). White blood cell (WBC) and neutrophil counts were greatest on day 6 [WBC = 43.8 +/- 13.9 x 10(9) L-1 (mean +/- 1 SD) and neutrophils = 36.6 +/- 12.8 x 10(9) L-1]. In contrast the CD34+ cell counts on day 6 (107 +/- 104 x 10(6) L-1) were less than on day 5 (128 +/- 136 x 10(6) L-1) (P = 0.048) but still greater than on day 4 (60.7 +/- 40.2 x 10(6) L-1) (P < 0.0001). The CD34+ cell counts of 10 donors were measured 2, 4 and 6 h after the fifth injection to determine if the counts increased further between days 5 and 6. The number of CD34+ cells in the blood on day 5 2 h after the fifth injection (193 +/- 277 x 10(6) L-1) was greater than the number prior to the injection (158 +/- 190 x 10(6) L-1), 4 h post-injection (139 +/- 158 x 10(6) L-1) and 6 h post-injection (170 +/- 236 x 10(6) L-1), but the differences were not significant (P = 0.29, 0.25 and 0.45). The number of CD34+ cells in the blood of 12 people were measured before and after the fourth G-CSF dose. Prior to the day 4 injection the CD34+ count was 61 +/- 40 x 10(6) L-1. At 2, 4 and 6 h the counts were 60 +/- 40, 61 +/- 29 and 64 +/- 30 x 10(6) L-1, respectively, and the differences were not significant (P = 0.99, P = 0.98, and P = 0.73). In conclusion, when healthy volunteers are given daily G-CSF injections, the number of mobilized CD34+ cells was the greatest on day 5, slightly less on day 6 and the least on day 4. If only one PBSC component is needed, PBSCs can be collected on day 5 after only 4 days of G-CSF. If PBSC components are collected on both days 5 and 6, the fifth dose can be given either before or after the collection of the first PBSC component.

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