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Biomedical subjects

W N Erber

Publications and source records attributed to W N Erber.

At least 37 records · Page 2Linked to original sources
PubMed1998

Monocyte activation in platelet concentrates.

BACKGROUND AND OBJECTIVES: Cytokines in platelet concentrates contribute to febrile transfusion reactions. Activated monocytes are a major source of inflammatory cytokines, however the role of monocytes in cytokine production in platelet concentrates has not been clarified. This study undertook to quantitate monocytes, determine whether monocyte activation occurs and identify an association with IL-6 and IL-1beta concentrations in platelet concentrates. MATERIALS AND METHODS: 17 platelet concentrates were analysed for total leucocyte and monocyte counts, CD14 and CD16 monocyte-associated antigen expression and IL-1beta and IL-6 measurements on days 1, 2, 3, 4 and 5. RESULTS: Monocytes in all platelet concentrates expressed increased levels of CD14 and CD16 from day 1 of storage. 10/17 platelet concentrates had elevated IL-6 levels by day 3. Platelet concentrates with IL-6 levels above 15 pg/ml on day 5 had monocyte counts between 0.14 and 15.6 x 10(6)/unit on day 1, while those with IL-6 levels below 15 pg/ml had low monocyte counts of < 0.01 to 1.2 x 10(6)/unit on day 1. CONCLUSION: Monocytes present in platelet concentrates exhibit features of activation. Monocyte activation is present following the preparation of platelet concentrates, implicating the manufacturing process in its development. Increased IL-6 and IL-1beta levels during platelet concentrate storage are commonly associated with a higher monocyte count. However, no direct association could be identified between the extent of monocyte activation and the level of cytokine release.

Blood Platelets↗
PubMed1997

An enhanced immunocytochemical method for staining bone marrow trephine sections.

AIMS: The detection of cellular antigens in fixed decalcified bone marrow trephine (BMT) sections depends on the method of processing, the nature of the antigen and antibody, antigen retrieval techniques, and the sensitivity of the immunocytochemical method. This study evaluated a tyramide enhanced avidin-biotin immunostaining method on formalin fixed decalcified BMT sections to determine whether the method could detect previously undetectable antigens. METHODS: Nineteen BMT biopsies from a range of haematological disorders were evaluated with 43 antibodies to haemopoietic antigens using horseradish peroxidase and alkaline phosphatase detection methods, using the tyramide enhanced avidin-biotin immunostaining method. RESULTS: Compared with standard avidin-biotin immunostaining methods the tyramide enhanced immunostaining method showed enhanced signal intensity, gave positive labelling for antigens that require pretreatment by other methods, and previously unreactive antigens were detected. Primary antibodies could be used at up to 200 times higher dilutions. CONCLUSION: The tyramide enhanced immunostaining method, while retaining specificity, is highly sensitive and enables an increased number and range of antigens to be detected than previously possible. The method could be applied to BMT sections for the routine diagnosis and classification of haematological disorders.

Antibodies, Monoclonal↗
PubMed1996

Use of unrefrigerated fresh whole blood in massive transfusion.

OBJECTIVE: To establish the efficacy of transfusion of fresh unrefrigerated whole blood in surgical patients with ongoing massive bleeding despite adequate blood-component replacement therapy and adequate surgical haemostasis. DESIGN: A retrospective review of patients who received fresh unrefrigerated whole-blood transfusions, noting blood usage and outcome. SETTING: A tertiary care teaching hospital with a major cardiac surgical and trauma service. PATIENTS: All patients (n=11) receiving fresh unrefrigerated whole-blood transfusions from March 1992 to February 1995. RESULTS: Mean blood usage in the 24 hours before the decision to transfuse fresh unrefrigerated whole blood was 16.5 units of packed cells (range, 6-27), 17.1 units of platelets (8-32), 14.5 units of fresh frozen plasma (6-26) and 13.5 units of cryoprecipitate (4-36). After the transfusion of fresh whole blood there was an immediate and substantial reduction in the rate and volume of blood loss in all patients. This was sustained in seven patients, who had a successful outcome; the other four patients died within 24 hours from recurrent uncontrollable haemorrhage. The reduction in blood usage in the 24 hours after the transfusion of fresh whole blood was statistically significant in the surviving patients but not in the patients who died. None of the surviving patients showed evidence of viral seroconversion six months after the transfusion. CONCLUSIONS: Our study provides preliminary evidence that there is a role for transfusion of fresh unrefrigerated whole blood in surgical patients with unresponsive life-threatening haemorrhage.

Adult↗
PubMed1996

The detection of rhodamine 123 efflux at low levels of drug resistance.

Although many cell models of multidrug resistance (MDR) have been developed, most have been high-resistance models which generate up to 100-fold increases in drug resistance. However, the drug concentrations required to achieve these levels of resistance are much higher than those found in vivo. In this paper we describe the development of a cell model that reflects the resistance levels that are likely to be found clinically. We then investigated the methods used to detect MDR1 expression at these low levels of drug resistance. We demonstrated that the immunological and PCR-based methods are unable to detect increased MDR1 expression in cells with a < 5.2- and 6.5-fold increase in vinblastine resistance, respectively, in our drug-resistant sublines. The rhodamine 123 (Rh 123) efflux assay was able to discriminate the vinblastine-sensitive parent cells from all the vinblastine-resistant sublines, including cells with a 1.7-fold increase in resistance. However, this assay is non-specific to the MDR1 gene and may detect the activity of other drug efflux proteins such as the MDR-associated protein (MRP). Our results show that the Rh 123 efflux assay is able to detect the activity of drug efflux proteins such as the P-glycoprotein, MRP or other efflux systems at the low levels of drug resistance that are likely to be attained in vivo.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Ultrastructural evidence of intestinal mucosal macrophage activation after bone marrow transplantation.

Previous studies have demonstrated upregulation of intestinal mucosal macrophage CD16 (an Fc receptor for IgG) in bone marrow transplant (BMT) recipients with graft-versus-host disease (GVHD). We sought to determine whether there was ultrastructural evidence of mucosal macrophage activation in allogeneic BMT recipients and relate appearances to those seen in autologous BMT patients and to immunohistological findings. Sigmoid colonic mucosal biopsies from five allogeneic and three autologous BMT patients were taken prior to, 30 days after transplant and, in three of the allogeneic patients, 120 days after transplant. These were examined by immunohistochemistry and electron microscopy. Immunohistological analysis revealed upregulation of lamina propria macrophage CD16 after transplant in all patients except one autologous BMT recipient; there were no such changes in total macrophage numbers. Ultrastructural evidence of lamina propria macrophage activation was prominent after both allogeneic and autologous BMT. There was an increase in nuclear size accompanied by increased euchromatin and larger nucleoli. In the cytoplasm there were increased numbers of lysosomes, many of which were small and cylindrical, and cytoplasmic flaps were prominent. Phagosomes were less numerous after transplant. These data confirm that after BMT intestinal mucosal macrophages become activated. However changes in macrophage ultrastructure specific to patients at risk of developing clinical GVHD are lacking.

Adult↗
PubMed1996

Antigen retrieval by microwave oven heating for immunohistochemical analysis of bone marrow trephine biopsies.

Immunohistochemical analysis of bone marrow trephine (BMT) biopsies with monoclonal antibodies for the analysis of hemopoietic disorders has been hindered by the fixation and decalcification regimens which mask or destroy tissue antigens. This study evaluated the effect of microwave oven treatment on the quality of immunostaining of fixed decalcified trephine biopsies. The aim was to establish whether this method of pre-treatment would enable additional antigens to be detected. Fifty-eight monoclonal and 4 polyclonal antibodies to hemopoietic antigens were assessed to compare no tissue pre-treatment, proteolytic (trypsin) enzyme digestion and microwave oven heating. The microwave heating of the sections was performed by placing them in a boiling solution of 0.01M tri-sodium citrate for a total of 10 mins. Following microwave heating 14 antibodies that previously showed no reactivity in BMT biopsies gave positive staining and 9 antibodies previously known to detect antigens in the absence of pre-treatment gave enhanced staining. Other antibodies showed no staining improvement with microwave heating and some failed to give a positive reaction by any of the pre-treatment methods. Antigen retrieval utilizing microwave oven heating can expose antigenic sites for antibody binding in bone marrow trephine sections. However not all antigens are retrieved and there is variation between epitopes on the one molecule and their ability to be exposed by microwave heating. Utilizing antigen retrieval methods, the range of antibodies applicable to BMT sections is greatly expanded enabling the immunophenotypic analysis of the majority of hemopoietic disorders.

Antibodies, Monoclonal↗
PubMed1995

Immunohistochemical changes in sigmoid colon after allogeneic and autologous bone marrow transplantation.

AIM: To determine whether there are characteristic immunohistological changes in the colonic mucosa in acute graft versus host disease (GvHD). METHODS: Consecutive allogeneic (n = 11) and autologous (n = 11) bone marrow transplant recipients underwent endoscopic biopsy of sigmoid mucosa before transplant and on day 30 post-transplant. Immunohistochemical staining and quantitation of intraepithelial and lamina propria mononuclear cells were undertaken using a panel of monoclonal antibodies and a Streptavidin-biotin alkaline phosphatase staining technique. RESULTS: In the allogeneic group (nine of whom had clinical acute GvHD) there was a fivefold increase in lamina propria CD16+ mononuclear cells (3.1 +/- 4.3 to 17.0 +/- 12.2 per 100 lamina propria nucleated cells), compared with autologous transplant recipients in whom this rise was twofold (5.5 +/- 4.6 to 10.6 +/- 7.1 per 100 lamina propria nucleated cells). The CD16+ mononuclear cells had morphological appearances of tissue macrophages, but in neither the allogeneic nor autologous groups was there an increase in total macrophage numbers (CD14+). In patients with acute GvHD the lamina propria CD4+:CD8+ lymphocyte ratio fell (1.97 +/- 1.12 to 1.07 +/- 1.01), primarily because of a fall in the number of lamina propria CD4+ lymphocytes. In both allogeneic and autologous groups there was a fall in intraepithelial lymphocyte numbers, but there was no change in CD19+ (B cell), CD25+ (interleukin-2 receptor positive) or CD56+ (natural killer) cell numbers. CONCLUSION: Following bone marrow transplantation, there appears to be upregulation of lamina propria tissue macrophage CD16 (an Fc receptor for IgG), a change which is more noticeable after allogeneic transplantation and which may be related to the development of acute GvHD. In patients with acute GvHD there was a fall in the lamina propria CD4+:CD8+ lymphocyte ratio. If these changes are functionally important, they may have significant implications for understanding the pathogenesis of GvHD.

Acute Disease↗
PubMed1995

The synthesis of a peanut agglutinin-ricin A chain conjugate: potential as an in vitro purging agent for autologous bone marrow in multiple myeloma.

The lectin peanut agglutinin (PNA) and CD19 monoclonal antibody have been covalently linked to magnetic beads and utilized in an in vitro purging system for autologous bone marrow in multiple myeloma (MM). An alternative to mechanical purging involves the use of immunotoxins to provide specifically targeted cellular toxicity; however, no studies to date have examined the utility of a lectin-ricin A chain (RCA) combination as a purging agent in MM. Initially, we studied the internalization of PNA by target cells (Raji) using flow cytometry. The surface fluorescence intensity of PNA-treated Raji cells was reduced upon incubation at 37 degrees C, and subsequent studies with fixed cells detected the endocytosed PNA. Complete internalization occurred within 120 minutes, indicating the potential of PNA as a purging agent. We manufactured a novel PNA-RCA conjugate and demonstrated its strong and specific binding to PNA reactive cell targets. Subsequent experiments assessed the toxicity of the conjugate to Raji cells and normal bone marrow progenitor cells. 3H-leucine uptake assays showed that PNA-RCA was capable of reducing protein synthesis in Raji cells and that the toxic effects were specific. In addition, at concentrations of conjugate achieving greater than 99% selective cytotoxicity for Raji cells, adequate CFU-GM were preserved in normal marrow. These studies suggest that PNA-RCA may be of value as an in vitro purging agent for MM.

Bone Marrow Purging↗
PubMed1995

Intestinal mucosal mononuclear cell chimaerism after sex-mismatched allogeneic bone marrow transplantation.

The contribution of haemopoietic cell chimaerism to the pathogenesis of GVHD after BMT is unclear. This report raises the possibility that donor lymphocyte-recipient macrophage chimaerism may occur shortly after allogeneic marrow engraftment and hence might contribute to the development of GVHD. Immunohistological studies of intestinal mucosa in an allogeneic BMT patient, who did not engraft, revealed an almost complete absence of lymphocytes 30 days after transplant, but preservation of mucosal macrophage numbers. Subsequently, combined immunohistology-Y chromosome in situ hybridization studies were performed in two female BMT recipients of male donor marrow. These studies revealed that between 25 and 40% of macrophages and between 25 and 40% of T lymphocytes were of donor origin during the first 6 months after transplant. In conclusion, whilst the immunohistological studies of intestinal mucosa from a patient who failed to engraft suggest that donor lymphocyte-recipient macrophage ('split') chimaerism may occur shortly after marrow engraftment, the subsequent in situ hybridization studies revealed 'mixed' chimaerism in the two sex-mismatched BMT recipients.

Adult↗
PubMed1994

Cross-matched platelets in bone marrow transplantation.

Antibodies to HLA-antigens remain a problem in multiply-transfused patients. Over a 2 yr period 44 bone marrow transplant recipients were screened at weekly intervals for the presence of HLA-antibodies using a solid phase red cell adherence technique. An adaptation of this method was used to provide cross-matched random donor platelets (XMRDP) when screening proved positive. Twelve of the 44 patients were antibody positive, 6 prior to transplantation and 6 following the transplant. Those 4 patients who developed an antibody following the transplant had a significant increase in platelet increments following the change from random donor platelets (RDP) to XMRDP even though only one patient was refractory to platelets at the time the antibody was first detected. No significant bleeding occurred during the study period. The use of routine platelet antibody screening followed by platelet cross-matching allows excellent support of thrombocytopenic patients without requiring HLA-typed blood donors and avoiding clinical platelet refractoriness.

Adolescent↗
PubMed1994

Unique immunophenotype of acute promyelocytic leukaemia as defined by CD9 and CD68 antibodies.

Twenty-one cases of acute promyelocytic leukaemia (FAB M3) demonstrating t(15,17) chromosomal translocation were studied in detail by immunocytochemical techniques using a panel of monoclonal antibodies. A characteristic myeloid phenotype of the leukaemic cells, co-expression of CD9 and CD68 antigens and absence of HLA-DR, was noted in all cases. Although the cellular morphology of acute promyelocytic leukaemia provides the most rapid means for diagnosis, this unique leukaemic cell phenotype provides confirmatory diagnostic evidence. In view of the new therapeutic options and prognosis in acute promyelocytic leukaemia, the detection of cases with atypical morphology based on this unique phenotype would be of value. In addition, the phenotype of acute promyelocytic leukaemia is similar to that of basophils and mast cells and raises the possibility that the leukaemic cells may have undergone a degree of basophilic differentiation.

Antigens, CD↗
PubMed1993

In situ hybridization of immunoglobulin light chain mRNA on bone marrow trephines using biotinylated probes and the APAAP method.

A non-radioactive in situ hybridization method has been established for the detection of mRNA for the constant region of light chain immunoglobulin genes in routinely processed bone marrow trephines. The method utilizes a cocktail of biotinylated synthetic oligonucleotide probes to kappa or lambda mRNA. The method entails dewaxing of the paraffin-embedded sections, proteinase K treatment and overnight hybridization with the biotinylated probe. Detection of probe hybridization was performed by 2 immunocytochemical detection methods, utilizing either streptavidin-alkaline phosphatase or monoclonal anti-biotin followed by the alkaline phosphatase: anti-alkaline phosphatase (APAAP) labelling system. The new fuchsin/naphthol phosphate substrate yielded the strongest signal with specific localization of the hybridization signal to positive cells. Morphological preservation was excellent enabling both polyclonal and monoclonal plasma cells to be detected in bone marrow trephines. We conclude that this in situ hybridization method is no more difficult than standard immunohistochemical techniques and can be used in routine diagnostic laboratories.

Biopsy↗
PubMed1992

Some characteristics of blood shed into the Solcotrans postoperative orthopaedic drainage/reinfusion system.

OBJECTIVE: To assess the suitability of blood shed into the Solcotrans orthopaedic autotransfusion system as a source of autologous blood for transfusion. DESIGN: Blood samples were taken from patients after surgery and from shed blood within the Solcotrans units. SETTING: Surgery was performed at a public hospital. PATIENTS: All six patients underwent total knee replacements. MAIN OUTCOME MEASURES: Measurements were made of haemoglobin, haematocrit, platelets, pH, potassium, plasma haemoglobin, fibrinogen, D-dimer, plasminogen activator, thromboplastin and fibrinopeptide A. The non-activated partial thromboplastin time was estimated. Shed blood was compared with homologous whole blood to assess the thrombogenic potential of shed blood in vitro. RESULTS: The haemoglobin and haematocrit levels of the shed blood were significantly lower than venous blood (P = 0.008). Levels of potassium in shed blood were normal although there was significant haemolysis. Shed blood was depleted of clotting factors, with increased levels of D-dimer (16-128 g/L). Activation of the coagulation pathway within the shed blood was shown by a shortened non-activated partial thromboplastin time (90-120 s), and detectable levels of thromboplastin. Propionibacterium acnes was isolated from one of the units. CONCLUSION: Reinfusion of large volumes of shed blood should probably be avoided, but use of the Solcotrans orthopaedic transfusion system in conjunction with other autologous transfusion practices can reduce the patient's requirement for homologous blood.

Blood Coagulation Tests↗
PubMed1992

Peanut agglutinin (lectin from Arachis hypogaea) binding to hemopoietic cells: an immunophenotypic study using a biotin streptavidin technique.

The lectin peanut agglutinin (PNA) was used to study the surface carbohydrate expression of galactose beta 1, 3, N-acetylgalactosamine by normal and malignant hemopoietic cells. Immunostaining was performed using biotinylated PNA and a streptavidin-alkaline phosphatase staining technique on 78 patients. The study was undertaken to enlarge on previous reports of lectin binding to cells of hemopoietic origin and to establish the potential role of biotinylated PNA as a component of an immunotoxin for in vitro purging of bone marrow in patients with multiple myeloma. In normals only monocytes, macrophages, centroblasts and plasma cells showed reactivity. Of the hematological malignancies, all cases of multiple myeloma were positive and non-Hodgkin's lymphoma cases with a large cell component had positive centroblasts. Two of 5 cases of acute myelomonocytic leukemia, one case of chronic myelomonocytic leukemia and one case of pleomorphic T cell non-Hodgkin's lymphoma showed PNA positive neoplastic cells. The reactivity of biotinylated PNA with centroblasts and plasma cells suggests that it may be of potential value when linked to a streptavidin-ricin conjugate in the in vitro purging of bone marrow of patients with multiple myeloma prior to autologous bone marrow transplantation.

Bacterial Proteins↗
PubMed1990

Human leucocyte differentiation antigens: review of the CD nomenclature.

In February 1989 the 4th International Workshop and Conference on Human Leucocyte Differentiation Antigens was held in Vienna. At this meeting the structure and function of recognized cellular antigens was reviewed and new antigens defined. In addition, information was obtained on the molecular genetics of antigens from gene cloning and sequencing. The CD (clusters of differentiation) nomenclature was extended, giving a total of 78 CD clusters, and some previously described CD groups revised and sub-divided. In this paper new information on the structure and function of leucocyte antigens presented at the meeting will be discussed. In addition, descriptions of the newly defined CD clusters and changes to established clusters will be outlined.

Antibodies, Monoclonal↗
PubMed1989

Use of APAAP technique on paraffin wax embedded bone marrow trephines.

The immunoalkaline phosphatase (APAAP) technique was applied to the labelling of decalcified sections of formalin fixed, paraffin wax embedded bone marrow trephine biopsy specimens. A panel of monoclonal antibodies reactive with haemopoietic and epithelial antigens, which survive routine formalin fixation, was assessed on 72 cases of haematological malignancy (including acute and chronic leukaemias and lymphomas) showing bone marrow infiltration. The APAAP method showed clear distinct labelling of antigen positive cells without loss of antigens due to decalcification. Both normal or reactive single cells present in the sample and neoplastic cell populations could be identified morphologically and their antigenic phenotype and cellular origin, whether lymphoid or myeloid, established. The application of the APAAP method to routinely prepared paraffin wax embedded trephines has many advantages over the assessment of specially prepared cryostat sections of bone marrow.

Alkaline Phosphatase↗
PubMed1988

Abnormal neutrophils in acute myeloid leukemia and myelodysplastic syndrome.

Neutrophils and band forms from patients with acute myeloid leukemia and myelodysplastic syndrome were stained for the presence of myeloperoxidase using a cytochemical method (diaminobenzidine/hydrogen peroxide) and the alkaline phosphatase--anti-alkaline phosphatase immunocytochemical procedure (using monoclonal anti-myeloperoxidase). Neutrophils and bands were also stained for elastase and lactoferrin using monoclonal and polyclonal antibodies, respectively. Subpopulations of neutrophils and bands from cases of acute myeloid leukemia and myelodysplasia exhibited a qualitative and/or quantitative deficiency in myeloperoxidase. In addition, a quantitative decrease in elastase and/or lactoferrin staining was detected. Thus, neutrophils and bands from patients with acute myeloid leukemia and myelodysplastic syndrome have a defect in one or more of the constituents of primary and/or secondary granules. These defects are consistent with the view that abnormal neutrophils and bands are derived from a malignant clone of myeloid precursor cells.

Cytoplasmic Granules↗