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Biomedical subjects

G J Buffone

Publications and source records attributed to G J Buffone.

At least 55 records · Page 3Linked to original sources
PubMed1987

Transferrin analysis by immunofixation as an aid in the diagnosis of cerebrospinal fluid otorrhea.

Laboratory diagnosis of cerebrospinal fluid leakage has been unreliable and has required expensive, labor-intensive radiographic procedures. Recently, using protein electrophoresis and immunofixation, the presence of an isoform of transferrin present only in cerebrospinal fluid has been identified. We describe the value of this simple test in a patient with recurrent meningitis in whom repeated radiographic studies failed to demonstrate a leak.

Adolescent↗
PubMed1986

Conjugated bilirubin versus direct bilirubin in neonates.

Conjugated and direct bilirubin were measured in 288 samples from 107 neonates less than 15 days old. Retrospective analysis of the medical records showed that 53 neonates were hepatobiliary-normal, 42 patients had no obvious evidence of hepatobiliary disease but had received total parenteral nutrition, and 12 were clearly hepatobiliary-abnormal. Neither the mean values nor the distributions of either the conjugated bilirubin, as measured by a multilayered slide, or the direct bilirubin, measured by a solution diazo procedure, differed when comparing the hepatobiliary-normal population to that receiving total parenteral nutrition. However, as would be predicted, the hepatobiliary-abnormal population differed significantly from both of these groups using either direct bilirubin or conjugated bilirubin results. Samples obtained from hepatobiliary-abnormal neonates were reviewed in chronologic sequence if direct bilirubin results differed from conjugated bilirubin with respect to classification of normality/abnormality. In two of three neonates with developing cholestasis, conjugated bilirubin exceeded its upper limit of normal earlier than did direct bilirubin. Conjugated bilirubin returned to normal earlier than direct bilirubin for two neonates with an improving clinical status. Conjugated bilirubin measurement was judged to be more responsive to developing or resolving cholestasis then direct bilirubin. In addition, conjugated bilirubin measurement from the slide is known to agree well with that determined by high performance liquid chromatography fractionation of bilirubin and is less susceptible to interference than is direct bilirubin measurement, characteristics that recommend its use over direct bilirubin analysis in a clinical setting.

Biliary Tract Diseases↗
PubMed1986

Liquid chromatography used in diagnosis of a rare hemoglobin combination: hemoglobin S/LeporeBoston.

"High-performance" liquid chromatography (HPLC), applied to hemoglobin analysis, is decidely more sensitive and gives better resolution than do routine electrophoretic methods. Here we present a case with a rare double heterozygote hemoglobin S/LeporeBoston, originally diagnosed as homozygous hemoglobin S by routine electrophoretic methods. Using a gradient elution weak cation-exchange HPLC technique, we could separate hemoglobin S and hemoglobin LeporeBoston and make the correct diagnosis. This case demonstrates how HPLC can be a useful adjunct to routine electrophoresis.

Adult↗
PubMed1985

Characterization and evaluation of immunochemical methods for the measurement of fecal alpha 1-antitrypsin.

Measurement of alpha 1-antitrypsin in feces has been proposed as a method of diagnosing a protein-losing enteropathy. This approach makes use of an endogenous marker rather than radioisotopically labeled materials such as 51CrCl3 or 131albumin to measure protein clearance. The validity of using fecal alpha 1-antitrypsin measurement as a reflection of protein loss through the gastrointestinal tract has been demonstrated by several investigators. The authors report here the characterization of excreted alpha 1-antitrypsin and an evaluation of the immunochemical methods used to measure this protein. They find alpha 1-antitrypsin to be excreted both as a protease-antiprotease complex and in a form that is relatively unaltered compared with serum alpha 1-antitrypsin. The proportion of alpha 1-antitrypsin excreted as a complex was found to vary from patient to patient. Formation of the protease-antiprotease complex was found to decrease the apparent alpha 1-antitrypsin concentration when radial immunodiffusion or immunonephelometry were used. The observed bias was greater for radial immunodiffusion. When these methods were applied to a newborn population at risk for necrotizing enterocolitis, radial immunodiffusion was found to have better sensitivity and a higher predictive value for a positive result than the nephelometric method. The use of fecal alpha 1-antitrypsin for diagnosis of protein-losing enteropathy appears to be best accomplished by radial immunodiffusion.

Electrophoresis, Polyacrylamide Gel↗
PubMed1985

High-performance liquid chromatography in the diagnosis of hemoglobinopathies and thalassemias. Report of three cases.

High-performance liquid chromatography is a technic that has recently been applied to the diagnosis of hemoglobinopathies and thalassemias. Its advantages over other methods include increased sensitivity, resolution and simplicity, as well as speed. In this report, the authors present an HPLC procedure that uses a weak cation exchange column and a gradient elution system for the diagnosis of hemoglobinopathies and thalassemias. The authors illustrate the utility of this procedure by reporting three cases in which the technic enabled them to make the correct diagnosis, which by traditional methods would have been missed or equivocal.

Child↗
PubMed1985

Unconjugated hyperbilirubinemia is overestimated in neonates with cholestasis. A more reliable method is proposed.

Unconjugated bilirubin was determined in 458 serum samples from 160 neonatal and pediatric patients by using two approaches. The first approach was to calculate indirect bilirubin by subtracting direct bilirubin, as measured by a diazo solution assay, from total bilirubin quantified by a Jendrassik-Grof procedure. The second approach was actually to measure the unconjugated bilirubin fraction using a multilayered slide. For samples containing little or no conjugated bilirubin, correlation between the calculated indirect bilirubin value and the unconjugated bilirubin measured by the slide was judged acceptable. Samples containing increased levels of conjugated bilirubin, however, yielded discrepancies between the approaches including differences of up to 5-10 mg/dL and more. Moreover, the magnitudes of the differences correlated with the amounts of conjugated bilirubin present in the samples. The unconjugated bilirubin values given by the slide were found to correlate more closely than the calculated indirect values with results obtained by an HPLC procedure that may be regarded as the most reliable method available. Therefore, we find that the slide provides a more accurate measure of unconjugated bilirubin than does the indirect bilirubin value in specimens from pediatric patients having evidence of cholestasis. The authors also believe that the magnitude of the difference in the values could make a difference in therapeutic strategies.

Age Factors↗
PubMed1985

Rapid radiometric assay used to assess lactoferrin in granulocytes.

We describe a rapid, precise, and sensitive radiometric assay for human lactoferrin. In this typical "sandwich"-type assay, anti-human lactoferrin is adsorbed onto a polystyrene sphere and bound lactoferrin is detected by the subsequent binding of 125I-labeled anti-human lactoferrin. The assay is accurate for lactoferrin concentrations of 5 to 1500 micrograms/L and takes about 2.5 h to complete. The within-assay and interassay variations (CV) are 5% and 13%, respectively. Neither lysozyme nor heparin, substances that form complexes with lactoferrin, interfered with lactoferrin measurement by this method. The assay has been applied to the measurement of lactoferrin in polymorphonuclear leukocytes of both healthy adults and neonates. We found significantly (p less than 0.001) less lactoferrin in the latter, an abnormality that may be related to known functional deficits of polymorphonuclear leukocytes during the newborn period.

Adult↗
PubMed1984

Evaluation of the Kodak EKTACHEM clinical chemistry slide for the measurement of bilirubin in newborns.

Measurement of neonatal bilirubin using the Jendrassik-Grof method (x) and the EKTACHEM NBIL assay (y) was compared over a 6-month period in a total of 1191 specimens from 483 patients less than 30 days of age. Linear regression analysis of the data yields a slope of 0.937, an intercept of 0.387, and Sy,x of 0.55 and a correlation coefficient of 0.983 for a total of 1032 specimens from patients less than or equal to 14 days of age and a slope of 1.090, an intercept of 0.002, an Sy,x of 1.03 and a correlation coefficient of 0.950 for a total of 159 specimens from patients greater than 14 days of age. The best correlation between EKTACHEM NBIL assay and the reference Jendrassik-Grof method was observed in samples from patients less than or equal to 14 days of age. Data from patients older than 14 days showed a higher proportional bias and a lower correlation coefficient between the methods. High performance liquid chromatographic analysis demonstrated that patients greater than 14 days of age had a higher incidence of elevated delta-bilirubin. Linearity extends to 200 mg/l. The NBIL assay provides a rapid, precise micromethod that is less sensitive than the Jendrassik-Grof method to the in vitro photo degradation of bilirubin and is not subject to the interference from hemoglobin and lipids. Because delta-bilirubin is not measured by this method, it is only recommended for newborns less than or equal to 14 days of age.

Bilirubin↗
PubMed1984

Generation of chemotactic activity in serum by Haemophilus influenzae type b.

Studies were performed to characterize chemotactic activity generated by Haemophilus influenzae type b (HiTb) in serum or elaborated independent of serum. Neutrophil aggregometry, Sephadex G-75 gel chromatography, and anti-C5 neutralization studies were used to demonstrate that the complement fragment C5a represented the major chemotactic moiety derived from HiTb-serum interactions. HiTb elaborated minimal chemotactic activity independently. Maximal C5a generation by HiTb as measured by neutrophil response in chemotaxis, shape change, and aggregation assays required specific antibody to the capsular polysaccharide, polyribosyl ribitol phosphate (PRP). Significantly more C5a was generated in pooled normal human serum containing high titers of anti-PRP (determined by an enzyme-linked immunosorbent assay) than in hypogammaglobulinemic serum. Furthermore, C5a generated in hypogammaglobulinemic serum reconstituted with purified high-titer immunoglobulin G, hyperimmune rabbit serum or heat-inactivated normal human serum was comparable to that generated in normal human serum. Absorption of antibody with PRP versus whole HiTb showed a contribution by non-PRP-directed antibody. As shown with the use of C4-deficient guinea pig serum, C5a generation occurred via the alternative complement pathway in nonimmune serum, and activation of the alternative complement pathway was facilitated by specific anti-PRP. C5a generation in test sera was proportional to its opsonic activity for HiTb as assessed by a luminol-chemiluminescence assay. Overall low levels of C5a activity were observed in 13 pediatric patient serum samples obtained during the acute phase of HiTb meningitis, and no pulmonary symptoms or radiographic abnormalities consistent with a leukocyte aggregation syndrome were observed in these patients.

Antibodies, Bacterial↗
PubMed1984

Abnormalities of polymorphonuclear leukocyte function associated with a heritable deficiency of high molecular weight surface glycoproteins (GP138): common relationship to diminished cell adherence.

Investigations of polymorphonuclear leukocyte (PMN) function were performed in a 5-yr-old white female with delayed umbilical cord separation, impaired pus formation, and a severe defect of PMN chemotaxis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated an almost total deficiency of a high molecular weight glycoprotein(s) (GP138) in the granule and membrane fractions of the patient's cells, and NaB3H4-galactose oxidase labeling demonstrated the absence of a major glycoprotein complex on the surface of her PMNs. Monoclonal antibodies (MAb) were employed in flow cytometry experiments to demonstrate that two previously characterized glycoproteins (Mo1 and LFA1) were undetectable on the surface of the patient's PMNs and monocytes. Immunoprecipitation of 125I-labeled patient cells with subunit specific MAbs confirmed that the alpha-subunits of Mo1 (155 kD) and LFA1 (177 kD) and their common beta-subunit (94 kD) were totally deficient. Functional analyses of patient PMNs demonstrated severe impairment of adherence- and adhesion-dependent cell functions including spreading, aggregation, orientation in chemotactic gradients, antibody-dependent cellular cytotoxicity, and phagocytosis of particles (Oil-Red-0-paraffin, zymosan) selectively opsonized with C3-derived ligands. Patient PMNs demonstrated a normal capacity to rosette with IgG or C3b-coated sheep erythrocytes, but rosette formation with C3bi-coated erythrocytes was profoundly diminished. Adhesion-independent functions including shape change, N-formyl-methionyl-leucyl-3H-phenylalanine binding, and O-2 generation or secretion elicited by soluble stimuli were normal. Membrane fluidity, surface charge, and microtubule assembly were also normal. These findings provide new evidence that critical PMN surface glycoproteins are required to facilitate multiple adhesion-dependent cellular functions of the inflammatory response.

Adult↗
PubMed1983

High-performance liquid chromatography of human hemoglobins on a new cation exchanger.

We have investigated the use of a high-performance liquid chromatographic (HPLC) column packed with a unique weak cation exchanger prepared by coating silica with poly(aspartic acid) for hemoglobin analysis. The complete separation of hemoglobin Bart's F, A0, A2, S, C, D, E, G, SG, Winnipeg and Sealy was achieved by gradient elution within 30 min. The high resolution made it possible to distinguish hemoglobin variants such as Bart's, AC, AD, AE, AG, AS, ASG, CC, SC, SS, Winnipeg, Sealy and beta-chain variants with thalassemia such as S/beta +, S/beta 0 and S(C)-beta + thalassemia. Comparison of DEAE-cellulose column chromatography and our HPLC method for the quantitation of hemoglobin A2 yielded a good correlation. Hemoglobins A2, C and E are completely resolved on PolyCAT A columns in contrast to both cellulose acetate electrophoresis and DEAE-cellulose column chromatography. The high resolution of the system and the accuracy of the method combined with complete automation make this procedure useful for diagnosis of hemoglobin disorders in both a research and clinical laboratory environment.

Adult↗
PubMed1983

Impaired chemotaxigenesis by type III group B streptococci in neonatal sera: relationship to diminished concentration of specific anticapsular antibody and abnormalities of serum complement.

A chemotaxigenesis (CTG) assay employing adult or neonatal sera, type III group B streptococci (GBS) and polymorphonuclear leukocytes (PMNs) was designed to evaluate the role of PMN mobilization in the pathogenesis of type III GBS infection in neonates. Generation of C5a in healthy adult sera with moderate-high (3-40 micrograms/ml) or low (less than or equal to 2 micrograms/ml) levels of specific anticapsular antibody was confirmed by PMN aggregometry and by the neutralization of CTG by goat anti-human C5. CTG was significantly (P less than 0.001) greater in high as compared to low specific antibody-containing adult sera; stepwise increases in CTG occurred when specific IgG was added to untreated, but not heat-inactivated, hypogammaglobulinemic serum. Immunospecificity of CTG was shown by a failure of type III GBS to generate C5a in heterologous (type Ia) high antibody sera. Mean CTG values in three high and 16 low antibody-containing sera from healthy term neonates were 24% and 62% of high (P less than 0.001) and low (P less than 0.01) antibody adult sera, respectively. The addition of both complement and specific IgG to low antibody-containing neonatal sera was required to enhance their CTG activity to high antibody adult values. CTG by type III GBS in neonatal sera-neonatal PMN mixtures was only 25% (high antibody sera) and 14% (low antibody sera) of values for paired maternal sera mixtures reacted with adult PMNs (P less than 0.001). These studies demonstrate that CTG by type III GBS in neonatal sera is markedly diminished and that low concentrations of specific anticapsular antibody and abnormalities of complement function contribute to impaired PMN mobilization in human neonates.

Adult↗