Biomedical subjects
G L Hortin
Publications and source records attributed to G L Hortin.
Critical values, panic values, or alert values?
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Yield of laboratory tests for case-finding in the ambulatory general medical examination.
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Progress toward preparation of universal donor red cells.
Coupling of an inert polymer to the surface of red cells was examined as a potential means of covering blood group antigens and producing cells that could serve as universal donor cells for transfusion. Effective blockade of red cell antigens was achieved with N-hydroxysuccinimide-activated esters of polyethylene glycol. It was possible to block all antigens tested, but lower concentrations of reactants were required to block peptide-defined antigens than carbohydrate-defined antigens. Red cells remained intact after modification but were significantly damaged. Our results demonstrate the feasibility of antigenic blockade of red cells, but there is a need to reduce damage during coupling reactions to produce viable red cells.
Beyond traditional quality control. How to check costs and quality of point-of-care testing.
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Lipemia interference with a rate-blanked creatinine method.
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Win the productivity battle.
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Lancets for capillary blood collection.
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Multiple biochemical blood testing.
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Managing information from bedside testing.
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Analytical artifacts in hematocrit measurements by whole-blood chemistry analyzers.
Compact analyzers suited to near-patient testing estimate hematocrit by measuring the conductivity of undiluted blood. We evaluated the accuracy of hematocrit determination of one such analyzer (Instrumentation Laboratory BGE analyzer) against an automated cell counter (EPC) and packed cell volume (PCV) microhematocrit. When specimens (n = 34) from outpatient and ward patients were analyzed with all three methods, the BGE analyzer correlated well with both EPC and PCV hematocrit determinations (BGE = 1.00 PCV + 0.3%, S(y)/x = 2.0%), suggesting that all three methods are similar in performance for most patients. However, a patient with increased plasma osmolality showed significant decreases in BGE and PCV hematocrits relative to the EPC method. The differences in hematocrit measurements could be reproduced by adding solutes to blood in vitro or by modifying the plasma osmolality of rats in vivo. Samples from patients undergoing cardiac surgery, whose blood had large changes in protein concentration, showed discrepancies between hematocrits by conductivity and other methods; similar effects could be produced by changes in protein concentration or in vitro addition of polyethylene glycol. We conclude that conductivity measurements provide accurate hematocrit results for physiologically normal subjects but not for some intensive-care and surgical patients.
Rapid measurement of serum pancreatic amylase.
A simple, rapid assay for the pancreatic isoenzyme of human serum amylase was developed. The assay utilized an immunoabsorbent prepared by coating latex beads with a monoclonal antibody specific for pancreatic amylase. Treatment of patient serum with immunoabsorbent removed pancreatic amylase, and measurement of residual amylase activity with standard total amylase methodology allowed estimation of the pancreatic amylase content. Extraction efficiency of pancreatic amylase was consistent at amylase concentrations up to 1,000 U/L (y = 0.97 x +16.7 U/L; r = 0.9995). The assay was standardized with purified pancreatic amylase added to neonatal serum (low endogenous activity). A comparison of patient specimen results with the results of a standard technique (cellulose acetate electrophoresis) yielded an excellent correlation (immunoabsorption result = 0.96 electrophoresis result + 1.2 U/L; r = 0.987). Salivary amylase did not interfere with the assay until levels exceeded 1,000 U/L. Daily analysis of a frozen serum pool yielded a coefficient of variation of 9.2% at mean pancreatic amylase value of 54 U/L (+/- 5 U/L). A normal range study found a strong influence of age, with pancreatic amylase levels increasing dramatically in the first 3 years of life, to stabilize at a range of 0-66 U/L thereafter.
Detection of autoantibodies to amylase by ELISA: comparison of detection of macroamylase and free autoantibody.
New ELISAs for detecting macroamylase or free autoantibodies to amylase were tested with 48 samples that had been characterized by gel chromatography and electrophoresis. The macroamylase ELISA, with anti-IgG or anti-IgA for detection, detected macroamylase in 28 of 33 samples known to contain macroamylase (85% sensitivity), whereas the ELISA for free autoantibody to amylase was positive for only 11 samples. Specificities of both ELISAs were 93%. Among 28 true positives detected with the macroamylase ELISA, 22 contained IgA, 3 contained IgG, and 3 contained both immunoglobulins. Detection of IgM added no true positives. ELISA responses (y) were proportional to log [macroamylase concentration by chromatography (x)] from 0 to 1200 U/L: y = 5.15 x + 1.66; r = 0.72; Sy x = 1.65. As new tools for detecting macroenzymes consisting of enzyme-autoantibody complexes, the ELISAs show that some autoantibodies are detected more sensitively as antibody-antigen complexes than as free antibody.
Changes in plasma amino acid concentrations in response to HIV-1 infection.
Plasma concentrations of 21 amino acids were determined for 20 control subjects and 20 subjects infected with human immunodeficiency virus type 1 (HIV). Compared with the control subjects, the HIV-infected group had lower cystine, tryptophan, and methionine (decreased 67%, 52%, and 32%, respectively, P < 0.001 for each) and increased taurine (230%, P < 0.001) and lysine concentrations (30%, P < 0.001). Other amino acid concentrations changed modestly. Amounts of cystine, tryptophan, methionine, taurine, and lysine did not differ significantly between subgroups of HIV-infected subjects with > 200 (n = 6) or < 200 (n = 14) CD4+ lymphocytes per microliter, suggesting that the concentrations decrease soon after infection and change little thereafter. Activation of metabolism of cystine to taurine may explain reciprocal changes in these amino acids and known depletion of cystine and glutathione. The selective changes in amino acid profiles observed during HIV infection differ from those recognized for malnutrition or other pathological processes.
Interference in ionized calcium measurements by heparin salts.
We determined the suitability of various heparin salts used for anticoagulation of whole-blood specimens for measurement of ionized calcium (iCa), blood gases, and electrolytes. We were particularly interested in a new heparin product containing both zinc and lithium cations (CNLZ heparin), in which the binding sites with greatest affinity for divalent cations are bound with zinc and low-affinity sites with lithium. In initial experiments Li heparin decreased iCa concentrations 0.07 mmol/L at the lowest heparin concentration (3000 units/L) and progressively lowered them at higher concentrations. Zn heparin initially increased iCa concentrations 0.06 mmol/L but progressively lowered them as the heparin concentration was increased. Li heparin interfered even when present in amounts (9 units per 3-mL syringe) minimally effective in preventing coagulation. Use of CNLZ heparin (36 units per 3-mL syringe; Zn 63-78 g/kg of heparin) largely eliminated interference of heparin in iCa measurements. In studies that included the effects of concentration of heparin through partial filling of syringes, specimens anticoagulated with CNLZ heparin compared well with unheparinized controls in measurements of iCa, blood gases, and electrolytes. Blood gases and iCa results on CNLZ-heparinized specimens from intensive-care-unit patients also compared well with specimens anticoagulated with a preparation of heparin (EB heparin) in which calcium has been added to balance the calcium-binding capacity. However, the presence of calcium in EB heparin significantly increased measured total calcium concentrations, whereas the new CNLZ heparin did not interfere in total calcium determinations.
Automated enzymatic analysis of inulin.
We have developed an automated enzymatic assay for quantitation of inulin in plasma and urine that can be performed on the Cobas FARA II. In the assay, inulinase hydrolyzes inulin to fructose, and sorbitol dehydrogenase converts fructose to sorbitol with consumption of NADH, which is detected by spectrophotometry. The method incorporates a sample blank (inactivated inulinase) for each specimen to subtract contributions of endogenous fructose. Recovery of fructose or inulin was near 100%, with linearity to 300 mg/L. The enzymatic assay (y) agreed well with an anthrone comparison method (x) for analysis of inulin in both urine specimens (y = 1.00x - 138; Sy/x = 714) and plasma specimens (y = 1.00x - 3.5; Sy/x = 5.5). Glucose at 300 mg/L yielded an apparent inulin value of 1.3 mg/L in the enzymatic assay, but reacted at nearly 10% equivalency in the anthrone assay. Interferences from sorbitol, mannitol, and xylitol were negligible. CVs for day-to-day precision studies were 1-4%. The automated enzymatic assay of inulin is faster and avoids the use of caustic reagents required by the classical anthrone method.
Evaluation of evacuated blood-collection tubes: effects of three types of polymeric separators on therapeutic drug-monitoring specimens.
The potential of three types of separator materials found in conventional blood-collection tubes for interference in therapeutic drug measurements was assessed. None of the separators (based on acrylic, silicone, or polyester polymers) had any significant effect on the concentrations of seven drugs (theophylline, digoxin, phenytoin, phenobarbital, gentamicin, ethanol, and cyclosporine) in blood specimens that were processed and analyzed promptly. Storage of specimens for 24 h resulted in an average 2.4% increase in theophylline values in specimens collected in tubes with the acrylic separator (P = 0.024); an average 8.1% decrease in phenytoin in specimens collected in tubes with the polyester-based separator (P < 0.001); and an average 4.2% decrease in phenobarbital in specimens collected in tubes with the polyester-based separator (P = 0.02). All other drug concentrations were not significantly affected. A small decrease in phenytoin (7.9%; P < 0.01) was seen when the specimen volume in 7-mL tubes containing polyester-based separator was reduced to 1.0 mL; all other drug concentrations were unaffected by partial filling of tubes. Paired blood specimens from pediatric patients, when collected in plain tubes and tubes containing acrylic separator, yielded no significant differences for theophylline, digoxin, tobramycin, phenytoin, or phenobarbital concentrations. The three commercially available separators had only small effects on therapeutic drug concentrations, and a newly developed separator based on an acrylic resin was suitably inert.
Recognition of substrates by tyrosylprotein sulfotransferase. Determination of affinity by acidic amino acids near the target sites.
The sulfation of proteins by tyrosylprotein sulfotransferase (TPST) is highly site-specific. In this study, we examined the sequence specificity of the target site for TPST by determining the kinetics of rat liver TPST with peptides related to the sequence of the C4 component of complement. The data obtained from this study demonstrate that selective elimination of negative charges from the -5 to +5 region of the acceptor tyrosine, either by removal or by isosteric substitution or the acidic amino acids in the region, produced a substantial change in the Km value, with relatively little effect on Vmax. Substitutions at -1 and +1 positions increase the Km value by 22- and 4-fold, respectively, whereas removal of the acidic amino acids from the -5, -4, or +4 positions increased the Km values by a factor of 2-4. The effect of elimination of an acidic amino acid on the Km value was constant and specific for its particular position in relation to tyrosine, and the effect of modification of more than one amino acid was multiplicative. This study provides evidence that: 1) acidic residues near tyrosines promote sulfation by increasing the affinity of enzyme-substrate binding and have little effect on catalytic rate; 2) the contribution of each acidic residue to affinity for TPST is independent and varies according to position relative to the acceptor tyrosine; and 3) the enzyme interacts with a segment of at least 4-5 residues on each side of the tyrosine, with the residues on the -1 and +1 positions being the most important determinants. In general, residues on the NH2-terminal side of the tyrosine have a greater effect on affinity for TPST.