Hemolysis assay

Another proposed in vitro assay for muscle irritancy for injectable formulations is the red blood cell hemolysis assay (Brown et al., 1989). Water-soluble formulations in a 1 2 ratio with freshly collected human blood are gently mixed for 5 min. The percentage of red blood cell survival is then determined by measuring differential absorbance at 540 nm this value is then compared to values for known irritants and nonirritants. Against a very small group of compounds (four), this assay reportedly accurately predicts muscle irritation. 

Reim, V. and Rohn, S. 2014. Characterization of saponins in peas (Pisum sativum L.) by HPTLC coupled to mass spectrometry and a hemolysis assay, Food Res. Int., article in press, http //dx.doi.Org/10.1016/j.foodres.2014.06.043. 

The hemolytic activity of PT has been compared to that of several snake venoms and a CTX prepared from Naja naja kaouthia, which was devoid of contaminating PLA2 (6,33). The CTX was slightly less active than PT in the hemolysis assay, and inhibition studies performed with iodinated PT (which was inactive) showed a competitive inhibition, indicat-... 

Effect of hemolysis on the assay of glucose by the fluorescence-hexokinase method without protein precipitation. 

There is no clinical disease state that is pathognomonic for lead exposure. The neurotoxic effects and hematopoietic effects of lead are well recognized. The primary biomarkers of effect for lead are EP, ALAD, basophilic stippling and premature erythrocyte hemolysis, and presence of intranuclear lead inclusion bodies in the kidneys. Of these, activity of ALAD is a sensitive indicator of lead exposure (Hemberg et al. 1970 Morris et al. 1988 Somashekaraiah et al. 1990 Tola et al. 1973), but the assay can not distinguish between moderate and severe exposure (Graziano 1994). Sensitive, reliable, well-established methods exist to monitor for these biomarkers however, they are not specific for lead exposure. Therefore, there is a need to develop more specific biomarkers of effect for lead. Recent data... 

Brydon and Roberts- added hemolyzed blood to unhemolyzed plasma, analyzed the specimens for a variety of constituents and then compared the values with those in the unhemolyzed plasma (B28). The following procedures were considered unaffected by hemolysis (up to 1 g/100 ml hemoglobin) urea (diacetyl monoxime) carbon dioxide content (phe-nolphthalein complex) iron binding capacity cholesterol (ferric chloride) creatinine (alkaline picrate) uric acid (phosphotungstate reduction) alkaline phosphatase (4-nitrophenyl phosphate) 5 -nucleotidase (adenosine monophosphate-nickel) and tartrate-labile acid phosphatase (phenyl phosphate). In Table 2 are shown those assays where increases were observed. The hemolysis used in these studies was equivalent to that produced by the breakdown of about 15 X 10 erythrocytes. In the bromocresol green albumin method it has been reported that for every 100 mg of hemoglobin/100 ml serum, the apparent albumin concentration is increased by 100 mg/100 ml (D12). Hemolysis releases some amino acids, such as histidine, into the plasma (Alb). 

Serum should be straw colored a pink coloration shows that hemolysis has taken place. This should not affect the performance of the antibodies during most assay procedures. 

Free fatty acids are elevated in the plasma of obese patients and are known to cause muscle and liver insulin resistance. The Wako HR series NEFA-HR(2) is an in vitro enzymatic colorimetric method assay for the quantitative determination of non-esterified fatty acids (NEFA) in serum. Perform the assay on serum collected from mice fasted for a period greater than 4 h, but less than 16 h. Perform the test on samples immediately after collection, without freezing. Also note that hemolysis in the serum samples may interfere with the assay. 

The serum should be yellowish. A reddish color is indicative of erythrocytes lysis (hemolysis) which may interfere with clinical chemistry assays based on colorimetric values. If this happens, you may need to adjust the speed at which the blood is collected and processed or other steps that may cause sheer and red blood cell lysis. 

Selectivity is the ability of an assay to measure the analyte of interest in the presence of other constituents in the sample. Because IAs are often performed without sample extraction, they are more prone to matrix interference than are chromatographic methods with extraction. Matrix interference could come from crossreactivity with structurally similar components in the sample, or from nonspecific binding to structurally dissimilar components in the matrix. The results are high background noise, loss of sensitivity, and inaccurate and nonreproducible data. Sometimes, the problem may only occur in a few exceptional patient samples that have structurally similar components such as unknown metabolites, or dissimilar components from samples with hyperlipidemia, hemolysis, complement components, rheumatoid factors, binding proteins, autoantibodies, and heterophilic anti-immunoglobulin Ab. 

Clinical pathology is conducted on each animal independently. The report from the consultant laboratory includes evaluation of total bilirubin, urea nitrogen, creatinine, alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), WBC, RBC, and platelet count, ft is important to note that the accuracy of most assays is affected by hemolysis. 

There is also increased erythrocyte hemolysis, which responds to synthetic antioxidants or selenium. The sensitivity of erythrocytes to chemically induced hemolysis can be used both as a biological assay of vitamin E in... 

Palytoxin hemolysis neutralization assay 0.3 pg cell palytoxin equivalents (isolated from Rangiputa)... 

Bignami, G.S. 1993. A rapid and sensitive hemolysis neutralization assay for palytoxin. Toxicon 3, 817-820. 

Both are abundant in skeletal muscle, myocardium, liver, and erythrocytes, so that hemolysis must be avoided, and in serum they may be assayed spectrophotometrically by their conversion of phosphate-buffered pyruvate to lactate (R6, W16) or oxalacetate to malate (S25) at the expense of added NADH2, when the rate of decrease of optical density at 340 m x thus measmes the serum activities of the respective enzymes. Recently, however, the reverse reaction has been found best for serum lactic dehydrogenase assay (A2a). In conventional spectrophotometric units the normal ranges are 100-600 units per ml for lactic dehydrogenase (W16) and 42-195 xmits per ml for malic dehydrogenase (S25) as before, one conventional spectrophotometric unit per ml = 0.48 pmoles/ minute/liter (W13). 

This soluble enzyme is specially abundant in skeletal muscle, only one quarter as plentiful in the myocardium and brain, and practically absent from other tissues (C13), so that hemolysis does not affect its activity in serum in the study of muscle disease this distribution offers great advantages. Its function is specifically the equilibration of creatine phosphate and ADP with creatine and ATP, with equilibrium heavily in favor of the latter compounds. Since its activity in serum is some million times lower than in skeletal muscle, serum assay offers certain difficulties three reliable methods, however, are available. 

E221 O Connor, J.E., Rueter, C.A., Stanton, C.A. and Lo, D.H. (1985). Thin-film enzyme assays on the Kodak Ektachem 700 analyzer avoid interference by li-pemia, hemolysis and bilirubin. Clin. Chem. 31, 1007, Abstr. 548. 

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