2 TBIL alias erythrocyte serum total bilirubin
3 TBIL physical examination 3. 1 exam name TBIL
3.2 biochemical examination of classified blood > pigment determination
3.3 Testing blood samples
3.4 TBIL( 1) improved caffeine (JG) method determination principle: serum bound bilirubin can directly react with diazonium reagent to generate azobilirubin; Under the same conditions, free bilirubin needs an accelerator to break the hydrogen bond of bilirubin and then react with diazonium reagent. Caffeine and sodium benzoate are used as accelerators, and sodium acetate can maintain pH and accelerate. Ascorbic acid (or sodium azide) destroys the residual diazo reaction, terminates the azo reaction combined with bilirubin measuring tube, and prevents the slow reaction of free bilirubin. The maximum absorbance was transferred from 530nm to 598nm by adding alkaline sodium tartrate, and the absorbance produced by non-bilirubin lutein and other red-brown lutein decreased to be negligible, which increased the sensitivity and specificity. The final green color is a mixture of blue alkaline azobilirubin and lutein formed between caffeine and sulfanilic acid.
(2) Bilirubin oxidase method: Bilirubin oxidase (BOD) catalyzes bilirubin oxidation to produce biliverdin, and biliverdin is further oxidized to produce lilac compounds. When the wavelength is 450nm, the decrease of absorbance (△A) is directly proportional to the serum bilirubin concentration.
3.5 reagent (1) modified caffeine (JG) method:
① Caffeine sodium benzoate reagent: weigh anhydrous sodium acetate 4 1.0g (or ch3coona 3h2o63.0g), sodium benzoate 38.0g and disodium ethylene diamine tetraacetate (EDTANa2)0.5g, dissolve them in about 500ml distilled water, then add caffeine 25.0g, stir to dissolve (after adding caffeine, it cannot be heated to dissolve), and make up to 6500 with distilled water. Filter with filter paper, put in a brown bottle and store at room temperature.
② Alkaline sodium tartrate solution: weigh 75.0g of sodium hydroxide and 263.0g of sodium tartrate (Na2C4H4 O6 H2O), dissolve them in distilled water to a constant volume of 1000ml, and mix them evenly. Put in plastic bottles and store at room temperature.
③5.0g/L sodium nitrite solution: weigh 5.0g sodium nitrite (nano 2), dissolve it in distilled water and dilute it to 100ml, mix it evenly, put it in a brown bottle and store it in the refrigerator for at least 3 months. Dilute with 10 times to 0.5g/L, and keep stable in the refrigerator for at least 2 weeks.
④5.0g/L sulfanilic acid solution: weigh 5.0g/L sulfanilic acid (NH2c6H4SO3H2O), dissolve it in 800ml distilled water, add 15ml concentrated hydrochloric acid, and make it up to 1000ml with distilled water.
⑤ Diazo reagent: Before use, mix 0.5 ml of 5.0 g/L sodium nitrite solution with 20 ml of 5.0 g/L sulfanilic acid solution.
⑥5.0g/L sodium azide solution: weigh 0.5g sodium azide, dissolve it in distilled water and dilute it to 100ml.
⑦ bilirubin standard solution
A general standard solution is prepared with free (unbound) bilirubin, so the diluent for preparing standard solution needs to contain albumin. Bovine serum albumin (40g/L) or human serum can be used instead of human serum albumin. The latter method is to collect fresh serum without hemolysis, jaundice and lipid turbidity, mix them and filter them with a bacterial filter if necessary. Take 1 ml of filtered serum, add 24 ml of fresh normal saline and mix well. When the wavelength is 4 14nm and the optical path 1cm, when the zero point is adjusted with physiological saline, the absorbance should be less than 0.100; The absorbance at 460 nm should be less than 0.04.
B. Bilirubin in standard solution must meet the following standards: at 25℃, the optical diameter of chloroform solution of pure bilirubin is10 0 01mm, the wavelength is 453mm, and its molar absorption coefficient should be in the range of 607001600; The molar absorption coefficient of azobilirubin measured by improved JG method should be 74380 866.
C. bilirubin storage standard solution,171μ mol/l (10mg/dl): accurately weigh qualified bilirubin10mg, add 1ml dimethyl sulfoxide, and stir with a glass rod to make a suspension. Add 2ml of 0.05mol/L sodium carbonate solution to completely dissolve bilirubin, transfer it to a 100ml volumetric flask, wash it with diluted serum for several times and merge it into the volumetric flask, slowly add 2ml of 0.1mol/L hydrochloric acid, and shake well while adding it (do not use force to avoid bubbles). Finally, make it up to 100ml with diluted serum. Avoid light as much as possible during preparation, and wrap the storage container with black paper. It is effective in the refrigerator at 4℃ for 3 days, but the standard curve should be made as soon as possible after preparation.
(2) bilirubin oxidase method:
①0. 1mol/L TrisHCl buffer (pH 8.2: weigh tris1.21g, sodium cholate 172.3mg, SDS432.6mg, dissolve in 90ml distilled water, and at room temperature (0.05 mg). The solution contains 4mmol/L sodium cholate and 150mmol/L sodium dodecyl sulfate.
②BOD solution: if it is freeze-dried, it should be reconstituted according to the instructions, but it should not be stored in the refrigerator for too long (about 1 week) after reconstitution. If it is liquid (possibly containing glycerol), it can be stored in the refrigerator or freezer for a long time. The enzyme activity of BOD storage solution is generally several thousand ~ 1 ~ 20000 U/L, and the enzyme activity of BOD working solution can be calculated according to the final concentration of BOD in the reaction solution reaching 0.3 ~ 1.0U/mL.
③ Bilirubin standard solution: 342μmol/L: prepared according to JG bilirubin determination method, or a standard solution that meets the requirements on the market.
3.6 Operation method (1) Modified caffeine (JG) method: operate according to table 1
After fully mixing, the wavelength is 600nm, the control tube is zeroed, and the absorbance of each tube is read; Or use distilled water to zero, read the absorbance of the measuring tube and the control tube, and use the difference between the absorbance of the measuring tube and the control tube (AUAC) to find out the corresponding bilirubin concentration on the standard curve.
(2) Bilirubin oxidase method: operate according to Table 2.
Immediately after adding BOD solution, mix it evenly, put it in a water bath at 37℃ for 5 minutes, use a spectrophotometer, adjust it to zero with distilled water at the wavelength of 450 or 460nm, and read the absorbance of each tube. The cuvette used for the control tube shall not be mixed with the cuvette of the non-control tube.
3.7 Total bilirubin in normal serum: 2 ~ 20μ mol/L (0.1~1.2mg/dl).
Bilirubin oxidase method and modified JG method (diazotization method) umbilical cord: premature infants: < 34 μ mol/L (< 2.0 mg/DL) full-term infants: < 34 μ mol/L (< 2.0 mg/DL) 0 ~ 1 day: premature infants: <137 μ mol/L. Dl) 3 ~ 5 days: premature infants: < 274 μ mol/l (< 16.0 mg/dl) full-term infants: < 205 μ mol/l (< 12.0 mg/dl) and then: premature infants: < 34 μ mol/l (< 2.0 mg/dl) L (0.2 ~ 1.0 mg/dl) Adult:1.7 ~17.1μ mol/l (0.1~1.0 mg/dl)
3.8 The clinical significance of the test results (1) in judging whether there is jaundice and its degree: STB17.1~ 34.2 μ mol/L is recessive jaundice; 34.2 ~ 17 1 μ mol/L is mild jaundice; 17 1 ~ 342 μ mol/L is moderate jaundice; > 342 μ mol/L is severe jaundice.
(2) judging the nature of jaundice: complete obstructive jaundice is 342 ~ 5 13 μ mol/L, incomplete obstruction 17 1 ~ 266.8 μ mol/L, and hepatocellular jaundice17.1~/kloc.
(3) Classification and differentiation of bilirubin types: Total bilirubin and unconjugated bilirubin are increased, which are seen in hemolytic jaundice, blood group incompatibility transfusion reaction, falciparum malaria, neonatal jaundice, etc. Unconjugated bilirubin mainly increased; The increase of total bilirubin and conjugated bilirubin is obstructive jaundice, such as gallstones, liver cancer, pancreatic head cancer and so on. , mainly the increase of bound bilirubin; Total bilirubin, conjugated bilirubin and unconjugated bilirubin all increase to hepatocellular jaundice, such as acute icteric hepatitis, chronic active hepatitis, liver cirrhosis and acute yellow liver necrosis.
(4) Used to judge the degree of liver cell injury and prognosis: In liver diseases, bilirubin concentration is obviously increased, which often reflects the serious liver cell injury and poor prognosis. However, a few subacute hepatitis can be free from jaundice. Cholestasis hepatitis, serum bilirubin is high, but liver cells are slightly damaged.
(5) It is helpful to understand the severity of neonatal hemolysis and make a reasonable treatment plan.
(6) STB of patients with aplastic anemia and chronic nephritis anemia decreased.
3.9 Note (1) Modified Caffeine (JG) Method:
① The determination of serum total bilirubin by this method is not affected by temperature change at 10 ~ 37℃. The color is very stable within 2 hours.
② This method has high sensitivity (molar absorption coefficient is 74380 866 L mol-1cm-1).
③ Mild hemolysis has no effect on this method, but severe hemolysis can reduce the determination result. The reason is that the products produced by the reaction between hemoglobin and diazonium reagent can destroy azobilirubin, and can also be oxidized to methemoglobin by nitrous acid, which interferes with the determination of absorbance.
④ Sodium azide can destroy diazotization reagent and stop azo reaction. Any quality control serum with sodium azide as preservative can cause incomplete azo reaction or even no color development.
⑤ Bilirubin is sensitive to light, and standards and specimens should be protected from light as much as possible.
⑥ Blood lipids and pigments interfere with the determination, and blood should be taken on an empty stomach as much as possible.
⑦ The absorbance of the sample control tube is generally very close. If the sample volume is small, the sample control tube can be omitted, and the absorbance of other sample control tubes can be referenced.
⑧ Specimens with bilirubin greater than 17 1μmol/L can reduce the sample dosage, or do it after diluting the serum with normal saline.
Pet-name ruby determination, so far there is no candidate reference method, and there is no recommended method in China. Different methods, different reaction times and different results. Short time, less free bilirubin reaction and incomplete bound bilirubin reaction; After a long time, the bound bilirubin reaction was complete, but some unbound bilirubin participated in the reaction. This is a difficult problem to weigh, and it is more complicated without bilirubin standard solution.
According to the method of Michaelsson and Gambino, Doumas first added 50 mol/L hydrochloric acid to the serum, then added diazonium reagent, and the reaction was 65438 100min, which not only prevented the reaction of unbound bilirubin, but also made the reaction of bound bilirubin more complete.
The method used by Doumas: 50 mol/L hydrochloric acid 0.8ml, serum 0.2ml, and mix well. Then add 0.4ml nitrogen reagent, mix well, leave it at room temperature for 10min, add 32g/L ascorbic acid 0. 1ml (or 20g/L NaN2 0. 1ml) and mix well. Finally, add 1.2ml alkaline sodium tartrate solution and 1.6ml caffeine reagent in turn. When unbound bilirubin is used as the standard solution, the operation sequence of the standard tube is the same as that of the JG method, but ascorbic acid should be added after the diazo reagent reaction, and finally 50 mnl/L hydrochloric acid should be added.
(2) bilirubin oxidase method:
The concentration of BOD can be determined according to the reaction speed of the products used in the determination of serum samples with hyperbilirubinemia or the standard of 342μmol/L, that is, whether the reaction can be completed within 5 minutes. It is reported that the final concentration of BOD in the reaction solution is 0.18 ~1.14u/ml.
(2) When there is enough active BOD in the reaction system, the reaction time is 5min(37℃), that is, △A reaches the maximum value in 5min. It is suggested to use high concentration serum samples or 342μmol/L standard solution to detect the reaction time according to the kit used.
③ Hemolytic samples can reduce the determination results, which is related to Hb content and bilirubin. HB < 1.0g/L has little effect, and the determination result of bilirubin is obviously low when HB >1.5g/l.
(4) In this method, the pH curve of enzyme activity changed little between pH 7.3 and 9.0, but the optimum pH was 8.0-8.2.
⑤ The method is simple in operation, high in specificity and wide in linear range, at least lower than 513 μ mol/L. ..
3. 10 related diseases