(HicN) method hemoglobin assay experiment

Summary

This experiment is mainly used for the determination of hemoglobin.

Operation method

(HicN) method hemoglobin assay experiment

Principle

Hb was oxidized to methemoglobin (Hi) by potassium hexacyanoferrate, and then combined with cyanide ion (CN) to produce stable cyanide methemoglobin (HiCN).The maximum absorption peak of HiCN was at 540 nm, and the millimolar absorption coefficient was 44,000 (mmol-cm) under specific conditions, and the concentration of hemoglobin (g/L) could be found from the absorbance.

Materials and Instruments

Blood Samples
Prabkin's Reagent Vinci's Reagent Macchi's Modified Reagent HiCN Conversion Solution
Spectrophotometer Syringe

Move

I. Experiments, reagents:

l, all of the (Prabkin) reagent

sodium bicarbonate 1.0g, potassium cyanide 0.05g potassium ferrocyanide 0.20g

distilled water 1000mL the reagent PH value of 8.5 and the role of hemoglobin slower, so the reagent and the blood mixture is generally left for 20min to colorimetry, the reagent is prone to turbidity, in recent years, has been not used. 2, the text of the Qi (Van) KamPen and zilstra) reagent

Potassium cyanide 0.05g potassium ferricyanide 0.2g nonionic surfactant 0.5-lml, potassium dihydrogen phosphate 0.14g distilled water 1000mL, correct the PH value to 7.0-7.4. 3, the horse (Matsabara) improved reagent in the Wenqi reagent with NaCl5.0g or 50g (common reagent) (strengthen the reagent) common reagent can slightly remove the turbidity caused by abnormal globulin, strengthen the reagent to improve the turbidity caused by a significant increase in white blood cells.

Second, the experimental method:

l, take finger blood or V blood 20ul, add to 5mL hemoglobin conversion solution, mixing, rest for 5 minutes, 2, with a spectrophotometer colorimetric, wavelength 540nm, with distilled water or blank conversion solution to adjust the zero, the determination of absorbance.

3, if you use in line with the WHO standard push of the spectrophotometer, can be based on the determination of the absorbance A directly calculated from the concentration of hemoglobin, Hb (g / L) = (A) = (A) = (g / L) = (A) = (A) = (A) = (g / L). L) = (Aλ540/44) x (64458/1000) x 251 = Ax367.7, of which Aλ540/HicN for the determination of the absorbance of the tube. 64458 for the molecular weight of Hb, 64458/l000 for the number of grams of Hb in the 10 mmoL / LHb solution, 251 for the blood dilution times, Hb curve for the R-value, the value of the fixed value, 50g/L 100g / L 150g / L 100g / L 150g / L and 200g / LHicN test solution in the 721-type spectrophotometer to fix the absorbance A were 0.13, 0.27, 0.405, 0.54

Cyanide high iron hemoglobin standard curve or according to the following formula to find the absorbance hemoglobin conversion constant (K value) K = qHb (g / L) qA = (50 + 100 + 150 + 200) / (0.13 + 0.27 + 0.405 + 0.54) = 371.75 V. Calculation: hemoglobin (g / L) = assay tube absorbance x 367.7 Reference value: adult male: 120-160g/L

Adult female: 110-150g/L

Newborn: 170-220g/L

Caveat

The KCN in the reagent of this method is highly toxic, and hyperleukocytosis and hyperglobulinemia tend to cause turbidity and slow conversion of HbCO during the assay.

Common Problems

Sources A Concise Manual of Operative Test Techniques and Hayakawa Applications, Laboratory Medicine and Clinics.

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