E.J. Van Kampen

Standardization of hemoglobinometry II. The hemiglobincyanide method

An easy and reliable (spectro-)photometric procedure is described for the determination of hemoglobin as hemiglobincyanide. The time needed for a determination has been shortened from 20 to 4 min by shifting the ph of the diluent from 8.6 to 7.2. This has been accomplished by substituting (140 mg/l) KH2PO4 for the NaHCO3 in the diluent conventionally used in the HiCN method. Turbidity through precipitation of plasma proteins is prevented by the addition of a small amount of a detergent (Sterox SE) which also promotes rapid lysis of the red cells.

Determination of Hemoglobin and Its Derivatives

Publisher Summary This chapter discusses that the measurement of the total hemoglobin concentration and the determination of the hemoglobin derivatives oxyhemoglobin (HbOz), carboxyhemoglobin (HbCO), sulfhemoglobin (SHb), and hemiglobin (Hi) in blood are undoubtedly the most frequent determinations in the clinical chemical laboratory. A clinical diagnosis is often made solely on the basis of hemoglobin determinations: anemia, when values are too low; polycythemia in cases of abnormally high values. It explains that when significant variations occur in the determination of the hemoglobin concentration, it becomes difficult to give a clear-cut definition of, for example, anemia. The chapter also highlights the general requirements for the HB determination and standardization—such—as the method of choice, dilution factor, absorption spectrum, specificity, and stability.

Standardization of hemoglobinometry: I. the extinction coefficient of hemiglobincyanide at λ = 540 mμ : ε540HiCN

Abstract The extinction coefficient of hemiglobincyanide (cyanmethemoglobin), e540HiCN at λ = 540 mμ has been established as n.o, on the basis of iron determinations (n = 259) of 21 hemoglobin preparations. Two methods for the determination of iron are used: a spectrophotometric method with α, α′-dipyridyl and a titrimetric method with titanous chloride. By means of CO-capacity determinations, a somewhat higher extinction coefficient (11.3) is obtained, e540HiCN = 11.0 is recommended for the standardization of hemoglobinometry.

A Multi-Wavelength Spectrophotometric Method for the Simultaneous Determination of Five Haemoglobin Derivatives

A method is described by which the concentration of deoxyhaemoglobin, oxyhaemoglobin, carboxyhaemoglobin, haemoglobin and sulfhaemoglobin in a human blood sample is determined by passing the haemolysate without air contact through a coarse filter and subsequently measuring the absorbance at lambda = 500, 569, 577, 620 and 760 nm. The ensuing set of equations is solved by matrix calculation with the aid of a simple computer program. The method has been tested by comparing it with conventional methods for the determination of the various haemoglobin derivatives separately.

Experience in the netherlands with an external quality control and scoring system for clinical chemistry laboratories

A procedure is described to judge the quality of clinical chemistry hospital laboratories in The Netherlands. In 1974, 45% of the laboratories took part in the national control scheme. A score system of the results, independent of the standard deviation, opens a provisional possibility to strive for (dynamic) reference laboratories. The Youden plots possibly will unveil systematic method-differences. Discrepancies in results seem to be caused as much by methods/instruments applied, as by unknown factors which are under investigation now.

Carboxyhaemoglobin. spectrophotometric determination tested and calibrated using a new reference method for measuring carbon monoxide in blood

The spectrophotometric determination of HbCO at lambda = 562 and 540 nm in the system HbCO/HbO2 was reinvestigated using a new reference method for measuring CO in blood. This reference method is based on the conversion of CO from HbCO into CO2 which is determined by titration. Plotting the absorbance ratio A562/A540 against the titrimetrically determined HbCO fractions of 46 blood samples demonstrated a linear relationship up to 90% HbCO and yielded more accurate values for the constants in the equation for calculating the HbCO fraction from A562/A540. The standard deviation of the differences between the spectrophotometric and the titrimetric method was 1.2% HbCO. It is shown that the influence of other haemoglobin derivatives and other possible sources of error is either negligible or can be prevented by simple precautions.

Urinary steroid spectra by gas chromatography

Abstract Application of standardized procedures of hydrolysis, extraction, injection technique, column and temperature programming in the gas-chromatographic separation of urinary steroids made it possible to characterize several endocrine disorders. From the urinary steroid spectra thus obtained, important information can also be acquired regarding the quantitative excretion of total 17-ketosteroids, 17-ketogenic steroids and, especially, of etiocholanolone and pregnanediol. Although these spectra are, in a way, the image of the absolute initial steroid excretion in urine—an excretion which in turn is relativized by the important excretion of steroids via the skin—they nevertheless provide important information in endocrine diseases in men.

Determination of total carbon dioxide in blood and plasma by means of the cediometer. Theory and experimental verification

Abstract The cediometer, an instrument for the determination of total carbon dioxide in blood and plasma samples, consists of a closed system incorporating an electrode chamber filled with a NaHCO 3 solution, a sample chamber in which the CO 2 of injected blood or plasma is set free with an acid reagent and a roller pump which circulates the CO 2 -containing gas until the P CO 2 , has become uniform throughout the system and a stable pH in the electrode chamber solution has been reached. The relationship between this pH and the total carbon dioxide content of the sample has been formulated exactly. For an arbitrarily chosen standard system with constant volumes of the three parts of the circuit, constant volume of the sample injected and constant temperature as well as constant pre-injection pH of the electrode chamber solution, the relationship of final pH and total carbon dioxide of the samples was calculated and presented in a table. This table can be used with any cediometer to convert the final pH into total carbon dioxide content. A simple calibration procedure using Na 2 CO 3 reference solutions can be used. The method is accurate within ± 1%, when the volumes of the various parts of the circuit do not differ by more than 10% from those of the standard system. The apparatus needs no special care and performs well under ordinary clinical conditions.

The pH-resistance of erythrocytes. I. An investigation into the cause of haemolysis.

Abstract An investigation was made into the influence of pH on the haemolysis of erythrocytes. Normally this haemolysis starts at pH 5.15 ± 0.09 (Δ = 0.323° C). This pH is unmistakably increased in a number of haematological diseases (haemolytic anaemia, leukaemia, pernicious anaemia, etc.) and is therefore of value as an aid in the diagnosis of these conditions. In nearly all cases, with a few exceptions as regards haemolytic anaemia, it was found that osmotic resistance was normal. Treatment with vitamin B12 for pernicious anaemia and the influence of splenectomy (haemolytic anaemia) were manifested by the decrease in initial pH. No anomalous haemolysis was found in association with hepatitis, cirrhosis of the liver, etc. The methods of investigation are described.

Spectral absorption of coproporphyrin in relation to the estimation in urine

Abstract A study has been made of the routine analysis of coproporphyrin and its precursor in urine, by means of the Beckman D.U. Spectrophotometer. It is shown that the quantitative estimation of these two compounds should preferably be carried out in 0.1 N HCl. Emax and λmax are highly dependent upon [H+]. A detailed three wavelengths method for the determination is given.