Results of routine determination of clinically significant hemoglobin derivatives by multicomponent analysis.

Our recently developed multiwavelength method for multi-component analysis of hemoglobin (Hb) derivatives (Clin Chem 1984;30:373-379) was adapted for routine use in the clinical chemical laboratory. The method was applied in 4066 determinations on blood specimens from patients awaiting major surgery (n = 3863) or visiting the outpatient department for pulmonary disease (n = 203). Mean total hemoglobin concentration was 141 (SD 14) g/L. The proportion of HbCO was slightly to moderately increased (1.5-10.0%) in 36.5% of all patients; in a few cases it was as high as 15%. Mean methemoglobin was 0.4% (SD 0.2%) in the surgical patients, but 1.5% (SD 0.8%) in the patients with pulmonary disease. In some patients of the latter group the proportion of methemoglobin amounted to 5%. Sulfhemoglobin was found less than 0.4% in all specimens. Interference by paraproteins and by increased concentrations of bilirubin and lipids in plasma was easily detected by means of the performance checks provided by the spectrophotometer (an HP 8451 UV/Vis). The method is equally suitable for measuring blood samples containing fetal hemoglobin.

Simultaneous spectrophotometric calibration of wavelength and absorbance in an interlaboratory survey using holmium oxide (Ho2O3) in perchloric acid as reference, compared with p-nitrophenol and cobaltous sulphate solutions (1978-1984).

The wavelength accuracy of ten different types of spectrophotometer was tested with holmium perchlorate solutions. It was found to be good, with mean deviations from the literature values of maximally 0.3 nm. Standard deviations over the entire spectral range were within 0.75 nm. The absorbance accuracy for different types of instruments was generally within 5%, except in the 287 nm range where higher deviations were found. The sharpness of the holmium peaks, in combination with band width and sensitivity of the instruments, troubled the majority of the participants. 150 spectrophotometers were involved in the surveys. Linearity of the spectrophotometers was tested with p-nitrophenol and cobaltous sulphate and found to be satisfactory.

Multicomponent analysis of hemoglobin derivatives with reversed-optics spectrophotometer.

We describe a method for the simultaneous determination of the five clinically relevant hemoglobin derivatives (Hb, HbO2, HbCO, Hi, SHb) in a blood sample by means of a reversed-optics spectrophotometer (Hewlett-Packard HP8450 A UV/Vis). A built-in computer program is used for multicomponent analysis in an overdetermined system, i.e., a system in which the number of independent equations used exceeds the number of unknowns to be determined. First, the spectra of the five hemoglobin derivatives are measured in a series of different human blood samples. Thereafter, the multicomponent method for the simultaneous determination of the five hemoglobin derivatives is tested by comparison with conventional methods for the separate determination of oxygen saturation, HbCO, Hi, and SHb fractions. The multicomponent (multiwavelength) method is sufficiently reliable, accurate, and easy to justify its use in physiological chemical research as well as its routine application in the clinical chemical laboratory.

Interlaboratory investigation on the CEA assay (Roche) with column filtration, dialysis and ultrafiltration techniques.

An interlaboratory study on the reproducibility of the CEA (Roche) RIA Test was carried out. Four different plasma pools of approximately 2, 3, 6, and 12 micrograms/l CEA were tested over a period of 4 weeks with 4 different lots of reagents in order to determine the interassay variances. At the same time we compared the lately introduced column technique with the dialysis and ultrafiltration method. Best results were obtained with the column technique which also showed best reproducibility. Only 1.4% of samples showed deviations greater than 5% between the mean of CEA duplicates and single CEA values, and these were omitted from the evaluation. On the other hand about 15% of the corresponding dialysis results showed deviations greater than 5% and were excluded from the evaluation. The methods compared showed a good correlation with a coefficient of 0.96, but the average values for the CEA determination, using the columns technique were lower than those obtained from dialysis. Interassay variances were greater for the dialysis procedures, i.e. 1.88 +/- 0.81, 3.25 +/- 0.83, 5.81 +/- 1.09, and 11-91 +/- 1.23 compared with 1.77 +/- 0.54, 2.63 +/- 0.68, 4.89 +/- 0.79, and 11.16 +/- 1.23 for the column technique. There were no systematic changes of the CEA values over the period of 4 weeks, thus giving optimal conditions for a follow up of patients.

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.

Ionic composition of arterial and mixed venous plasma in the unanesthetized dog.

The use of permanent catheters in the aorta and pulmonary artery permitted the establishment of normal values for hemoglobin concentration in blood and -or pH, PCO2, osmolality, and protein and electrolyte concentrations in the plasma of arterial and venous samples from unanesthetized, undisturbed dogs, and the comparison of the ionic composition of simultaneously taken arterial and venous samples. Arterial samples yielded the following mean values: CHb, 143 g liter-1; pHP, 7.427; PCO2, 32.5 mmHg; CPosmol, 295 mmol kg-1; CPpr, 73.1 g liter-1, CPNa+, 148.0; CPK+, 3.9; CPCa2+, 2.38; CPMg2+, 0.85; CPCl-, 116.0; CPHCO3-, 21.1; CPlact-, 1.4; CPphosph, 1.21; net cation equivalency, 16.4; and anion gap, 1.03 mmol liter-1 in eight male mongrel dogs with seven or eight samplings from each dog. The anion gap in arterial and venous plasma was small, indicating that the contribution of sulfate and organic acids to the ionic composition of dog plasma is quantitatively unimportant. In simultaneously taken arterial and venous samples the following significant arteriovenous differences were found: HP, +0.038; PCO2, -5.6 mmHg; CPosmol, -1.8 mmol kg-1; protein, -0.8 g liter-1; CPNa+, -1.0; CPK+, -0.1; CPCl-, +1.3; and CPHCO3-, -1.7 mmol liter-1. These differences are explained on the basis of the changes that occur in blood upon the addition of CO2 and the ensuing chloride and water shifts.

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.

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.

Quality control in haemoglobinometry with special reference to the stability of haemiglobincyanide reference solutions.

In haemoglobinometry grave errors are still being made even though an internationally accepted standardized method is available for the determination of the haemoglobin content of blood. Up to the present only haemiglobincyanide reference solutions have been available on a wide scale to check the measuring stage of the standardized haemiglobincyanide method. These reference solutions are shown to remain stable, under proper storage conditions, for more than 10 years. Concentrated haemoglobin solutions have become available recently, offering the possibility to control the dilution and conversion steps of the haemiglobincyanide method. Such a solution is shown to remain stable, under proper storage conditions, for at least a year. Using both haemiglobincyanide reference solutions and concentrated haemoglobin solutions, as well as having the possibility of checking the cyanide content of the reagent used, an acceptable intra-laboratory control program may now be set up.

Outflow from the left and the right coronary artery into the coronary sinus in the dog heart in situ.

The fractions of the left and the right coronary arterial flow determining coronary sinus flow (facs, facd) were measured in the open-chest dog. Both coronary arteries were isolated and perfused at the same pressure, while the sinus outflow was isolated and drained against the prevailing mean pressure in the right atrium. The fractions were determined by injecting 51Cr labeled erythrocytes into each coronary artery selectively and measuring the total resulting radioactivity in the sinus blood. In addition, they were estimated from flow measurements. The values of facs and facd were found to approximate 0.70 and 0.02 respectively. The variability between the different dog hearts was considerable and it was demonstrated that they were not intercomparable with respect to their coronary flow distribution.