Evaluation of a quality control material containing hemoglobin for blood gas and pH measurement.

A procedure for the preparation of a Stroma-Free Hemoglobin Solution (SFHS) is given. The stability of this SFHS containing Methemoglobin Reductase, can be improved by addition of NADH. The characteristics of the stable SFHS can be manipulated by varying independently the concentrations of bicarbonate and Inositol-Hexa-Phosphate. This way the desired acid-base behaviour and position of the Oxygen Hemoglobin Equilibrium Curve (OHEC) can be obtained. Three SFHS were prepared with acidotic, alkalotic or normal acid-base characteristics and all SFHS had an OHEC in the normal position (actual p50 3.33-3.87 kPa). Results show that: stability of SFHS is 1 year, xHi less than 2.5% after 1 year; p50 decreases about 15% per year; Hill's coefficient, nHill, is constant, but differed between levels; the mean values for nHill are 2.0 for the acidotic level, 2.2 for the normal and 2.4 for the alkalotic level; temperature coefficients for SFHS are: d(pH)/dt = -0.016 pH/degrees C, d(pCO2)/dt = 6.2%/degrees C and d(pO2)/dt = 7.2%/degrees C. Oxygen and carbon dioxide tonometered SFHS, of which the pH was measured with the reference method for pH measurement in blood was used on several blood gas analyzers to demonstrate the suitability for pH, pCO2 and pO2 measurement. The SFHS, which contained oxyhemoglobin, carboxyhemoglobin and methemoglobin, was also used as a control material for hemoglobin meters and CO-Oximeters. It is concluded that SFHS behaves blood-like with respect to pH, pCO2 and pO2 as well as total hemoglobin, oxygen saturation, carboxyhemoglobin and methemoglobin measurements. In contrast to hemoglobin-free aqueous control material, it buffers oxygen in a blood-like manner. Its shelflife is limited compared to the generally used aqueous control materials, but it is sufficient for repetitive use in clinical laboratories.

Quality control material containing hemoglobin for blood gas and pH measurement: preparation of stroma-free hemoglobin solution.

A method for the preparation of stroma-free hemoglobin solution suitable for quality control of blood gas and pH measurements as well as hemoglobinometry, is described. Several methods were compared for purification and lysis of red blood cells. For separation of stroma from hemoglobin solution tangential cross-flow filtration has been used. Diluted hemoglobin solutions were concentrated using various forms of ultrafiltration as well as other methods. A precipitate removing procedure is introduced in which the pH is increased temporarily to 8.0 and the ionic strength is enhanced by adding 130 mmol NaCl per litre stroma-free hemoglobin solution, to remove a precipitate that was observed during tonometry at 37 degrees C in the pH-range 7.4-8.0 and when electrolytes were added to create a plasma-like composition of stroma-free hemoglobin solution. Tests were designed to quickly detect turbidity and precipitate. During storage at 4 degrees C no methemoglobin was formed in contrast with two other types of stroma-free hemoglobin solution, which formed appreciable amounts of methemoglobin within 40 days.

Quality control material containing hemoglobin for blood gas and pH measurement: improvement of the stability of stroma-free hemoglobin solution.

In stroma-free hemoglobin solution (SFHS) formation of methemoglobin (hemiglobin; Hi) occurs over a period of some months, due to the fact that Hi reduction stops in hemolysates. SFHS should contain active hemoglobin (Hb), which is able to bind oxygen and should not contain inactive Hb (Hi, carboxyhemoglobin) which does not bind oxygen. Reversible binding of oxygen by Hb is only possible when the molecule is in its reduced (Fe++) form. In red blood cells (RBC) Hb is in the reduced form. The formation of Hi, which contains Fe as a result of Hb oxidation, is the first step in Hb degradation. This step is reversible in RBC. Previously, we have described the preparation of SFHS containing the methemoglobin reductase (MR) system of RBC. To improve the stability of SFHS, we first investigated the formation of Hi as a function of pH and ionic strength and quantified the MR activity in SFHS. Non-enzymatic Hi reduction was studied with substances as ascorbate and glutathione. Stimulation of MR by EDTA was tested. Inhibition of Hi formation was studied with nicotinic acid amide in the presence and absence of NADH. It is concluded that ascorbate and glutathione are not effective during extended periods of storage of SFHS, and that EDTA causes formation of large amounts of Hi. Nicotinic acid amide did not inhibit Hi formation. NADH, as a substrate for the MR system, is very effective in keeping Hi low.

Preparing a quality control material for blood gases, pH and hemoglobinometry using stroma-free hemoglobin solution.

Properties of a quality control material for blood gases and pH should be similar to normal human whole blood with respect to oxygen buffering and acid-base behaviour. A hemoglobin solution may potentially fulfill this. However, the drawbacks of such a solution are the high oxygen affinity (lowp50), especially when it is prepared from human blood, and the improper concentration of bicarbonate. Bicarbonate is added to human stroma-free hemoglobin solution (SFHS), prepared as described previously, to obtain the desired pH and pCO2 combinations. Tonometry was used to determine the appropriate concentration of bicarbonate, which is 22.5 mmol/L to obtain an acidotic, and 29 mmol/L for both an alkalotic, and normal pH and pCO2 combination. Inositolhexaphosphate (IHP) is added to SFHS containing bicarbonate to obtain a normal p50 (around 3.55 kPa). Tonometry was used to determine the molar ratio of IHP/Hb4 (mol/mol) at which this is achieved. The molar ratios of IHP/Hb4 are 1.52, 1.74 and 3.40 for preparations with an acidotic, normal and alkalotic pH, respectively. In human SFHS nHill is 2.55 in the absence of IHP,nHill is at minimum 1.71 at a molar ratio IHP/Hb4 of 1.86 and increases to 2.53 at a molar ratio IHP/Hb4 of 5.04 and higher. Because the p50 will decrease with chi Hi this was studied at molar ratios of IHP/Hb4 of 0, 2 and 4, which covers the range of ratios as used. At these molar ratios of 0, 2 and 4, the decrease in p50 is 0.017 kPa/%Hi, 0.023 kPa/%Hi and 0.028 kPa/%Hi, respectively. Because bovine Hb was p50 near that of normal human blood, it is also used. The oxygen affinity shows a small decrease (p50 increases from 3.05 to 5.27 kPa) on addition of IHP. In the absence of IHP, nHill is 2.51 and nHill is at maximum 3.35 at molar ratios IHP/Hb4 between 3.00 to 4.56. At higher molar ratios nHill decreases to 2.90.

A comparative study of the electrode systems of three pH and blood gas apparatus.

We present a comparative evaluation of the electrode systems of three modern blood gas analysers: IL-413, ABL-1 and AVL-937C. The response curves, accuracy and precision of the pH-, pCO2- and pO2-electrodes were established with tonometered blood and buffer solutions. pH values (range 6.8-7.8) measured on the AVL deviate (-0.03 pH for blood and +0.03 pH for buffer) from those of BMS2 Mk2; whereas on the IL and ABL analysers the pH values deviate by not more than 0.01 pH. The standard deviation was better than 0.005 pH. pCO2 values of blood and buffer (range 14-106 mm Hg) deviate from the calculated tonometer values by quantities ranging from 3 to 10 mm Hg. The average precision (CV)1) of the pCO2 measurement on each analyser was better than 1.8%. pO2 values of blood (range 0-130 mm Hg) did not differ by more than 3 mm Hg from the calculated values. Above 130 mm Hg a linear negative increasing difference was seen. For buffer solutions a linear relationship between pO2 difference and pO2 value was found over the whole range from zero up to 642 mm Hg: a positive difference below and a negative difference above the pO2 of the previous calibration; if the calibration pO2 is higher, the sample pO2 is shifted to a higher value. The average precision of the pO2 measurements was better than 3%. In the (patho)-physiological range the three instruments may provide suitable results for the clinician. Suggestions are made for standardization and improvement of the electrode systems.

Evaluation of ampouled tonometered buffer solutions as a quality-control system for pH, pCO2, and pO2 measurement.

In response to the need for an adequate quality-control system for blood-pH and blood-gas analyzers, we investigated the practical application of ampouled phosphate-bicarbonate-chloride solutions tonometered with mixtures of carbon dioxide, oxygen, and nitrogen. This system offers three discrete sets of pH, pCO2, AND PO2 values, which are consistent with normal and pathophysiologically high and low values. The stated values were based on the U.S. National Bureau of Standards scale for pH and on gas analysis for pCO2 and pO2. Influence of temperature, air contact, calibration gas, and storage was established. Internal and external quality control by means of these ampoules is presented. The system is stable, accurate, precise, and suitable for simultaneous quality control of pH, pCO2, and pO2 measurements.

Use of carbon dioxide- and oxygen-tonometered phosphate-bicarbonate-chloride-glycerol-water mixtures for calibration and control of pH, pCO2, and pO2 electrode systems.

Calibration of pH, PCO2, and PO2 electrode systems of modern blood-gas analyzers, designed with one sample cuvet for measurement, is mostly performed separately with buffer solutions of known pH, PCO2, and PO2 for doing such calibrations simultaneously, containing phosphate, bicarbonate, and chloride in glycerol-water mixtures as solvent. A method is suggested for computing the relation between pH and log PCO2 of these solutions in equilibrium with carbon dioxide gas. It is demonstrated that a solution of phosphate (Na2HPO4, KH2PO4, each 25 mmol/liter), bicarbonate (NaHCO3, 30 mmol/liter), and chloride (Nacl, 30 mmol/liter) in glycerol-water mixture (3/7 by vol) and equilibrated with CO2 in air (4 vol/100 vol) and CO2 in nitrogen (8 vol/100 vol), respectively, makes possible acurate and simultaneous calibration of the pH, PCO2, PO2 electrodes of a Corning Model 165 blood-gas analyzer. Similar solutions may also be used for quality-control of blood-gas measurement.