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.