Evaluation of protein containing quality control materials for blood gas analysis.

Protein containing quality control (QC) material in ampoules for blood gas, pH and electrolyte analysers has been manufactured using buffered protein (Bovine Serum Albumine, BSA) solution with sodium bicarbonate and chloride salts. For comparison a similar QC material but without protein was manufactured. Results obtained with ampouled QC material depend on pre-analytical effects, on matrix effects and on the stability of the material. Pre-analytical variation occurs with closed and/or opened ampoules. The shaking rate of the ampoule must be high (vortexing) and the duration of shaking long enough (15 seconds) to give good reproducibility. Temperature coefficients of protein containing controls are equal to those of protein free controls when incubated at different temperatures. Vigorously shaking of the ampoule gives a protein foam layer resulting in stable values for pH, pCO2 and pO2 during maximally 6 minutes after opening of the ampoule. Concerning matrix effects the CO2 buffer capacity of protein containing QC material is slightly higher compared to that of protein free QC materials as determined by tonometry with CO2/air gas mixtures and measuring pH and plotting log pCO2 vs. pH. The O2 buffer capacity measured as the bias on a properly functioning blood gas analyser is smaller than the bias of protein free QC material. The protein containing quality control is stable for at least 12 months when stored refrigerated at 2-6 degrees C and 28 days at room temperature.

The need for protein containing quality control materials for blood pH and electrolyte analyzers.

In clinical chemistry quality control refers to monitoring of precision and accuracy of the performance of analytical methods. Calibration solutions and (matrixed) control solutions are used in transferring accuracy between definitive method, reference method and field methods. For this purpose aqueous (protein-free), protein-containing and serum-based types of quality control materials having different matrices are available. Here are presented differences in behaviour between aqueous (protein-free) and protein-containing materials. Potentiometry with an electrochemical cell is an often used field method to determine pH and activity of electrolytes with an Ion-Selective Electrode (ISE). Basically, measured e.m.f.'s of calibrators and sample are translated into the activity of the ionic species of the sample by means of the Nernst equation. Besides the standard e.m.f. (E(zero)) of the electrochemical system, the measured e.m.f. includes the ISE-membrane e.m.f. (EISE) and depends on electrolyte type and its concentration and Eij depends on the composition and geometry of the salt bridge. Both EISE and Eij depend on the sample matrix. Protein-containing samples cause a negative bias on the e.m.f. at the liquid junction and a positive bias at the ISE. When EISE and Eij are compared to the mV span of the reference interval, the effects are large for Ca2+, Na+ and Cl-. Exactly the same effects exist for H+, K+, Li+ and Mg2+ but the inaccuracy is less critical for these ions. It may be concluded that only protein containing controls can detect an error that occurs only with measurements on plasma specimens. In practice this means that calibration is checked with protein-free solutions, but that measurements in plasma are best checked with protein-containing solutions.

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.

The effect of high-dose fentanyl anaesthesia on P50 in man.

The effect of high dosage fentanyl anaesthesia on P50 was studied in patients undergoing coronary bypass surgery. After induction of fentanyl anaesthesia 100 micrograms X kg-1 with pancuronium muscle relaxation P50 changed significantly (P less than 0.005) from 3.40 +/- 0.12 to 3.33 +/- 0.13 kPa. This anaesthesia technique decreases P50 in vivo, but this has more theoretical than practical importance.

Platelet preservation. III. The influence of dimethylsulfoxide and cooling conditions on serotonin uptake velocity and response to hypotonic stress of human platelets.

1. Serotonin uptake velocity of human platelets was found to be reversibly inhibited by the presence of dimethylsfulfoxide (DMSO). 2. Incubation of platelet concentrates in the presence of DMSO for 90 minutes at room temperature decreased serotonin uptake velocity and response to hypotonic stress. 3. The serotonin uptake velocity was not substantially influenced by the temperature of incubation with DMSO. 4. The best recovery of serotonin uptake velocity after cryopreservation in the presence of DMSO was observed when DMSO was added quickly before cooling and removed quickly after thawing. 5. No significant differences were observed in serotonin uptake velocity and response to hypotonic stress cryopreservation in the presence of DMSO when cooling rates were varied from 2 to 10 degrees C/min.