Hemoglobin hafnia: alpha 2 (beta 116 (G18) His----Gln)2; a new hemoglobin variant mistaken for glycated hemoglobin.

Isoelectric focusing of hemolysate from patients with diabetes mellitus is routinely performed to measure their level of HbA1c (glycated hemoglobin). For a 6 year old boy with diabetes mellitus this analysis showed an HbA1c fraction of approx. 50%, which is very unlikely to occur. The possibility of a hemoglobin variant was considered, and by HPLC-separation the presence of two different beta-chains was shown. One tryptic fragment was found to deviate from the normal, and amino-acid analysis and sequence determination revealed the following amino-acid substitution: beta 116 His----Gln. It is the first reported mutation at this position. Functional studies showed almost normal behaviour, consistent with the fact that the affected persons are without any symptoms. In a family survey we found five nondiabetic members with an abnormality similar to the proband. For the variant we chose the name Hafnia, which is Latin for Copenhagen.

Hypophosphatemia and acute respiratory failure in a diabetic patient.

A previously healthy 48-year-old male developed diabetic ketoacidosis and severe hypophosphatemia. Within a few hours, acute respiratory insufficiency developed with a marked discrepancy between the pulmonary pathology and the very poor oxygenation seen. We argue that this was due to the effect of hypophosphatemia on respiratory muscle- and heart function and P50, leading to impaired oxygen delivery.

Direct reading glucose electrodes detect the molality of glucose in plasma and whole blood.

It is the activity that determines the direction of chemical processes, transport, etc. and thus provides the clinically more relevant information. Direct reading glucose electrodes consume glucose at a rate proportional to the glucose activity in the sample. The activity equals the molality (mmol glucose per kg water), so results from direct reading glucose electrodes must differ from the conventionally measured glucose concentration. This was observed in 159 whole blood samples which gave higher results from a direct reading glucose electrode than by our conventional method (y = 1.21x - 0.37 mmol/l). However, adjustment for the different water concentration due to salt, plasma proteins, and hemoglobin occupying space, gave results equal to the concentrations (y = 1.00x - 0.28 mmol/l, r = 0.997). Furthermore, results for samples with constant glucose concentration and varying albumin concentration correlated with the albumin concentration (r = 0.989), but not after adjustment for water concentration (r = 0.037, n.s.).

Diode-array spectrophotometry for simultaneous measurement of hemoglobin pigments.

A prototype oxygen saturation meter was used to measure the concentrations of deoxygenated hemoglobin (rHb), oxyhemoglobin (HbO2), carboxyhemoglobin (HbCO), methemoglobin (MetHb), and sulfhemoglobin (SHb) in 35 microliter blood. Simultaneous absorbance measurements at 535, 560, 577, 622, 636, and 670 nm permitted the composition of any hemoglobin pigment mixture to be determined more accurately, precisely and easily than before. The inclusion of 670 nm, where the hemoglobin pigments have low absorption coefficients, allowed correction for turbidity.

Spectrophotometric determination of hemoglobin pigments in neonatal blood.

Fetal and adult hemoglobin pigments have slightly different light absorption coefficients. Blood from newborns therefore gives inaccurate results with a direct spectrophotometric determination of hemoglobin pigments (Radiometer's OSM3), if the absorption coefficients for adult blood are used. We determined an absorption coefficient matrix for hemoglobin pigments in twenty full term newborns' blood. This matrix yielded results accurate to within 0.2% in 40 further newborns, but the accuracy of the results varied with the individual ratio of fetal to total hemoglobin, which was 80 +/- 5% (SD) in the examined samples. The OSM3's inaccuracy with the adult absorption coefficients can be used to directly estimate the ratio of fetal to total hemoglobin in an infant.

IFCC document stage 3, draft 1, dated 1989 02 01. An approved IFCC recommendation. IFCC method (1988) for tonometry of blood: reference materials for pCO2 and pO2. International Federation of Clinical Chemistry Scientific Division. Committee on pH, Blood Gases and Electrolytes.

A reference method for tonometry of blood is described. The document covers the theory of tonometry, the materials and equipment needed, and essential aspects of the tonometry procedure for blood. The partial pressures of oxygen and carbon dioxide in tonometered blood are accurately known and therefore this blood is recommended for assessing the accuracy of blood gas analyzers. Tonometry of blood samples from patients may also be used in the determination of acid-base quantities and hemoglobin-oxygen affinity, e.g. p50.

Oxygen monitoring in the newborn.

Current techniques for measuring oxygenation in newborn infants with respiratory insufficiency are reviewed, as well as the consequences of variable and high fetal hemoglobin (HbF) fractions on both the measurement of sO2 and FCOHb and the application of Siggaard-Andersen's oxygen status algorithm to newborn infants. A procedure involving tonometry of blood is described for measuring FHbF in newborn infants' blood.

Accurate measurements of hemoglobin oxygen saturation, and fractions of carboxyhemoglobin and methemoglobin in fetal blood using Radiometer OSM3: corrections for fetal hemoglobin fraction and pH.

The differences in the visible absorption spectra between fetal and adult oxyhemoglobin and carboxyhemoglobin result in errors in the measurements og hemoglobin oxygen saturation (SO2) and carboxyhemoglobin fraction (FCOHb) in fetal blood, if not corrected for the actual fetal hemoglobin fraction (FHbF) in the sample. In 11 fully oxygenated umbilical cord blood samples (mean FHbF = 77%), we found a mean positive bias in SO2 of 4.7%, and in FCOHb of 2.7%, when measured with a dedicated spectrophotometer (OSM3, Radiometer A/S, Denmark), and using the matrix of absorption coefficients for adult hemoglobin. Accurate measurements were obtained by using OSM3's correction for FHbF in the blood specimen after measurement of FHbF by OSM3. The effects of plasma pH on the measurements of SO2 and FCOHb in fully oxygenated fetal blood were found to be similar to those found for adult blood. From plasma pH 7.05 to 8.02, measured SO2 increased 1.3% and FCOHb 0.6%. Correction for the pH of fetal blood samples should be considered when calibrating OSM3 and in connection with research studies. The effects of FHbF and pH on the measurement of methemoglobin fraction (FMetHb) were less than 0.2%, and can be ignored. FHbF measured by OSM3 at pH 7.4 is about 14% too high compared to alkali denaturation rate method. However, the presence of a metabolic acidemia, which is common in fetal blood specimens, decreases this bias, so that for example in our study, FHbF, measured by OSM3 and uncorrected for pH changes was on average only 6% too high. We recommend that OSM3's factor of 18.6 is reduced to 16.4, and that correction is made for pH.

Hemoglobinopathies in Danish families.

Based on cases referred for investigation, as well as a questionnaire sent to all medical and pediatric departments in Denmark, 48 cases of hemoglobinopathy in 15 families of Danish ancestry are reviewed. 18 Danes in six families have been identified as having beta-thalassemia, and remarkably one - a homozygote - has beta-thalassemia intermedia requiring treatment with iron-chelation therapy. A further 36 Danes in 9 families have a hemoglobin variant: five unstable hemoglobins (Volga, Niteroi, and three unidentified), one hereditary methemoglobinemia (M-Arhus), one polycythemia (Ty Gard) and 2 asymptomatic (Athens-Georgia and Hafnia). Although rare in Danish families, a hemoglobinopathy should be considered in families with an unexplained chronic hemolytic anemia, cyanosis or polycythemia.

Arterial oxygen status determined with routine pH/blood gas equipment and multi-wavelength hemoximetry: reference values, precision, and accuracy.

We measured pH, pCO2, pO2, oxygen saturation, total hemoglobin concentration, and fractions of carboxy- and methemoglobin in arterial blood samples from 35 healthy adults. We used a new algorithm to calculate active hemoglobin concentration, total oxygen concentration, actual half-saturation tension, 2,3-diphosphoglycerate concentration, estimated functional shunt, oxygen extraction tension px (for extracting 2.3 mmol of oxygen per liter of blood, values below 4.5 kPa indicating risk of tissue hypoxia), and the oxygen compensation factor Qx (the factor by which the cardiac output should rise to maintain a normal mixed venous pO2 of 5.0 kPa, factors above 1.5 indicating an extra burden on the heart). Analytical precision was evaluated by duplicate determinations. The accuracy of the half-saturation tension was evaluated by comparison with values for simultaneously drawn venous blood, the accuracy of the calculated concentration of 2,3-diphosphoglycerate by comparison with direct enzymatic measurements. We conclude that all the variables may be determined with sufficient accuracy and precision in healthy adults, provided the oxygen saturation is less than 0.97 and the measurements are performed according to the highest state of the art.

The oxygen status of the arterial blood revised: relevant oxygen parameters for monitoring the arterial oxygen availability.

The new generation of very accurate multi-wavelength oximeters, e.g. OSM3, for in vitro measurement of the hemoglobin oxygen saturation, total hemoglobin concentration, and carboxy- and methemoglobin fractions opens new aspects of oxygen monitoring. Combined with the data from the blood gas analyzer (e.g. ABL300) these very accurate measurements allow the calculation of several derived oxygen parameters on the basis of a set of newly developed calculation algorithms. The traditional parameters obtained from an arterial sample are the oxygen tension (pO2) and the hemoglobin oxygen saturation (sO2). Clinical examples illustrate that the pO2 and the sO2 even in combination may give misleading information. The new algorithm calculates three extra oxygen parameters. 1) The oxygen extraction tension, px, defined as the tension required to extract 2.3 mmol of oxygen per liter blood. It signals the mixed venous pO2 level on the assumption that the arterio-venous oxygen difference is normal (2.3 mmol/L). 2) The concentration of extractable oxygen, cx, defined as the concentration of oxygen extracted at a tension of 5.0 kPa. 3) The oxygen compensation factor, Qx, derived as (2.3 mmol/L)/cx. It may be interpreted as the increase in cardiac output necessary to maintain a normal mixed venous pO2 of 5 kPa. These three parameters indicate the oxygen availability of the blood and summarize important properties of the arterial blood in relation to oxygen supply of the tissues, including the arterial pO2, the 'active' hemoglobin concentration (equivalent to the oxygen capacity), and the hemoglobin oxygen affinity (p50). The set of data measured with the blood gas analyzer, e.g. the ABL300 combined with the data measured with the OSM3 contains much more information than is routinely utilized. This information is extracted and summarized by our calculation algorithm. Omitting the calculation of the extra oxygen parameters involves a risk of losing valuable information.

Relation between pH and ionized calcium in vitro and in vivo in man.

We investigated the in vitro and in vivo relation between pH and ionized calcium during acute respiratory pH changes. The in vitro slope delta 1gcCa2+/delta pH determined in 20 healthy volunteers after equilibration at two different pCO2 values was -0.22 +/- 0.04 (mean +/- 2 SD) for whole blood and -0.24 +/- 0.04 (mean +/- SD) for serum. The in vivo slope delta 1gcCa2+/delta pH during acute respiratory changes in 10 healthy volunteers was -0.17 +/- 0.04 (mean +/- 2 SD) for capillary blood. A mathematical formula which describes the relation between pH and ionized calcium is presented.

Fiber-optic chemical sensors (Gas-Stat) for blood gas monitoring during hypothermic extracorporeal circulation.

Measurements of pO2, pCO2 and pH by optical fluorescence microsensing technology has recently become available for monitoring blood gases during extracorporeal circulation ECC). We have compared simultaneous measurements with fiber-optic sensors (Gas-Stat, Bentley) and electrochemical sensors (ABL-4, Radiometer) on discrete samples. In 10 patients undergoing coronary artery bypass grafting during hypothermic (25 degrees C) ECC and hemodilution (hemoglobin concentration 4 mmol.l-1) arterial and venous pO2, pCO2 and pH were measured in-line in the extracorporeal circuit at the actual blood temperature. Simultaneous and anaerobically collected blood samples in glass syringes were analyzed within five minutes at 37 degrees C in the ABL-4. Linear regression analysis of the values at actual temperature shows the following equations: Gas-Stat = Y, ABL-4 = X: pO2 (kPa): Y = 1.04 X + 0.5 r = 0.95 n = 136; pCO2 (kPa): Y = 0.71 X + 1.5 r = 0.79 n = 136; pH: Y = 0.788 X + 1.590 r = 0.76 n = 136. The advantage of the Gas-Stat is continuous monitoring of blood gas parameters during ECC. The present study shows that measurements of pO2, pCO2 and pH with fiber-optic chemical sensors may be reliable. The differences between the two principles of measurement may be due to unknown factors interfering with the in-line measurements or to variations in sensitivity and stability of the individual sensor.

Pulse oximetry versus transcutaneous pO2 in sick newborn infants.

A pulse oximeter (Ohmeda Biox 3700) and two transcutaneous systems (Radiometer TCM3) were applied simultaneously to 18 newborn infants with respiratory insufficiency. All infants had either an umbilical catheter placed in the mid thoracic aorta or a radial artery catheter. The average monitoring time was 2 hours. Arterial blood pO2, pCO2 and pH (Radiometer ABL300), arterial sO2, HbCO and metHb (Radiometer OSM3), erythrocyte 2,3 DPG concentration, and fetal hemoglobin fraction (alkali denaturation kinetic method) were measured. Using arterial sO2 and pO2 as reference, the analytical bias of pulse oximetry (-0.5 +/- 1.0%, mean +/- 1 SD) corresponded in magnitude, when converted to pO2, to that of transcutaneous - pO2 (0.6 +/- 1.4 kPa for combined O2-CO2 electrode and -0.1 +/- 2.3 kPa for single O2 electrode). Transcutaneous pCO2 showed the smallest bias (0.3 +/- 0.3 kPa). Both pulse oximetry and transcutaneous pO2 electrodes were good as trend monitors detecting rapid changes in the infants' oxygenation status. The pulse oximeter offers certain advantages in not requiring calibration or heating. The variations in the levels of fetal hemoglobin fraction (44 to 97%), pH (7.27 to 7.49), pCO2 (3.3 to 6.8 kPa) and 2,3 diphosphoglycerate concentration (1.6 to 5.9 mmol/l) between the infants studied, resulted in a variable pO2-sO2 relation (p50 2.5 to 3.5 kPa). This presents difficulties in interpreting sO2 values in sick newborn infants, and we therefore recommend caution in using a pulse oximeter to apply strict limits for avoiding hypoxia and hyperoxia in this population.

Guidelines for routine measurement of blood hemoglobin oxygen affinity. International Federation of Clinical Chemistry, Scientific Division, Committee on pH, Blood Gases and Electrolytes.

Two methods for the routine determination of blood hemoglobin oxygen affinity are described. Both methods use whole blood and do not require special equipment, tonometry or special gas mixtures. The first method consists of a one-point determination of p50, and requires only 200 microL to 400 microL of whole blood, therefore making it suitable for the pediatric population. The second method uses multiple points, thereby establishing both the shape and position of the hemoglobin oxygen equilibrium curve between 10 and 99% oxygen saturation. Interpretation of p50 is discussed in relation to evaluation of patients with hemoglobinopathies and as a parameter in estimating availability of oxygen to the tissues.

[Oxygen status of arterial blood including uncompensated mixed venous oxygen tension and cardial oxygen compensation factor. Reevaluation on the basis of 250 arterial punctures]. / Arterieblodets oxygenstatus, herunder ukompenseret blandetvenøs oxygentension og kardial oxygenkompensationsfaktor. Nyvurdering på basis of 250 arteriepunkturer.

In arterial blood from 250 patients we measured pH, pco2, and po2 (electrochemically) together with total-hemoglobin concentration, oxygen saturation, carboxy- and methemoglobin fractions (spectrometrically). With a previously published algorithm we calculated the effective hemoglobin concentration, total-oxygen concentration, half saturation tension, erythrocyte 2,3-diphosphoglycerate concentration, and two new oxygen parameters: uncompensated mixed venous oxygen tension and cardiac oxygen compensation factor. 11% of the patients have normal arterial oxygen tension, but nevertheless risk of tissue hypoxia judged from the two new oxygen parameters. This is due to a low hemoglobin concentration and/or low half saturation tension (increased hemoglobin oxygen affinity). Some patients have decreased arterial oxygen tension but normal uncompensated mixed venous oxygen tension (15%) or normal cardiac oxygen compensation factor (9%). This is due to a high hemoglobin concentration and/or increased half saturation tension. The latter varies from 2.6 to 5.2 kPa (ref.: 3.3-3.9 kPa); 36% have decreased, 27% increased values. The 2,3-diphospho-glycerate concentration varies from 2.0 to 7.9 mmol/l (ref.: 3.6-5.1 mmol/l); 14% have decreased, 30% increased values. Uncompensated mixed venous oxygen tension varies from 1.8 to 5.7 kPa (ref.: 4.5-5.5 kPaf). The cardiac oxygen compensation factor varies from 0.9 to infinity (ref.: 0.8-1.6). We conclude that the variation in the different oxygen parameters is so significant that it justifies routine calculation for all arterial blood samples where the measurement on a conventional blood gas analyzer is supplemented with measurement on one of the new multi-wavelength hemoximeters. The calculation algorithm permits calculation of all the oxygen parameters for the majority of arterial samples (84%) where the oxygen saturation is less than or equal to 0.970.