Bilirubin Interference with Oximetry

Một phần của tài liệu Endogenous Interferences in Clinical Laboratory Tests (Trang 35 - 41)

Bilirubin may cause an interference in the measurement of the different forms of hemoglobin by absorbing light at the same wavelengths as that of hemoglobin. The active portion of the hemoglobin molecule is the heme unit, which is a porphyrin ring. It contains one atom of iron, which may be in the ferrous (Fe2+) or ferric (Fe3+) oxidation state. In order to bind oxygen (O2), the iron must be in the ferrous state. On binding oxygen, there is a tendency for the iron to become oxidized from the ferrous to the ferric state, which is known as methemoglobin . Normal individuals reduce the ferric form of iron back to the ferrous form by means of the enzyme NADH-cyto- chrome-b5 reductase [3]. The blood of normal persons contains approximately 1.5 % methemoglobin.

Hemoglobin provides a way for the blood to capture oxygen in the lungs and transport it back to the tissues. Lung diseases, both acute and chronic, demonstrate decreased concentrations of oxyhemoglobin . Determination of oxyhemoglobin and percent saturation of hemoglobin by oxygen represent key measurements in the care of many acutely ill patients and that information is critical in reducing morbidity and mortality. The percent saturation of hemoglobin by oxygen can be determined by both pulse oximetry and co-oximetry. Pulse oximetry is an in vivo, non-invasive technique where the oxy and deoxy forms of hemoglobin are measured through the skin. Co- oximetry measures oxy and deoxy forms of hemoglobin, but requires a whole blood sample.

One can distinguish between the different forms of hemoglobin by using oxime- try. There are two types of analyzers for oximetry, the pulse form and the co-oximeter.

Both of these types of devices work on the principle of using multiple wavelengths of light to measure the different forms. Both oxyhemoglobin and deoxyhemoglobin absorb light at the same wavelengths, but the amount of light absorbed varies depending on the wavelength, which is expressed as the molar absorptivity or molar extinction coefficient.

3.3 Bilirubin Interference with Oximetry       23

One could measure the total amount of hemoglobin at 431 nm, which has an extinc- tion coefficient of 528,600 (extinction coefficients are in units of per Mole/L for a pathlength of 1 cm). The other forms of hemoglobin absorb light at wavelengths very close to 431 nm with nearly similar extinction coefficients (Fig. 3.1) [3].

wavelength (nm) 600000

500000 400000 300000 200000 100000

0 400 500 600

hemoglobin absorbance spectra

300 700

extinction coefficient

oxyhemoglobin deoxyhemoglobin

wavelength (nm) 60000

50000 40000 30000 20000 10000 0 -10000

300 350 400 450 500 550 600

extinction coefficient bilirubin

650 700

Fig. 3.1: Absorbance spectra for oxyhemoglobin, deoxyhemoglobin and bilirubin.

The extinction coefficients are too high, near 431 nm, to be very useful; however, one can measure the hemoglobin molecule absorption at slightly above 500 nm to dis- criminate among the varying hemoglobin forms. At a wavelength of 555 nm, deoxyhe- moglobin has an extinction coefficient of 54,520, oxyhemoglobin an extinction coeffi- cient of 36,815, carboxyhemoglobin and methemoglobin have extinction coefficients that are distinct from these other species. The absorption maximum in the 500–700 nm region for oxyhemoglobin is 578 nm, while 621 is the absorption maximum for meth- emoglobin, with carboxyhemoglobin showing absorbance maxima at 541 nm and 577 nm [4]. By measuring the peaks for deoxyhemoglobin, oxyhemoglobin, carboxyhe- moglobin and methemoglobin at three other wavelengths, one can distinguish among the varying forms, because the peaks occur at different wavelengths and the extinc- tion coefficient are different.

3.3.1 Co-oximetry Interference

By measuring the absorbance at four carefully chosen wavelengths, one can discern among the four species of hemoglobin. In some methods, one measures the total hemoglobin as cyanohemoglobin, so a fifth wavelength is added. To find the concen- trations of the various species one solves the set of simultaneous equations given by

(3.2)

where C represents the concentration of each hemoglobin species, ε represents the extinction coefficient for that species at that particular wavelength, λ, and Aλ rep- resents the absorbance at that particular wavelength. The equations represent five equations and five unknowns, and can be directly solved for the concentration of each species. The problem is that bilirubin can also absorb light in the region of the selected wavelengths and thus masquerade as one of the hemoglobin species.

An example of this type of interference occurs in oximetry where it has been noted that pulse oximetry is often not affected by bilirubin, but co-oximetry is [5]. Beall and Moorthy reported a case that is interesting. A patient was treated for nodular scleros- ing Hodgkin’s lymphoma with several rounds of chemotherapy and whole body irra- diation followed by autologous bone marrow transplantation. He developed hepatic venous occlusive disease, which resulted in hepatic failure with bilirubin concentra- tions ranging between 633–770 μmol/L and respiratory failure requiring intubation and mechanical ventilator support. The patient was monitored with continuous pulse oximetry and blood gas analysis. The blood gas analysis included determination of hemoglobin oxygen saturation by co-oximetry.

The staff were able to maintain the patient’s arterial pO2 in a range of 92–

133  mmHg, which should be sufficient for normal oxygenation of hemoglobin;

however, the results from the co-oximeter of the blood gas unit were lower than expected, with hemoglobin saturations of 88–93 % (IL 282 Co-oximeter from Instru- mentation Laboratory). These results for the co-oximeter do not correlate well with the arterial pO2, obtained on the same specimen as used for the co-oximetry deter- mination of hemoglobin saturation. Further evidence helped to discern this discrep- ancy. The staff recorded values for the hemoglobin saturation from pulse oximetry using two different devices, the Nellcor N100c and the Ohmeda Biox 3700® [5]. The results from pulse oximetry gave results of hemoglobin saturation of 98–99 %, which is within the normal range and consistent with the arterial pO2 measurements.

Additional information from the co-oximeter showed a slight increase in the frac- tion of hemoglobin represented as carboxyhemoglobin , in this case the values ranged

C1"11+C2"21+C3"31+C4"41+C5"51=A1,

C1"12+C2"22+C3"32+C4"42+C5"52=A2,

C1"13+C2"23+C3"33+C4"43+C5"53=A3,

C1"14+C2"24+C3"34+C4"44+C5"54=A4,

C1"15+C2"25+C3"35+C4"45+C5"55=A5,

3.3 Bilirubin Interference with Oximetry       25

from 2.4–2.9 % and increased fractions measured as methemoglobin , in this case the values ranged from 3.2–11.9  % [5]. Differences besides the specimen, transcutane- ous nonvasive for pulse oximetry and an arterial blood sample for co-oximetry are required to explain these differences.

The basis for measurements of oxygen saturation depends on the differences in extinction coefficient s at various wavelengths for deoxyhemoglobin and oxyhemo- globin. Oxyhemoglobin absorbs less light in the longer wave, red region, near 660 nm, than does deoxyhemoglobin [6]. Because deoxy hemoglobin absorbs a consider- able amount of light in the 660 nm range, associated with red color, its color does not appear as red as oxyhemoglobin. The color of the substance is determined by the wavelengths that the light reflects. If a substance, such as oxyhemoglobin, absorbs more light in the blue, yellow and green regions of the spectrum , it reflects more red light and therefore looks red. Carboxyhemoglobin, the form of hemoglobin which has combined with carbon monoxide, CO, instead of oxygen, absorbs even less light in the red region, and thereby appears even brighter red than oxyhemoglobin.

3.3.2 Pulse Oximetry

A pulse oximeter works by synchronizing the absorbances with the arterial pulse [6].

In the skin, the capillaries dilate in response to the pulse and synchronizing the meas- urements with the pulse gives the most reliable results. In pulse oximetry, absorb- ances are measured at two absorbances, one at 660 nm, at which the absorbance of oxyhemoglobin is less than that of deoxyhemoglobin, and at a much longer wave- length. The secondary measurement of the absorbance is typically done at a wave- length between 815 and 940 nm. In this region of the spectrum, the absorbance of the oxyhemoglobin is slightly greater than that of deoxyhemoglobin [6]. Pulse oximeters are normalized to account for variation in skin absorbance from person to person.

By examining a ratio of the absorption of light in the red region (R) and the infra- red region (IR), pulse oximetry can account for variations from person to person. The ratio is calculated as

(3.3)

Manufacturers calibrate the pulse oximeters empirically by observing the ratio from a group of normal volunteers and comparing them with results obtained by a co-oxime- ter [6]. Subjects are asked to breathe hypoxic gas mixtures so that a calibration curve may be generated. Around an oxyhemoglobin saturation of 85 %, the absorbance at the red and infrared wavelengths measures are about the same, the ratio of these two absorbances would be 1.0. Using a standard or calibration curve provides for a robust relationship because there is a large difference in the values for the ratio; at 99  % Ratio (660 : 815940)= R

IR.

oxygenation, the ratio is around 0.4, while at 50 % it is around 2.0 [6]. Bilirubin does not absorb much light at either of the two wavelengths chosen for measurement with pulse oximeters; therefore, bilirubin is fairly unlikely to cause an interference with this methodology.

Pulse oximeters do have limitations though. Because pulse oximeters measure light at only two wavelengths, they can only distinguish two different species of hemoglobin. At 660 nm, methemoglobin absorbs light in a fashion similar to oxy- hemoglobin [6]. Carboxyhemoglobin absorbs similarly to oxyhemoglobin as well.

Caution must be used in depending on pulse oximetry alone, because it cannot provide information about these two important species of hemoglobin. Frequently, acutely ill patients have their oxygen saturation determined by both methods.

The IL 282 Co-oximeter (Instrumentation Laboratories) used in the case measured light at four wavelengths, 535, 585, 594 and 626 nm. Even though bilirubin exhibits its absorption peak at 450 nm, the bilirubin absorption band extends its tail into the 535–585 nm range, adding its absorption to that measured for hemoglobin. Based on the set of equations used to calculate deoxyhemoglobin, oxyhemoglobin, carboxyhe- moglobin and methemoglobin, the presence of bilirubin in the specimen will add to the apparent absorbance of carboxyhemoglobin and methemoglobin, which is why the carboxyhemoglobin and methemoglobin were overestimated in the co-oximeter.

In the co-oximeter, the percent hemoglobin saturation by oxygen is determined by dividing the calculation fraction of oxyhemoglobin by the total hemoglobin. Total hemoglobin is determined from the fractions for deoxyhemoglobin, carboxyhemo- globin and methemoglobin. Because the calculations for the co-oximeter are per- formed differently than for the pulse oximeter, bilirubin may cause an interference with co-oximeters not seen with pulse oximeters. Many co-oximeters today use an increased number of wavelengths, so that they can calculate the contribution to absorbance from bilirubin and subtract it from the total, thus avoiding bilirubin inter- ference .

Pulse oximetry is utilized by transmitting light through skin, usually a finger.

New techniques in oximetry provide a noninvasive method assessing cerebral oxygen saturation using dual-wavelength near-infrared spectrophotometry (NIRS) [7]. NIRS has been used for monitoring cerebral oxygenation during carotid endarterectomy, acute heart failure and orthotopic liver transplantation [7]. NIRS works by measuring absorbance in the cerebral tissue at 733 and 809 nm [7]. The wavelength at 733 nm provides a measure of deoxygenated hemoglobin, while the wavelength at 809 nm provides a sum of deoxygenated and oxyhemoglobin.

3.3.3 Cerebral Oximetry

Cerebral oximetry employs infrared light which can penetrate the tissues. The selec- tion of the two wavelengths, one near 730 nm and the other near 810 nm allows for

3.3 Bilirubin Interference with Oximetry       27

maximal tissue penetration, with the light being scattered back from the skin, the skull, and up to 15 mm of cerebral tissue [8]. The operation of orthotopic liver trans- plantation is divided into four phases: dissection, anhepatic, reperfusion and end.

Patients requiring liver transplantation are likely to be jaundiced. During the reperfu- sion phase a rise in the cerebral oxygen saturation is expected; however, in patients with elevated bilirubin this increase in the cerebral oxygen saturation is blunted [7].

Examination of the hemoglobin oxygen saturation showed no effect of bilirubin in arterial or venous samples; however, examination of the cerebral oxygen satura- tion demonstrated a negative interference with increasing concentrations of blood bilirubin [7]. It is suspected that rather than bilirubin deposited in the cutis, it is biliverdin, the oxidative product of bilirubin, deposited in the cutis that is interfer- ing with the cerebral oxygen saturation measurement [7]. The absorption of light by biliverdin changes depending on the orientation that the molecule takes: in nonpolar solvents it takes on a ring form and absorbs light in the ultraviolet region, around 350 nm, but in polar solvents or attached to proteins, it takes on a straight chain form with four connected pyrrole groups and shifts its light absorbance to the visible region [9].

3.3.4 Interference with Methemoglobin

Falsely elevated values for methemoglobin , caused by bilirubin when using co-oxi- metry, can cause erroneous diagnosis of methemoglobinemia. Methemoglobinemia is defined when the hemoglobin in the red blood cells possess greater than 1 % hemo- globin, and even though a small amount of methemoglobin is normal and does not pose a health risk, elevated fractions of methemoglobin decrease the oxygen carry- ing and delivery capacity of the blood and may pose a risk, resulting in a functional anemia [10]. Methemoglobin is formed when the iron in normal hemoglobin is oxi- dized from the ferrous to the ferric form. When this occurs the skin become cyanotic or blue in appearance; neurologic and cardiac symptoms begin when the fraction increases above 15 % because of hypoxia and death occurs for fractions of 70 % or above [10].

The normal method for reduction of ferric iron to ferrous iron involves the adenine dinucleotide (NADH)-dependent reduction, called the diaphorase pathway, the enzyme cytochrome b5 reductase playing the major role in the pathway [10]. The diaphorase pathway reduces 95–99 % of the methemoglobin, while another enzyme system, the nicotinamide adenine dinucleotide phosphate (NADPH)-dependent methemoglobin reduction accounts for the rest; this enzyme uses glutathione and glucose-6-phosphate dehydrogenase (G6PD) to reduce methemoglobin to hemo- globin [10]. Hereditary methemoglobinemia is a rare condition and is most commonly found among Native American tribes such as the Navajo, Athabascan Alaskans and the Yakutsk people of Siberia; it involves a deficiency in cytochrome b5 reductase [10].

Most cases of methemoglobinemia are acquired through exposure to drugs or toxins,

such as benzocaine, with infants, and especially premature infants being the most susceptible [10].

Acquired methemoglobinemia requires treatment and is caused by several recog- nized agents, such as nitrites, nitrates, chlorates, and dapsone [10]. There are several tests used in the diagnosis and management of methemoglobinemia, including pulse oximetry, co-oximetry, potassium cyanide test, as well as complete blood count. Co- oximetry is the best way to quantify the fraction of methemoglobin, but bilirubin can cause falsely elevated results, unless the co-oximeter has additional wavelengths that can detect the presence of bilirubin. Once the diagnosis is suspected, a search for the cause prompts the ordering of additional tests such as a complete blood count, hemo- globin M by electrophoresis, mass spectrometry and DNA sequencing.

Hemoglobin Barts is a variant hemoglobin where the alpha (α) chains are absent and the gamma (γ) chains form tetramers and it is associated with hydrops fetalis, occurs in the cord blood of healthy neonates and neonates with hemoglobin S, a-thalassemia and other hemoglobinopathies [11]. High-performance liquid chroma- tography (HPLC) is the most common method used to identify hemoglobin variants and there are several reports that the presence of hemoglobin can produce a peak in the HPLC procedure that is mistaken for hemoglobin Barts [11, 12]. Hemoglobin Barts elutes within the first 0.5 minutes after injection and a peak identified as bilirubin appeared in 0.2 minutes from the time of injection [11]. In a series of 8,000 hemo- globin chromatograms, 90 chromatograms were identified with an unusually large peak in the first 0.2 minutes after injection, and of these 90 patients, 86 had hemo- globin SS, one had hemoglobin AS and three had hemoglobin AA. The height of these peaks ranged from 3 % to more than 46.5 %. When these peaks were compared against the concentration of bilirubin, a positive interference was revealed with a correlation coefficient of 0.87 [11]. When serum samples with more than 513 μmol/L of bilirubin were taken and analyzed by chromatography using the HPLC analyzer, the chromatograms showed a single peak at 0.2 minutes of injection [11]. This study clearly elucidated that bilirubin could cause an interference with the HPLC method for hemoglobin variants and showed several useful techniques to prove the inter- ference .

Một phần của tài liệu Endogenous Interferences in Clinical Laboratory Tests (Trang 35 - 41)

Tải bản đầy đủ (PDF)

(156 trang)