Year: 2018
Type: article
Abstract: Our laboratory encountered strikingly dark brown plasma from 2 different patients within a 24-h period. Patient 1 was a 52-year-old woman. She reported waking up one morning with nausea and multiple episodes of nonbloody, nonbilious emesis and diarrhea. She subsequently developed stinging pain in her anterior mid-abdomen. On inspection, she discovered 2 areas of dusky discoloration with surrounding erythema of about 1–2 cm in diameter. She presented to an outside hospital (OSH)2 and was diagnosed with cellulitis and treated with antibiotics. She developed jaundice and a diffuse rash over her torso and lower extremities over the next 3 days. She was admitted to our institution with concern for necrotizing fasciitis. Blood samples were sent to our laboratory and extremely dark brown plasma was noted (Fig. 1). Patient 2 was a 47-year-old man who was riding a stationary bicycle when he lost consciousness, fell off, and began to seize. He was transported to an OSH, where it was discovered that he had subarachnoid hemorrhage. At our institution, he underwent endovascular coiling and ventriculostomy. Blood samples were sent to our laboratory and dark brown plasma was identified (Fig. 1). The cause for these discolorations was investigated. Hemolysis is the most common cause of dark red or brown plasma. Hemolysis releases hemoglobin (Hb) from red blood cells (RBCs) into plasma. All forms of Hb absorb light over the violet-to-green end of the visible spectrum (300–600 nm). Oxygenated Hb (OxyHb) has 3 absorption maxima (415, 540, and 570 nm), whereas the deoxygenated form (deoxyHb) has 2 (420 and 550 nm). Carboxyhemoglobin has an absorption spectrum similar to OxyHb, but with a slight left-shift. Hemolysis becomes apparent to the human eye at plasma Hb concentrations ≥50 mg/dL (1). However, visual inspection of hemolysis is an unreliable approach, even in the hands of well-trained laboratory personnel or with use of color standards for comparison (2, 3). In addition to inter-individual variability, visual inspection can further be complicated by concomitant icterus and vary by specimen type (plasma or serum) (2, 3). Thus, a determination of an automated hemolysis index is preferred to visual assessment (3). Dark plasma samples may be observed after lysis of RBCs containing methemoglobin (MetHb). MetHb is deoxyHb in which the heme iron (Fe2+) has been oxidized to the ferric (Fe3+) state (4). Unlike oxyhemoglobin, MetHb cannot bind oxygen. In healthy people, MetHb forms spontaneously owing to the presence of endogenous oxidants, but mechanisms exist to ensure that the proportion of Hb as MetHb remains at ≤1% (4). Although a MetHb concentration <1% is ideal, MetHb concentrations may be higher at baseline in some populations such as smokers or urban dwellers. MetHb broadly absorbs light with wavelength <700 nm, with an absorption peak at 650 nm. As more light is absorbed, the sample appears darker. Methemalbumin can also form as a consequence of hemolysis when either free heme or hematin forms a complex with albumin (5, 6). The complex, with an absorbance maximum around 410 nm, broadly absorbs light across the violet-to-green spectrum so the light that is transmitted is yellow-to-red and appears brown to the eye. Bilirubin is another possible cause of brown plasma in hemolyzed specimens. Bilirubin absorbs light over a narrower range (350–500 nm) than Hb, allowing more yellow light to be transmitted or reflected. Thus, icteric samples usually appear lighter than hemolyzed samples; however, samples may have both processes present, making visual detection more difficult (7). Myoglobin release after muscle damage is another major cause of dark-colored plasma (8). Myoglobin has an absorbance spectrum similar to Hb and also exists in oxygenated and deoxygenated forms. Myoglobin can appear in circulation and in urine after muscle tissue necrosis, such as rhabdomyolysis (8). Haptoglobin (Hp) is a serum protein that binds to Hb and directs its clearance via the reticuloendothelial system (6). Thus, a suppressed Hp concentration in a hemolyzed specimen suggests in vivo hemolysis, but it cannot exclude concomitant in vitro hemolysis. Hp concentrations remain in the normal range when hemolysis occurs solely in vitro. Moreover, potassium is usually increased after in vitro, but not in vivo, hemolysis. Other nonspecific hemolysis markers include lactate dehydrogenase (LDH) and bilirubin. LDH is abundant in RBCs and is released into the plasma after hemolysis. Bilirubin is a breakdown product of heme. When hemolysis occurs in vivo, both direct (conjugated) and indirect (unconjugated) bilirubin may be increased. When hemolysis occurs in vitro, only indirect bilirubin will be increased. Myoglobinuria may occur in patients with severe muscle damage. Myoglobin can masquerade as blood in urine owing to its dark color and because myoglobin cross reacts with the peroxidase reaction in qualitative urine strips (8). Thus, a positive blood urine dipstick without detectable RBCs is supportive of myoglobinuria. Serum creatine kinase (CK) and aldolase can be measured to distinguish muscle damage from hemolysis. If necessary, CK isoenzymes may be used to pinpoint the source of CK elevation. Hemolysis can cause spurious increases or decreases in the results of several laboratory tests (Table 1) (7). There is no universally established clinical guideline for reporting laboratory results from hemolyzed specimens. Thus, the management of hemolyzed specimens often varies according to institutional policy. Common approaches laboratories use to deal with hemolyzed specimens include collection of a new specimen, reporting results with a disclaimer urging clinicians to interpret results with caution, or reporting with correction based on an estimated degree of hemolysis (9). A proposal by Cadamuro et al. advocated reporting preanalytically altered laboratory results with a comment on potential deviations (10). However, collection of a new specimen is the most common approach, contingent upon patient availability and ease of access (9, 10). One important caveat with this approach is that recollection from a patient with ongoing hemolysis will not resolve the issue. Therefore, the decision of when to report or suppress results obtained from hemolyzed specimens should be customized to specific situations, and clinical laboratorians should clearly communicate with ordering clinicians when these issues arise (11). Influence of hemolysis on selected laboratory tests. Influence of hemolysis on selected laboratory tests. Patient 1 developed red urine and a urinalysis was notable for 3+ blood and 2+ albumin. Other relevant laboratory results were low Hb concentration of 4.5 g/dL [reference interval (RI), 12.1–15.1], positive Coombs test (C3 antibodies), low Hp concentration of 14 mg/dL (RI, 30–200), increased total bilirubin concentration of 4.7 mg/dL (RI, 0.1–1.2), increased serum LDH concentration of 738 U/L (RI, 100–250), and moderately increased myoglobin concentration of 114 μg/L (RI, ≤90). The high serum bilirubin and LDH concentration with the low Hp concentration suggested in vivo hemolysis, and the modestly increased myoglobin concentration was consistent with muscle necrosis. Overall, the extremely dark-colored plasma in this case was likely because of a combination of Hb, bilirubin, and myoglobin. The patient was diagnosed with systemic loxoscelism, a condition produced by the bite of a recluse spider (genus Loxosceles), based on the necrotic wound and hemolytic anemia. At the time of discharge, the patient's Hb was 11.5 g/dL and she had negative direct Coombs test result, normal bilirubin, and improving serum LDH concentrations. Interference in creatinine and CK tests was of highest concern for patient 1 because both tests involve measurement of absorbance (546 nm and 340 nm, respectively) but were needed to monitor renal function and muscle damage. To investigate interference in the creatinine method owing to the color, we divided the patient's specimen into 2 aliquots. We measured creatinine by the Roche Creatinine Plus II method with 1 aliquot and the Abbott i-STAT analyzer with the other. The Roche Creatinine Plus II makes use of an enzymatic colorimetric method, whereas the Abbott i-STAT measures creatinine using an amperometric method. The result was 1.3 mg/dL from both instruments, demonstrating that the color did not interfere in the Roche method. The possibility of interference in the Roche CK test was evaluated by mixing the patient's plasma with plasma from another patient with increased CK. The values for CK before and after 1:1 dilution with the plasma from patient 1 were 2932 U/L and 2627 U/L (after correcting for the dilution and the contribution of CK from patient 1). Although it is difficult to assess the effect of sample mixing and the calculated correction on the repeatability of the test method, these data may indicate a statistically significant but biologically irrelevant difference (based on the manufacturer's average %CV for repeatability of 1.5%). After discussing these results with the ordering clinician, it was determined that the modest effect on CK was not a clinical concern. Patient 2 was part of a phase II trial investigating the use of SanguinateTM in the treatment of patients with subarachnoid hemorrhage. Sanguinate is a polyethylene glycol-modified (PEGylated) bovine carboxyhemoglobin, which both provides carbon monoxide to encourage vasodilation and serves as an oxygen transfer agent superior to Hb alone (12). The presence of the PEGylated carboxyhemoglobin explained the dark color. The patient's clinical team was aware of the possible interference in laboratory tests and did not express any concern regarding test results. Artificial blood substitutes are associated with increased mortality and myocardial infarction, likely owing to vasoconstriction secondary to the nitric-oxide scavenging properties of cell-free Hb (13). Sanguinate is designed to circumvent this and other problems associated with blood substitutes (12). In patients receiving Sanguinate, clinical laboratories should be alert to the possibility of abnormal test results for colorimetric methods. Interference with other methods is less likely, although further studies are needed to investigate this possibility. As such, strong communication between the laboratory and clinicians is critical to ensure high-quality and clinically significant test results. A common cause of dark brown plasma is hemolysis, particularly after lysis of red cells containing methemoglobin. Loxoscelism, a less frequently encountered cause of hemolysis, is highlighted in one of the cases. Dark brown plasma can also be due to the presence of myoglobin in the blood or the biopharmaceutical Sanguinate, PEGylated Bovine Carboxyhemoglobin. Hb freed during hemolysis can cause spectral interference in methods that use spectrophotometry in an analyte-dependent manner because Hb can absorb light at different wavelengths (415, 540, and 570 nm). There is no single test to determine the cause of dark brown plasma. However, laboratories should combine multiple tests to determine the cause of colored plasma to decide whether to report or suppress test results. There is no universally established clinical guideline on how to handle hemolysis-affected laboratory results. Thus, management may vary according to institutional policy. outside hospital hemoglobin red blood cell oxygenated Hb deoxygenated Hb methemoglobin Haptoglobin lactate dehydrogenase creatine kinase reference interval polyethylene glycol-modified.
Institution: Washington University in St. Louis
Language: English
Subfield: Physiology
Field: Medicine
Domain: Health Sciences
Open Access status: bronze
OpenAlex ID: W2902555810