Which of the following values can be used to indicate the presence of a hemolytic anemia?

Basic Information

Hemolysis

Hemolysis occurs around streptococcal colonies growing on blood agar plates because of production of the cytolytic toxin streptolysin. Streptolysin causes transmembrane pore formation in red blood cells resulting in osmotic lysis. Three patterns of hemolysis occur

α-Hemolysis: growth on blood plates causes incomplete destruction of blood cells. This produces dark green discoloration around bacterial colonies, reflecting the presence of biliverdin and other hemoglobin breakdown products.

β-Hemolysis: growth on blood plates causes complete destruction of blood cells, resulting in transparency of the region surrounding bacterial colonies.

γ-Hemolysis: no observable destruction of blood cells surrounding bacterial colonies.

Pathogenic equine streptococci are hemolytic and most are β-hemolytic. Non-hemolytic streptococci may be isolated from the equine upper respiratory tract (URT) but are regarded as commensals without pathogenic potential

8.2 Hemolysis

Hemolysis is a known interference of select cTnI and cTnT immunoassays [82–85]. Hemolysis is the breakdown of erythrocytes with subsequent release of intracellular contents. Frequently, clinical specimens are contaminated due to hemolysis with rates up to 20% in specimens collected from patients in the Emergency Department [86]. Presence of hemolysis can cause either positive or negative biases in cTn measurement which is assay specific (Fig. 5; [85]). Florkowski et al. showed that in samples containing cTn concentrations approximately at the 99th percentile medical decision limit, the Roche hs-cTnT assay exhibited a negative interference up to 50% with increasing hemolysis, whereas the Vitros ECi TnI assay (Ortho Clinical Diagnostics, Rochester, NY, USA) showed a positive interference up to 576% with increasing hemolysis [82]. The mechanism of hemolysis interference with cTn measurement remains unclear; it has been suggested that the release of hemoglobin and proteases from erythrocytes upon lysis may cause interference with the detection method or anti-cTn antibody recognition of degraded cTn fragments [25,83,87]. Considering the heterogeneity of cTn assays and unknown mechanism of hemolysis interference, each central laboratory must perform interference studies to evaluate the effect of hemolysis on cTn measurements. Hemolysis presence in serum or plasma specimens can be visually identified as a pink to red color, when hemoglobin concentrations are > 0.2 g/dL [88]. However, visual examination of specimen color is extremely subjective. Advancements in central laboratory analyzers typically support automated spectrophotometric detection of hemoglobin, commonly referred to as the H-index, to consistently detect hemolysis and assist laboratories in the determination of whether specimens are acceptable for analysis. Unfortunately, the H-index cannot be applied for detection of hemolysis in whole-blood specimens which is the common specimen type used for POC cTn testing. Central laboratories must implement acceptance/rejection criteria for hemolyzed specimens to assure reliable cTn measurement and educate care givers regarding the importance of collecting high-quality blood specimens as well as cautious interpretation if whole-blood specimens are utilized and the cTn test system cannot detect hemolysis.

9.2.2 Haemolysis in gel test

Haemolysis in gel test (HIGT), as a serological assay for the detection of B. abortus, was developed by Ruckerbauer et al. (1984). The test sera were added to the wells in agarose gel in veronal buffer containing guinea pig complement and J-antigen negative bovine red blood cells (5% suspension in PBS, pH 7.20). These RBCs were sensitized in alkali treated smooth lipopolysaccharide from B. abortus biotype 1 (strain 413) at a concentration of 250 μM of suspension. After the incubation (primary incubation: 4 °C for 18 h; secondary incubation: 37 °C for 2 h), the zones of haemolysis were measured and compared with the quality control. Usually, a minimum seropositive threshold would exhibit a zone of haemolysis of 6 mm.

Experiments carried out while comparing the HIGT with CFT and STAT revealed that the former was more sensitive in detecting the infection at an earlier period than the other two tests in B. abortus biotype 1 infection; moreover, the assay was less sensitive for biotype 4 infection (Ruckerbauer et al., 1984). On comparing HIGT with radioimmunoassay (RIA), Nielsen, Heck, Stiller, and Rosenbaum (1983) reported that HIGT was equally able to detect antibodies against brucellosis using a sensitive test such as RIA. Further, killed B. abortus strain 1119-3 treated with hot phenol/water was used to extract the soluble fraction of crude lipopolysaccharide antigen in order to perform HIGT assay. This modified version of HIGT was reported to have comparable results with the earlier method (Dillender, Buening, & McLaughlin, 1984). Although HIGT has reported a very high specificity when tested in non-vaccinated cattle, a low specificity was observed among vaccinated cattle (Dohoo et al., 1986).

Factors Influencing Hemolysis

Hemolysis may be influenced by (a) the basic medium employed, (b) the kind of blood used, (c) the length of the incubation period, (d) the atmosphere (anaerobiosis may result in a reduction of hemolysis), (e) location of the colonies on the plates, (f) concentration of blood, and (g) depth of the agar. Some difference may be noted between surface and subsurface colonies.

Sheep blood in trypticase soy agar is recommended for a careful study of hemolysis but, for practical purposes, media containing other kinds of blood are satisfactory.

Other causes of intravascular hemolysis

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Microangiopathic hemolysis can occur following vessel thrombosis and is a potential sequel to chronic DIC.

Liver failure can result in hemolysis with the proposed pathogenesis being changes in RBC lipoprotein as a result of increased bile acids.

Snake bite envenomation can result in hemolysis in addition to altered coagulation.

Bacterial exotoxins may result in hemolysis and are most commonly seen in septicemic neonates.

Leptospira spp. infections although rare may also result in hemolysis.

Heavy metal intoxication.

Intravenous administration of hypotonic fluids, poorly diluted DMSO or excessively rapid administration of appropriately diluted DMSO can all result in intravascular hemolysis.

1 Introduction

Hemolysis, or the abnormal breakdown of circulating red blood cells (RBCs), is a major clinical problem that can be a disease process unto itself, or manifest as a component of another disorder. When RBCs are pathologically destroyed, patients typically present with symptoms of anemia such as chest pain, shortness of breath, and fatigue. While these findings are relatively easy for clinicians to recognize and link to decreased circulating RBC mass, the establishment of pathologic hemolysis (and subsequently a hemolytic disorder) is almost entirely dependent on assays performed in the hospital laboratory. Furthermore, the analyses performed are not limited to one area of the lab, but spread across multiple divisions including Hematology, Urinalysis, Blood Bank/Transfusion Medicine, Clinical Chemistry, and Immunology.

Because of the critical role that the lab plays in assessment of hemolytic disorders, and because there are a very wide variety of assays used across the spectrum of clinical pathology, the evaluation for hemolysis can be a complex task. Therefore, the aim of this chapter is to provide a thorough and up-to-date review on the laboratory investigation of pathologic hemolysis and hemolytic disorders. We will first introduce basic concepts on the pathophysiology of hemolysis, which directly inform testing and test interpretation. Subsequently, we will examine assays available for hemolysis evaluation on a laboratory-by-laboratory basis, evaluating the strengths, limitations, and potential future directions for each of these assays.

6 HELLP: Diagnosis and Clinical Characteristics

HELLP has been reported to affect 0.17–0.85% of all live births, and occurs most commonly in a slightly older population than preeclampsia, with a mean age of 25 years.108 In 70% of cases, HELLP is diagnosed antepartum, primarily between 27 and 37 weeks of gestation; 30% of cases develop postpartum.113 The percentage of nulliparous patients ranges from 52% to 81%, a lower proportion than those with preeclampsia.108,114 HELLP syndrome, particularly when it develops early in pregnancy,115,116 may be associated with antiphospholipid antibodies in some patients.117,118 Patients with HELLP characteristically present with nonspecific complaints, including nausea, fatigue, and malaise. Right upper quadrant and epigastric pain is the most common symptom, occurring in 86–92% of patients; in occasional patients right upper quadrant pain may precede the onset of liver enzyme abnormalities.108,114 Thus, patients with HELLP may be mistakenly diagnosed with a primary gastrointestinal disorder, particularly since only 50–70% of these patients have hypertension at the time of presentation; the differential diagnosis in a patient with such manifestations is wide (Table 44-2). Patients may have accompanying edema, and 5–15% have no or minimal proteinuria; 15% may have neither hypertension nor proteinuria.106,108 Patients who present with HELLP postpartum usually do so within several days after delivery,119 with 6% of these individuals having no antepartum signs of preeclampsia.108

Table 44-2. Differential Diagnosis of the HELLP Syndrome

The classic liver lesion in HELLP syndrome consists of periportal or focal parenchymal necrosis, with periportal hemorrhage and fibrin deposition in the liver sinusoids.120,121 Necrosis may arise from liver ischemia and may contribute to the characteristic right upper quadrant pain.122 Dissection of necrosis into the liver capsule may lead to the development of subcapsular hemorrhage and hepatic rupture.119

Single Radial Hemolysis

For SRH tests, sheep erythrocytes, which have previously been incubated with influenza virus, are mixed with guinea pig complement and incorporated in agarose gels (Fig. 13-6). Heat-inactivated serum samples are then added to wells cut into the gel, and the antibody titer is determined based on the zone of hemolysis induced by diffusion of the antibody-positive sample from the well.138,204,205 An increase of 50% or 25 mm2 is considered evidence of recent infection.202 Although more labor intensive than HI assays, SRH tests have been shown to be more reproducible than HI tests.202 Since it was found that the level of antibody measured by SRH after vaccination correlates well with the level of protection, SRH may also be used to predict the level of antibody-mediated immunity and determine the need for revaccination.72,73,165

10.3.4 Hemolysis Profiling

Hemolysis is a dangerous condition where blood cells burst in circulation, which can ultimately promote jaundice and anemia. Many natural and synthetic nanoparticles have elicited hemolytic action and hence, preclinical investigation of hemolytic activity is necessary for newly developed nanomedicine with anticipated delivery modes having blood contact. For example, mesoporous silica nanoparticles are known to cause hemolysis of red blood cells (RBCs) (Paula et al., 2012). The nanosize and unique physicochemical properties can lead to interactions of nanoparticles with RBCs. Therefore, the standard of pharmacological screening needs to be enhanced with respect to nanoparticles (Neun and Dobrovolskaia, 2011).

To perform a hemolysis assay, nanoparticles are incubated in the blood. If hemolysis occurs due to the properties of the nanoparticles, hemoglobin is released from damaged cells, and by using reagents this released hemoglobin is converted to red-colored cyanmethemoglobin. The undamaged RBCs and nanoparticles are separated by centrifugation and the concentration of cyanmethemoglobin within the supernatant is measured using spectrometric analysis. From the cyanmethemoglobin concentration, hemoglobin concentration can be determined. These results are compared with a negative control group to determine percentage hemolysis due to the nanoparticles (Fig. 10.5).