Which of the following is least likely to cause hemolysis in a collected blood sample?

Anemia Due to Hemolysis

Hemolysis indicates a reduction in the survival of red cells in the circulation that is not due to bleeding. In this sense, some reduction in red cell survival is an element in many hematologic syndromes. Decreased red cell survival is a component of the anemia observed in renal disease, the anemia of inflammation, and the anemia of liver disease (distinct from spur cell anemia). Microangiopathic hemolytic morphology is occasionally seen in disseminated intravascular coagulation. However, these disorders are not considered “hemolytic anemias” in the same sense as the disorders discussed in this section. The disorders discussed here are those in which the primary etiology of anemia is hemolysis, with further details about the specific disorders of hemolysis inChapters 151,152,153, and154.

Table 149-7 outlines laboratory studies that raise suspicion of a hemolytic anemia and those that confirm it. These are not specific to any etiology of hemolytic anemia. The distinction between extravascular hemolysis (which includes hemolysis in the spleen) and intravascular hemolysis is helpful in understanding these disorders at the pathophysiologic level. However, it is not absolute in terms of diagnostic testing. While the haptoglobin tends to become lower in intravascular hemolysis, it is generally decreased in extravascular hemolysis also. Hemoglobinemia and hemoglobinuria are specific for intravascular hemolysis.

Table 149-8 lists causes of hemolysis, using a popular classification that separates etiologies that are (1) entirely external to the red cell (e.g., immunologic, mechanical, or toxic processes); (2) defects that are intrinsic to the red cell membrane (e.g., hereditary spherocytosis or spur cell anemia), and (3) processes primarily related to red cell metabolism or to abnormalities of hemoglobin. The enzymatic defects producing hemolysis are typically related to oxidative metabolism and particularly to oxidative stress such as that induced by medications. Specific syndromes of hemolytic anemias are discussed inChapters 151 through 154Chapter 151Chapter 152Chapter 153Chapter 154. The goal of this section is to outline a general approach to hemolytic anemias that can then be expanded upon in the chapters devoted to specific syndromes.

Figure 149-2 outlines an approach to the diagnosis of the various classes of hemolytic anemia syndromes. In hemolytic anemia, the examination of the peripheral smear is extremely important and likely to yield important information. Identification of the characteristic microangiopathic changes of thrombotic thrombocytopenic purpura immediately provides direction to life-saving therapy. Similarly, congenital defects in the intrinsic proteins of the red cell membrane produce specific morphologic features, which can lead rapidly to a confirmable diagnosis. If the morphology on the peripheral blood smear is normal or shows only a small number of spherocytes, then evaluation for an immunologic etiology of hemolysis needs to be pursued. In general, autoimmune hemolytic anemia is associated with an abnormal direct antiglobulin (Coombs) test. In individuals with a previous transfusion history or a history of pregnancy in which fetal-maternal transfusion may have occurred, the possibility of other immune hemolytic mechanisms arises, and an antibody screen (also called the indirect antiglobulin test) should be ordered. If negative, metabolic or toxic causes of hemolysis should be considered.

HELLP Syndrome

HELLP syndrome defines a preeclampsia variant defined by Hemolysis, Elevated Liver transaminases, and Low platelets. Thrombocytopenia occurring in HELLP syndrome is generally more severe than with uncomplicated preeclampsia. It has been reported that the rate of fall of the platelet count is a predictor of the eventual severity of HELLP, with women whose counts decrease by > 50,000/μL per day having a higher probability of developing moderate to severe thrombocytopenia (an absolute platelet count <100,000/μL).34 There also appears to be a correlation between the extent of thrombocytopenia and the degree of liver dysfunction in women with HELLP syndrome. The platelet nadir is usually reached approximately 24 h postpartum, with normalization occurring over the next 6–11 days.34–36 As with preeclampsia, the thrombocytopenia of the HELLP syndrome reflects a multifactorial pathogenesis with platelet activation by contact with damaged endothelium, platelet consumption secondary to thrombin generation, and microangiopathic hemolysis.

Fragmentation Hemolysis

SeeTable 484.2 inChapter 484.

Red blood cell (RBC) destruction may occur in hemolytic anemias because ofmechanical injury as the cells traverse a damaged vascular bed. Damage may be microvascular when RBCs are sheared by fibrin in the capillaries during intravascular coagulation or when renovascular disease accompanies thehemolytic-uremic syndrome (HUS) (seeChapter 538.5) orthrombotic thrombocytopenic purpura (TTP) (seeChapter 511.5). Larger vessels may be involved inKasabach-Merritt syndrome (giant hemangioma and thrombocytopenia; seeChapter 532) or when a replacement heart valve is poorly epithelialized. The blood film shows manyschistocytes, or fragmented cells, as well aspolychromatophilia, reflecting the reticulocytosis (seeFig. 485.4F inChapter 485). Secondary iron deficiency may complicate the intravascular hemolysis because of urinary hemoglobin and hemosiderin iron loss (seeFig. 484.2 inChapter 484).Treatment should be directed toward the underlying condition, and the prognosis depends on the effectiveness of this treatment. The benefit of transfusion may be transient because the transfused cells are destroyed as quickly as those produced by the patient.

It is critical to determine the precise etiology of the fragmentation hemolysis because the treatment depends on the underlying problem (Table 492.1).Acquired TTP results from an antibody to an enzyme (ADAMTS13) that regulates the size of von Willebrand multimers. The lack of this enzyme results in a marked increase in multimer size and a resultantthrombotic microangiopathy.Congenital TTP can result in the inability to produce adequate amounts of the enzyme ADAMTS13 and results in the same pathophysiology. The treatment for acquired TTP involves plasmapheresis to remove the antibody and replace the ADAMTS13. The treatment for congenital TTP involves scheduled plasma infusions to replace the ADAMTS13. In contrast, HUS results fromShiga toxin produced byEscherichia coli 0157 and may not be helped by plasmapheresis.Atypical HUS involves activation of the alternative complement pathway and can be treated witheculizumab (anti-C5), an inhibitor of the complement pathway. Plasmapheresis may reduce the RBC fragmentation and improve the platelet count but has little effect on the tissue (kidney) vasculopathy and thus is not usually recommended.Pneumococcal-induced HUS results from neuraminidase produced by the bacteria, which damages the membranes of the RBCs and the kidney, exposing theT-antigen. Plasma contains natural antibody to the T-antigen, producing hemolysis, renal damage, and a thrombotic microangiopathy. Thus, patients with T-antigen activation from suspected pneumococcal-induced HUS should not be given plasma infusions, because this will significantly exacerbate the RBC hemolysis and can lead to life-threatening anemia.

Hemolysis

Hemolysis is characterized by a compensatory reticulocytosis. The unconjugated bilirubin is elevated, as is the lactate dehydrogenase (LDH), reflecting RBC breakdown. Decreased levels of haptoglobin, a hemoglobin-binding protein that is rapidly cleared from the circulation, is a more specific indicator of RBC breakdown. There are several classification schemes of hemolysis: immune versus nonimmune, hereditary versus acquired, but perhaps the most practical categorization of hemolysis is the site of destruction: intravascular versus extravascular. Intravascular hemolysis is characterized by breakdown of RBCs in the peripheral circulation: shistocytes and fragmented cells are visible on the peripheral blood smear (Fig. 46.10). There is evidence of free hemoglobin in the serum and free hemoglobin or hemosiderin in the urine (i.e. the ‘blackwater fever’ of Plasmodium falciparum malaria or acute hemolysis in G6PD deficiency). Extravascular hemolysis is characterized by removal of circulating RBCs in the spleen. Microspherocytes are evident on the peripheral smear. RBC membrane defects and sickle cell anemia result in extravascular hemolysis and a portion of the anemia of thalassemia major (although best characterized as ineffective erythropoiesis) is due to extravascular hemolysis.

G6PD Deficiency and Hemolysis.

Most G6PD-deficient individuals lead perfectly normal lives and will, for the most part, be unaware of their inherited condition. However, G6PD deficiency may be associated with severe hemolytic episodes with resultant jaundice and anemia, following exposure to a hemolytic trigger. Classically, these episodes often occur after ingestion of or contact with the fava bean (favism). Medications and chemical substances may be suspected, but sometimes no offending trigger is identified. Infection may play a role in the pathogenesis of acute hemolysis.

In neonates, extreme hemolytic hyperbilirubinemia may develop suddenly and without previous warning. Some identifiable substances associated with neonatal hemolysis include naphthalene (used to store clothes), herbal medicines, henna applications, or menthol-containing umbilical potions. Frequently, the trigger cannot be recognized and the hemoglobin concentration may not drop, leading to the erroneous diagnosis that hemolysis is not occurring. There can, however, be no other viable explanation for the exponential increase in TB to dangerous levels. G6PD deficiency may, therefore, be the one reason that kernicterus may not be completely preventable. Exchange transfusion may be the only recourse. Early hospital discharge with delayed follow-up may place these patients at risk for severe sequelae.

In a Nigerian neonatal cohort, G6PD-deficient and -intermediate (presumable heterozygotes) had higher TB, lower hematocrit values, and a greater need for phototherapy during the first postnatal week than G6PD-normal counterparts, suggestive of increased hemolysis.20 Frequently, hematologic indices typical of hemolysis in older children and adults, including falling hemoglobin and hematocrit values and increasing reticulocyte counts, may be absent despite a clinical picture of hemolysis. However, studies of endogenous CO formation, reflective of the rate of heme catabolism, have demonstrated an important role of increased hemolysis in association with this condition. Significantly higher levels of COHb have been reported in Nigerian G6PD-deficient neonates who developed kernicterus compared with neonates who were hyperbilirubinemic but did not develop signs of kernicterus.

More frequently and less life threatening, G6PD-deficient neonates may have a moderate form of jaundice, which occurs at a rate several-fold that of controls. The jaundice usually responds to phototherapy, although exchange transfusion may also be necessary. These infants have a low-grade hemolysis that cannot be implicated as the primary icterogenic factor.135 Diminished bilirubin conjugation has been shown to be of major importance in the pathogenesis of the hyperbilirubinemia. An intriguing interaction has been noted between G6PD deficiency and a noncoding area (TA)7 promoter polymorphism in the gene encoding UGT1A1.138 This polymorphism, also known as UGT1A1*28, is associated with Gilbert syndrome. The incidence of a TB of at least 15 mg/dL (257 µmol/L) increased in a stepwise, dose-dependent fashion in G6PD-deficient neonates who were heterozygous or homozygous, respectively, for the polymorphism. This effect was not seen in the G6PD-normal control group. Furthermore, G6PD deficiency alone, in the absence of the promoter polymorphism, did not increase the incidence of hyperbilirubinemia over and above that of G6PD-normal counterparts. In Asians, in whom the (TA)7 promoter polymorphism is rare, a similar interaction was noticed between G6PD deficiency and coding area mutations of theUGT1A1 gene.115 In a recent study of African-American neonates, although 20.6% had variations in bothUGT1A1 andSLCO1B1 genes, these genetic variants did not have an effect on the incidence of hyperbilirubinemia.238 This may be because very few neonates in that cohort were G6PD-deficient, confirming the concept of a gene interaction necessary to influence the incidence of hyperbilirubinemia.

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.

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).

Hemolysis

Hemolysis will elevate the concentration of any constituent of erythrocytes and may slightly dilute constituents present in low levels in erythrocytes. It becomes significant when the serum concentration of hemoglobin surpasses 20 mg/dL. Typically, hemolysis elevates aldolase, acid phosphatase, isocitrate dehydrogenase, lactate dehydrogenase, potassium, magnesium, ALT, hemoglobin, and phosphate [49,52]. AST is elevated slightly [52]. Colorimetric assays are also affected by the increased absorbance of light by Hgb in the 500- to 600-nm range [49]. The effects of hemolysis are further discussed in Chapter 5.

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).

14.4.1 The hemolysis

Hemolysis can be evaluated via exposure of stent materials under in vitro, in vivo, and ex vivo conditions. In vitro conditions are used to evaluate materials as well as stents. Hemolysis experiments were conducted in accordance with ISO 10993-4:2009 and ASTMF756-00 [1,14].

In vivo and ex vivo assessments in animal models or during clinical trials are possible. Justification can be made for either of the following study designs. The test stent is compared to reference stent materials with known acceptable levels of hemolysis; furthermore the significant consequences was evaluated according to clinical application.