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Brief introduction on red blood cell biology

Although a comprehensive description of red blood cell (RBC) biology is beyond the scope of this review, a brief introduction is useful to assist readers in understand-ing the material presented. RBCs (erythrocytes) are the most common type of blood cells and represent the

leading oxygen delivering mechanism to the peripheral tissues. In the adult life RBCs originate from the bone marrow and display a physiological life span of 90–120 days before undergoing degradation by phagocytosis in the reticuloendothelial system of the spleen, liver, and bone marrow1,2. Erythropoiesis (the production of RBCs)

Review aRticle

Hemolyzed specimens: a major challenge for emergency departments and clinical laboratories

Giuseppe Lippi1, Mario Plebani2, Salvatore Di Somma3, and Gianfranco Cervellin4

1U.O. Diagnostica Ematochimica, Azienda Ospedaliero-Universitaria di Parma, Parma, Italy, 2U.O. Pronto Soccorso e Medicina d’Urgenza, Azienda Ospedaliero-Universitaria di Parma, Parma, Italy, 3Dipartimento di Medicina d’Emergenza, Ospedale Sant’Andrea, Università “La Sapienza”, Roma, Italy, and 4Dipartimento di Medicina di Laboratorio, Università degli Studi di Padova, Padova, Italy.

abstractThe term hemolysis designates the pathological process of breakdown of red blood cells in blood, which is typically accompanied by varying degrees of red tinge in serum or plasma once the whole blood specimen has been centrifuged. Hemolyzed specimens are a rather frequent occurrence in laboratory practice, and the rate of hemolysis is remarkably higher in specimens obtained in the Emergency Department (ED) as compared with other wards or outpatient phlebotomy services. Although hemolyzed specimens may reflect the presence of hemolytic anemia, in most cases they are due to preanalytical sources related to incorrect procedures or failure to follow procedures for collection, handling and storage of the samples; some of these are typical of the ED. Since hemolyzed specimens are often an important cause of relationship, economic, organizational and clinical problems between the ED and the clinical laboratory, it is essential to develop effective processes for systematically identifying unsuitable specimens (e.g. by using the hemolysis index), differentiating in vitro from in vivo hemolysis, troubleshooting the potential causes, and maintaining good relations between the clinical laboratory and the ED.

Keywords: hemolysis; hemolyzed specimens; laboratory testing; hemolytic anemia

abbreviations: AIHA, autoimmune hemolytic anemia; CAS, cold agglutinin syndrome; CLL, chronic lymphocytic leukemia; DAT, direct antiglobulin test; DIC, disseminated intravascular coagulation; ED, emergency department; Epo, erythropoietin; G6PD, glucose-6-phosphate dehydrogenase; GPSC, Global Preanalytical Scientific Committee; HbS, hemoglobin S; HS, hereditary spherocytosis; HUS, hemolytic uremic syndrome; ICU, intensive care unit; IFCC, International Federation of Clinical Chemistry and Laboratory Medicine; IV, intravenous; LDH, lactate dehydrogenase; MAHA, microangiopathic hemolytic anemia; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; MCV, mean corpuscular volume; NADPH, nicotinamide adenine dinucleotide phosphate; PCH, paroxysmal cold hemoglobinuria; PNH, paroxysmal nocturnal hemoglobinuria; RBC, red blood cell; RCV, reference change value, difference between two consecutive measurements of one analyte in a person and representing a significant change in health status; SCA, sickle cell anemia; SCD, sickle cell disease; SLE, systemic lupus erythematosus; TTP, thrombotic thrombocytopenic purpura; VWF, von Willebrand factor; WHO, World Health Organization

Address for Correspondence: Prof. Giuseppe Lippi, U.O. Diagnostica Ematochimica, Azienda Ospedaliero-Universitaria di Parma, Via Gramsci, 14, 43100 - Parma, Italy. E-mail: glippi@ao.pr.itReferee: Prof. Dr. Joris Delanghe, Laboratory Clinical Chemistry, Ghent University Hospital, Ghent, Belgium

(Received 09 June 2011; revised 17 June 2011; accepted 17 June 2011)

Critical Reviews in Clinical Laboratory Sciences, 2011; 48(3): 143–153© 2011 Informa Healthcare USA, Inc.ISSN 1040-8363 print/ISSN 1549-781X onlineDOI: 10.3109/10408363.2011.600228

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lasts ∼1 week and is regulated by a two-step process by the hormone erythropoietin (Epo), which can stimu-late production and maturation of the cells from their precursors (stem cells), as well as prevent apoptosis of immature RBCs (reticulocytes) through a process known as neocytolysis (in normal condition, reticulocytes rep-resent ∼1% of circulating RBCs). Human RBCs have a diameter of 6–8 µm and a thickness of 2 µm, with a final volume of nearly 90–95 fL. The unique biconcave shape of the RBC optimizes the rheological properties of blood in the large vessels (i.e. amplification of laminar flow and minimization of platelet scatter). The mean number of RBCs in adult life is typically 4.2–6.2 × 1012/L in males and 3.8–5.5 × 1012/L in females1,2.

Definition of hemolysis

The term hemolysis (or haemolysis) designates the pathological process of breakdown of RBCs in blood, which is typically accompanied by various (visible) degrees of red tinge in serum or plasma once the whole blood specimen has been centrifuged. The term hemo-lysis per se does not distinguish between clinical or artifactual causes. The former case, which is essentially triggered by a variety of medical conditions, is known as in vivo hemolysis or hemolytic anemia, whereas the lat-ter, which is due to a variety of extra-clinical problems arising from collection to analysis of the specimen, is usually referred to as in vitro or spurious hemolysis3. Regardless of the cause, in the clinical laboratory prac-tice hemolyzed specimens may be classified accord-ing to the concentration of free hemoglobin in serum or plasma, as shown in Table 1. Hemolysis is typically detected by scrutiny when the concentration of free hemoglobin in centrifuged specimens (in serum or plasma) exceeds 0.3 g/L, which reflects the breakdown of ∼ 0.2% of all RBCs in a sample with a mean hemoglo-bin concentration of 140 g/L3.

epidemiology of hemolyzed specimens

Although the chance of analytical errors has substan-tially decreased over the past decades due to advances in technology and computer science, uncertainty and lack of standardization of several manually-intensive activities of the preanalytical phase still carry a signifi-cant risk of error. Several studies and critical reviews of the literature clearly attest that preanalytical activities

are the leading source of mistakes in laboratory testing, representing up to 70% of all errors arising through-out the total testing process. Among these errors, in vitro hemolysis is reportedly the most prevalent for both inpatient and outpatient samples, as well as for both stat and routine testing. Although the overall prevalence of hemolyzed specimens received in clinical laboratories varies widely according to the geographical area, the type of facility and the clinical setting, it can be estimated at ∼2–3% of all samples received3. A recent international survey carried out under the auspices of the Working Group on Laboratory Errors and Patient Safety (WG-LEPS) of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC)4 and the Global Preanalytical Scientific Committee (GPSC)5 involving 391 clinical laboratories worldwide has reported that the percentage of hemolyzed speci-men ranges between 1 to 5% in the vast majority (over 60%) of clinical laboratories, and of these, the largest percentage comes from the ED (up to 53%), followed by pediatric departments (16%) and intensive care units (ICU) (7%)6. The frequency of hemolyzed specimens referred from the ED is also remarkably higher than that reported for other inpatients departments, and ranges consistently between 3% and 12.4%7–10. It may not be surprising that the likelihood of obtaining unsuitable samples increases substantially in those settings where blood collection responsibility is outside the direct control of the laboratory staff, where there might be less compliance to protocols for specimen collection and handling. Among hemolyzed specimens, most (up to 95%) are characterized by a slight degree of hemolysis (free hemoglobin <0.3 mg/L), whereas only 5% exceeds the clinically significant threshold (free hemoglobin ≥0.3 mg/L)7.

In vivo causes of hemolysis

Numerous medical conditions lead to in vivo hemoly-sis, and it is therefore crucial for the emergency physi-cian to distinguish between in vivo and in vitro causes of a hemolyzed blood sample as well as to identify timely and accurately the underlying cause of hemolytic anemia. In vivo hemolysis is defined as the premature destruction of RBCs within the circulation, which can lead to hemolytic anemia when bone marrow activ-ity is unable to compensate for the amount of RBC breakdown11. The clinical significance of the hemolytic process depends mostly on whether the onset of hemo-lysis is progressive or abrupt, as well as on the severity (degree) of RBC destruction. Patients with mild hemo-lysis may be asymptomatic, whereas anemia can be life-threatening in severe cases and can be accompanied by dyspnoea, angina and cardiopulmonary decompensa-tion. Hemolytic anemias account for approximately 5% of all anemias. The clinical presentation may reflect the underlying cause, which represents the final converging pathway of a kaleidoscope of hereditary and acquired

Table 1. Laboratory classification of hemolyzed specimens.

ClassificationFree hemoglobin in

serum or plasmaPredictable tinge of the specimen

Non-hemolyzed ≤0.05 g/L YellowSlightly hemolyzed ≥0.05–0.3 g/L Yellow to slightly pinkMildly hemolyzed ≥0.3–0.6 g/L Pink to slightly redModerately hemolyzed

≥0.6–2.0 g/L Slightly red

Grossly hemolyzed ≥2.0 g/L Red to brown

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disorders. The etiology of premature RBC destruction is varied (more than 200 causes have been described so far) and includes intrinsic erythrocyte membrane defects, abnormal hemoglobins, erythrocyte enzymatic defects, immune destruction of erythrocytes, toxic or immune effects exerted by drugs, mechanical injury, and hypersplenism (Table 2). In cases of mild hemo-lysis, the patient may present with normal hemoglobin values, since increased RBC production would com-pensate for the rate of destruction. Patients with mild hemolysis may occasionally experience marked ane-mia when the RBC production in the bone marrow is transiently suppressed by viral (e.g. parvovirus B19) or other infections, resulting in uncompensated hemolysis (aplastic hemolytic crisis)3,11.

Depending on the site, in vivo hemolysis can be divided between intravascular and extravascular. The former condition occurs in association with pathologies such as prosthetic cardiac valves, hereditary RBC disor-ders (e.g. glucose-6-phosphate dehydrogenase [G6PD] deficiency, hereditary spherocytosis, sickle cell anemia [SCA], thrombotic thrombocytopenic purpura [TTP], disseminated intravascular coagulation [DIC], and par-oxysmal nocturnal hemoglobinuria [PNH])12.

Extravascular hemolysisExtravascular hemolysis, characterized by RBC break-down occurring in the spleen and other reticuloendothe-lial organs, is typical of autoimmune hemolytic anemia (AIHA) and hereditary spherocytosis. Nevertheless, both intra- and extravascular- hemolysis can occasion-ally occur concomitantly, such as in the case of SCA. Acquired hemolytic conditions can be due to immune disorders, toxic chemicals and drugs, antiviral agents (e.g. ribavirin)13, physical damage of the erythrocytes and infections.

AIHAThe AIHA have traditionally been classified into three categories: autoimmune, alloimmune, and drug-related. AIHA may result from warm or cold autoantibody types, whereas mixed types rarely occur11,14–16. Patients with AIHA typically develop autoantibodies against RBCs. Alloimmune-mediated hemolytic anemia is character-ized by specific autoantibodies triggered by and only reactive against allogeneic RBCs and their antigens, so that the patient’s RBCs are not damaged. Warm-type autoantibody-mediated hemolysis is predominantly extravascular and occurs in the spleen. Warm-type AIHA display a 2:1 female to male ratio, and has no racial predilection. Nearly half of warm-type AIHA cases can be labeled as primary or idiopathic, while secondary cases are most frequently associated with lymphoproliferative disorders (in about 50% of cases) or systemic autoimmune diseases (e.g. systemic lupus erythematosus, SLE). Chronic lymphocytic leukemia (CLL), the most common lymphoproliferative disorder associated with warm-type AIHA, accounts for up to 25% of cases17,18. AIHA may also occur after allogeneic hematopoietic stem cell transplantation, with a 3-year cumulative incidence of 4.4%19. AIHA is typically clas-sified into two types: an IgG or “warm” type (optimally active at 37°C) and an IgM or “cold” type (optimally active at 4°C). The vast majority of warm autoantibodies are IgG and can be identified with the direct Coombs test (also known as the direct antiglobulin test [DAT]), whereas in cold-type AIHA, the autoantibodies are typically IgM and most strongly hemolytic at tempera-tures between 0°C and 4°C. The presence of cold-type autoantibodies triggers clumping or agglutination of RBCs, which may be observed on the peripheral blood smear. This IgM-mediated disease is associated with complement fixation on the RBC surface and conse-quent activation of the complement cascade. As such, hemolysis can occur in both the extravascular and the intravascular spaces after massive autoantibody pro-duction and complement activation. The Kupffer cells of the liver, rather than splenic macrophages, are mostly responsible for the extravascular RBC destruction20. The two most common cold-type AIHA disorders are cold agglutinin syndrome (CAS) and paroxysmal cold hemo-globinuria (PCH). Fifty percent of secondary cold-type

Table 2. Leading causes of in vivo hemolysis.Inherited hemolytic anemias1. Defects in hemoglobin production a. Thalassemias b. Sickle cell disease2. Defects of red blood cell membrane production a. Hereditary spherocytosis b. Hereditary elliptocytosis c. Paroxysmal nocturnal hemoglobinuria (PNH)3. Defective red cell metabolism4. Glucose-6-phosphate dehydrogenase deficiency5. Pyruvate kinase deficiencyAcquired hemolytic anemias1. Immune-mediated causes a. Mycoplasma pneumoniae infection (cold agglutinin disease) b. Autoimmune hemolytic anemia (AIHA) c. Autoimmune diseases (systemic lupus erythematosus and

chronic lymphocytic leukemia)2. Hypersplenism3. Burns4. Infections a. Malaria b. Babesiosis c. Clostridium5. Mechanical damage in the circulation a. Disseminated intravascular coagulation (DIC) b. Hemolytic uremic syndrome (HUS) c. Thrombotic thrombocytopenic purpura (TTP) d. Prosthetic cardiac valves e. HELLP (hemolysis, elevated liver enzymes and low platelets)

syndrome6. Transfusion of blood from a donor with a different blood type7. Drugs, toxins and other miscellaneous causes

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AIHA cases are associated with lymphoproliferative disorders, while infections represent the second leading cause. CAS accounts for up to one-third of all AIHA cases and is typically mediated by IgM autoantibodies that are directed against the I/i blood group antigens21. PCH is determined by a biphasic IgG autoantibody (the Donath-Landsteiner antibody) that is directed against the P antigen system on RBCs. This potent autoantibody fixes complement after binding to RBCs not only at lower temperatures, but also at normal physiologic tempera-tures, causing clinically significant hemolysis even in the presence of low autoantibody titers. The leading symp-toms including high fever, chills, headache, abdominal cramps, nausea and vomiting, diarrhea, leg and back pain, cold urticaria and Raynaud phenomenon, which typically develops with cold exposure. Several drugs can also trigger AIHA, especially penicillin and related antibiotics such as ampicillin and methicillin, some cephalosporins, and antimalarial drugs22–24.

Intravascular hemolysisTTP and HUSMicroangiopathic anemia, due to a significant degree of intravascular hemolysis, is observed in patients with DIC, hemolytic uremic syndrome (HUS) and TTP25. Both TTP and HUS involve platelet aggregation in the microvascu-lar circulation that is mediated by von Willebrand factor (VWF); this causes various degree of thrombocytopenia, and is more frequently associated with a platelet count <20,000/L. Microangiopathic hemolytic anemia (MAHA) or schistocyte-forming hemolysis develops when RBCs are fragmented as they pass through occluded arterioles and capillaries. TTP and HUS are each characterized by specific clinical symptoms, but the differential diagno-sis can be challenging since clinical overlap may occur. TTP is typically more frequent in adults, whereas HUS is more common in children. The former condition is associated with more evident neurologic deficits and deposition of platelet aggregates in a broader systemic distribution, whereas HUS affects more specifically the kidney26. Fragmented erythrocytes (schistocytes) and some degree of hemolysis also occur in patients with defective prosthetic cardiac valves27.

Sickle cell diseaseAmong the congenital disorders of RBCs, sickle cell dis-ease (SCD) is one of the most frequent diseases leading to hemolytic anemia. According to recent statistics of the World Health Organization (WHO), its estimated prevalence varies from 0.07% in Europe up to 10.7% in Africa28. SCD, also known as hemoglobin S (HbS) disease, is caused by the substitution of the amino acid valine for glutamine at position 6 in the hemoglo-bin β-chain. Due to this mutation, deoxygenated HbS polymerizes, so that RBCs are deformed and assume the characteristic sickled appearance. This abnormal morphology is associated with premature RBC destruc-tion, but also with increased blood viscosity that finally

leads to microvasculature obstruction. The final effect is chronically ongoing hemolysis and episodic periods of vascular occlusion, resulting in tissue ischemia that affects most organs and systems29. The hemolytic pro-cess may worsen in the presence of infections and the hemoglobin level rapidly decreases, whereas the reticu-locyte count increases in parallel with RBC destruction, although it may not balance the increased hemolysis. The defect is inherited as an autosomal recessive trait, but the disease is most frequently observed in homozy-gotes (HbSS). Conversely, patients with sickle cell trait (HbAS, heterozygous) may be characterized by a nor-mal RBC life span and are usually asymptomatic, except in the presence of severe physiologic stress, when they may suffer an acute pain crisis, splenic infarction, or cerebrovascular complications.

Glucose-6-phosphate dehydrogenase deficiencyG6PD deficiency is caused by inherited defects of this enzyme, which is responsible for the production of nic-otinamide adenine dinucleotide phosphate (NADPH), an essential cofactor for maintaining glutathione in its reduced state. Since G6PD activity is the only source of NADPH that prevents oxidative damage to intra-erythrocytic hemoglobin, the severity of G6PD disease mainly depends on the extent of enzyme deficiency. Patients severely affected display <10% of normal enzyme activity, while those moderately affected have 10–60% of normal activity30. G6PD-deficient RBCs are highly susceptible to oxidative stress and the resulting oxidization of the hemoglobin sulfhydryl groups causes its precipitation within the cell. Oxidative damage can also occur at the RBC membrane, producing both extravascular and intravascular hemolysis. Some drugs (e.g. acetanilide, furazolidone, nitrofurantoine, isobu-tyl nitrite, nalidixic acid, ciprofloxacin, norfloxacin, chloramphenicol, vitamin K analogues, sulfonamides, and antimalarials), naphthalene, and foods (e.g. fava beans) can typically trigger hemolysis in G6PD-deficient patients. The affected RBCs are then cleared from the circulation by the spleen. The precipitated hemoglobin is reflected by the presence of Heinz bod-ies on the peripheral blood smear31.

Hereditary spherocytosisHereditary spherocytosis (HS) results from an erythrocyte membrane defect and is the most prevalent hereditary hemolytic anemia among people of northern European descent. Molecular abnormalities in the cytoskeleton of the cell membrane cause an abnormal shape of RBCs, which hence undergo an increased rate of destruction associated with a compensatory increase in production from the bone marrow32.

Laboratory aspectsThe diagnosis of hemolytic anemia always requires careful interpretation of clinical signs and laboratory data. Hemolysis is always associated with a release of

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hemoglobin, potassium, and erythrocytic enzymes. An increase in indirect bilirubin and urobilinogen is due to the catabolism of free hemoglobin. In general, the diagnosis of hemolysis is corroborated by the pres-ence of spherocytes or schistocytes on the peripheral blood smear, a variable decrease in the hematocrit, an increase in the reticulocyte count (reflecting enhanced RBC production), increased unconjugated (indirect) bilirubin secondary to excessive hemoglobin catabo-lism, increased lactate dehydrogenase (LDH) (a non-specific marker of RBC destruction), and decreased haptoglobin, a serum protein that binds free hemoglo-bin and serves as a sensitive marker of intravascular hemolysis. The major role of this protein is in fact the binding of free hemoglobin within the bloodstream, to limit hemoglobin-induced oxidative damage33. The haptoglobin–hemoglobin complexes formed upon breakdown of RBCs are cleared by hepatocytes through a transmembrane receptor as well as by monocytes and macrophages through a specific high-affinity scav-enger receptor34. Since haptoglobin is an acute phase reactant, the presence of concomitant infection, other reactive states, or chronic hemolysis may confound the diagnostic reasoning by increasing the synthesis of the protein.

Although an increased reticulocyte count is com-monplace in hemolytic anemia, it is not specific, since increased RBC production is also associated with blood loss or bone marrow response to iron, vitamin B12, or folate deficiencies. Moreover, the reticulocyte count may be normal or low in patients with bone marrow sup-pression despite severe ongoing hemolysis. High mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) are also suggestive for spherocytosis. Serum LDH elevation is commonplace in hemolysis, but it lacks diagnostic specificity because the enzyme is ubiquitous and is also released in a variety of neoplastic or ischemic disorders. LDH isoenzymes 1 and 2 are more specific for RBC destruction, but they are also released from damaged myocardiocytes. Increased concentration of unconjugated bilirubin is frequently observed in patients with in vivo hemolysis, although it also characterizes Gilbert’s syndrome. In the presence of hemolytic anemia, the concentration of indirect bilirubin is usually less than 70 umol/L, so that higher values reflect the concomitant presence of impaired liver function. The urine free hemoglobin test clearly reflects hemoglobi-nuria, which occurs with intravascular hemolysis when the amount of free hemoglobin exceeds the concentra-tion of available haptoglobin in the bloodstream. The urines may be dark in the presence of hemoglobinuria, but myoglobinuria, and porphyria as well as other con-ditions are associated with dark urines. Taken together, increased LDH and decreased haptoglobin are the most sensitive general tests for diagnosing and distinguishing in vitro from in vivo hemolysis3.

When in vivo hemolysis is suspected, some addi-tional simple tests in combination with clinical history,

physical examination, peripheral blood smear, and the aforementioned laboratory investigations are necessary for the differential diagnosis of the various conditions causing in vivo hemolysis35. The complete blood count documents anemia and abnormalities in leukocyte counts, whereas the platelet count helps to rule out an underlying infection or hematologic malignancy, since its value is within the reference range in most hemolytic anemias. Thrombocytopenia can occur in SLE, CLL and microangiopathic hemolytic anemia (defective pros-thetic cardiac valves, TTP, HUS, and DIC). Peripheral smear and morphologic examination may reveal the presence of polychromasia (reflecting RBC immaturity), reticulocytosis and spherocytes (suggesting congenital spherocytosis or AIHA), and schistocytes (fragmented RBCs, suggesting TTP, HUS, or mechanical damage), but can also help diagnose a concomitant underlying hematologic malignancy associated with hemolysis (e.g. CLL). RBC indices are also useful. A low mean cor-puscular volume (MCV) and MCH are consistent with a microcytic hypochromic anemia, which may occur in chronic intravascular hemolysis (e.g. PNH). High MCV is consistent with a macrocytic anemia, usually due to megaloblastic anemias or liver disease. The direct Coombs test, or DAT, is usually positive in patients with AIHA, but it may be negative in up to 10% of cases36. A high titer of anti-I antibody may be found in myco-plasmal infections and a high titer of anti-i antibody may be observed in hemolysis associated with infec-tious mononucleosis. An anti-P cold agglutinin can be detected in paroxysmal cold hemoglobinuria. A G6PD screen is useful to diagnose a deficiency of this enzyme, but results can be normal when the reticulocyte count is elevated because immature RBCs contain a consider-able amount of G6PD. A Heinz body preparation also helps to detect G6PD deficiency. Bacterial infection-related cases of hemolysis can be ruled out by cultur-ing and a number of serologic tests, while a relatively new tool for distinguishing viral from bacterial infec-tion is the measurement of serum procalcitonin levels37.

In vitro causes of hemolysis

A variety of factors associated with collection, handling, transportation, processing, and storage of blood speci-mens have been associated with a high risk of producing hemolyzed specimens, especially in the ED (Table 3). Understandably, most of these preanalytical problems arise from incorrect procedures and/or materials for collecting blood, whereas transportation, processing and storage issues account for only a minority of cases. With respect to the causes of spurious hemolysis in the ED, Burns and Yoshikawa examined the various factors involved in the blood drawing process to identify those carrying an increased risk of hemolysis9. Analysis of the data showed that the leading factors contributing to mechanical hemolysis in blood samples collected in

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the ED were: drawing blood from a distal arm versus antecubital fossa (p = 0.0054), the use of small bore (i.e. 22 gauge) versus a larger bore (i.e. 20 gauge) plastic can-nula (p = 0.010), the collection of less than a half-full tube versus greater than or equal to a half-full tube (p = 0.016), tourniquet placing extended to >2 min versus ≤2 min (p = 0.016), and the use of a plastic versus metal cannula (p = 0.016). Interestingly, collection of blood by a syringe produced the same extent of hemolysis as that observed by using an evacuated tube system (p = 1.00). Although blood collection with a syringe and transfer of blood into the primary tube is firmly discouraged due to the high biological risk arising from needlestick injuries, the low amount of hemolysis associated with this procedure is not surprising in as much as it would prevent the well-known risk of mechanical breakdown of RBCs due to the combination of evacuated blood collection systems with

cannulas (due to excessive aspiration force of the vacuum through the cannula), which is commonplace in EDs for limiting the number of repeated venipunctures (e.g. for establishing the kinetics of cardiac biomarkers in patients with suspected acute myocardial infarction). In contrast, in another study, Ong et al. identified that the use of vacuum tubes was associated with the highest chance of producing spurious hemolysis, while additional impor-tant causes were use of IV cannulae rather than repeated venipunctures for blood sampling, reduced sample vol-ume, and prolonged interval from sampling to analysis38. Lowe et al. carried out a prospective, cross-over study of blood collection techniques in a large community teaching hospital ED, and reported that the rate of hemo-lysis was much higher in intravenous catheter samples (5.6%) versus venipuncture samples (0.3%; p < 0.05)39. Similar figures were reported by Kennedy et al. (13.7% hemolysis in samples obtained through the IV catheter versus 3.8% in those obtained with separate vacuum tube venipuncture)40, and Grant (20% hemolysis and test can-cellation in blood drawn through IV catheters versus <1% in blood samples collected by straight needle)41. In a sep-arate study aimed at identifying the factors related with hemolysis rates in blood samples drawn from IV sites in the ED, the leading causes of spurious hemolysis included device placement in the right hand/forearm and ante-cubital area, IV catheter with a caliber <22 gauge, blood drawing categorized as difficult, number of attempts for IV placement, small blood tube size and discharge diag-noses of respiratory, gastrointestinal, reproductive, der-matologic, and endocrine diseases42. Recently, a critical review was carried out to pool results of observational, descriptive, comparative, and experimental studies com-paring hemolysis between specimens collected from IV catheters and those collected from direct (straight needle) venipunctures in the ED. The rate of hemolysis varied considerably among the different studies, but occurred at a remarkably lower rate when blood samples were collected via venipuncture (0%–3.8%) as compared with those obtained via IV catheters (3.3%–77%)43. The leading factors associated with hemolysis were classified as (i) anatomic and physiological: right hand, forearm, or antecubital space, smaller distal veins, discharge diag-noses (respiratory, gastrointestinal, reproductive, der-matological, endocrine disease); (ii) equipment: plastic, smaller, and new IV catheters, partial catheter obstruc-tions, laboratory tube of larger size; and (iii) technical: difficult catheter placements, difficulty collecting blood, multiple or unsuccessful attempts to place IV catheters, partial filling of the primary vacuum tubes and exces-sive force when aspirating blood or filling tubes with a syringe.

Detection of hemolyzed specimens in clinical laboratories

Historically, hemolysis (either in vivo or in vitro) has been detected by visual inspection of the specimen after

Table 3. Potential causes of in vitro hemolysis.1. Patient-dependent a. Fragile or difficult veins b. Unsuitable vein location c. Collection from hematomas, lymphedema, and traumas2. Operator-dependent a. Lack of skill to perform venipuncture b. Poor knowledge of best practices for blood collection3. Sample collection a. Prolonged tourniquet placing (i.e. >3 min) b. Inappropriate removal of antiseptic solutions from the

phlebotomy area c. Unsatisfactory and/or repeated attempts to puncture the vein d. Use of small needles (i.e. <22 gauge) e. Blood collection with unsuitable devices (e.g. butterfly

needles catheters, cannulae, and IV devices) f. Partial obstruction of and/or coagulation of blood within the

collection system g. Traumatic collection h. Vein missed during venipuncture i. Underfilling or overfilling of tubes4. Sample handling a. Inappropriate mixing of tubes containing additives (e.g.

anticoagulants) b. Vigorous shaking of tube c. Forceful transfer of blood collected with syringes within the

primary tube5. Sample transportation a. Unsuitable transport modality (e.g. use of pneumatic tubes

and couriers) b. Unsuitable transport conditions (time, temperature,

duration, and humidity) c. Long delay before centrifugation6. Sample processing a. Unsuitable conditions of centrifugation (speed, time, and

temperature) b. Poor barrier integrity between cells and serum or plasma c. Re-spin of the specimen after centrifugation7. Sample storage a. Unsuitable storage conditions (e.g. time and temperature) b. Freezing and thawing of whole blood specimens

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centrifugation, when serum or plasma is separated from the corpuscular elements of blood (i.e. RBCs, leukocytes and platelets)3. A newer approach is the assessment of the so-called serum indices, which also includes the hemolysis index (HI). Several automated instruments are now equipped with automated HI detection systems, which involve the rapid spectrophotometric measure-ment of free hemoglobin in serum or plasma along with decision rules for the systematic handling of specimens based on tolerance tables for clinically relevant interfer-ence. The final output is a quantitative (concentration) or semi-quantitative (score) measure of free hemoglobin concentration in the sample, which can be compared with analyte-specific thresholds of interference and thereby enable the laboratory professional to identify the results that are most likely to be affected by the poor quality of the specimen3. This approach carries several advantages over visual inspection, since it is standard-ized and more reproducible, enhances the detection of slightly and mildly hemolyzed specimens, and increases the reliability of test results. It overcomes the inherent limits of visual inspection, which is arbitrary, non-stan-dardized, and subject to high inter-operator variability. Finally, the HI can also be used as a measure of preana-lytical quality (e.g. sub-optimal preanalytical conditions for the collection and handling of specimens across the healthcare facility)44,45 and becomes essential with pre-analytical workstations/laboratory automation systems where preparation (e.g. centrifugation) and aliquoting are serially connected to the analytical instrumentation, making the sample inaccessible to visual inspection.

interference of in vitro hemolysis on laboratory testing

Although a comprehensive description of the kaleido-scope of laboratory interferences due to in vitro hemo-lysis is outside the scope of this article (see review3), it is useful to outline the general issues. The most reliable definition of analytical interference is that provided by the IFCC: “analytical interference is the systematic error of measurement caused by a sample component, which does not, by itself, produce a signal in the measuring system46”. As such, free hemoglobin released from undue break-down of RBCs in blood can be considered an interefer-ent, in that it may introduce a significant bias when measuring some common analytes. In vitro hemolysis typically reflects a more generalized process of blood (i.e. white blood cells, platelets) and endothelial cell injury, whereby various constituents of these cells are released into the surrounding serum or plasma and lead to a vari-ety of interferences. There is general consensus that types of interference fall within four major areas: (i) biological, including spurious increases due to release in the blood of molecules with higher intracellular than extracellu-lar concentration; spurious decreases due to release of intraerythrocytic water and dilution of molecules with a higher extracellular than intracellular concentration; and

perturbation of physiological hemostasis due to release of thromboplastin-like material from the RBC membrane that can trigger activation of primary and secondary hemostasis; (ii) spectrophotometric, which is due to increased optical absorbance or change in blank value for laboratory techniques encompassing measurements at those wavelengths where hemoglobin more strongly absorbs (i.e. 415, 540, and 570 nm); (iii) chemical, which is mainly due to the pseudoperoxidase activity of hemo-globin molecules; and (iv) other, which include spectral overlap in chemical reactions of free hemoglobin in serum or plasma3.

One important aspect is to define reliable limits for establishing whether a certain degree of hemolysis in the specimen would produce a clinically significant bias in the test system. Unfortunately, the specific reagent package inserts often contain limited infor-mation on interferences. Moreover, the findings of individual studies published so far on this topic cannot be easily generalized. Since the bias of in vitro hemoly-sis is substantially analyte-, concentration-, method-, instrument- and degree of hemolysis-dependent, the potential interference should be considered highly variable and ideally should be investigated on a local basis. Accordingly, the HI decision rules provided by the manufacturer should also be validated locally, thus enabling the laboratory to identify analyte-specific thresholds of interference that most probably affect test results. This can be done by spiking plasma or serum samples with serial dilutions of blood cells lysate, per-forming the analysis and then identifying the thresholds of hemolysis (i.e. free hemoglobin) where the interfer-ence becomes clinically meaningful (e.g. when the bias exceeds either the desirable specifications for impreci-sion, inaccuracy, and total allowable error calculated from data on within-subject and between-subject bio-logic variation, or the reference change value (RCV)47). Irrespective of the advantages to locally assessing the interferences of hemolysis with the aim to establish a more reliable decision making process, some basic issues can be translated into a practical approach. As such, Figure 1 provides an accepted description of the hemolysis thresholds that delimit the clinical reliability of some (stat) test results according to the current scientific literature.

Management of hemolyzed specimens from the emergency department

The management of hemolyzed specimens has plagued laboratory activity for a long time, and has engaged the minds of both laboratory professionals and clini-cians. On the one hand, the inability to provide results on an unsuitable sample is frustrating for the labora-tory professional, and on the other hand, such samples cause substantial delays in clinical decision making (i.e. establishing or ruling out a diagnosis, or deciding on a therapy). It is also an important source of poor

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relations between the ED and the clinical laboratory. As such, good cooperation and collaboration between the clinicians and the laboratory is essential to limit or overcome issues arising from poor relationships, and is also effective in troubleshooting problems and identify-ing the underlying cause of hemolysis without the need for performing further investigations in the vast major-ity of cases7. Once a good relationship between the ED and the laboratory has been established, the next step is to plan a joint strategy to manage results obtained on hemolyzed specimens. The importance of this concept is further emphasized by the evidence that healthcare staff traditionally overestimate the effect of hemolysis on test results (e.g. potassium measurement) and may use an incorrect approach to the problem (e.g. faulty thinking) that may further lead to diagnostic delays or misdiagnoses48.

It is clear that actions to limit the burden of in vitro hemolysis should focus on prevention. On the assump-tion that spurious hemolysis is preventable in most cases, the dissemination of best practice recommendations for collection, handling, processing and storage of blood specimens is essential3. Additional efforts for the appro-priate training of phlebotomists should be devised, along with continuing education and information on the most frequent causes of in vitro hemolysis (Table 3). In one study showing the success of such a strategy, the devel-opment of an educational program in a tertiary hospital ED reduced sample hemolysis from 19.8% to 4.9%, a

finding that translated into a remarkable cost saving of $556 US per day40. Regarding the most frequent cause of hemolyzed specimens in the ED, blood collection via venipuncture rather than through IV catheters may be one of the most effective ways to reduce the chance of producing hemolyzed specimens38–43, although this practice may be uncomfortable for the patient due to repeated venipunctures and time consuming for the ED staff. Nevertheless, when specimens need to be col-lected by IV devices or butterfly needles (e.g. in patients with unsuitable venous access or fragile veins), appro-priate procedures on how to collect blood using these devices should be provided. At present, the so-called discard method seems to be the preferred one to obtain suitable specimens for testing, and encompasses: (i) flushing the catheter or other IV device prior to obtain-ing the specimen, (ii) removal of at least three times the catheter volume to clear the tubing from infusion liquid, and (iii) labeling one tube as discard prior to drawing the discard sample, to avoid the potential for confusing the discard sample with the actual blood samples for analy-sis49. Other important issues include the use of a connec-tor with an eclipse needle with pre-attached holder49, as well as the reduction of the interval from sampling to analysis, since delayed centrifugation of the specimen is a well-recognized cause of spurious hemolysis3,38–43. It seems advisable to monitor the effectiveness of local practices to reduce the rate of hemolyzed specimens by systematically reviewing the number of unsuitable

Figure 1. Thresholds of free hemoglobin in the specimen that may produce a clinically meaningful bias in stat laboratory testing.

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specimens obtained from the ED, preferably by using a highly sensitive approach such as the HI44.

The next step is ruling out hemolytic anemia, since the presence of this condition may reflect, mirror or underlie a serious, even life-threatening disorder. As such, the laboratory personnel should alert the staff of the ED (either the clinician or other healthcare staff ) in a timely manner that the sample is hemolyzed. Although hemolytic anemia has been reported to account for a minority of the hemolyzed samples received in clinical laboratories (2–3%), one-third of hemolytic anemias may be missed or may not be suspected by clinicians even though they reflect pathologies requiring urgent management; in the study of Carraro et al., 44% of in vivo hemolyzed specimens detected by the laboratory staff were associated with prolonged extracorporeal circulation during cardiac surgery, 19% with transfu-sion reactions, 12% with acute ethanol toxicosis, and 6% with necrotic-hemorrhagic pancreatitis and rhab-domyolysis7. When hemolytic anemia is suspected, a reliable approach for the differential diagnosis of in vitro hemolysis involves haptoglobin testing, which is now available on several laboratory instruments used for stat testing. A reduced (or depleted) level of haptoglobin generally mirrors the presence of a hemolytic syndrome and can be used as a reliable marker of intravascular hemolysis. When interpreting haptoglobin values for distinguishing between spurious hemolysis and hemo-lytic anemia, a problem may arise from the presence of congenital anhaptoglobinemia. This is a rare condition in Caucasians (∼0.1%), but its prevalence is substantially higher in (West) Africans50,51. Moreover, the reference values for serum haptoglobin are phenotype-dependent

and reportedly lower in Africans (e.g. the lower limit of 0.12 g/L in a Black Zimbabwean population is much lower than the 0.3 g/L proposed interim international reference limit) due to the presence of the Hp 2-1 M phenotype (unknown amongst Europeans) and the pre-viously mentioned increased prevalence of congenital anhaptoglobinemia50,51. An additional challenge may be transient CD163 pathway impairment (e.g. in patients undergoing therapy with the immunotoxin gemtu-zumab ozogamicin or following an acute HIV-1 retroviral syndrome), which leads to temporary impaired hemo-globin scavenging and may be associated with increased HI, absence of decreased haptoglobin levels, presence of circulating complexes in serum and decreased hemopexin and α-1-microglobulin concentrations52,53.

Along with patient history and clinical examination, additional useful parameters for ruling out a hemolytic syndrome include normal values of indirect bilirubin and reticulocytes3.

the challenge of synthetic blood substitutes

An emerging challenge for clinical laboratories as well as for EDs and ICUs is the development of synthetic blood substitutes, which are based on polymeric hemoglobin analogues or RBC-mimicking synthetic biomaterials54. The leading indication for these compounds is the treat-ment of acute and life-threatening hemorrhages, although it is predictable that their significant advantages over con-ventional hemotransfusions (larger supply, lower risk of blood-borne pathogen transmission, no risk of immune incompatibility, and extended survival of stored materi-als) may soon broaden their application to the treatment

Figure 2. Tentative strategy to deal with hemolyzed specimens from the emergency department.

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of other more frequent forms of chronic anemia (i.e. SCD, cancer, chronic renal disease, etc.)54. Since artificial blood is intensely colored and is used in high concentrations, it generates a significant analytical interference with current technologies in use in most laboratories, and a large amount of laboratory testing may be unreliable on patients transfused with such products55–57. While studies in the scientific literature may provide useful guidelines, individual laboratories should assess these interferences in light of the myriad of testing methods in use, differ-ences in the various hemoglobin-based blood substitute products, and institutional-specific opinions as to what constitutes significant interference.

conclusion

Hemolyzed specimens are a common occurrence in laboratory practice, with the vast majority of them being referred from the ED 3. From a basic laboratory standpoint, whatever strategy is undertaken on this frequent source of unsuitable specimens, it should be guided by the consid-eration that the practice of blindly providing questionable laboratory data to clinicians without considering how the information will be used (or misused) is a risk, since it can lead to inaccurate and potentially misleading clini-cal decision making (Figure 2). Since spurious hemolysis may seriously interfere with some (stat) laboratory tests, thereby introducing an unsuitable bias in test results, it seems reasonable to suppress all those test results that are most influenced by the relative degree of hemolysis (e.g. the HI) of the sample (Figure 1). In addition, the labora-tory staff should request the recollection of the sample. Alternative strategies, including correction of test results for the degree of hemolysis by using specific equations or reporting of all test results along with the inclusion of interpretative comments within the laboratory report are now firmly discouraged for a variety of clinical and ana-lytical reasons previously reviewed 3.

Declaration of interest

The authors stated that there are no conflicts of interest regarding the publication of this article. Research fund-ing: None declared. Employment or leadership: None declared.

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