Rodak's Hematology Clinical Principles and Applications

PART IV

Erythrocyte Disorders

CHAPTER 25

Extrinsic defects leading to increased erythrocyte destruction-nonimmune causes

Elaine M. Keohane

OUTLINE

Microangiopathic Hemolytic Anemia

Thrombotic Thrombocytopenic Purpura

Hemolytic Uremic Syndrome

HELLP Syndrome

Disseminated Intravascular Coagulation

Macroangiopathic Hemolytic Anemia

Traumatic Cardiac Hemolytic Anemia

Exercise-Induced Hemoglobinuria

Hemolytic Anemia Caused by Infectious Agents

Malaria

Babesiosis

Clostridial Sepsis

Bartonellosis

Hemolytic Anemia Caused by Other Red Blood Cell Injury

Drugs and Chemicals

Venoms

Extensive Burns (Thermal Injury)

Objectives

After completion of this chapter, the reader will be able to:

1. Describe the general pathophysiology and clinical laboratory findings in microangiopathic hemolytic anemia, including the characteristic red blood cell morphology.
2. Compare and contrast the pathophysiology, clinical symptoms, and typical laboratory findings in thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, HELLP (hemolysis, elevated liver enzymes, and low platelet count) syndrome, and disseminated intravascular coagulation.
3. Explain the pathophysiology and typical laboratory features of traumatic cardiac hemolytic anemia and exercise-induced hemoglobinuria.
4. Describe the life cycle of Plasmodium,including the hepatic and erythrocytic cycles, and the insect vector.
5. Explain the pathophysiologic mechanisms in P. falciparuminfection that lead to anemia and neurologic manifestations.
6. Differentiate the five Plasmodiumspecies affecting humans based on the geographic distribution, characteristic morphology on a peripheral blood film, the extent of parasitemia, and the length of the erythrocytic cycle.
7. Describe the proper specimen collection and procedure for performing a thin and thick blood film examination.
8. Compare and contrast Babesiaspecies and Plasmodium species in terms of geographic distribution, clinical symptoms of infection, and morphology.
9. Describe the pathophysiology, laboratory findings, and peripheral blood morphology in hemolytic anemia due to clostridial sepsis, bartonellosis, drugs, chemicals, venoms, and extensive burns.
10. Given the history, symptoms, laboratory findings, and a representative microscopic field from a peripheral blood film of a patient with suspected extrinsic, nonimmune hemolytic anemia, discuss possible causes of the anemia and indicate the data that support the conclusions.

CASE STUDY

After studying the material in this chapter, the reader should be able to respond to the following case study:

A 24-year-old woman was brought to the emergency department with a 2-day history of fever, chills, excessive sweating, nausea, and general malaise. Because she had recently returned from a 3-week family trip to Ghana in Western Africa, the treating physician ordered a CBC and examination of thin and thick peripheral blood films. The following are the patient’s laboratory results:

	Patient Results	Reference Interval
WBCs (×109/L)	11.0	4.5–11.0
HGB (g/dL)	8.7	12.0–15.0
HCT (%)	25	35–49
MCV (fL)	92	80–100
Platelets (×109/L)	176	150–450

Inclusions were noted on the thin and thick peripheral blood films (Figures 25-1 and 25-2).

Based on the results of the CBC and peripheral blood films, the patient was treated with oral quinine sulfate and doxycycline.

1. Identify the inclusions present on the thin and thick peripheral blood films.
2. What is the likely diagnosis for this patient?
3. What clues in the history support this diagnosis?
4. What other forms might be found on the peripheral blood films in this disease?
5. What are the pathophysiologic mechanisms for the anemia in this disease?

FIGURE 25-1 Thin peripheral blood film for the patient in the case study (×1000).

FIGURE 25-2 Thick peripheral blood film for the patient in the case study (×1000). Source: (Courtesy Linda Marler, Indiana Pathology Images.)

Extrinsic hemolytic anemias comprise a diverse group of disorders in which red blood cells (RBCs) are structurally and functionally normal, but a condition outside of the RBCs causes premature hemolysis. The extrinsic hemolytic anemias can be divided into conditions with nonimmune and immune causes. A common feature in the nonimmune extrinsic hemolytic anemias is the presence of a condition that causes physical or mechanical injury to the RBCs. This injury can be caused by abnormalities in the microvasculature (microangiopathic) or the heart and large blood vessels (macroangiopathic), infectious agents, chemicals, drugs, venoms, or extensive burns. The nonimmune disorders causing hemolytic anemia are discussed in this chapter and are summarized in Box 25-1. In immune hemolytic anemia, hemolysis is mediated by antibodies, complement, or both, and these conditions are covered in Chapter 26. Examination of a peripheral blood film is important in suspected extrinsic hemolytic anemias, because observation of abnormal RBC morphology, such as schistocytes, spherocytes, or the presence of intracellular organisms, provides an important clue to the diagnosis.

BOX 25-1

Extrinsic Conditions Causing Nonimmune Red Blood Cell Injury and Hemolytic Anemia

Microangiopathic hemolytic anemia

Thrombotic thrombocytopenic purpura

Hemolytic uremic syndrome

HELLP syndrome

Disseminated intravascular coagulation

Macroangiopathic hemolytic anemia

Traumatic cardiac hemolytic anemia

Exercise-induced hemoglobinuria

Infection

Malaria

Babesiosis

Clostridial sepsis

Bartonellosis

RBC injury due to other causes

Chemicals

Drugs

Venoms

Extensive burns

HELLP, Hemolysis, elevated liver enzymes, and l ow platelet count; RBC, red blood cell.

Microangiopathic hemolytic anemia

Microangiopathic hemolytic anemias (MAHAs) are a group of potentially life-threatening disorders characterized by RBC fragmentation and thrombocytopenia. The RBC fragmentation occurs intravascularly by the mechanical shearing of RBC membranes as the cells rapidly pass through turbulent areas of small blood vessels that are partially blocked by microthrombi or damaged endothelium.^{1, 2} Upon shearing, RBC membranes quickly reseal with minimal escape of hemoglobin, but the resulting fragments (called schistocytes) are distorted and become rigid.¹ The spleen clears the rigid RBC fragments from the circulation through the extravascular hemolytic process (Chapter 23).¹ Laboratory evidence of the hemolytic anemia includes a decreased hemoglobin level, increased reticulocyte count, increased serum indirect (unconjugated) bilirubin, increased serum lactate dehydrogenase activity, decreased serum haptoglobin level, and increased urine urobilinogen. In some cases, the fragmentation is so severe that intravascular hemolysis occurs with varying amounts of hemoglobinemia, hemoglobinuria, and markedly decreased levels of serum haptoglobin.¹ The presence of schistocytes on the peripheral blood film is a characteristic feature of microangiopathic hemolytic anemia. The RBC shearing may also produce helmet cells and, occasionally, microspherocytes. Polychromasia and nucleated RBCs may also be present on the blood film, depending on the severity of the anemia.

Thrombocytopenia is also a feature of microangiopathic hemolytic anemia; it is due to the consumption of platelets in thrombi that form in the microvasculature.³ Thus these disorders are sometimes called thrombotic microangiopathies.^{4, 5}

The major microangiopathic hemolytic anemias include thrombotic thrombocytopenic purpura (TTP), hemolytic uremic syndrome (HUS), HELLP (hemolysis, elevated liver enzymes,low platelet count) syndrome, and disseminated intravascular coagulation (DIC).^{1, 4, 5} TTP and HUS can be difficult to differentiate because they have overlapping clinical and laboratory findings (Box 25-2). Definitive diagnosis, however, is critical because they have different etiologies and require different treatments.³ This chapter provides an overview of these conditions. TTP, HUS, and HELLP syndrome are covered in more detail in Chapter 40. DIC is covered in more detail in Chapter 39.

BOX 25-2

Typical Laboratory Findings in TTP and HUS

Hematologic

Decreased hemoglobin

Decreased platelets

Increased reticulocyte count

Peripheral blood film

Schistocytes

Polychromasia

Nucleated red blood cells (severe cases)

Biochemical

Markedly increased lactate dehydrogenase activity*

Increased serum total and indirect bilirubin

Decreased serum haptoglobin level

Hemoglobinemia

Hemoglobinuria

Proteinuria, hematuria, casts^†

HUS, Hemolytic uremic syndrome; TTP, thrombotic thrombocytopenic purpura.

*From systemic ischemia and hemolysis; more commonly found in TTP.

^†From acute renal failure; more commonly found in HUS.

Thrombotic thrombocytopenic purpura

Thrombotic thrombocytopenic purpura is a rare, life-threatening disorder characterized by the abrupt appearance of microangiopathic hemolytic anemia, severe thrombocytopenia, and markedly elevated serum lactate dehydrogenase activity.^{2, 6} Neurologic dysfunction, fever, and renal failure may also occur, but they are not consistently present.^{2, 6} TTP is most commonly found in adults in their fourth decade, but it can present at any age.^{5, 6} There is a higher incidence in females than in males.⁵

TTP is caused by a deficiency of the von Willebrand factor-cleaving protease known as a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS-13).ADAMTS-13 regulates the size of circulating von Willebrand factor (VWF) by cleaving ultra-long VWF multimers (ULVWF) into shorter segments that have less hemostatic potential.^{6, 10}VWF multimers circulate in a folded conformation so that their cleavage sites for ADAMTS-13 (in the A2 domain) and binding sites for platelets (GP Ibα receptor in the A1 domain) are hidden.^{5, 6, 10} These sites normally become accessible only when the ULVWF multimer is “unrolled or stretched out,” which occurs (1) during its release from endothelial cells, (2) during passage through small blood vessels with very high shear forces, or (3) after binding to collagen in the subendothelium after vascular injury (Chapter 37).^{5, 6, 10} Once unrolled, ADAMTS-13 binds to the cleavage sites on the ULVWFs and cuts them into smaller multimers.^{6, 10} Thus ADAMTS-13 serves an important antithrombotic function by preventing VWF from excessively binding and activating platelets.⁶

When ADAMTS-13 is deficient, however, the hyperreactive ULVWF multimers adhere to the endothelial cells of the microvasculature, where they readily unroll as a result of hydrodynamic shear forces.^{5-7, 10} Platelets are then able to bind to the A1 domains of the ULVWF multimers, and platelet aggregation is triggered.^{5, 6} The platelet-VWF microthrombi accumulate in and block small blood vessels, leading to severe thrombocytopenia; ischemia in the brain, kidney, and other organs; and hemolytic anemia due to RBC rupture as they pass through blood vessels partially blocked by microthrombi.^{2, 3, 7} The intravascular hemolysis along with the extensive tissue ischemia result in a striking increase in serum lactate dehydrogenase activity that is characteristic of TTP.⁵

TTP can be idiopathic, secondary, or inherited. Idiopathic TTP has no known precipitating event.^{5, 6} In idiopathic TTP, autoantibodies to ADAMTS-13 inhibit its activity, causing a severe deficiency.^{5, 8, 9} These autoantibodies are usually of the IgG class but can be IgM or IgA.

Secondary TTP can be triggered by infections, pregnancy, surgery, trauma, inflammation, and disseminated malignancy, possibly by depressing the synthesis of ADAMTS-13.^{3, 5} Other conditions may induce an inhibitory reaction to ADAMTS-13, including hematopoietic stem cell transplantation; autoimmune disorders; human immunodeficiency virus (HIV); andcertain drugs, such as quinine, ticlopidine, and trimethoprim. ^{, 11} Secondary TTP is very heterogeneous, and the mechanisms that trigger the TTP pathophysiology are not completely clear.³

Inherited TTP, also called Upshaw-Schülman syndrome, is a severe ADAMTS-13 deficiency caused by mutations in the ADAMTS13 gene.^{5, 6, 12} Over 75 different mutations have been identified, and symptomatic individuals are either homozygous for one of the mutations or compound heterozygous for two different mutations.^{5, 12, 13} Inherited TTP may present in infancy or childhood with recurrent episodes throughout life; however, some patients may not be symptomatic until adulthood after their system is stressed by pregnancy or a severe infection.

Typical initial laboratory findings in all types of TTP include a hemoglobin level of 8 to 10 g/dL, a platelet count of 10 to 30 × 10⁹/L, and schistocytes on the peripheral blood film (Figure 25-3).⁶ After the bone marrow begins to respond to the anemia, polychromasia and nucleated RBCs may also be present on the blood film. The white blood cell count is often increased, and immature granulocytes may appear. The bone marrow shows erythroid hyperplasia and a normal number of megakaryocytes. Hemoglobinuria occurs when there is extensive intravascular hemolysis. Various amounts of protein, RBCs, and urinary casts may also be present in the urine, depending on the extent of the renal damage. Results of coagulation tests are usually within the reference interval, which differentiates TTP from DIC (covered below). An elevation of the serum indirect bilirubin level does not occur for several days after an acute onset of hemolysis, but the serum lactate dehydrogenase activity will be markedly elevated and the serum haptoglobin level will be reduced.

FIGURE 25-3 Peripheral blood film from a patient with thrombotic thrombocytopenic purpura. Note the schistocytes and a nucleated red blood cell (×1000).

In idiopathic and inherited TTP, the ADAMTS-13 activity is usually severely reduced to less than 5% to 10% of normal.^{4, 6, 7} In secondary TTP, the ADAMTS-13 deficiency is not as severe. ADAMTS-13 autoantibodies can be detected in idiopathic TTP but are absent in inherited TTP.⁶

Approximately 80% to 90% of patients with idiopathic TTP respond favorably to plasma exchange therapy due to the removal of the offending ADAMTS-13 autoantibody and infusion of replacement ADAMTS-13 enzyme from donor plasma.^{6, 11} Therefore, it is important that this type of TTP be recognized and quickly treated with plasma exchange therapy to avoid a fatal outcome. Corticosteroids are also administered to suppress the autoimmune response.^{5, 6} Approximately one third of patients who respond to plasma exchange experience recurrent episodes of TTP.⁴ Rituximab (anti-CD20) is effective in suppressing an autoantibody response in some patients with relapsing TTP.⁶ Patients with secondary TTP generally do not respond well to plasma exchange, and the prognosis in these cases is poor, except when the TTP is related to autoimmune disease, pregnancy, or ticlopidine use.^{11, 13} Plasma exchange is not required in inherited TTP, which is treated by infusion of fresh frozen plasma to supply the deficient ADAMTS-13 enzyme.^{3, 4, 6}

Hemolytic uremic syndrome

HUS is characterized by microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure from damage to endothelial cells in the glomerular microvasculature.^{3, 5} There are two general types: typical and atypical HUS. Typical HUS (Shiga toxin-associated HUS or stx-HUS) is caused by bacteria that produce Shiga toxin and is preceded by an episode of acute gastroenteritis, often with bloody diarrhea.⁵ Atypical HUS (aHUS) is caused by unregulated activation of the alternative complement pathway.^{3, 5} Patients with HUS have the typical laboratory findings of microangiopathic hemolytic anemia discussed previously. However, the platelet count is only mildly to moderately decreased, and evidence of renal failure is usually present, including an elevated level of serum creatinine, proteinuria, hematuria, and the presence of hyaline, granular, and RBC casts in the urine (Box 25-2).

The stx-HUS type comprises 90% of cases of HUS.^{5, 14} The most common cause is infection with Shiga toxin-producing Escherichia coli (STEC), such as serotype O157:H7, but strains of toxin-producing Shigella have also been implicated.^{4, 5} Stx-HUS occurs most often in young children but can be found in patients of all ages. Patients initially have acute gastroenteritis, often with bloody diarrhea, and after approximately 5 to 13 days develop oliguria and other symptoms of renal damage.⁵ About one fourth of patients also develop neurologic manifestations.^{5, 14}

1. coliand Shigellaserotypes implicated in HUS release Shiga toxins (Stx-1 and Stx-2, also called verotoxins) that are absorbed from the intestines into the plasma. The toxins have an affinity for the Gb3 glycolipid receptors (CD77) on endothelial cells, particularly those in the glomerulus and brain.^{4, 5, 15} The toxin is transported into the endothelial cells, where it inhibits protein synthesis and causes endothelial cell injury and eventual apoptosis.^{5, 15} The Shiga toxin, together with many cytokines secreted as a result of the infection, also induces changes in endothelial cells that are prothrombotic, including expression of tissue factor, adhesion molecules, and secretion of increased amounts of ULVWF multimers.^{4, 5, 15} The endothelial cell damage can cause stenosis (narrowing) of small blood vessels which can be exacerbated by activation of platelets and formation platelet-fibrin thrombi.^{3, 5, 15} The resultant blockages in the microvasculature of the glomeruli results in acute renal failure.^{3, 15} Endothelial damage and microthrombi can also occur in the microvasculature of the brain and other organs.^{3, 5} There is no specific treatment for stx-HUS, but patients are provided supportive care as needed, including hydration, dialysis, and transfusions.^{14, 15} The symptoms usually resolve spontaneously in 1 to 3 weeks, and the prognosis is favorable for most patients.¹⁴

Atypical HUS comprises about 10% of cases of HUS and can first present in infancy, childhood, or adulthood.^{5, 16} The characteristic feature is uncontrolled activation of the alternative complement system, which causes endothelial cell injury, activation of platelets and coagulation factors, and formation of platelet-fibrin thrombi that obstruct the microvasculature in the glomerulus and other organs.^{3, 5} Approximately 50% to 70% of aHUS patients have inherited mutations in genes that code for components of the alternative complement pathway or its regulatory proteins.^{5, 16} Inactivating mutations have been identified in genes for complement regulatory proteins, including complement factor H, complement factor I, membrane cofactor protein, and thrombomodulin.^{3, 5, 16} Activating mutations have been identified in the genes for complement factor B and C3.^{3, 5, 16} An acquired form of aHUS is associated with autoantibodies against complement factor H and accounts for approximately 5% to 10% of cases.⁵ In the remaining cases, no mutation or autoantibodies have been identified.⁵ aHUS may be triggered by hematopoietic stem cell therapy, pregnancy, infection, inflammation, surgery, or trauma.³ Plasma exchange and plasma infusion have limited efficacy in aHUS.^{5, 16} In recent studies, therapy with eculizumab (antibody to C5) has improved platelet counts and renal function in aHUS patients and may become the therapy of choice.¹⁶

Differential diagnosis of aHUS and TTP is difficult due to the similarities in their clinical presentation and initial laboratory findings. Both are life-threatening disorders that require rapid action to prevent a fatal outcome, with plasma exchange most beneficial for TTP, and eculizumab more likely to benefit patients with aHUS.^{5, 16} Assays for ADAMTS-13 activity currently lack sufficient sensitivity and specificity and are not available in all laboratories.¹⁶ DNA analysis for complement system gene mutations is available in specialized laboratories but the results are not timely enough to be used in initial therapy decisions.¹⁶ More sensitive, specific, and rapid tests are needed for definitive diagnosis of these two conditions.

Hellp syndrome

HELLP syndrome is a serious complication in pregnancy and is named for its characteristic presentation of hemolysis, elevated liver enzymes, and low platelet count. It occurs in less than 1% of all pregnancies but develops in approximately 10% to 20% of pregnancies with severe preeclampsia, most often in the third trimester.¹⁷ The exact pathogenesis is not known. In preeclampsia, abnormalities in the development of placental vasculature result in poor perfusion and hypoxia. As a result, antiangiogenic proteins are released from the placenta that block the action of placental growth factors, including vascular endothelial growth factor.¹⁸ Continued vascular insufficiency of the placenta results in maternal endothelial cell dysfunction, which leads to platelet activation and fibrin deposition in the microvasculature, particularly in the liver.¹⁸

Anemia, biochemical evidence of hemolysis, and schistocytes on the peripheral blood film are found as in the other microangiopathies. The platelet count is less than 100 × 10⁹/L; counts falling below 50 × 10⁹/L indicate a worse prognosis.^{18, 19} The serum lactate dehydrogenase activity is elevated, which reflects the hepatic necrosis as well as the hemolysis. The serum aspartate aminotransferase activity can be markedly elevated due to the severe hepatocyte injury. The low platelet count and increased serum lactate dehydrogenase and aspartate aminotransferase activity are major diagnostic criteria for the HELLP syndrome and are used to assess the severity of the disease.^{17, 18} The prothrombin time and the partial thromboplastin time are within the reference interval, which distinguishes the HELLP syndrome from DIC. Therapy includes delivery of the fetus and placenta as soon as possible, along with supportive care to control seizures, hypertension, and fluid balance. The mortality rate is 3% to 5% for the mother and 9% to 24% for the fetus.¹⁸

Disseminated intravascular coagulation

Disseminated intravascular coagulation (DIC) is characterized by the widespread activation of the hemostatic system, resulting in fibrin thrombi formation throughout the microvasculature. The major clinical manifestations are organ damage due to obstruction of the microvasculature and bleeding due to the consumption of platelets and coagulation factors and secondary activation of fibrinolysis. DIC is a complication of many disorders, such as metastatic cancers, acute leukemias, infections, obstetric complications, crush or brain injuries, acute hemolytic transfusion reactions, extensive burns, snake or spider envenomation, and chronic inflammation (Table 39-14).

Thrombocytopenia of varying degrees is a consistent finding. Only about half of the patients have schistocytes on the peripheral blood film.²⁰ The prothrombin time and partial thromboplastin time are prolonged, the fibrinogen level is decreased, and the level of D-dimer is increased in DIC, which distinguish it from the other microangiopathies. Tables 39-15 and39-16 contain the primary and specialized tests used in the diagnosis of DIC.

Macroangiopathic hemolytic anemia

Traumatic cardiac hemolytic anemia

Mechanical hemolysis can occur in patients with prosthetic cardiac valves due to the turbulent blood flow through and around the implanted devices.^{18, 21} The hemolysis is usually mild, and anemia does not generally develop due to compensation by the bone marrow.²¹ Severe hemolysis is rare and is usually due to paravalvular leaks in prosthetic cardiac valves.²¹Hemolysis can also occur in patients with cardiac valve disease prior to corrective surgery.¹⁸ The anemia that occurs in severe cases is usually normocytic but can be microcytic if iron deficiency develops due to chronic urinary hemoglobin loss.

Depending on the severity of the hemolysis and the ability of the bone marrow to compensate for the reduced RBC life span, patients can be asymptomatic or present with pallor, fatigue, and even heart failure.²¹ On the peripheral blood film, schistocytes are a characteristic feature due to the mechanical fragmentation of the RBCs (Figure 25-4). The reticulocyte count is increased, but the platelet count is within the reference interval. Serum lactate dehydrogenase activity and levels of serum indirect bilirubin and plasma hemoglobin are elevated, and the serum haptoglobin level is decreased. Hemoglobinuria may be observed in severe hemolysis. Hemosiderinuria and a decreased level of serum ferritin occur with chronic hemoglobinuria due to the urinary loss of iron.

FIGURE 25-4 Peripheral blood film for a patient with traumatic cardiac hemolytic anemia. Note the presence of schistocytes (×1000).

Surgical repair or replacement of the prothesis may be required if the anemia is severe enough to require transfusions. For patients with hemoglobinuria, iron supplementation is provided to replace urinary iron loss. Folic acid may also be required, because deficiencies can occur due to the increased erythropoietic activity in the bone marrow.²¹

Exercise-induced hemoglobinuria

RBC lysis, with an increase in free plasma hemoglobin and a decrease in serum haptoglobin level, has been demonstrated in some individuals after long-distance running and to a lesser extent after intensive cycling and swimming,^{1, 22-24} but frank hemoglobinuria after exercise is a rare occurrence.²⁵ Exercise-induced hemoglobinuria has been reported mainly in endurance runners but has also been observed after strenuous hand drumming.^{22, 26} Various causes have been proposed, including mechanical trauma from the forceful, repeated impact of the feet or hands on hard surfaces;^{22, 23, 26} increased RBC susceptibility to oxidative stress;²⁷ and exercise-induced alterations in membrane cytoskeletal proteins.^{28, 29}

Exercise-induced hemoglobinuria does not usually cause anemia unless the hemoglobinuria is particularly severe and recurrent.^{1, 25} Laboratory findings include a decreased level of serum haptoglobin, an elevated level of free plasma hemoglobin, and hemoglobinuria observed after strenuous exercise. Patients also have a slight increase in mean cell volume (MCV) and reticulocyte count. Schistocytes are not present on the peripheral blood film except in rare cases.^{25, 26} Exercise-induced hemoglobinuria is a diagnosis of exclusion, and other possible causes of hemolysis and hemoglobinuria should be investigated and ruled out.²⁵ There is no treatment for the disorder other than minimizing the physical impact on the feet with padding in shoes, running on softer terrain, or, if hemolysis is severe, discontinuing the activity.

Hemolytic anemia caused by infectious agents

Malaria

Malaria is a potentially fatal condition caused by infection of RBCs with protozoan parasites of the genus Plasmodium. Most human infections are caused by P. falciparum and P. vivax, butP. ovale, P. malariae, and a fifth species, P. knowlesi, also infect humans. P. knowlesi, a natural parasite of macaque monkeys, is easily misdiagnosed as P. malariae by microscopy, because the organisms are difficult to distinguish morphologically. Since 2004, hundreds of microscopically identified cases of P. malariae infection in Malaysia were actually found to be caused by P. knowlesi when polymerase chain reaction (PCR) assays were used, including tests on archival blood films from 1996.^30-32 With the use of molecular techniques, P. knowlesi malaria is now known to be widespread in Malaysia, and cases have been reported in other areas of Southeast Asia.^30-32

Prevalence

Approximately 3.4 billion people live in areas in which malaria is endemic and are at risk for the disease.³³ Worldwide in 2012, there were an estimated 207 million cases of malaria, with 627,000 deaths, mostly in children younger than 5 years of age.³³ The majority of deaths were in Africa (90%), followed by Southeast Asia (7%) and the Eastern Mediterranean region (3%).³³ The World Health Organization is coordinating a major global effort to control and eliminate malaria. These efforts include implementation of indoor chemical spraying, distribution of millions of insecticide-treated sleeping nets in high-risk areas, and promotion of policies for appropriate treatment in regions of endemic disease.³³ An estimated 42% reduction in mortality rates from malaria occurred worldwide between 2000 and 2012, and the goal is to reduce mortality rates by 75% by 2015.³³

In 2011, 1925 cases of malaria were diagnosed in the United States, and almost all of the cases were associated with travel to a malaria-endemic country.³⁴ P. falciparum and P. vivax cause most of the infections seen in the United States.

Plasmodium life cycle

Malaria is transmitted to humans by the bite of an infected female Anopheles mosquito. During a blood meal, sporozoites from the salivary gland of the mosquito are injected into the skin and migrate into the bloodstream of the human host. The sporozoites rapidly leave the circulating blood and invade hepatic parenchymal cells to begin exoerythrocytic schizogony, the parasite’s asexual cycle. After 5 to 16 days (depending on the species), hepatic cells rupture, with each cell releasing tens of thousands of merozoites into the bloodstream to invade circulating RBCs, thus beginning erythrocytic schizogony.³⁵ Inside the erythrocyte, the merozoite grows and metabolizes the hemoglobin. The merozoite becomes a ring form, which grows into a mature trophozoite, then into an immature schizont (chromatin dividing), and finally into a mature schizont that contains merozoites. The merozoites are released from the erythrocytes into the bloodstream and invade other RBCs to continue the asexual cycle. As the infection continues, the cycles often recur at regular intervals as all the individual parasitic cycles become synchronous; this produces paroxysms of fever and chills at a frequency that varies according to malaria species. Resting stages of P. vivax and P. ovale, called hypnozoites,can remain dormant in the liver and produce a relapse months or years later.^{36, 37}

Some merozoites enter RBCs and form male and female gametocytes (sexual stages). Gametocytes are ingested by an Anopheles mosquito when it takes in a blood meal. The female gamete is fertilized by the male gamete in the mosquito gut to produce a zygote, which becomes an ookinete that migrates to the outer wall of the mosquito midgut and develops into an oocyst. The oocyst produces sporozoites that are released into the hemocele and migrate to the salivary glands of the mosquito. When the mosquito takes in a blood meal, the sporozoites are inoculated into the human host.

Other modes of transmission include congenital infection and transmission by blood transfusion, organ transplantation, or sharing of syringes and needles, but malaria acquired by these routes and local mosquito-transmitted malaria occur infrequently in the United States.³⁴

Pathogenesis

If an individual is bitten by infected mosquitos, the clinical outcome may be (1) no infection, (2) asymptomatic parasitemia (the patient has no symptoms, but parasites are present in the blood), (3) uncomplicated malaria (the patient has symptoms and parasitemia but no organ dysfunction), or (4) severe malaria (the patient has symptoms, parasitemia, and major organ dysfunction).^{33, 36} The clinical outcome depends on parasite factors (species, number of sporozoites injected, multiplication rate, virulence, drug resistance), host factors (age, pregnancy status, immune status, previous exposure, genetic polymorphisms, nutrition status, coinfection with other pathogens, and duration of infection), geographic and social factors (endemicity, poverty, and availability of prompt and effective treatment), and other as yet unknown factors.^{35, 36, 38} In areas of high Plasmodium transmission, most individuals develop immunity, and the major risk groups for severe malaria are children younger than 5 years of age and women in their first pregnancy.³⁹ Even in these risk groups, severe malaria is infrequent.³⁶ On the other hand, immunity is low or nonexistent in individuals living in regions with low Plasmodium transmission and in travelers to regions where malaria is endemic, so all age groups are at risk for severe malaria.³⁹ Most cases of severe malaria are due to P. falciparum; however, P. vivax and P. knowlesi can also cause severe disease.^{31, 35} Infection with P. malariae or P. ovale, however, is usually uncomplicated and benign. Hyperparasitemia, defined as greater than 2% to 5% of the total RBCs parasitized, is usually present in severe malaria.³⁹Major complications of severe malaria include respiratory distress syndrome, metabolic acidosis, circulatory shock, renal failure, hepatic failure, hypoglycemia, severe anemia (defined as a hemoglobin level below 5 g/dL),³⁹ poor pregnancy outcome, and cerebral malaria.³⁵ Even with treatment, the fatality rate of severe malaria is 10% to 20%.³⁹

The causes of anemia in malaria include direct lysis of infected RBCs during schizogony, immune destruction of infected and noninfected RBCs in the spleen, and inhibition of erythropoiesis and ineffective erythropoiesis.^{37, 38} The destruction of noninfected RBCs contributes significantly to the anemia. In the invasion process, parasites shed proteins that bind to infected as well as noninfected RBCs.⁴⁰ These proteins may change the RBC membrane in noninfected cells, allowing adherence of immunoglobulins and complement and thus enhancing their removal by the spleen.^{40, 41} In addition, parasite proteins may also cause oxidative damage, resulting in decreased RBC survival.⁴²

Malaria parasites metabolize hemoglobin, forming toxic hemozoin or malaria pigment. When RBCs lyse in schizogony, the hemozoin and other toxic metabolites are released, which results in an inflammatory response and cytokine imbalance.⁴⁰ Abnormal levels of tumor necrosis factor-α and interferon-γ result in inhibition of erythropoiesis as well as ineffective erythropoiesis; increased levels of interleukin-6 stimulate hepcidin production in the liver, which decreases the iron available to developing RBCs (Chapter 20).⁴⁰ In areas where malaria is endemic, poor nutrition and coinfection with hookworm or HIV contribute to the anemia, inflammation, and cytokine imbalance.^{38, 40}

36. falciparumis unique and particularly lethal in that infected RBCs adhere to endothelial cells in the microvasculature of internal organs, including the brain, heart, lung, liver, kidney, dermis, and placenta.^{35, 36}This contributes to the pathogenesis by obstructing the microvasculature and decreasing oxygen delivery to organs and by protecting the parasite from clearance by the spleen.^{35, 36} In the placental microvasculature, adherence of infected RBCs to endothelial cells results in local inflammation that can cause severe maternal anemia, decreased fetal growth, premature delivery, and increased risk of fetal loss.^{35, 36} In the brain microvasculature, adherence of infected RBCs to endothelial cells can cause lethal cerebral malaria. Infected RBCs express a parasite protein on their membranes, called Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1). PfEMP1 mediates binding of infected RBCs to cell receptors, particularly CD36 on platelets and some endothelial cells.^{35, 43} Reasons why some strains are more likely to cause severe cerebral malaria is a subject of intense research. Adherence of infected RBCs in the brain has been attributed to a complex formed by VWF released by cytokine-stimulated endothelial cells, platelets bound to the VWF, and infected RBCs bound to platelet CD36.⁴⁴ A recent mechanism was proposed whereby a specific variant of PfEMP1, expressed on the surface of RBCs infected with certain strains of P. falciparum, specifically binds to endothelial protein C receptor (EPCR) on endothelial cells lining the microvessels in the brain.⁴⁵ This binding prevents the activation of protein C (an inhibitor of activated factors V and VIII), creating a local hypercoagulable state in the brain. The result is fibrin deposition and parasite sequestration in the brain microvasculature and symptoms of severe cerebral malaria.⁴⁵

The ability of the parasite to invade RBCs affects the extent of the parasitemia and the severity of the disease. P. vivax and P. ovale can only invade reticulocytes, and P. malariae can only invade older RBCs. On the other hand, P. falciparum and P. knowlesi are able to invade RBCs of all ages and thus can lead to very high levels of parasitemia.^{35, 37} P. vivax requires Duffy antigens on RBCs for invasion, so individuals lacking Duffy antigens are resistant to infection with P. vivax. The expansion of the Duffy-negative population in West Africa seems to be an effective genetic adaptation because P. vivax infection is almost nonexistent in West Africa.³⁵

Polymorphisms in genes for the α and β hemoglobin chains (Hb S, Hb C, Hb E, and α- and β-thalassemia) and for glucose-6-phosphate dehydrogenase (G6PD) protect individuals from developing severe falciparum malaria. Although individuals with these polymorphisms become infected, they do not develop serious complications.³⁶ The reason for this protection has not been completely elucidated. Enhanced phagocytosis of ring forms (sickle cell trait, β-thalassemia trait, and G6PD deficiency),⁴⁶ decreased expression of PfEMP1 on infected erythrocytes causing reduced endothelial adherence (sickle cell trait, Hb C disease and trait),^{47, 48} and decreased RBC invasion (Hb E trait)⁴⁹ may contribute to this protective effect. Over thousands of years, the evolutionary pressure of P. falciparum has resulted in a higher frequency of these polymorphisms in populations located in high-prevalence malaria regions, including sub-Saharan Africa, the Middle East, and Southeast Asia (Figure 27-3). Polymorphisms in cytokines and cellular receptors are also being investigated as modulators of malaria severity.³⁵

Clinical and laboratory findings

The clinical symptoms of malaria are variable and can include fever, chills, rigors, sweating, headache, muscle pain, nausea, and diarrhea. In severe malaria, jaundice, splenomegaly, hepatomegaly, shock, prostration, bleeding, seizures, or coma may occur.³⁹ In patients with chronic malaria or with repeated malarial infections, the spleen may be massively enlarged.

During fevers, the WBC count is normal to slightly increased, but neutropenia may develop during chills and rigors. In chronic malaria with anemia, the reticulocyte count is decreased due to the negative effect of the inflammation on erythropoiesis. In severe malaria, one or more of the following laboratory features are found: metabolic acidosis, decreased serum glucose (less than 40 mg/dL), increased serum lactate, increased serum creatinine, decreased hemoglobin level (less than 5 g/dL), hemoglobinuria, and hyperparasitemia.³⁹

Microscopic examination

Malarial infection can be diagnosed microscopically by demonstration of the parasites in the peripheral blood. Optimally, blood should be collected before treatment is initiated.^{37, 50} At least two thick and two thin peripheral blood films should be made as soon as possible after collection of venous blood in ethylenediaminetetraacetic acid (EDTA) anticoagulant.⁵⁰Alternatively, blood films can be made directly from a capillary puncture. Wright-Giemsa stain is used for visualization of the parasites.³⁷ Thick blood films concentrate the parasites and are ideal for initial screening of peripheral blood. They are stained with a water-based Wright-Giemsa stain without methanol fixation to lyse the RBCs (Box 25-3). Thin blood films are used for species identification and determination of the percent parasitemia; they are stained after methanol fixation. The percent parasitemia is determined by counting the number of parasitized RBCs (asexual stages) among 500 to 2000 RBCs on a thin peripheral blood film and converting to a percentage.⁵¹ At least 300 fields on the thick and thin blood films should be examined with the 100× objective before a negative result is reported.^{37, 51} Multiple samples taken at 8- to 12-hour intervals may be needed because the number of circulating parasites may vary with the timing of the erythrocytic schizogony.⁵⁰ Microscopy can detect 5 to 20 parasites per microliter of blood, or 0.0001% parasitemia.³⁷ A negative result for a single set of thick and thin peripheral blood films does not rule out a diagnosis of malaria.³⁷ Malarial parasite detection and species identification require experienced laboratory personnel. A platelet lying on top of an erythrocyte in a thin blood film may be confused with a malarial parasite by an inexperienced observer (Figure 25-5).

FIGURE 25-5 Peripheral blood film with a platelet on top of a red blood cell (A) compared with an intraerythrocytic Plasmodium vivax ring form (B) (×1000).

BOX 25-3

Thick Film Preparation for Malaria

To make a thick film, place three small drops of blood close together near one end of the slide. With one corner of a clean slide, stir the blood for about 30 seconds to mix the three drops over an area approximately 1 to 2 cm in diameter. Allow the film to dry thoroughly. Stain the film using a water-based Giemsa stain. (The water-based stain lyses the red blood cells. Thin blood films are fixed in methyl alcohol to preserve the red blood cells so they do not lyse.) In a thick film, more parasites are seen in each field.

Plasmodium vivax.

1. vivax is widely distributed and causes 40% of human malaria cases worldwide.³⁹It is the predominant species in Asia and South and Central America, but also occurs in Southeast Asia, Oceania, and the Middle East.⁵²It is rare in Africa and virtually absent in West Africa.⁵² The early ring forms are delicate with a red chromatin dot and blue-staining cytoplasm. As the trophozoite grows, the RBC becomes enlarged. Schüffner stippling appears in all stages except the early ring forms. The growing trophozoite is ameboid in appearance and has fine light brown hemozoin pigment. The mature trophozoite almost fills the RBC (Figure 25-6). In an immature schizont, red chromatin begins to divide into two or more dots, and the mature schizont has 12 to 24 merozoites (Figure 25-7). Gametocytes are round with blue cytoplasm and light brown pigment; they have either centrally located (microgametocyte) or eccentrically located (macrogametocyte) red chromatin. The length of the erythrocytic cycle is 44 to 48 hours.³⁷ P. vivax is difficult to eradicate because the hypnozoite forms remain dormant in the liver and may cause a relapse.

FIGURE 25-6 Two Plasmodium vivax trophozoites in a thin peripheral blood film. Note that the infected red blood cells are enlarged and contain Schüffner stippling, and the trophozoites are large and ameboid in appearance.

FIGURE 25-7 Plasmodium vivax schizont in a thin peripheral blood film. Note the number of merozoites and the presence of brown hemozoin pigment. Source: (Courtesy Linda Marler, Indiana Pathology Images.)

Plasmodium ovale.

1. ovale is found mainly in West Africa and India.⁵²The young ring forms are larger and more ameboid than those of P. vivax,and as the trophozoite grows, the RBCs become enlarged, oval, and fringed (Figure 25-8). Schüffner stippling is present in all stages, including early ring forms. In the schizont stage, the RBCs are oval, and the parasite is round and compact. The mature schizont has 8 to 12 merozoites. Gametocytes are smaller than those of P. vivax but have a similar appearance. The length of the erythrocytic cycle is 48 hours.³⁷

FIGURE 25-8 Two Plasmodium ovale trophozoites in a thin peripheral blood film. Note that the infected cells are enlarged, are oval, have fringed edges, and contain Schüffner stippling. Source: (Courtesy Linda Marler, Indiana Pathology Images.)

Plasmodium malariae.

1. malariae is found worldwide but at low frequency. The highest prevalence is in East Africa and India.⁵²The ring stage is often smaller and wider than that of P. vivax,although the two may be indistinguishable. In growing trophozoites, the cytoplasm forms a characteristic narrow band across the cell and contains dark brown pigment (Figure 25-9). RBCs do not become enlarged, and there is no stippling. The mature schizont has 6 to 12 merozoites and often forms a rosette around clumped pigment. Gametocytes are similar to those of P. vivax but are smaller. The length of the erythrocytic cycle is 72 hours.³⁷

FIGURE 25-9 Plasmodium malariae band form in a thin peripheral blood film. Source: (Courtesy Linda Marler, Indiana Pathology Images.)

Plasmodium falciparum.

1. falciparum is the predominant species in sub-Saharan Africa, Saudi Arabia, Haiti, and the Dominican Republic.⁵²It is also found in Asia, Southeast Asia, the Philippines, Indonesia, and South America.⁵²P. falciparum can produce high parasitemia (greater than 50% of RBCs infected) due to its ability to invade RBCs of all ages. Only ring forms and crescent- or banana-shaped gametocytes are observed in the peripheral blood. RBCs with trophozoites and schizonts adhere to the endothelial cells in various organs and do not circulate. Ring forms are small and can be easily missed. One or more rings may occupy a given cell, with some rings located at the cell periphery (Figure 25-1). The crescent-shaped gametocytes have deep blue cytoplasm with brownish pigment and red chromatin near the center and are easily recognized on blood films (Figure 25-10). The length of the erythrocytic cycle is 36 to 48 hours.³⁷

FIGURE 25-10 Plasmodium falciparum crescent-shaped gametocyte in a thin peripheral blood film (×1000). Source: (Courtesy Linda Marler, Indiana Pathology Images.)

Plasmodium knowlesi.

1. knowlesi is widespread in Malaysia, and cases have been reported in Myanmar, the Philippines, and Thailand.⁵²In the early trophozoite stage, it can look similar to P. falciparumwith high parasitemia and multiple ring forms in one cell. In the growing trophozoite, the cytoplasm forms a band across the cell, similar to P. malariae (Figure 25-11). Mature schizonts have an average of 16 merozoites and do not form rosettes.³⁷ The length of the erythrocytic cycle is only 24 hours, so infected patients can rapidly develop a high level of parasitemia and severe malaria; thus prompt diagnosis and treatment are critical.³¹ Molecular methods may be required for definitive diagnosis. Figure 25-12 illustrates four species of Plasmodium.

FIGURE 25-11 Plasmodium knowlesi ring forms (A) and a ring form and trophozoite (B) in a thin peripheral blood film (×1000). Source: (Courtesy Wadsworth Center Laboratories, New York State Department of Health, New York, NY.)

FIGURE 25-12 Malarial parasites from four species of Plasmodium. Source: (From Diggs LW, Sturm D, Bell A: Morphology of human blood cells, ed 5, Abbott Park, Ill, 1985, Abbott Laboratories. Permission has been granted with approval of Abbott Laboratories, all rights reserved by Abbott Laboratories, Inc.)

Other tests for diagnosis

Fluorescent dyes may be used to stain Plasmodium species; they are sensitive for parasite detection but are not useful for speciation.³⁷ Molecular-based tests (such as PCR) can be used for detection and speciation of malarial parasites and are especially helpful in cases of mixed infections, low parasitemia, and infection with P. knowlesi. A rapid antigen test, the BinaxNOW^®Malaria Test, has been approved by the Food and Drug Administration for use in the United States.⁵³ It is based on the detection of P. falciparum histidine-rich protein II and genericPlasmodium aldolase.⁵³ The sensitivity of the test is low when there are fewer than 100 parasites per microliter of blood.^{37, 53} Therefore, thick and thin blood film microscopy should be done with the antigen test.⁵³

Treatment

Chloroquine or hydroxychloroquine is used for treatment of all malaria, except for disease caused by strains of P. falciparum and P. vivax acquired from areas known to harbor chloroquine-resistant organisms.⁵⁴ For infection with the chloroquine-resistant strains, combination therapy (use of two drugs with different mechanisms of action) is recommended, including atovaquone-proguanil, quinine sulfate plus doxycycline or tetracycline, or mefloquine.⁵⁴ Another choice for resistant P. falciparum is artemether-lumefantrine.⁵⁴ Primaquine is also administered in P. vivax and P. ovale infections to eradicate the hypnozoites in the liver and prevent relapse.^{39, 54} Prior to administration of primaquine, testing for G6PD deficiency is recommended because patients with moderate to severe deficiency can develop hemolytic anemia after primaquine treatment (Chapter 24).^{39, 54} There is growing concern about the widespread resistance of P. falciparum to chloroquine, the increase in chloroquine-resistant P. vivax strains, and the possible emergence and spread of resistance to other antimalarial drugs.³⁹ Transfusion therapy is used for severe anemia, including exchange transfusion for patients with a level of parasitemia greater than 10% infected RBCs. Because infections with P. falciparum or P. knowlesi have the potential for a rapid, fatal course, patients must be treated without delay after confirmation of the diagnosis. Intensive research is under way to develop an effective vaccine.

Babesiosis

Babesiosis is a tick-transmitted disease caused by intraerythrocytic protozoan parasites of the genus Babesia. There are hundreds of species of Babesia, but only a few are known to cause disease in humans. B. microti is the most common cause of babesiosis in the United States, where it was originally called Nantucket fever because the first cluster of cases was found on Nantucket Island, off Massachusetts, in 1969 and the early 1970s.^{55, 56} The sexual cycle of B. microti occurs in the tick, Ixodes scapularis, whereas its asexual cycle primarily occurs in the white-footed mouse, the reservoir host in the United States.⁵⁷ Humans are incidental hosts and become infected after injection of sporozoites during a blood meal by infected ticks. OtherBabesia species, such as B. duncani and B. divergens, can be found sporadically in humans.^{56, 57} Babesia may also be transmitted by transfusion of RBCs from asymptomatic donors. Between 1979 and 2009, 159 cases of transfusion-transmitted babesiosis were identified.⁵⁸ Congenitally acquired babesiosis has been reported, but it is rare.^{57, 59}

Geographic distribution

The areas in which B. microti is endemic in the United States are southern New England, New York State, New Jersey, Wisconsin, and Minnesota.^{56, 57} B. duncani occurs in northern California and Washington State; B. divergens–like organisms are found in Missouri, Kentucky, and Washington state; and B. divergens and B. venatorum occur in Europe.^{56, 60} Isolated cases of babesiosis have also been reported in Asia, Africa, Australia, and South America.^{56, 60}

Clinical findings

The incubation period for B. microti infection can range from 1 to 9 weeks.⁵⁶ Infection is asymptomatic in perhaps a third of individuals, so the exact prevalence is unknown.^{56, 61} In other individuals, B. microti causes a mild to severe hemolytic anemia.^{56, 57} The patient usually experiences fever and nonrespiratory flu-like symptoms, including chills, headache, sweats, nausea, arthralgias, myalgia, anorexia, and fatigue, that last from several weeks to months. Jaundice, splenomegaly, or hepatomegaly may be present. Some individuals progress to severe, life-threatening disease due to acute respiratory failure, congestive heart failure, renal shutdown, liver failure, central nervous system involvement, or disseminated intravascular coagulation. Severe disease may occur at any age, but it is more common in individuals over 50 years of age.^{1, 56} Immune deficiency due to asplenia, malignancy, immunosuppressive drugs, or HIV infection increases the risk of severe disease.^{1, 56, 57} The overall mortality rate is less than 10% but is likely higher in immunocompromised individuals.^{56, 62}

Laboratory findings and diagnosis

Evidence of hemolytic anemia is usually present in symptomatic infection, including decreased hemoglobin level, increased reticulocyte count, decreased serum haptoglobin level, and bilirubinemia. Leukopenia, thrombocytopenia, hemoglobinuria, and proteinuria may also be present, along with abnormal results on renal and liver function tests.⁵⁶

The diagnosis of babesiosis is made by demonstration of the parasite on Wright-Giemsa–stained thin peripheral blood films. Babesia appear as tiny rings or occasionally as tetrads inside the RBCs. The ring forms may be round, oval, or ameboid; they have a dark purple chromatin dot and a minimal amount of blue cytoplasm surrounding a vacuole. Multiple rings can be found in one RBC (Figures 25-13 and 25-14). Tetrads may also appear in a “Maltese cross” formation. Babesia can be distinguished from P. falciparum by the pleomorphism of their ring forms, absence of hemozoin pigment and gametocytes, and occurrence of extraerythrocytic forms. Parasitemia may be low (fewer than 1% of RBCs affected) in early infections, but as many as 80% of RBCs may be infected in asplenic patients.⁶³ In cases of low parasitemia, babesiosis can be diagnosed by detection of IgG and IgM antibodies to B. microti by indirect immunofluorescent antibody assay.⁵⁷ The sensitivity of the assay ranges from 88% to 96%.⁶⁴ Definitive species identification requires PCR-based methods.^{56, 57}

FIGURE 25-13 Babesia microti ring forms in a peripheral blood film. Note the varying appearance of the ring forms and the presence of multiple ring forms in individual red blood cells (×1000).

FIGURE 25-14 Babesia microti ring and tetrad forms in a peripheral blood film (×1000).

Treatment

Babesiosis is treated with combination therapy using azithromycin/atovaquone or clindamycin/quinine.^{56, 57} Exchange transfusion is used when greater than 10% of RBCs are infected or when there is evidence of major organ failure.⁵⁶ Asymptomatic infections usually require no treatment.^{56, 57}

Clostridial sepsis

Sepsis with massive intravascular hemolysis, which is often fatal, is a rare complication of infection with Clostridium perfringens, an anaerobic gram-positive bacillus. C. perfringens grows very rapidly (7-minute doubling time) and produces an α-toxin with phospholipase C and sphingomyelinase activity that hydrolyzes RBC membrane phospholipids.^{65, 66} The RBCs become spherical and extremely susceptible to osmotic lysis, which results in sudden, massive hemolysis and dark red plasma and urine.^{1, 65, 67} The hematocrit may drop to below 10%.^{1, 67}The intravascular hemolysis can trigger DIC and renal failure. Spherocytes, microspherocytes, and toxic changes to neutrophils can be observed on a peripheral blood film.^{1, 65, 67} Some conditions that increase the risk of clostridial sepsis and hemolytic anemia include malignancies (genitourinary, gastrointestinal, and hematologic), solid organ transplantation, postpartum or postabortion infections, biliary surgery, acute cholecystitis, and deep wounds.^{1, 65, 68} Rapid therapy with transfusions, antibiotics, and fluid management is required. The prognosis is grave, and many patients die despite intensive treatment.

Bartonellosis

Human bartonellosis (Carrión disease) is transmitted by the bite of a female sandfly and is endemic in certain regions of Peru, Ecuador, and Colombia.^{69, 70} It is caused by Bartonella bacilliformis, a small, pleomorphic, intracellular coccobacillus that adheres to RBCs and causes hemolysis.^{1, 71} The bacteria produce a protein called deformin that forms pits or invaginations in the RBC membrane.^{1, 71} There are two clinical stages: the first stage is characterized by acute hemolytic anemia (called Oroya fever after a city in the Peruvian Andes); the second or chronic verruga stage is characterized by the eruption of skin lesions and warts on the extremities, face, and trunk. The acute phase begins with fever, malaise, headache, and chills, followed by pallor, jaundice, general lymphadenopathy, and, less commonly, hepatosplenomegaly.^69-71 Over several days, there is rapid hemolysis, with the hematocrit dropping below 20% in two thirds of patients.^{69, 71} Polychromasia, nucleated RBCs, and mild leukocytosis with a left shift are observed on the peripheral blood film. The mortality rate is approximately 10% for hospitalized patients and 90% for those who are untreated.^{69, 70} Diagnosis is made by blood culture and observation of bacilli or coccobacilli on the erythrocytes on a Wright-Giemsa–stained peripheral blood film. During the acute phase, 80% of the RBCs can be involved.¹ Serologic diagnosis with indirect immunofluorescent antibody or immunoblotting has a sensitivity of 89%.⁷⁰ The acute stage is treated with transfusions and ciprofloxacin or chloramphenicol.⁷⁰

Carrión disease was named for a Peruvian medical student, Daniel Alcides Carrión, who in 1885 inoculated himself with fluid from a wart of a patient in the chronic stage of infection. He subsequently developed fatal hemolytic anemia similar to that in Oroya fever, which linked the two stages of the disease to the same agent.^{71, 72}

Hemolytic anemia caused by other red blood cell injury

Drugs and chemicals

Hemolytic anemia of varying severity may result from drugs or chemicals that cause the oxidative denaturation of hemoglobin, leading to the formation of methemoglobin and Heinz bodies.¹ Examples of agents that can cause hemolytic anemia in individuals with normal RBCs include dapsone, a drug used to treat leprosy and dermatitis herpetiformis,^{1, 73} and naphthalene, a chemical found in mothballs.⁷⁴ Individuals deficient in G6PD are particularly sensitive to the effects of oxidative agents. For example, primaquine can cause hemolytic anemia in G6PD-deficient individuals (Chapter 24).³⁹

The typical laboratory findings include a decrease in hemoglobin level, increase in the reticulocyte count, increase in serum indirect bilirubin, and decrease in serum haptoglobin level. In severe drug- or chemical-induced hemolytic anemia, Heinz bodies (denatured hemoglobin) may be observed in RBCs. Heinz bodies can only be visualized with a supravital stain, and they appear as round, blue granules attached to the inner RBC membrane (Figure 14-11). Exposure to high levels of arsine hydride, copper, and lead can also cause hemolysis.^{1, 75}

Venoms

Envenomation from contact with snakes, spiders, bees, or wasps can induce hemolytic anemia in some individuals. The hemolysis can occur acutely or be delayed 1 or more days after a bite or sting.^{1, 75} The severity of the hemolysis depends on the amount of venom injected, and in severe cases, renal failure and death can result. Some mechanisms by which venoms can induce hemolysis are direct disruption of the RBC membrane, alteration of the RBC membrane that results in complement-mediated lysis, and initiation of DIC.⁷⁵ Hemolytic anemia has been reported after bites from poisonous snakes (e.g., some cobras and pit vipers) and the brown recluse spider (Loxosceles reclusa and Loxosceles laeta), and after multiple stings (50 or more) by bees or wasps.^{1, 75-79}

Extensive burns (thermal injury)

Warming normal RBCs to 49° C in vitro induces RBC fragmentation and budding.¹ Likewise, patients with extensive burns manifest similar RBC injury with acute hemolytic anemia. Schistocytes, spherocytes, and microspherocytes are observed on the peripheral blood film (Figure 25-15), but the damaged RBCs are usually cleared by the spleen within 24 hours of the burn injury.⁷⁵ In addition to resulting from the acute hemolysis, the anemia associated with extensive burns is also caused by blood loss during surgical excision and grafting of the burn wounds, nutritional deficiency, impaired metabolism, and anemia of chronic inflammation.⁸⁰ Overheating blood in malfunctioning blood warmers prior to transfusion can also result in RBC fragmentation and hemolysis of the donor RBCs.¹

FIGURE 25-15 Peripheral blood film for a patient with extensive burns. Note the presence of schistocytes, microspherocytes, and spherocytes (×1000).

Summary

• A common feature of the nonimmune extrinsic hemolytic anemias is the presence of a condition that causes physical or mechanical injury to the RBCs. These conditions include microangiopathic hemolytic anemia; macroangiopathic hemolytic anemia; some infections; exposure to certain drugs, chemicals, or venoms; and extensive burns.
• Microangiopathic hemolytic anemia is characterized by the shearing of RBCs as they pass through small blood vessels partially blocked by microthrombi. Fragmented RBCs (calledschistocytes) are formed, and the premature RBC destruction results in hemolytic anemia. Ischemic injury to the brain, kidney, and other organs also occurs. Thrombocytopenia also occurs as a result of consumption in the microthrombi. The major microangiopathic hemolytic anemias are thrombotic thrombocytopenic purpura (TTP), hemolytic uremic syndrome (HUS), HELLP (hemolysis, elevated liver enzymes, low platelet count) syndrome, and disseminated intravascular coagulation (DIC).
• TTP is a rare disorder found predominantly in adults and characterized by microangiopathic hemolytic anemia, severe thrombocytopenia, a markedly increased serum lactate dehydrogenase activity, and variable symptoms of fever, neurologic dysfunction, and renal failure. Idiopathic TTP is due to autoimmune antibodies to the VWF-cleaving protease ADAMTS-13 causing a severe functional deficiency. Secondary TTP is associated with stem cell transplantation, disseminated cancer, pregnancy, and use of certain drugs. Inherited TTP is due to mutations in the ADAMTS13gene.
• HUS is characterized by microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure. Typical HUS (stx-HUS) comprise 90% of cases, is found predominantly in young children, and is caused by toxin-producing strains of E. coli. Atypical HUS (aHUS) is due to inherited mutations in genes coding for complement components and regulators or autoantibodies to complement factor H. It also occurs secondary to organ transplantation, cancer, pregnancy, HIV infection, and some drugs.
• HELLP syndrome is a serious complication of pregnancy presenting with microangiopathic hemolytic anemia, thrombocytopenia, and elevated levels of liver enzymes.
• DIC is due to the widespread intravascular activation of the hemostatic system and formation of fibrin thrombi; coagulation factors and platelets are consumed in the thrombi, and secondary fibrinolysis occurs. Schistocytes are found in about half of the cases.
• Macroangiopathic hemolytic anemia is caused by traumatic cardiac hemolysis (RBC fragmentation from damaged or prosthetic cardiac valves) or exercise-induced hemolysis (mechanical trauma from forceful impact on feet or hands or from strenuous exercise). The platelet count is normal in both conditions; schistocytes are seen only in traumatic cardiac hemolysis.
• Infections associated with hemolytic anemia due to invasion of RBCs include malaria and babesiosis. Hemolysis in bartonellosis is due to attachment of the bacteria to red blood cells and production of a lytic protein. Hemolysis in clostridial sepsis is due to the production of α-toxin.
• In malaria, severe anemia is due to direct lysis of infected RBCs, immune destruction of infected and uninfected RBCs in the spleen, and inhibition of erythropoiesis.
• Plasmodium(five species) and Babesia organisms are identified by the morphology of their intraerythrocytic stages on a Wright-Giemsa–stained peripheral blood film. Plasmodiumspecies are transmitted to humans by mosquitoes, whereas a tick is the vector for Babesia.
• Hemolytic anemia can also be caused by injury to RBCs by drugs, chemicals, venoms, and extensive burns (thermal injury). In patients with extensive burns, schistocytes, spherocytes, and microspherocytes are observed on the peripheral blood film.

Now that you have completed this chapter, go back and read again the case study at the beginning and respond to the questions presented.

Review questions

Answers can be found in the Appendix.

1. Which one of the following is a feature found in allmicroangiopathic hemolytic anemias?
2. Pancytopenia
3. Thrombocytosis
4. Intravascular RBC fragmentation
5. Prolonged prothrombin time and partial thromboplastin time
6. Typical laboratory findings in TTP and HUS include:
7. Schistocytosis and thrombocytopenia
8. Anemia and reticulocytopenia
9. Reduced levels of lactate dehydrogenase and aspartate aminotransferase
10. Increased levels of free plasma hemoglobin and serum haptoglobin
11. The pathophysiology of idiopathic TTP involves:
12. Shiga toxin damage to endothelial cells and obstruction of small blood vessels in glomeruli
13. Formation of platelet-VWF thrombi due to autoantibody inhibition of ADAMTS-13
14. Overactivation of the complement system and endothelial cell damage due to loss of regulatory function
15. Activation of the coagulation and fibrinolytic systems with fibrin clots throughout the microvasculature
16. Which of the following tests yields results that are abnormal in DIC but are usually within the reference interval or just slightly abnormal in TTP and HUS?
17. Indirect serum bilirubin and serum haptoglobin
18. Prothrombin time and partial thromboplastin time
19. Lactate dehydrogenase and aspartate aminotransferase
20. Serum creatinine and serum total protein
21. Which one of the following laboratory results may be seen in BOTH traumatic cardiac hemolytic anemia and exercise-induced hemoglobinuria?
22. Schistocytes on the peripheral blood film
23. Thrombocytopenia
24. Decreased serum haptoglobin
25. Hemosiderinuria
26. Which of the following species of Plasmodiumproduce hypnozoites that can remain dormant in the liver and cause a relapse months or years later?
27. P. falciparum
28. P. vivax
29. P. knowlesi
30. P. malariae
31. Which one of the following is nota mechanism causing anemia in P. falciparum infections?
32. Inhibition of erythropoiesis
33. Lysis of infected RBCs during schizogony
34. Competition for vitamin B₁₂in the erythrocyte
35. Immune destruction of noninfected RBCs in the spleen
36. Which Plasmodiumspecies is widespread in Malaysia, has RBCs with multiple ring forms, has band-shaped early trophozoites, shows a 24-hour erythrocytic cycle, and can cause severe disease and high parasitemia?
37. P. falciparum
38. P. vivax
39. P. knowlesi
40. P. malariae
41. One week after returning from a vacation in Rhode Island, a 60-year-old man experienced fever, chills, nausea, muscle aches, and fatigue of 2 days’ duration. A complete blood count (CBC) showed a WBC count of 4.5 × 10⁹/L, hemoglobin level of 10.5 g/dL, a platelet count of 134 × 10⁹/L, and a reticulocyte count of 2.7%. The medical laboratory scientist noticed tiny ameboid ring forms in some of the RBCs and some tetrad forms in others. These findings suggest:
42. Bartonellosis
43. Malaria
44. Babesiosis
45. Clostridial sepsis
46. What RBC morphology is characteristically found within the first 24 hours following extensive burn injury?
47. Macrocytosis and polychromasia
48. Burr cells and crenated cells
49. Howell-Jolly bodies and bite cells
50. Schistocytes and microspherocytes
51. A 36-year-old woman was brought to the emergency department by her husband because she had experienced a seizure. He reported that she had been well until that morning, when she complained of a sudden headache and malaise. She was not taking any medications and had no history of previous surgery or pregnancy. Laboratory studies showed a WBC count of 15 × 10⁹/L, hemoglobin level of 7.8 g/dL, a platelet count of 18 × 10⁹/L, and schistocytes and helmet cells on the peripheral blood film. Chemistry test results included markedly elevated serum lactate dehydrogenase activity and a slight increase in the level of total and indirect serum bilirubin. The urinalysis results were positive for protein and blood, but there were no RBCs in the urine sediment. Prothrombin time and partial thromboplastin time were within the reference interval. When the entire clinical and laboratory picture is considered, which of the following is the most likely diagnosis?
52. HUS
53. HELLP syndrome
54. TTP
55. Exercise-induced hemoglobinuria

References

1. Price E.A, Schrier S.S. Extrinsic nonimmune hemolytic anemias. In: Hoffman R, Benz E.J, Jr Silberstein L.E, et al. HematologyBasic Principles and Practice 6th ed. Philadelphia : Saunders, an imprint of Elsevier 2013; 628-637.
2. Moake J.L. Thrombotic microangiopathies. N Engl J Med; 2002; 347:589-600.
3. Tsai H-M. Thrombotic thrombocytopenic purpura and the atypical hemolytic uremic syndrome. Hematol Oncol Clin N Am; 2013; 27:565-584.
4. Moake J. Thrombotic microangiopathiesmultimers, metalloprotease, and beyond. Clin Transl Sci; 2009; 2:366-373.
5. McCrae K.R, Sadler J.E, Cines D.B. Thrombotic thrombocytopenic purpura and the hemolytic uremic syndrome. In: Hoffman R, Benz E.J, Jr Silberstein L.E, et al. HematologyBasic Principles and Practice 6th ed. Philadelphia : Saunders, an imprint of Elsevier 2013; 1925-1939.
6. Crawley J.T.B, Scully M.A. Thrombotic thrombocytopenic purpurabasic pathophysiology and therapeutic strategies. Hematology Am Soc Hematol Educ Program 2013; 292-299.
7. Moake J.L, Rudy C.K, Troll J.H, et al. Unusually large plasma factor VIIIvon Willebrand factor multimers in chronic relapsing thrombotic thrombocytopenic purpura. N Engl J Med; 1982; 307:1432-1435.
8. Furlan M, Robles R, Galbusera M, et al. Von Willebrand factor–cleaving protease in thrombotic thrombocytopenic purpura and the hemolytic-uremic syndrome. N Engl J Med; 1998; 339:1578-1584.
9. Tsai H-M, Lian E C-Y. Antibodies to von Willebrand factor–cleaving protease in acute thrombotic thrombocytopenic purpura. N Engl J Med; 1998; 339:1585-1594.
10. Zhang Q, Zhou Y.F, Zhang C.Z, et al. Structural specializations of A2, a force-sensing domain in the ultralarge vascular protein von Willebrand factor. Proc Natl Acad Sci U S A; 2009; 106:9226-9231.
11. Kremer Hovinga JA, Vesely SK, Terrell DR, et al. Survival and relapse in patients with thrombotic thrombocytopenic purpura. Blood; 2010; 115:1500-1511.
12. Lotta L.A, Garagiola I, Palla R, et al. ADAMTS13 mutations and polymorphisms in congenital thrombotic thrombocytopenic purpura. Hum Mutat; 2010; 31:11-19.
13. Verbeke L, Delforge M, Dierickx D. Current insight into thrombotic thrombocytopenic purpura. Blood Coagul Fibrinolysis; 2010; 21:3-10.
14. Scheiring J, Rosales A, Zimmerhackl L.B. Clinical practicetoday’s understanding of the haemolytic uraemic syndrome. Eur J Pediatr; 2010; 169:7-13.
15. Petruzziello T.N, Mawji I.A, Khan M, et al. Verotoxin biologymolecular events in vascular endothelial injury. Kidney Int; 2009; 75:S17-S19.
16. Cataland S.R, Wu H.F. Diagnosis and management of complement mediated thrombotic microangiopathies. Blood Reviews; 2014; 28:67-74.
17. Haram K, Svendsen E, Abildgaard U. The HELLP syndromeclinical issues and management. BMC Pregnancy Childbirth; 2009; 9:1-15.
18. Baker K.R, Moake J. Hemolytic anemia resulting from physical injury to red cells. In: Lichtman M.A, Kipps T.J, Seligsohn U, Kaushansky K, Prchal J.T. Williams Hematology.8th ed. New York : McGraw-Hill 2010 Available at: http://accessmedicine.mhmedical.com.libproxy2.umdnj.edu/content.aspx?bookid=358& Sectionid=39835868 Accessed 16.04.14.
19. Mihu D, Costin N, Mihu C.M, et al. HELLP syndrome—a multisystemic disorder. J Gastrointest Liver Dis; 2007; 16:419-424.
20. Labelle C.A, Kitchens C.S. Disseminated intravascular coagulationtreat the cause, not the lab values. Cleve Clin J Med; 2005; 72:377-397.
21. Shapira Y, Vaturi M, Sagie A. Hemolysis associated with prosthetic heart valves. a review. Cardiol Rev; 2009; 17:121-124.
22. Davidson R.J.L. Exertional haemoglobinuriaa report on three cases with studies on the haemolytic mechanism. J Clin Pathol; 1964; 17:536-540.
23. Telford R.D, Sly G.J, Hahn A.G, et al. Footstrike is the major cause of hemolysis during running. J Appl Physiol; 2003; 94:38-42.
24. Selby G.B, Eichner E.R. Endurance swimming, intravascular hemolysis, anemia, and iron depletion. New perspective on athlete’s anemia. Am J Med; 1986; 81:791-794.
25. Shaskey D.J, Green G.A. Sports haematology. Sports Med; 2000; 29:27-38.
26. Tobal D, Olascoaga A, Moreira G, et al. Rust urine after intense hand drumming is caused by extracorpuscular hemolysis. Clin J Am Soc Nephrol; 2008; 3:1022-1027.
27. Smith J.A, Kolbuch-Braddon M, Gillam I, et al. Changes in the susceptibility of red blood cells to oxidative and osmotic stress following submaximal exercise. Eur J Appl Physiol; 1995; 70:427-436.
28. Beneke R, Bihn D, Hütler M, et al. Haemolysis caused by alterations of a- and b-spectrin after 10 to 35 min of severe exercise. Eur J Appl Physiol; 2005; 95:307-312.
29. Yusof A, Leithauser R.M, Roth H.J, et al. Exercise-induced hemolysis is caused by protein modification and most evident during the early phase of an ultraendurance race. J Appl Physiol; 2007; 102:582-586.
30. Singh B, Lee K.S, Matusop A, et al. A large focus of naturally acquired Plasmodium knowlesi infections in human beings. Lancet; 2004; 363:1017-1024.
31. Cox-Singh J, Davis T.M.E, Lee K.S, et al. Plasmodium knowlesi malaria in humans is widely distributed and potentially life-threatening. Clin Infect Dis; 2008; 46:165-171.
32. Lee K.S, Cox-Singh J, Brooke G, et al. Plasmodium knowlesi from archival blood filmsfurther evidence that human infections are widely distributed and not newly emergent in Malaysian Borneo. Int J Parasitol; 2009; 39:1125-1128.
33. World Health Organization. World malaria report 2013. Geneva : WHO Press 2013 Available at: http://www.who.int/malaria/publications/world_malaria_report_2013/report/en/Accessed 19.04.14.
34. Centers for Disease Control and Prevention. Malaria facts. Retrieved from Available at: http://www.cdc.gov/malaria/about/facts.xhtml2014 Accessed 20.04.14.
35. Miller L.H, Baruch D.I, Marsh K, et al. The pathogenic basis of malaria. Nature; 2002; 415:673-679.
36. Wellems T.E, Hayton K, Fairhurst R.M. The impact of malaria parasitismfrom corpuscles to communities. J Clin Invest; 2009; 119:2496-2505.
37. Garcia L.S. Malaria. Clin Lab Med; 2010; 30:93-129.
38. Ekvall H. Malaria and anemia. Curr Opin Hematol; 2003; 10:108-114.
39. World Health Organization. Guidelines for treatment of malaria. 2nd ed. Geneva : WHO Press 2010.
40. Haldar K, Mohandas N. Malaria, erythrocytic infection, and anemia. Hematology Am Soc Hematol Educ Program2009; 87-93.
41. Waitumbi J.N, Opollo M.O, Muga R.O, et al. Red cell surface changes and erythrophagocytosis in children with severe Plasmodium falciparum anemia. Blood; 2000; 95:1481-1486.
42. Griffiths M.J, Ndungu F, Baird K.L, et al. Oxidative stress and erythrocyte damage in Kenyan children with severe Plasmodium falciparum malaria. Br J Haematol; 2001; 113:486-491.
43. Craig A, Scherf A. Molecules on the surface of the Plasmodium falciparum infected erythrocyte and their role in malaria pathogenesis and immune evasion. Mol Biochem Parasitol; 2001; 115:129-143.
44. Bridges D.J, Bunn J, van Mourik J.A, et al. Rapid activation of endothelial cells enables Plasmodium falciparum adhesion to platelet-decorated von Willebrand factor strings. Blood; 2010; 115:1472-1474.
45. Aird W.C, Mosnier L.O, Fairhurst R.M. Plasmodium falciparum picks (on) EPCR. Blood; 2014; 123:163-167.
46. Ayi K, Turrini F, Piga A, et al. Enhanced phagocytosis of ring-parasitized mutant erythrocytesa common mechanism that may explain protection against falciparum malaria in sickle trait and beta-thalassemia trait. Blood; 2004; 104:3364-3371.
47. Fairhurst R.M, Baruch D.I, Brittain N.J, et al. Abnormal display of PfEMP-1 on erythrocytes carrying haemoglobin C may protect against malaria. Nature; 2005; 435:1117-1121.
48. Cholera R, Brittain N.J, Gillrie M.R, et al. Impaired cytoadherence of Plasmodium falciparum–infected erythrocytes containing sickle hemoglobin. Proc Natl Acad Sci U S A; 2008; 105:991-996.
49. Chotivanich K, Udomsangpetch R, Pattanapanyasat K, et al. Hemoglobin Ea balanced polymorphism protective against high parasitemias and thus severe P. falciparum malaria.Blood; 2002; 100:1172-1176.
50. Centers for Disease Control and Prevention. Diagnostic procedures—blood specimensspecimen collection. Retrieved from Available at:http://www.cdc.gov/dpdx/diagnosticProcedures/blood/specimencoll.xhtml Accessed 20.04.14.
51. Centers for Disease Control and Prevention. Diagnostic procedures—blood specimensmicroscopic examination. Retrieved from Available at:http://www.cdc.gov/dpdx/diagnosticProcedures/blood/microexam.xhtml Accessed 20.04.14.
52. Centers for Disease Control and Prevention. Malaria map. Retrieved from Available at: http://cdc-malaria.ncsa.uiuc.eduAccessed 20.04.14.
53. Centers for Disease Control and Prevention. Diagnostic procedures—blood specimensdetection of parasite antigens. Retrieved from Available at:http://www.cdc.gov/dpdx/diagnosticProcedures/blood/antigendetection.xhtml Accessed 20.04.14.
54. Centers for Disease Control and Prevention (2013, July 1). Guidelines for treatment of malaria in the United States. Retrieved from Available at:http://www.cdc.gov/malaria/resources/pdf/treatmenttable.pdfAccessed 20.04.14.
55. Ruebush T.K, II Juranek D.D, Spielman A, et al. Epidemiology of human babesiosis on Nantucket Island. Am J Trop Med Hyg; 1981; 30:937-941.
56. Vannier E, Krause P.J. Update on babesiosis. Interdiscip Perspect Infect Dis; 2009; 2009:984568.
57. Centers for Disease Control and Prevention. Babesiosis. Retrieved from Available at: http://www.cdc.gov/babesiosis/Accessed 20.04.14.
58. Centers for Disease Control and Prevention. Babesiosis and the U.S. blood supply. Available at: http://www.cdc.gov/parasites/babesiosis/resources/babesiosis_policy_brief.pdfAccessed 20.04.14.
59. Sethi S, Alcid D, Kesarwala H, et al. Probable congenital babesiosis in infant, New Jersey, USA. Emerg Infect Dis; 2009; 15:788-791.
60. Hildebrandt A, Gray J.S, Hunfeld K.P. Human babesiosis in Europewhat clinicians need to know. Infection; 2013; 41:1057-1072.
61. Krause P.J, McKay K, Gadbaw J, et al. Increasing health burden of human babesiosis in endemic sites. Am J Trop Med Hyg; 2003; 68:431-436.
62. Meldrum S.C, Birkhead G.S, White D.J, et al. Human babesiosis in New York Statean epidemiological description of 136 cases. Clin Infect Dis; 1992; 15:1019-1023.
63. Blevins S.M, Greenfield R.A, Bronze M.S. Blood smear analysis in babesiosis, ehrlichiosis, relapsing fever, malaria, and Chagas disease. Cleve Clin J Med; 2008; 75:521-530.
64. Krause P.J, Telford S.R, 3rd. Ryan R, et al. Diagnosis of babesiosisevaluation of a serologic test for the detection of B. microti antibody. J Infect Dis; 1994; 169:923-926.
65. Kapoor J.R, Montiero B, Tanoue L, et al. Massive intravascular hemolysis and a rapid fatal outcome. Chest; 2007; 132:2016-2019.
66. Urbina P, Flores-Díaz M, Alape-Girón A, et al. Phospholipase C and sphingomyelinase activities of the Clostridium perfringens (a-toxin. Chem Phys Lipids; 2009; 159:51-57.
67. Merino A, Pereira A, Castro P. Massive intravascular haemolysis during Clostridium perfringens sepsis of hepatic origin. Eur J Haematol; 2009; 84:278-279.
68. Barrett J.P, Whiteside J.L, Boardman L.A. Fatal clostridial sepsis after spontaneous abortion. Obstet Gynecol; 2002; 99:899-901.
69. Maguina C, Gotuzzo E. Bartonellosis new and old. Infect Dis Clin North Am; 2000; 14:1-22.
70. Huarcaya E, Maguina C, Torres R, et al. Bartonellosis (Carrion’s disease) in the pediatric population of Peruan overview and update. Braz J Infect Dis; 2004; 8:331-339.
71. Lichtman M.A. Hemolytic anemia resulting from infections with microorganisms. In: Lichtman M.A, Kipps T.J, Seligsohn U, Kaushansky K, Prchal J.T. Williams Hematology.8th ed. New York : McGraw-Hill 2010 Available at: http://accessmedicine.mhmedical.com.libproxy2.umdnj.edu/content.aspx?bookid=358& Sectionid=39835870 Accessed 16.04.14.
72. Garcia-Caceres U, Garcia F.U. Bartonellosis. An immunodepressive disease and the life of Daniel Alcides Carrion. Am J Clin Pathol; 1991; 95:S58-S66.
73. Ranawaka R.R, Mendis S, Weerakoon H.S. Dapsone-induced haemolytic anemia, hepatitis and agranulocytosis in a leprosy patient with normal glucose-6-phosphate-dehydrogenase activity.Lepr Rev; 2008; 79:436-440.
74. Lim H.C, Poulose V, Tan H.H. Acute naphthalene poisoning following the non-accidental ingestion of mothballs. Singapore Med J; 2009; 50:e298-e301.
75. Bull B.S, Herrmann P.C. Hemolytic anemia resulting from chemical and physical agents. In: Lichtman M.A, Kipps T.J, Seligsohn U, Kaushansky K, Prchal J.T. Williams Hematology.8th ed. New York : McGraw-Hill 2010 Available at: http://accessmedicine.mhmedical.com.libproxy2.umdnj.edu/content.aspx?bookid=358& Sectionid=39835869 Accessed 16.04.14.
76. Athappan G, Balaji M.V, Navaneethan U, et al. Acute renal failure in snake envenomationa large prospective study. Saudi J Kidney Dis Transpl; 2008; 19:404-410.
77. McDade J, Aygun B, Ware R.E. Brown recluse spider (Loxosceles reclusa) envenomation leading to acute hemolytic anemia in six adolescents. J Pediatr; 2010; 156:155-157.
78. Kolecki P. Delayed toxic reaction following massive bee envenomation. Ann Emerg Med; 1999; 33:114-116.
79. Paudel B, Paudel K. A study of wasp bites in a tertiary hospital of western Nepal. Nepal Med Coll J; 2009; 11:52-56.
80. Posluszny J.A, Gamelli R.L. Anemia of thermal injurycombined acute blood loss anemia and anemia of critical illness. J Burn Care Res; 2010; 31:229-242.