erythrocyte destruction
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Introduction to Increased Destruction of Erythrocytes
Excessive Fragmentation (Intravascular) Hemolysis
Clinical Features
Laboratory Findings
Tests of Accelerated Red Blood Cell Destruction
Tests of Increased Erythropoiesis
Laboratory Tests to Identify Specific Hemolytic Processes
Differential Diagnosis
Classification
Hemolysis
Normal Macrophage-Mediated Hemolysis and Bilirubin Metabolism
Plasma Hemoglobin Salvage During Normal Fragmentation Hemolysis
Excessive Macrophage-Mediated (Extravascular) Hemolysis
Objectives
Define hemolysis and recognize its hallmark clinical findings.
Differentiate a hemolytic disorder from hemolytic anemia by definition and recognition of laboratory findings.
Discuss methods of classifying hemolytic anemias and apply the classification to an unfamiliar anemia.
Describe the processes of fragmentation (intravascular) and macrophage-mediated (extravascular) hemolysis, including sites of hemolysis, catabolic products, and time frame for the appearance of those products after hemolysis.
Describe protoporphyrin catabolism (bilirubin production), including metabolites and their sites of production and excretion.
Describe mechanisms that salvage hemoglobin and heme during fragmentation hemolysis.
Describe changes to bilirubin metabolism and iron salvage systems that occur when the rate of fragmentation or macrophage-mediated hemolysis increases.
Identify, explain the diagnostic value, and interpret the results of laboratory tests that indicate increased hemolysis and erythropoiesis.
Differentiate between hemolytic anemias and other causes of increased erythropoiesis given laboratory or clinical information.
Differentiate between hemolytic anemias and other causes of bilirubinemia given laboratory or clinical information.
Interchange conjugation terminology for bilirubin fractions with Van den Bergh reaction terminology.
Explain the principle of the Van den Bergh reaction to quantitate and fractionate bilirubin in body fluids.
Case Study
A 34-year-old woman admitted to the hospital for a vaginal hysterectomy.
Excessive menstrual bleeding.
Preoperative laboratory test results within reference intervals.
No excessive blood loss during or after surgery.
Recovery was uneventful except for some expected pain.
Three days after surgery, abdominal pain and "root beer"-colored urine.
CBC revealed a hemoglobin level of 5.8 g/dL.
Questions:
What process is indicated by the root beer-colored urine?
What laboratory tests can be used to differentiate the cause of the hemolysis?
Based on the patient's clinical presentation, predict the results expected for each test listed for question 2.
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Classification
Many anemias have a hemolytic component.
Hemolysis alone does not cause anemia in certain conditions.
Anemias with a secondary hemolytic component.
Hemolysis is the primary feature in some anemias.
Classification:
Acute versus chronic
Inherited versus acquired
Intrinsic versus extrinsic
Intravascular versus extravascular
Fragmentation versus macrophage-mediated
Table 20.1 shows a noncomprehensive list of hemolytic anemias categorized in this way.
Focus on the mechanism of hemolysis, specifically fragmentation and macrophage-mediated hemolytic conditions.
Acute versus chronic hemolysis delineates the clinical presentation.
Chronic hemolysis may be punctuated over time with hemolytic crises that cause anemia.
Glucose-6-phosphate dehydrogenase deficiency is an example of chronic hemolysis.
Classification of Selected Hemolytic Anemias by Primary Cause and Type of Hemolysis:
Extrinsic defects
Intrinsic defects
Hereditary conditions
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Acute hemolytic events can occur due to antimalarial drugs
Compensation returns when the drug is withdrawn
Chronic conditions can result in severe anemia
Bone marrow cannot generate cells fast enough to compensate
Thalassemia major is an example of a condition with chronically low oxygen-carrying capacity
RBC production is brisk, but each cell has an inadequate complement of one type of globin chain
Functional hemoglobin production is decreased overall
Inherited hemolytic conditions, such as thalassemia, are passed to offspring by mutant genes
Acquired hemolytic disorders develop in individuals who acquire an agent or condition that lyses RBCs
Malaria is an example of an infectious disease causing hemolytic disorders
Hemolytic disorders can be intrinsic or extrinsic
Intrinsic defects are in the RBC itself, while extrinsic defects are caused by external agents
Intrinsic defects include abnormalities of the RBC membrane, enzymatic pathways, or the hemoglobin molecule
Extrinsic hemolytic conditions are caused by substances in plasma or conditions affecting the circulatory system
Most intrinsic defects are inherited, while most extrinsic ones are acquired
HEMOLYSIS Normal Macrophage-Mediated Hemolysis and Bilirubin Metabolism
Detection of hemolysis depends on the detection of RBC breakdown products, such as bilirubin
RBCs live approximately 120 days and undergo metabolic and chemical changes
Macrophages recognize these changes and phagocytize aged erythrocytes, leading to macrophage-mediated hemolysis
The spleen, bone marrow, liver, lymph nodes, and circulating monocytes are involved in this process
Majority of RBC degradation occurs inside macrophages
Hemoglobin is hydrolyzed into heme and globin, with globin further degraded into amino acids
Iron is released from heme and recycled to needy cells
Protoporphyrin component is catabolized and the products are processed in the liver
Bilirubin and urobilinogen are excretory products derived from the protoporphyrin component of heme
Introduction to Increased Destruction of Erythrocytes
Blood circulation in the liver
Blood enters the liver lobule at the periphery via a hepatic artery and portal vein
Blood from each vessel enters the sinusoids between rows of hepatocytes
Sinusoids have a loose structure, allowing blood to percolate through the lobule
Blood drains into progressively larger veins that leave the liver to enter the inferior vena cava
Bilirubin metabolism in the liver
Bilirubin formed in macrophages enters the liver sinusoid via the hepatic artery
In the liver sinusoid, bilirubin dissociates from albumin
Bilirubin is carried into the hepatocyte by organic anion transporter (OAT) proteins
Inside the hepatocyte, bilirubin is bound to glutathione S-transferase for transport to the endoplasmic reticulum
Unconjugated bilirubin is joined with glucuronic acid by UDP glucuronosyltransferase to form conjugated bilirubin
Conjugated bilirubin is excreted by hepatocytes into the canaliculi by multi-drug resistant protein 2 (MRP2)
Conjugated bilirubin continues down to the common bile duct and eventually into the intestines
Structure of the liver lobule
Small ducts called canaliculi run from the central vein outward between hepatocytes
Canaliculi receive bile excreted by hepatocytes and channel it toward the exterior of the lobule
Canaliculi join into larger bile ducts that collect the bile into the common bile duct
Normal Catabolism of Hemoglobin
Macrophages lyse ingested red blood cells (RBCs) and separate hemoglobin into globin chains and heme components
Amino acids from the globin chains are reused
Heme is degraded to iron and protoporphyrin
Iron is returned to the blood to be reused
Protoporphyrin is degraded to unconjugated bilirubin
Catabolism of Heme to Bilirubin
In cells containing heme oxygenase, iron is removed from heme and the protoporphyrin ring is opened up to form biliverdin
Biliverdin is converted to unconjugated bilirubin by biliverdin reductase
Unconjugated bilirubin is secreted into the blood and binds to albumin for transport to the liver
UGT1A1 adds glucuronic acid to form conjugated bilirubin when it enters the hepatocyte
Visualizing the Color Changes of Hemoglobin Degradation
The degradation of heme can be seen in bruises or in the sclera of the eye after a vascular bleed
Initially, extravasated but deoxygenated blood gives the injury a purple-red appearance
As hemoglobin is degraded, the color changes to a greenish hue due to biliverdin
Ultimately, the color becomes yellow due to bilirubin
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Hepatocytes conjugate bilirubin in the blood entering the sinus
They are not always able to export all of it into the bile duct
Conjugated bilirubin is exported into sinusoidal blood via MRP3
Conjugated bilirubin is absorbed by downstream hepatocytes
OATP1B1 protein in the sinusoidal membrane facilitates absorption
Absorbed, conjugated bilirubin can be exported into canaliculi by downstream hepatocytes
BOX 20.2 Laboratory Testing for Serum Bilirubin
Total serum bilirubin level is the sum of conjugated and unconjugated forms
Most of the total bilirubin is composed of the unconjugated form in transit from macrophages to the liver
Typical reference intervals:
Total serum bilirubin level: 0.5-1.0 mg/dL
Direct (conjugated) serum bilirubin level: 0-0.2 mg/dL
Indirect (unconjugated) serum bilirubin level: 0-0.8 mg/dL
Conjugated bilirubin reacts well in water-based spectrophotometric assay
Unconjugated bilirubin requires alcohol to promote solubility in water
Total bilirubin level is measured by adding a solubilizing reagent
Direct bilirubin is measured alone without the solubilizing agent
Indirect bilirubin level is calculated by subtracting the direct value from the total value
Figure 20.3 Normal Macrophage-Mediated Hemolysis
In a macrophage, hemoglobin is degraded to heme and unconjugated bilirubin
Unconjugated bilirubin binds to albumin for transport to the liver
Hepatocytes convert unconjugated bilirubin to conjugated bilirubin
Conjugated bilirubin leaves the liver in bile and enters the small intestine
Bacteria in the large intestine convert conjugated bilirubin to urobilinogen
Most urobilinogen is excreted in the stool, some is reabsorbed in the portal circulation
A small component of reabsorbed urobilinogen is filtered and excreted in the urine
Note: This transcript provides information about the metabolism and testing of bilirubin, as well as the normal process of macrophage-mediated hemolysis.
Page 6: Introduction to Increased Destruction of Erythrocytes
Conjugated bilirubin is oxidized by gut bacteria into urobilinogen
Urobilinogen is further oxidized to stercobilin and similar compounds that give stool its brown color
Some conjugated bilirubin and urobilinogen molecules are absorbed into the blood from the intestines
Absorbed by osmosis into the blood of the portal circulation
Bilirubin derivatives enter the liver via the portal veins and flow into the sinusoidal blood
Most of the conjugated bilirubin in the sinusoidal blood is absorbed into hepatocytes and recycled into bile
Urobilinogen is similarly recycled into bile
A small amount of direct bilirubin and urobilinogen remains in the blood entering the central vein
Filtered at the renal glomerulus and excreted in urine
Urobilin, a stool derivative of urobilinogen, gives urine its yellow color
Plasma Hemoglobin Salvage During Normal Fragmentation Hemolysis
Fragmentation hemolysis is the result of trauma to the RBC membrane, causing hemoglobin to spill into plasma
Approximately 10% to 20% of normal RBC destruction is via fragmentation
Hemoglobin is filtered through the glomerulus, leading to potential iron loss
Free hemoglobin and heme can scavenge nitric oxide and cause oxidative damage to cells
Haptoglobin-hemopexin-methemalbumin system salvages hemoglobin iron and prevents oxidation reactions
Hemoglobin binds to haptoglobin in plasma, avoiding filtration at the glomerulus and saving iron from urinary loss
Haptoglobin-hemoglobin complex is taken up by macrophages, where iron is salvaged and protoporphyrin is converted to unconjugated bilirubin
Hemopexin binds to free plasma hemoglobin, preventing oxidant injury to cells and tissues
Hemoglobinemia can lead to falsely elevated hemoglobin values in blood tests
Haptoglobin and hemopexin have therapeutic applications in reducing tissue damage during fragmentation hemolysis
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Unconjugated bilirubin is produced from the degradation of hemoglobin (Hb) and haptoglobin (Hpt) complex
The complex of hemoglobin-haptoglobin (Hb-Hpt) binds to CD163 on macrophages in various organs
The complex is internalized into the macrophage, releasing the hemoglobin dimer
The released hemoglobin dimer is degraded to heme, releasing iron and converting the protoporphyrin ring to unconjugated bilirubin
Haptoglobin is degraded
Unconjugated bilirubin is bound to albumin and processed through the liver
Free hemoglobin released into the blood with fragmentation forms methemoglobin
Hemopexin binds to free methemoglobin and forms a complex
The hemopexin-methemoglobin complex binds to CD91 on hepatocytes
The complex is internalized into the hepatocyte, releasing iron and converting the protoporphyrin ring to unconjugated bilirubin
Hemopexin is recycled to the blood
Figure 20.4 Normal Fragmentation Hemolysis
A small number of red blood cells lyse within the circulation, forming schistocytes and releasing hemoglobin (Hb) into the blood, mostly as a/b dimers.
Haptoglobin (Hpt) binds a hemoglobin dimer in a complex.
The hemoglobin-haptoglobin complex binds to CD163 on the surface of macrophages in various organs.
The complex is internalized into the macrophage, where the hemoglobin dimer is released.
The hemoglobin dimer is degraded to heme, releasing iron and converting the protoporphyrin ring to unconjugated bilirubin.
Haptoglobin is degraded.
Unconjugated bilirubin released into the blood is bound to albumin and processed through the liver.
Free hemoglobin released into the blood with fragmentation forms methemoglobin.
Hemopexin binds free methemoglobin into a complex.
The hemopexin-methemoglobin complex binds to CD91 on the surface of hepatocytes.
The complex is internalized into the hepatocyte.
Iron is released from the methemoglobin, and the protoporphyrin ring is converted to unconjugated bilirubin.
Note: Metheme can also bind to albumin, forming metheme-albumin, but this complex is temporary because metheme is rapidly transferred to hemopexin. (Adapted from Brunzel, N. A. [2013]. Fundamentals of Urine and Body Fluid Analysis. [3rd ed., p. 141, Figure 7-8]. St. Louis: Elsevier.)
Introduction to Increased Destruction of Erythrocytes
Hemopexin as a therapy for metheme toxicity (Page 8)
Hemopexin binds to ligand nonspecific CD91 receptor, LRP1, on hepatocytes
Hemopexin-metheme complex is internalized by endocytosis
Fate of internalized heme is still being researched
Hemopexin is recycled to blood from the hepatocyte
Metheme-albumin system for iron salvage (Page 8)
Albumin acts as a carrier for metheme in plasma
Metheme is rapidly transferred to hemopexin when available
Hemopexin-metheme complex travels to the liver for processing
Sequential and simultaneous salvage systems (Page 8)
Prevailing view: Haptoglobin binds hemoglobin, then hemopexin binds metheme, and then albumin binds metheme
Recent theories suggest these systems work simultaneously, especially during accelerated hemolysis
Hemopexin acts to prevent heme toxicity at all times
Renal handling of iron (Page 8)
Proximal tubular cells can reabsorb iron in various forms during excessive hemolysis
Iron staining of tubular cells in urinary sediment shows presence of hemosiderin
Small amount of filtered transferrin is salvaged by transferrin receptors on proximal tubular cells
Ferroportin on basolateral membrane of proximal tubular cells allows transfer of salvaged iron back into the blood
Excessive Macrophage-Mediated (Extravascular) Hemolysis
Increased macrophage-mediated hemolysis (Page 8)
Many hemolytic anemias result from increased macrophage-mediated hemolysis
Senescent RBCs and pathologic processes lead to expression of surface markers for removal by macrophages
If affected cells increase beyond normal senescence removal, anemia develops
Mechanisms of macrophage-mediated hemolysis (Page 8)
Excessive oxidation of hemoglobin leads to increased formation of Heinz bodies, which bind to RBC membrane and cause changes detected by macrophages
Intracellular parasites, complement, or immunoglobulins on RBC surface can also trigger macrophage-mediated hemolysis
Ingested RBCs are lysed within phagolysosomes in macrophages, contents are processed within the macrophage
Spherocytes and survival in circulation (Page 8)
Macrophage-mediated hemolysis often occurs in the spleen and liver, where macrophages possess receptors for senescent RBC markers
Macrophages may ingest a portion of the RBC membrane, leaving the remainder to reseal and form spherocytes
Spherocytes have shortened survival due to rigidity and inability to traverse the splenic sieve, may be fully ingested or mechanically lyse
Bilirubin levels in macrophage-mediated hemolytic anemias (Page 8)
Total plasma bilirubin level rises as RBCs are lysed prematurely
Increase in unconjugated bilirubin due to liver processing of increased load, producing more conjugated bilirubin
Increased urobilinogen forms in intestines, absorbed by portal circulation and excreted by kidney
Increased direct bilirubin absorbed into portal circulation but reprocessed through hepatocytes and into bile
Increased urobilinogen detectable in urine, unconjugated bilirubin bound to albumin and cannot pass through glomerulus
Role of hemopexin in macrophage-mediated hemolysis (Page 8)
Hemopexin may play an important role in preventing heme toxicity to macrophages when they ingest multiple erythrocytes
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Main ideas:
Excess macrophage-mediated hemolysis
Laboratory findings indicating accelerated red blood cell destruction in fragmentation versus macrophage-mediated hemolysis
Supporting details:
More than the usual number of red blood cells are ingested each day by macrophages
Increased amount of unconjugated bilirubin is produced and released into the blood
Increased unconjugated bilirubin is presented to the liver, resulting in increased amount of conjugated bilirubin
Increased conjugated bilirubin in the intestine leads to increased urobilinogen formation and excretion in the stool
Increased urobilinogen in the intestine results in increased urobilinogen reabsorbed into the blood
Increased urobilinogen in the blood leads to increased urobilinogen filtered and excreted in the urine
Comparison of laboratory findings indicating accelerated red blood cell destruction in fragmentation versus macrophage-mediated hemolysis
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Main ideas:
Excess fragmentation hemolysis: the role of macrophages
Excessive fragmentation (intravascular) hemolysis
Excess fragmentation hemolysis: the role of the liver
Supporting details:
Increased number of red blood cells lyse by fragmentation, releasing more hemoglobin into the blood
Haptoglobin binds the increased hemoglobin dimers, forming more complexes
Hemoglobin-haptoglobin complexes are taken up by macrophages bearing the CD163 receptor
Increased amount of hemoglobin dimers is released from the complexes, degraded to heme, and converted to unconjugated bilirubin
Unconjugated bilirubin is transported to the liver and processed as with excess macrophage-mediated hemolysis
Excessive fragmentation hemolysis is characterized by the appearance of hemoglobin in the plasma and the development of (met)hemoglobinemia
Iron salvage proteins form complexes with their respective ligands
Degradation of haptoglobin is accelerated compared to normal
If the amount of hemoglobin released from lysing red blood cells exceeds the capacity of haptoglobin, unbound free hemoglobin is rapidly oxidized, forming methemoglobin
Hemopexin binds to methemoglobin and is captured by the CD91 receptor on hepatocytes
The complex is internalized by the hepatocyte, iron is released, and the protoporphyrin ring is converted to unconjugated bilirubin
A small amount of hemopexin is recycled to the blood, while most is degraded
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Unconjugated bilirubin is produced during heme degradation in the liver
Circulates in the blood
Conjugated bilirubin is produced in the liver and excreted in the urine and feces
Urobilinogen levels are increased in both urine and feces
Urine bilirubin levels are negative
Hemoglobinuria occurs when excess hemoglobin is reabsorbed into the circulation
Schistocytes are fragmented red blood cells
Excess hemolysis leads to hemoglobinuria
Page 13: Fate of Iron Removed from Salvaged Hemoglobin in the Kidney
Iron salvaged from absorbed hemoglobin can be transported into the circulation
Binds to transferrin for transport
Excess iron is stored as ferritin and some is converted to hemosiderin
Hemosiderin can be detected in urine sediment using the Prussian blue stain
Haptoglobin and hemopexin levels decrease when there are high levels of metheme
Hemoglobin and heme can appear in the urine (hemoglobinuria) if the salvage capacity of plasma proteins is exceeded
Iron-containing proteins are absorbed into proximal tubular cells
Proximal tubular cells possess receptors for reabsorption of iron-containing proteins
Proximal tubular cells can catabolize heme and salvage iron for export to the plasma
Bilirubin conjugation and disposition in proximal tubular cells is uncertain
Excess iron in proximal tubular cells is stored as ferritin and some is converted to hemosiderin
Hemosiderin can be detected in urine sediment using the Prussian blue stain
Elevated levels of plasma indirect bilirubin and urinary urobilinogen are measurable
Time course of findings assists with differential diagnosis
Rise in reticulocytes several days later would also be seen after an acute onset
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Clinical Features
Hemolysis may be the primary cause of anemia
Symptoms: fatigue, dyspnea, dizziness
Signs: pallor, tachycardia
Increase in plasma bilirubin leads to yellow tinge in plasma and body tissues
Jaundice: yellow color of skin and sclera
Icterus: yellow plasma and tissues
Jaundice can occur in conditions other than hemolysis
Jaundice in hemolysis is called hemolytic jaundice or prehepatic jaundice
Unconjugated bilirubin can lead to brain damage in newborns (kernicterus)
Frequency or constancy of jaundice provides clues to the cause
Splenomegaly and gallstones can develop with chronic hemolysis
Bone deformities can occur in children with chronic hemolysis
Acute hemolysis can be confused with an acute infectious process
Acute fragmentation hemolysis can lead to renal failure
Laboratory Findings
Tests of Accelerated Red Blood Cell Destruction
Bilirubin: increased unconjugated bilirubin in hemolysis
Urine Hemoglobin and Urine Hemosiderin: indicative of fragmentation hemolysis
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Elevated plasma glucose levels lead to an increase in glycated hemoglobin value.
Coexistence of hemolysis with diabetes leads to falsely lowered glycated hemoglobin values.
This is a recognized problem in the interpretation of glycated hemoglobin values for glucose control.
RBC survival assay using random labeling of blood with chromium-51 radioisotope is the reference method for RBC survival studies.
Blood is collected, mixed with the isotope, and returned to the patient.
The labeled cells reflect normal peripheral blood.
Disappearance of the label from the blood is measured over time.
Lactate dehydrogenase activity is often increased in patients with fragmentation hemolysis.
This is due to the release of the enzyme from ruptured RBCs.
Other conditions such as myocardial infarction or liver disease can also cause increases in lactate dehydrogenase.
Increased erythropoiesis is observed in hemolytic processes.
Hypoxia associated with hemolysis leads to increased erythropoiesis.
Laboratory findings indicating increased erythropoiesis include an increase in circulating reticulocytes and nucleated RBCs.
A complete blood count (CBC) can provide clues to the cause of hemolysis.
Hemoglobin, hematocrit, and number of RBCs reflect whether hemolysis has caused anemia.
Spherocytes are seen with macrophage-mediated hemolysis, while schistocytes are noted with fragmentation hemolysis.
Haptoglobin can be quantified to indicate fragmentation hemolysis.
A substantial decline in serum haptoglobin level indicates fragmentation hemolysis.
Low haptoglobin levels suggest hemolysis but may also be due to impaired synthesis caused by liver disease.
Hemopexin may demonstrate a low value in hemolysis.
Hemopexin assays may be more valuable for detection of hemopexin depletion in conditions such as sepsis.
Tests to determine the rate of endogenous carbon monoxide production have been developed.
Carbon monoxide is produced in the first step of heme breakdown by heme oxygenase.
Increased carbon monoxide production has been detected in some patients with hemolytic anemia.
Glycated hemoglobin levels can be used as an indicator of RBC survival.
Glycated hemoglobin increases over the life of a cell as it is exposed to plasma glucose.
The glycated hemoglobin level is usually decreased in chronic hemolytic disease.
There is significant variability in RBC lifespan among hematologically normal individuals, making the use of glycated hemoglobin for detection of hemolysis problematic without baseline values.
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Complete Blood Count and Red Blood Cell Morphology
Peripheral blood film evaluation is crucial
Increase in poly-chromatic RBCs (reticulocytes) and nucleated RBCs represents bone marrow compensation for hemolysis or blood loss
Schistocytes are expected with excessive fragmentation hemolysis
Spherocytes may be seen with macrophage-mediated processes
Additional morphologic changes to RBCs may point toward the cause of the hemolysis
Increase in mean cell volume (MCV) is usually seen with extreme compensatory reticulocytosis resulting from larger, prematurely released "shift" reticulocytes
MCV may not increase to greater than the reference interval but rather may be more than the baseline value for that patient
Exceptions occur if the hemolytic condition itself involves smaller cells that counter the increased volume of the reticulocytes
Red cell distribution width (RDW) can be expected to rise with reticulocytosis
Addition of larger reticulocytes can be expected to extend the range of cell volumes
Leukocytosis and thrombocytosis may accompany acute hemolytic anemia
Considered reactions to the hemolytic process
Low platelet counts in association with other signs of hemolysis may indicate a platelet-consuming microangiopathic process
Reticulocyte Count
Most commonly used test to detect accelerated erythropoiesis
Expected to rise during hemolysis or hemorrhage
All measures of reticulocyte production should rise: absolute reticulocyte count, relative reticulocyte count, reticulocyte production index, and immature reticulocyte fraction
Association of reticulocytosis with hemolysis is strong
Exceptions occur during aplastic crises of some hemolytic anemias and in some immunohemolytic anemias with hypoplastic marrow
Bone Marrow Examination
Usually not necessary to diagnose hemolytic anemia
If conducted, bone marrow examination will reveal erythroid hyperplasia that results in peripheral blood reticulocytosis
Cellularity of bone marrow should be determined on a core biopsy specimen for a more accurate assessment
Laboratory Tests to Identify Specific Hemolytic Processes
Appearance of spherocytes or schistocytes on a peripheral blood film can point to a hemolytic cause for anemia
Other abnormalities found on the film may help reveal the specific disorder causing the hemolysis
Other tests for diagnosis of specific types of hemolytic anemia are discussed in subsequent chapters
RBC Morphology Hemolytic Disorders
Spherocytes: Hereditary spherocytosis, IgG-mediated immune hemolytic anemia, thermal injury to RBCs
Elliptocytes (ovalocytes): Hereditary elliptocytosis
Acanthocytes: Abetalipoproteinemia, severe liver disease (spur cell anemia)
Burr cells: Pyruvate kinase deficiency, uremia
Schistocytes: Microangiopathic hemolytic anemia, traumatic cardiac hemolytic anemia, IgM-mediated immune hemolytic anemia
Erythrophagocytosis: Damage to RBC surface, especially due to complement-fixing antibodies
RBC agglutination: Cold agglutinins, immunohemolytic disease
Hematologic Findings Indicating Accelerated Red Blood Cell Production
Increased absolute reticulocyte count, immature reticulocyte fraction, reticulocyte production index
Rising mean cell volume (compared with baseline)
Polychromasia, nucleated RBCs
(Table 20.3)
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Differential diagnosis of hemolytic anemias incorporates several intersecting lines of deduction.
Rapid decrease in hemoglobin concentration can signal hemolysis when hemorrhage and hemodilution have been ruled out.
Jaundice and reticulocytosis provide additional confirmation of a hemolytic cause.
Elevated urinary urobilinogen level strengthens the conclusion.
RBC morphology and haptoglobin levels can assist in differentiating fragmentation from a macrophage-mediated cause.
In chronic hemolysis, persistence of hemoglobinemia, hemoglobinuria, decreased serum haptoglobin level, indirect bilirubinemia, and reticulocytosis can be expected.
Hemolytic anemias must be differentiated from other anemias associated with bilirubinemia, reticulocytosis, or both.
Anemia with reticulocytosis but without bilirubinemia is expected during recovery from hemorrhage not treated with transfusion or with effective treatment of deficiencies such as iron deficiency.
Anemia that results from hemorrhage into a body cavity is characterized by reticulocytosis during recovery and bilirubinemia as a result of catabolism of the hemoglobin in the hemorrhaged cells.
Anemias associated with ineffective erythropoiesis, such as megaloblastic anemia, are essentially hemolytic, with the cell death occurring in the bone marrow.
Bilirubinemia and elevated serum lactate dehydrogenase levels are to be expected, but the reticulocyte count is low.
Differential diagnosis may rely on negative results of tests for increased cell destruction or accelerated production.
Figure 20.7 Hemolysis Timelines:
Example of fragmentation (intravascular) hemolysis timeline.
Hemoglobin is detectable in plasma and urine for a period very soon after the hemolysis occurs.
Haptoglobin levels will drop as the plasma hemoglobin rises.
Protoporphyrin of salvaged hemoglobin will be processed to bilirubin that will rise after the hemolytic event.
Reticulocyte indices will also rise several days after the hemolytic event.
Example of macrophage-mediated (extravascular) hemolysis timeline.
Evidence of hemolysis is delayed until bilirubin and reticulocytes rise.
Contents of the red blood cells do not enter the blood.
TABLE 20.5 Differential Diagnosis of Hemolytic Anemias Versus Other Causes of Indirect Bilirubinemia and Reticulocytosis:
Differential diagnosis of hemolytic anemias versus other causes of indirect bilirubinemia and reticulocytosis.
Provides a comparison of hemolytic anemias with acute and chronic fragmentation and macrophage-mediated hemolysis, as well as acute hemorrhage.
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Review Questions:
The term hemolytic disorder in general refers to a disorder in which there is:
Increased destruction of RBCs in the blood, bone marrow, or spleen
Excessive loss of RBCs from the body
Inadequate RBC production by the bone marrow
Increased plasma volume with unchanged red cell mass
RBC destruction that occurs when macrophages ingest and destroy RBCs is termed:
Extracellular
Macrophage mediated
Intra-organ
Extrahematopoietic
A sign of hemolysis that is typically associated with both fragmentation and macrophage-mediated hemolysis is:
Hemoglobinuria
Hemosiderinuria
Hemoglobinemia
Elevated urinary urobilinogen level
Summary:
A hemolytic disorder is a condition in which there is increased destruction of red blood cells (RBCs) and a compensatory acceleration in RBC production by the bone marrow.
A hemolytic anemia develops when the bone marrow is unable to compensate for the shortened survival of the RBCs.
Hemolytic anemias can be classified as acute or chronic, intravascular or extravascular, acquired or inherited, intrinsic or extrinsic, and fragmentation or macrophage mediated.
Most RBC death in healthy individuals occurs via macrophages of the spleen and liver. A small amount occurs by fragmentation due to mechanical trauma.
Hemoglobin from RBCs is converted to heme and globin within macrophages. Heme is further degraded to iron, carbon monoxide, and unconjugated bilirubin. The bilirubin is secreted into the blood, where it binds to albumin and is transported to the liver. In the liver, bilirubin is conjugated with glucuronic acid and excreted as bile into the intestines, and it is converted to urobilinogen by intestinal bacteria. Some urobilinogen is reabsorbed into the portal circulation and reexcreted through the liver. A small amount of urobilinogen remains in the blood, and it is excreted by the kidney into the urine.
In macrophage-mediated (extravascular) hemolytic anemia, there is an increase in unconjugated bilirubin in the plasma and an increase in urobilinogen in the stool and urine. Spherocytes may be seen on the peripheral blood film.
Signs of fragmentation (intravascular) hemolysis include (met) hemoglobinemia, (met)hemoglobinuria, and hemosiderinuria. Serum haptoglobin is markedly decreased or absent. Schistocytes may be seen on the peripheral blood film.
Jaundice can result from increased serum unconjugated bilirubin during any hemolytic anemia.
Major clinical features of chronic inherited hemolytic anemia are varying degrees of anemia, jaundice, splenomegaly, and the development of cholelithiasis. In children, bone abnormalities may develop as a result of accelerated erythropoiesis.
Laboratory studies providing evidence of hemolytic anemia include tests for increased RBC destruction and compensatory increase in the rate of erythropoiesis. Elevated serum indirect bilirubin level with a normal serum direct bilirubin level suggests accelerated RBC destruction. A moderate to marked decrease in serum haptoglobin level suggests a fragmentation cause of hemolysis. The reticulocyte count is the most commonly used laboratory test to identify accelerated erythropoiesis, including an elevation of the immature reticulocyte fraction. Other tests that are specific to a particular diagnosis also may be needed.
Hemolytic anemias must be differentiated from other anemias with reticulocytosis, including the post-acute hemorrhage state and recovery from iron, vitamin B12, or folate deficiency, and from those with bilirubinemia, such as with internal bleeding.
Differential Diagnosis of Hemolytic Anemias Versus Other Causes of Indirect Bilirubinemia and Reticulocytosis:
Hemoglobin Level: Rapidly dropping
Indirect Bilirubinemia: Absent
Reticulocytosis: Absent
Spherocytes or Schistocytes: Absent
Hemodilution: Absent
Recovery from hemorrhage:
Hemoglobin Level: Rising
Indirect Bilirubinemia: Absent or declining
Reticulocytosis: Present
Spherocytes or Schistocytes: Absent
Treated anemia (iron, vitamin B12, or folate deficiency):
Hemoglobin Level: Rising
Indirect Bilirubinemia: Absent or declining
Reticulocytosis: Present
Spherocytes or Schistocytes: Absent
Hemorrhage into a body cavity:
Hemoglobin Level: Rapidly dropping
Indirect Bilirubinemia: Delayed
Reticulocytosis: Delayed
Spherocytes or Schistocytes: Absent
Ineffective erythropoiesis (e.g., megaloblastic anemia):
Hemoglobin Level: Dropping
Indirect Bilirubinemia: Persistent
Reticulocytosis: Absent
Spherocytes or Schistocytes: Absent
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An elderly white woman with worsening anemia
Decrease of approximately 0.5 mg/dL of hemoglobin each week
Pale skin and yellow eyes
Extreme fatigue and inability to complete daily tasks without napping
Tiredness on exertion
Tests for accelerated erythropoiesis
Urine urobilinogen level
Hemosiderin level
Reticulocyte count
Glycated hemoglobin level
A 5-year-old girl with darkening urine and mild anemia
Diagnosed with pneumonia and prescribed antibiotics
No history of anemia or family history of hematologic disorder
Mild anemia, polychromasia, and a few schistocytes
Personal and family history of mild hemolytic anemia
Elevated levels of total and indirect serum bilirubin
Elevated urinary urobilinogen
Decreased serum haptoglobin level
Elevated reticulocyte count and polychromasia on peripheral blood film
Spherocytes on peripheral blood film
Major fraction of bilirubin in plasma
Unconjugated bilirubin secreted by the liver
Urobilinogen reabsorbed from the intestines
Macrophage-secreted indirect bilirubin
Direct bilirubin conjugated by hepatocytes
Anemia worsening over several months
Decline in hemoglobin level
Polychromasia and anisocytosis on peripheral blood film
Elevated reticulocyte count and red cell distribution width (RDW)
Normal levels of total bilirubin, indirect fractions, and urinary urobilinogen
Test results expected with chronic fragmentation hemolysis
Increased serum haptoglobin
Positive Prussian Blue stain in urine sediment
References:
Crosby, W. H., & Akeroyd, J. H. (1952). The limit of hemoglobin synthesis in hereditary hemolytic anemia. Am J Med, 13, 273–283.
Hillman, R. S., & Henderson, P. A. (1969). Control of marrow production by the level of iron supply. J Clin Invest, 48(3), 454–460.
Cui, Y., Konig, J., Leier, I., et al. (2001). Hepatic uptake of bilirubin and its conjugates by the human organic anion transporter SLC21A6. J Biol Chem, 276, 9626–9630.
Keppler, D. (2014). The roles of MRP2, MRP3, OATP1B1, and OATP1B3 in conjugated hyperbilirubinemia. Drug Metab Dispos, 42, 561–565.
Sticova, E., & Jirsa, M. (2013). New insights in bilirubin metabolism and their clinical implications. World J Gastroent, 19(38), 6398–6407.
Keppler, D., & Kartenbeck, J. (1996). The canalicular conjugate export pump encoded by the cmrp/cmoat gene. Prog Liver Dis, 14, 55–67.
van de Steeg, E., Wagenaar, E., van der Kruijssen, C. M., et al. (2010). Organic anion transporting polypeptide 1a/1b-k