Hemolytic Anemias

Congenital and Acquired Hemolytic Anemia

Life span:

Neonatal RBC 60-80 days

Adult: 120 days

Above replace “rubri” with “erythro”

Normal Erythrocyte Indices

Indices	Adult
Hemoglobin	10-16g/dL
MCV	75-99fL
MCH	26-32pg
MCHC (=MCH/MCV)	30-36gm/dL

Hemolytic Anemia

Intrinsic/inherited

Membrane (Spherocytes, Elliptocytes, Stomatocytes)

Enzyme (Anaerobic glycolyis, Pentose Phosphate Shunt)

Hemoglobin (Unstable, Methemaglin)

Extrinsic:

Antibodies

Toxins

Mechanical

Intravascular (in circulation)

Extra vascular ( liver and spleen)

Anemia, reticulocytosis (>75,000). SBR up. AST (Anti Specific) ?ALT, Haptoglobin and Hemopexin down.

Erythrocyte Metabolism

No nucleus (cell division), mitochondria (oxidative phosphorylation), ribosome (protein synthesis)

Glucose is the primary energy source (facilitated glucose transport)

Anaerobic respiration absence of cellular machinery.

Requires anaerobic glycolysis for ATP production (Embden-Meyerhof enzymatic pathway).

Energy needs for:

Maintanece of cation (Na/K) gradient

Protection from oxidative damage

Maitainance of 2+ ferrous (reduced) iron

Embden-Meyerhof enzymatic pathway generates ATP

Phosphoglycerate Kinase

X-linked

Less ATP therefore unable to regulate water and cations (Na/K ATPase pump)

Pyruvate Kinase

Autosomal Recessive

Less ATP therefore unable to regulate water and cations (Na/K ATPase pump)

	Pyruvate Kinase Deficiency Pyruvate kinase deficiency: post-splenectomy. Acanthocytes marked by the arrows may be increased. Polychromatophilic macrocytes (reticulocytes) increase dramatically after splenectomy even though hemolysis usually lessens. Reticulocyte percentages from 40% to 90% after splenectomy have been observed. A Howell-Jolly body is seen in one red cell, characteristic of the post-splenectomy state. Several apparent knizocytes (asterisk) are evident. Autosomal recessive Affects RBC, EBC, liver RBC have ½ life span Only 1 ATP made see above Unable to maintain water and cation balance affects RBC shape: Echinocytes Extravascular hemolysis destroyed in the liver and spleen (therefore partial response to splenectomy) 2,3-DPG high (because block is below this) Greater O2 release at low Hb concentrations
	G6PD Deficiency Most common genetic mutation (>100 million worldwide) X-linked >400 missense mutations Results in less reducing power (entry into HMP (or PPP) shunt Intravascular hemolyiss with drugs (sula + others), Naphthalene (moth balls), infections. Assay G6PD activity after acute crisis Blister cells Hemobolinuria (using hem dip, no RBC)
	G6PD Deficiency Distribution

The pentose phosphate pathway (PPP; also called Phosphogluconate Pathway, or Hexose Monophosphate Shunt [HMP shunt]) is a process that serves to generate NADPH and the synthesis of pentose (5-carbon) sugars. There are two distinct phases in the pathway. The first is the oxidative phase, in which NADPH is generated, and the second is the non-oxidative synthesis of 5-carbon sugars. This pathway is an alternative to glycolysis. While it does involve oxidation of glucose, its primary role is anabolic rather than catabolic. For most organisms it takes place in the cytosol.

The primary functions of the pathway are:

· To generate reducing equivalents, in the form of NADPH, for reductive biosynthesis reactions within cells.

· To provide the cell with ribose-5-phosphate (R5P) for the synthesis of the nucleotides and nucleic acids.

· Although not a significant function of the PPP, it can operate to metabolize dietary pentose sugars derived from the digestion of nucleic acids as well as to rearrange the carbon skeletons of dietary carbohydrates into glycolytic/gluconeogenic intermediates.

Located exclusively in the cytoplasm, the pathway is one of the three main ways the body creates molecules with reducing power, accounting for approximately 60% of NADPH production in humans.

One of the uses of NADPH in the cell is to prevent oxidative stress. It reduces the coenzyme glutathione, which then converts reactive H₂O₂ into H₂O. If absent, the H₂O₂ would be converted to hydroxyl free radicals, which can attack the cell.

Significantly, erythrocytes utilize the reactions of the PPP to generate large amounts of NADPH used in the reduction of glutathione

It is also used to generate hydrogen peroxide for phagocytes.

The overall reaction for this process is:

Glucose 6-phosphate + 2 NADP⁺ + H₂O → ribulose 5-phosphate + 2 NADPH + 2 H⁺ + CO₂

Erythrocytes and the Pentose Phosphate Pathway

• The predominant pathways of carbohydrate metabolism in the red blood cell (RBC) are glycolysis, the PPP and 2,3-bisphosphoglycerate (2,3-BPG) metabolism.
• Glycolysis provides ATP for membrane ion pumps and NADH for re-oxidation of methemoglobin.
• PPP supplies the RBC with NADPH to maintain the reduced state of glutathione.
• The inability to maintain reduced glutathione in RBCs leads to increased accumulation of peroxides, predominantly H₂O₂, that in turn results in a weakening of the cell membrane and concomitant hemolysis. Accumulation of H₂O₂ also leads to increased rates of oxidation of hemoglobin to methemoglobin that also weakens the cell wall.
• Glutathione removes peroxides via the action of glutathione peroxidase. The PPP in erythrocytes is essentially the only pathway for these cells to produce NADPH. Any defect in the production of NADPH could, therefore, have profound effects on erythrocyte survival.

Several deficiencies in the level of activity (not function) of glucose-6-phosphate dehydrogenase have been observed to be associated with resistance to the malarial parasite, Plasmodium falciparum, among individuals of Mediterranean and African descent. The basis for this resistance is the weakening of the red cell membrane (the erythrocyte is the host cell for the parasite) such that it cannot sustain the parasitic life cycle long enough for productive growth.

When G6PD converts G6P into G6G NADP gains an electron (reduced) in the form of ·H to become NADPH which acts on GS:SG to be reduced to 2GSH. GSH donates (reduces) a ·H to HO:OH to make HOH and HOH

G6PD

Phosphoglycerate Kinase

Pyruvate Kinase

2,3-DPG shifts oxygen dissociation curve to right (i.e. reduced affinity for oxygen)

Pyrimidine-5'-Nucleotidase (or uridine monophosphate hydrolase (UMPH1)) Deficiency

Blood film. The coarse basophilic stippled cells are characteristic of this red cell enzyme deficiency. Causes congenital hemolytic anemia. Physiologically important during reticulocyte maturation. The clinical observation that patients with P5N deficiency show an increased incubated Heinz body formation and a positive ascorbate cyanide test, strongly suggests that this enzymopathy may be associated with a disturbance in the HMS shunt. N.B. lead inhibits pyrimidine-5'-nucleotidase hence causes basophilic stippling.

Unstable Hemoglobins

• Is a Chronic nonspherocytic hemolytic anemia (CNSHA)
• Unstable hemoglobins are prone to oxidative denaturation even in the presence of a normal G6PD system.
• Autosomal dominant and of variable severity.
• Most patients have a mild chronic hemolytic anemia with splenomegaly, mild jaundice, and pigment (calcium bilirubinate) gallstones. Less severely affected patients are not anemic except under conditions of oxidative stress.
• The diagnosis is made by the finding of Heinz bodies and a normal G6PD level.
• Hemoglobin electrophoresis is usually normal, since these hemoglobins characteristically do not have a change in their migration pattern (however extra bands can sometimes be seen).
• These hemoglobins precipitate in isopropanol.
• Usually no treatment is necessary. Patients with chronic hemolytic anemia should receive folate supplementation and avoid known oxidative drugs. In rare cases, splenectomy may be required.
• Most of mutations in Hem pocket (See below)

Historically, these variants were named unstable hemoglobins because they precipitated when they were incubated for 1 hour at 50°C in contrast to HbA, which remains stable at this temperature.

Other tests were later described to detect unstable Hbs such as incubation at higher temperature (65°C) and kinetics measurements of the precipitation.

One of the best tests consists in incubating the lysate at 37°C in a buffer containing 17 % isopropanol during a length of time insufficient to precipitate Hb A.

Hemoglobin	Substitution
Torino	3 PheVal
Hasharon^* (Sinai, Sealy)	47 AspHis
Iwata	87 HisArg
Petah Tikva	100 AlaAsp
Freiburg	23 Val deleted
Riverdale-Bronx	24 GlyArg
Yokohama	31 LeuPro
Castilla	32 LeuArg
Perth^* (Abraham Lincoln)	32 LeuPro
Philly	35 TyrPhe
Hammersmith	42 PheSer
Bucuresti^* (Louisville)	42 PheLeu
Niteroi	42-44, or 43-45 Phe, Glu, Ser deleted
Duarte	62 AlaPro
Zürich	63 HisArg
Bristol	67 ValAsp
Sydney	67 ValAla
Mizuho	68 LeuPro
Seattle	70 AlaAsp
Christchurch	71 PheSer
Shepherd's Bush	74 GlyAsp
Bushwick	74 GlyVal
Buenos Aires^* (Bryn Mawr)	85 PheSer
Santa Ana	88 LeuPro
Redondo	92 HisAsnAsp
St. Etienne^* (Istanbul)	92 HisGln
Gun Hill	91-95 or 92-96 or 93-97 Leu, Cys, Asp, His deleted
Köln^* (Ube I)	98 ValMet
Djelfa	98 ValAla
Presbyterian	108 AsnLys
Shelby (Deaconess)	131 GlnLys
North Shore	134 ValGlu
Coventry	141 Leu deleted
Tak	Elongation of -chain C-terminus
Cranston	Elongation of -chain C-terminus
La Grange	101 GluLys
Poole	130 TrpGly

Approximately 200 different mutations are known as leading to a decreased stability but only about half of those are responsible for clinical disorders. Only a few of the variants classified today in this group are present in a sufficient amount in the lysate to be detected under the historical experimental conditions.

Hereditary Stomatocytosis: Stoma = mouth

As many as 3% of RBCs may be stomatocytes in a normal smear. Stomatocytes are erythrocytes with an elongated (mouth-like) area of central pallor. An occasional cell of this type might be seen as a non-specific finding in a variety of situations, such as regenerative anemias, liver disease, and lead poisoning. Stomatocytes can also be an artifact in a blood smear that is too thick. Erythrocytes have intracellular hemoglobin, 2-3,diphosphoglycerate (2,3-DPG), and ATP, which all exert osmotic pressure across the semipermeable cell membrane. By transporting Na^+ and K^+ ions across the cell membrane, red cells can adjust the intracellular concentration of these cations and regulate intracellular hydration. Any disturbances in membrane cation permeability alter cellular hydration and can cause numerous effects, including hemolysis.

Overhydrated hereditary stomatocytosis (OHS):

• Also called hydrocytosis.
• An abnormally increased cation influx results in swollen erythrocytes, hemolysis, and stomatocytes.
• In OHS, the major defect is a marked asymmetric increase in passive Na⁺ and K⁺ permeability. The influx of Na⁺ exceeds the loss of K⁺, causing a net influx of water, overhydration, and swelling. The resulting hydrocytosis leads to increased osmotic fragility and decreased deformability, with consequent hemolysis.
• The underlying gene mutation is unknown, and the observed decrease in stomatin, or protein 7.2b, is thought to be a trafficking alteration.

Dehydrated hereditary stomatocytosis (DHS)

• Net loss of cations and water results in dehydrated hereditary stomatocytosis (DHS), which is also called xerocytosis.
• Change in the relative membrane permeability to K⁺. Efflux of K⁺ is increased 2-fold to 4-fold and results in cation depletion, with decreased intracellular osmolality and water loss.

• The xerocytes formed are shear-sensitive and prone to membrane fragmentation in response to metabolic stress, with subsequent hemolysis.

• Mapped to the 16q23-24 genetic locus

Pseudohyperkalemia (FP)

• Asymptomatic or rarely shows mild macrocytosis.
• When erythrocytes are cooled to room temperature or lower (eg, after phlebotomy), the net K⁺ leak is greater than expected and results in factitious hyperkalemia.
• Most cases of FP have been mapped to the 16q23-24 genetic locus.

Cryohydrocytosis (CHC)

CHC has been linked to mutations in the band 3 chloride-bicarbonate exchanger AE1.

Mortality/Morbidity

• Morbidity in these disorders depends on the severity of the hemolytic anemia.
• The risks for neonatal hyperbilirubinemia with kernicterus are similar to those of other hemolytic anemias.
• Exchange transfusion is occasionally required.
• Aplastic crises associated with parvovirus infection occur, infrequent.
• Both OHS and DHS are associated with a significant risk of serious thrombosis after splenectomy, reason unknown.
• Most patients with OHS have chronic low-grade anemia punctuated by recurrent episodes of more severe anemia and jaundice. Other patients have a much milder disease. Iron overload, regardless of transfusion status, is now well recognized.
• Most patients with DHS are asymptomatic but experience mild-to-moderate hemolytic anemia, which is generally well compensated.
• Hydrops fetalis and neonatal ascites have been reported in a few kindreds. Exchange transfusions are occasionally required. Even simple transfusions carry risks of infection, allergic reactions, and febrile or hemolytic transfusion reactions.

Medical Care

Patients should also receive folate supplementation if they have significant ongoing hemolysis.

Hereditary or Congenital Spherocytosis

• Jaundice at 24hrs is ALWAYS pathological
• Autosomal Dominant
• 5-20% reticulocytosis (supra vital stain)
• Micorspherocytosis
• Splenomegaly, Gallstones
• 1/3 spontaneous mutations

	Schematic representation of the red cell membrane cytoskeleton and alterations leading to spherocytosis and hemolysis. Mutations weakening interactions involving α-spectrin, β-spectrin, ankyrin, band 4.2, or band 3 all cause the normal biconcave red cell to lose membrane fragments and adopt a spherical shape. Such spherocytic cells are less deformable than normal and therefore become trapped in the splenic cords, where they are phagocytosed by macrophages.
	A normal red cell is 6-8 µm in diameter. As the relative amount of hemoglobin in the red cell decreases or increases, the area of central pallor will decrease or increase accordinglySpherocytes are red blood cells that are almost spherical in shape. They have no area of central pallor like a normal red blood cell. Large spherocytes (macrospherocytes) are seen in hemolytic anemia. Small spherocytes (microspherocytes) are sometimes seen in severe burn cases. A variety of spherical forms are seen in hereditary spherocytosis.
	Osmotic fragility test. Immediate, 24hrs and 48hrs Difficulties in interpretation in neonate due to high reticulocytes, transfusion

Other Membrane Disorders

	Hereditary Elliptocytosis (Camels have elliptocytes) · African American Variant mild · Asian severe · Mutations in spectrin
	Hereditary Pyropoikilocytosis Thermal insensitivity Bizarre shapes, fragmented Moderate hemolytic anemia

Methemoglobinemia

Methemoglobinemia is diagnosed when the percentage of methemoglobin (metHb) exceeds 1% in the blood. Methemoglobin differs from normal hemoglobin in that the oxygen-carrying ferrous (+2) iron in the heme groups has been oxidized to ferric (+3) iron. Methemoglobin is characterized by increased oxygen affinity, resulting in a functional anemia and failure to deliver oxygen to the body's tissues.

The classic presentation of methemoglobinemia is cyanosis in the presence of a normal alveolar partial pressure of oxygen (PaO2), with brown- or chocolate-colored blood that does not become red on exposure to oxygen. Additional symptoms such as shortness of breath, anxiety, palpitations, and confusion occur as the level of metHb increases.

Methemoglobinemia is a misnomer, because metHb is only increased within the red blood cells and is not dissolved in the plasma. Methemoglobinemia can be hereditary or acquired. Acquired methemoglobinemia is usually secondary to medications or various exogenous exposures.

The major enzymatic system involved is adenine dinucleotide (NADH)–dependent methemoglobin reduction. This has also been called the diaphorase pathway. Cytochrome b5 reductase plays a major role in this process by transferring electrons from NADH to methemoglobin, which results in the reduction of methemoglobin to hemoglobin. This enzyme system is responsible for the removal of 95-99% of the methemoglobin that is produced under normal circumstances.

Abnormal hemoglobins can also cause methemoglobinemia. These abnormal hemoglobins are called hemoglobin M (Hb M) because they are associated with methemoglobinemia. In most of them, a tyrosine replaces the histidine residue, which binds heme to globin. This replacement displaces the heme moiety and permits oxidation of the iron to the ferric state. Then, hemoglobin M is more resistant to reduction by the methemoglobin reduction enzymes previously described. The end result is a functionally impaired hemoglobin with a decreased affinity for oxygen.

Most cases of methemoglobinemia are due to excessive production of methemoglobin following exposure to oxidant drugs, chemicals, or toxins. This increased production of methemoglobin overwhelms the physiologic regulatory mechanisms previously discussed. These agents can cause an increase in methemoglobin levels either by ingestion or by absorption through the skin. Such agents fall into 2 general categories: nitrites or aromatic amines. Dapsone and benzocaine are common causes for methemoglobinemia.

The clinical course of hereditary forms of methemoglobinemia is generally benign. However, individuals with type IIb5 cytochrome reductase deficiency are an exception to this rule. These persons have a markedly shortened life expectancy primarily due to multiple neurologic complications.

• Symptoms are proportional to the level of methemoglobin.
• Less than 10% methemoglobin – No symptoms
• 10-20% methemoglobin – Skin discoloration only (most notably
  on mucus membranes)
• 20-30% methemoglobin – Anxiety, headache, dyspnea on
  exertion
• 30-50% methemoglobin – Fatigue, confusion, dizziness,
  tachypnea, palpitations
• 50-70% methemoglobin – Coma, seizures, arrhythmias, acidosis
• Greater than 70% methemoglobin – Death

Treatment with Methylene blue

Splenectomy and Hemolytic Anemia

Indications

Splenomegaly, hypersplenism, gallstones, gallstones, jaundice,growth retardation, transfusion dependence.

Preoperative immunization with peumovax

Antibiotic prophylaxis

Acquired Hemolytic Anemia

Alloantibodies

Autoantibodies

Other (Drug, toxin, burns, compliment PNH)

Neonatal Alloimmune Hemolytic Anemia

Outcome determinants:

ABO

Isohemagl;utanins, usually IgM

Mild A+B not fully formed at birth

Kell: Kills

Duffy: Dies

Kidd Kills

Lewis Lives as Lewis not present on fetal cells.

Warm AIHA

	Warm AIHA Antibodies on surface because of +/- C3 (lack) are taken up by spleninc RES Works at 37C IgG agains Rh and others
	PCH agains P-antiigen · Avoid Cold · Steroids · C3 fixation · After viral infection
	Cold Agglutinins · Avoid Cold · C3 fixation · Liver RBC destruction · 4C · L,i · Avoid cold · Plasmapheresis
	Paroxysmal Nocturnal Hemoglobinemia Structure of GPI anchor: The biochemical defect in PNH occurs at the first step in the production of the GPI anchor: at the transfer of the glucosamine to the phosphatidylinositol.
	The pig-a gene The pig-a (phosphatidylinositol glycan complementation class A) gene is found on the X chromosome and the protein it produces is responsible for the first step in the production of the GPI anchor. Over 20 other genes involved in GPI production have now been described but these are not involved in PNH. In all reported cases of PNH there is one or more abnormality of the pig-a gene. The abnormalities are extremely diverse and result in blood cells with either total (Type III cells) or partial (Type II cells) deficiency of GPI-linked proteins.
	Missing GPI-linked proteins All proteins attached to the cell membrane via the GPI anchor have been found to be missing from PNH blood cells. The two missing proteins thought to cause the clinical manifestations of the disease are: 1. CD55 (DAF: decay accelerating factor) 2. CD59 (MIRL: membrane inhibitor of reactive lysis) Both proteins are involved in the protection of cells from the action of complement (a protein involved in the immune system that acts to break cells down). In the absence of CD55 and CD59, blood cells are vulnerable to attack from complement; red blood cells are destroyed prematurely and platelets undergo changes that increase the risk of blood clotting. The growth advantage of PNH cells over normal cells could explain why PNH is related to a condition called Aplastic Anaemia in which the bone marrow fails to produce blood cells. Its cause is unknown, but it is likely that the marrow stem cells are altered by an unknown factor (perhaps a virus or chemical). As a result these early blood cells are recognized as foreign by the immune system. The subsequent immune attack is probably mediated via GPI-linked proteins. This situation would favour the growth of GPI-deficient cells (which would avoid immune attack) and the emergence of PNH.

Flow cytometry is now the laboratory investigation of choice. This method measures GPI-anchored proteins directly on blood cells but requires expertise for interpretation of results. At least two types of cells are studied, usually white cells and red cells, and the percentage of GPI-deficient cells are reported.

Granulocyte flow cytometry in PNH. Granulocytes are electronically selected (upper left plot: red R1 region), and analysed for expression of CD16, CD55 and CD66 cell-membrane proteins (lower dot-plots). Two cell populations are visible, a residual normal and the GPI-deficient PNH clone.

Red-cell flow cytometry in PNH. Red-cells are analysed for expression of CD55, CD59 and Glycophorin-A (CD235a; red-cell marker). The normal and GPI-deficient PNH red-cell populations (defined by CD55 and CD59) are visible in the histogram overlay plot. The lower right histogram shows three CD59-defined red-cell populations, Types I (normal), II (partial deficiency) and III (complete deficiency).

Patients in whom Aplastic Anaemia is the predominant disease, generally have small PNH clone sizes. Those with 'haemolytic' PNH, (haemoglobin in the urine, anaemia), usually have large clones, often near to 100% affected white blood cells.

The Ham's (acid hemolysin) test

Looks for increased fragility of red blood cells in mild acid.

Used to confirm the diagnosis of paroxysmal nocturnal hemoglobinuria (PNH).

Diagnosis of PNH can be confirmed by having a positive acidified serum test (Ham test). In acidified serum, complement is activated by the alternate pathway. It binds to red blood cells, and ruptures the abnormal PNH cells, which are unusually susceptible to complement. With newer methods of diagnosis, such as flow cytometry, this test has become less important in the diagnosis of PNH.

The Ham test is also positive in another rare disorder called congenital dyserythropoietic anemia, but in this case the sugar-water fragility test is negative. Furthermore, the clinical aspects of this disorder are not similar to PNH