Glucose-6-phosphate Dehydration Deficiency Nada Mohamed Ahmed , MD, MT (ASCP)i

Objectives Introduction Definition Factors cause G6PD deficiency Pathophysiology (mechanism) Symptoms Lab findings

HEMOLYTIC ANEMIA Hereditary Membrane defects PNH Intracorpuscular WITHIN THE RED CELL Extracorpuscular OUTSIDE THE RED CELL Hereditary Acquired AUTO-IMMUNE 1. Warm Ab 2. Cold Ab 3. Transfusion reactions 4. Drug associated NON-IMMUNE 1. Hypersplenism 2. Infections (Malaria),, 3-mechanical trauma to RBCs 4. Liver dz (Spur cell) 1. Membrane defects - Hereditary spherocytosis - Hereditary elliptocytosis - Hereditary pyropoikilocytosis - Hereditary stomatocytosis 2. Enzyme defects -G6PD 3. -Hemoglobin defects -.Hemoglbinopathies(sickle cell disorders) - Hb SS, CC, SC & S-B- - Thalassemias Membrane defects PNH

Hereditary Acquired 1- Abnormalities of RBC interior a. Enzyme defects A Simple Classification of Hemolytic Anemias 1- Abnormalities of RBC interior a. Enzyme defects b. Hemoglobinopathies & Thalassemia Major 2-RBC membrane abnormalities a. Hereditary spherocytosis, elliptocytosis etc b. Paroxysmal nocturnal hemoglobinuria c. Spur cell anemia 3- Extrinsic factors a. Hypersplenism b. Antibody : immune hemolysis c. Traumatic & Microangiopathic hemolysis d. Infections , toxins , etc Hereditary Intracorpuscular Acquired Extracorpuscularr

Introduction FUNCTION OF G6PD Regenerates NADPH, allowing regeneration of glutathione Protects against oxidative stress Lack of G6PD leads to hemolysis during oxidative stress- infection, medication, fava beans Oxidative stress leads to Heinz body formation, extravascular hemolysis

History & Definition It was discovered in 1950s after it was observed that some black soldiers receiving antimalarial drug primaquine developed hemolytic anemia. It is the most common red cell enzymopathy associated with hemolysis. Large no. of abnormal genes code for the G6PD The inheritance of either of them results in this disorder.

Definition X-linked disease , sesult in G6PD Deficiency (RBCs enzym deficiency ) asymptomatic unless Oxidative stress causes the RBC’s to break apart. Inherited –, Recessive

Genetics The G6PD gene is located on the X chromosome Thus the deficiency state is a sex-linked trait Affected males inherit the abnormal gene from their mothers who are usually carriers (heterozygotes)

G6PD Deficiency So, an effected father will transmit the disease to all of his daughters and none of his sons. Whereas, a carrier mother will transmit the disease to half of her children. Half of her daughter will be carriers and half of her sons will be directly affected with the condition.

factors cause G6PD deficiency: Individuals who have inherited one of the many G6PD mutations do not show clinical manifestation. Some of patients with G6PD develop hemolytic anemia if they are exposed or ingest any of the followings oxidizing agents: 1-Oxidant drugs: Remember “AAA”? Antibiotics : e.g. sulfamethoxazole Antimalarias : e.g. primaquine Antipyretics : e.g. acetanilid 2- Favism: The hemolytic effect of ingesting of fava beans is not observed in all individuals with G6PD deficiency but all patients with favism have G6PD deficiency

G6PD Deficiency Biochemistry – summary G6PD Glutathione NADP+ to NADPH Which is responsible for Free radicals Which damage blood cells G6PD Reduces Glutathione Which fights G6PD, under normal conditions, is responsible for the maintenance of Nicotinamide adenine dinucleotide phosphate (NADP+) and its conversation to NADPH. NADPH is responsible for regenerating glutathione and the removal of free radicals which cause damage to blood cells. Free radicals are molecules that are missing an electron. Generally, free radicals attack the nearest stable molecule, "stealing" its electron. When the "attacked" molecule loses its electron, it becomes a free radical itself, beginning a chain reaction. Once the process is started, it can cascade, finally resulting in the disruption of a living cell. Glutathione eliminates the free radical by donating the needed electron and converting the free radical into a more stable byproduct such as water (H2O).

Pathophysiology Oxidative denaturation of hemoglobin is the major cause of H. A in G6PD deficiency It is important in the conversion of glucose-6-P to phosphogluconate For subsequent production of NADPH & reduced glutathione (GSH) GSH protects enzymes & hemoglobin against oxidation by reducing H2O2 & free radicals

Pathophysiology H2O2 is generated normally in small amounts during normal red cell metabolism Larger amounts produced when an oxidant drug interacts with oxyhemoglobin Normal red cells have sufficient G6PD activity to maintain adequate GSH levels When deficient, red cells fail to produces sufficient G6PD to detoxify peroxide

Pathophysiology Hb is then oxidized to Hi, heme is liberated from globin & globin denatures producing Heinz bodies Heinz bodies attach to membrane sulfhydryl groups inducing cell rigidity At this point red cells can no longer traverse the splenic microcirculation Hence lysis occurs

Symptoms Persons with this condition do not display any signs of the disease until their red blood cells are exposed to certain chemicals in food or medicine, or to stress. Symptoms are more common in men and may include: Dark urine Enlarged spleen Fatigue Pallor Rapid heart rate Shortness of breath Yellow skin color (jaundice)

“quin” Drugs G6PD Deficiency Malaria Treatment chloroquine atovaquone-proguanil (Malarone®) artemether-lumefantrine (Coartem®) mefloquine (Lariam®) quinine quinidine doxycycline (used in combination with quinine) clindamycin (used in combination with quinine) primaquine “quin” Drugs These are some of the drugs that are typically used for malaria treatment. However, they are either contraindicated in G6PD patients, high risk, possibly dose restricted, not indicated for prophylactic treatment. The biggest problem is the rapid spread of drug resistant malarial strains.

G6PD Deficiency Hemolytic Anemia Diagnosis of G6PD Deficiency Hemolytic Anemia 1- CBC Bite cells, blister cells, irregular small cells, Heinz bodies, polychromasia G-6-PD level Bite” cells Spleen removes portion of RBC that had Heinz body, preventing intravascular hemolysis 2-Screening: Qualitative assessment of G6PD enzymatic activity (UV-based test) 3-Confirmatory test: Quantitative measurement of G6PD enzymatic activity 4-Molecular test: Detection of G6PD gene mutation

Special test Methemoglobin reduction test

Methemoglobin Reduction Test Sodium nitrite converts Hb (hemoglobin) to Hi (methemoglobin) Adding methylene blue should stimulate the pentose phosphate pathway, reducing methemoglobin In G6PD Deficiency, methemoglobin persists

Methemoglobin Reduction Test Normal blood → clear red color Deficient blood → brown color Deficient blood Normal blood

Clinical Manifestations The patient may experience an acute hemolytic crisis within hours of exposure to the oxidant stress Hemolytic crisis is self-limited even if the exposure continues & Only older cell population is rapidly destroyed A minority of patients are sensitive to exposure to fava beans (favism phenomenon)

Despite the name, most patients do not present this way. What is PNH? Paroxysmal – sudden onset Nocturnal – occuring at night (or early in morning upon awakeneing) Hemoglobinuria Despite the name, most patients do not present this way.

What is PNH? A rare and unusual acquired hematologic disorder characterized by Intravascular hemolysis Bone marrow failure (cytopenias) Thrombosis.

What is PNH? Intravascular RBCs break down result from Complement activation due to absence of CD55, CD59 Symptoms - passage of dark brown urine in the morning.

. PNH –laboratory features: - pancytopenia - chronic urinary iron loss - serum iron concentration decreased - hemoglobinuria - hemosiderinuria - positive Ham’s test (acid hemolysis test) - positive sugar-water test - specific immunophenotype of erytrocytes (CD59, CD55)