Sickle Cell Anemia Case Study

• Sickle cell anemia results from the inheritance of hemoglobin F and hemoglobin S, leading to hemoglobin SS.

Case History

• An 11-year-old female was diagnosed with sickle cell at birth.
• The patient had splenic sequestration at 6 months, treated with transfusions and partial splenectomy.
• At 19 months admitted to hospital with acute chest syndrome.
• The patient had recurrent reactive airway disease.
• At 4 suffered a life-threatening acute chest syndrome, admission to intensive care, and exchange transfusion.
• Red blood cell transfusions monthly for 6 months prevented recurrence of acute chest syndrome.
• During admission, the patient acquired Streptococcus pneumoniae sepsis and pneumonia.
• The patient received RBC transfusions monthly for 6 months. Hydroxyurea therapy was never initiated.
• The parents gave a history of a cough, decreased physical activity, and a fever of 38.8°C.
• Medications prescribed were penicillin, folic acid, and albuterol inhaler.
• Examination revealed a toxic-appearing child with rapid breathing, decreased activity, and intermittent sleepiness.
• Temperature was 38°C orally.
• Heart rate measured 158 beats per minute.
• Respiratory rate was 24-28 breaths per minute.
• Blood pressure was 90/40 mmHg.
• Oxygen saturation on room air was 99%.
• Physical examination showed icteric sclera, dry mucus membranes, and erythematous posterior pharynx.
• The patient had a grade II/VI systolic ejection murmur.
• The Liver edge was palpable 2cm below the costal margin.
• A fine scarlatiniform rash and lethargy were apparent.
• Hematocrit was 17.5%.
• Hemoglobin was 62 g/L.
• Reticulocyte count was 12.9%.
• Total leukocyte count was 9.1 x 10(9)/L.
• Neutrophils: 46%, Band forms: 25%.
• Platelet count: 1860 x 10(9)/L.
• Serum glucose was 2.8 mmol/L.
• Bicarbonate was 15 mmol/L.
• Serum urea nitrogen was 8.2 mmol/L.
• Creatinine was 115 mmol/L.
• Total bilirubin was 32.5 mmol/L.
• Urinalysis specific gravity was 1.005.
• The patient had trace bilirubin.
• A crossmatch was performed for 3 units of leukopoor and phenotypically matched packed red blood cells.
• A chest radiograph revealed atelectasis of the right lung base and mild cardiomegaly.
• An abdominal radiograph revealed hepatomegaly.
• The patient was treated with lactated Ringer's solution and ceftriaxone for presumed sepsis.
• Vancomycin was also given.
• Oxygen saturation decreased to 92% on room air, leading to arterial blood gas assessment.
• Arterial blood gas showed a pH of 7.29, pO2 of 6 kPa, pCO2 of 4.30 kPa, bicarbonate of 15 mmol/L, and oxygen saturation of 76%.
• Upon arrival to intensive care, the oxygen saturation suddenly decreased to 67%, and the patient became unresponsive.
• The patient was intubated and placed on a ventilator with 100% oxygen, given blood transfusions, but hemoglobin was still 17 g/L.
• It was noted the patient had an enlarging right upper quadrant mass.
• A blood count revealed a hemoglobin of 81 g/L, a platelet count of 17,000, and a leukocyte count of 12.7%.
• The Glucose was 2.8 mmol/L, Calcium was 2.00 mmol/L.
• Fresh frozen plasma, cryoprecipitate, platelet concentrates, and normal saline were administered.
• Vasopressors were used along with volume support.
• The patient had no urine output after catheter placement.
• The patient expired from septic shock.
• Gram-negative rods were on her blood smear.
• Blood culture grew penicillin-sensitive Streptococcus pneumoniae, in 6 hours.
• Autopsy revealed Streptococcus pneumoniae endocarditis of the right ventricle, focal ischemia of the left ventricle, bilateral pleural effusions, hepatic congestion with thrombosis, renal congestion, bilateral adrenal hemorrhage, and necrosis.
• Death was a result of septic shock from Streptococcus pneumoniae.

Diagnosis

• Diagnosis of sickle cell disease is based on hemoglobin type identification in patient's red cells, which is determined at newborn screening and confirmed later.
• Parental hemoglobin samples help establish a diagnosis with certainty.
• If only one parent is available, DNA methods can establish hemoglobin mutations in the newborn.
• Newborn screening started in the US in the early 1990s.
• Some states previously targeted ethnic groups for screening, missing 20%.
• Individuals are diagnosed due to a symptom of the disease prompting their physician to investigate sickle cell disease as a possible diagnosis.
• Almost all US states and some EU members require newborn screening for hemoglobinopathies.
• A blood spot via the Guthrie method on filter paper is used for initial screening using HPLC or isoelectric focusing.
• Both HPLC and Isoelectric focusing use the molecular charge on the hemoglobin molecule.
• Confirmatory testing includes hemoglobin electrophoresis and β-globin chain analysis by DNA testing.
• Separation of hemoglobin species depends on the net charge of the molecule ability to migrate through media under an electric field.
• Standard solid media include cellulose acetate at pH 8.2-8.6 and citrate agar at pH 6.0-6.2.
• Isoelectric focusing uses a pH gradient to separate hemoglobins at their isoelectric points.
• Various hemoglobins are not sharply separated using isoelectric focusing.
• HPLC uses a negatively charged stationary absorbent column.
• Different hemoglobin species are separated by charge and eluted by increasingly positive buffers.
• HPLC system is automated and computerized, identifying and quantitating hemoglobin.
• HPLC is most common for mass screening of newborns in the United States. Variant hemoglobin species detected by HPLC are confirmed by other methods.

Biochemical Perspectives

• Sickle cell disease was first diagnosed in 1904 by Ernest Irons.
• Irons observed “pear shaped elongated forms" on the blood smear of a patient with pneumonia.
• James Herrick followed the patient for 2.5 years.
• In 1910, Herrick published an article describing the blood findings for Mr. Noel in the Archives of Internal Medicine.
• Mason reviewed a case in the Journal of the American Medical Association, entitling it "Sickle Cell Anemia" in 1922 -The description of the shape of the red cells seen by Dr. Irons.
• Mason promulgated the misconception that this disease was exclusively seen in persons of African origin.
• Sydenstricked observed blood smears of Caucasian and African American children in 1923.
• Neel correctly concluded sickle cell anemia was a disease with Mendelian inheritance in 1949.
• Pauling, Itano, Singer, and Wells published an article in Science: "Sickle Cell Anemia: A Molecular Disease" in 1949.
• Itano used electrophoresis and showed a slight charge difference between three hemoglobins.
• There must be a conformational change in the hemoglobin molecule for the molecules to align and change the shape of the red cell.
• Ingram was able to separate hemoglobin A from hemoglobin S in 1956 and showed that hemoglobin S had a positive charge relative to hemoglobin A.
• In 1957, Ingram showed there was more valine and less glutamic acid in hemoglobin S.
• Morotta showed in 1977 there was a one-nucleotide change of an adenine for a thymine, a residue in the β-globin gene: GAG to GTG.

Genetics of Sickle Cell Anemia

• In the late 1940s and 1950s, some African Researchers were unable to find evidence of a familial pattern of inheritance for the ailment.
• Lehmann and Raper described a community population in Uganda to have predicted 10% would have sickle cell anemia in 1949 and 1956.
• These early studies in Africa sampled populations after young children had died.
• The homozygous condition for sickle cell disease leads to early mortality, but the heterozygous condition confers a survival advantage.
• This is termed a balanced polymorphism.
• Sickle cell disease occurs in areas where Plasmodium falciparum malaria is common.
• Heterozygosity for sickle hemoglobin S with normal hemoglobin A confers a selective advantage.
• Mutation for sickle hemoglobin occurred at least three times in africa and once on the indian subcontinent.
• Understanding of the hemoglobin gene clusters occurring on chromosomes 11 and 16 and their expression are required.
• Max Perutz is most responsible for elucidating the structure and function of the hemoglobin molecule.
• Two similar gene clusters code for the two globin proteins.
• The β-globin gene cluster is found on chromosome 11 (11p15.4), and the a-globin gene cluster is found on chromosome 16 (16p13.3).
• The globin gene clusters are highly conserved.
• In both the a- and β-genes there are three exons and two introns.
• Within the β-globin gene cluster, there are five functional genes and one pseudogene.
• Within the a-gene cluster, there are three functional genes and two pseudogenes.
• Alpha-gene mutations involve deletions, duplications, and triplications.
• In β-gene, point deletions predominate.
• The order of expression during fetal development occurs from the 5' end to the 3' end.
• There are two ẞ-like globin switches, epsilon to gamma and gamma to beta, and one a-like globin switch, zeta to alpha.
• Promoter regions control each globin gene and share conserved sequences that bind transcription factors.
• Erythroid Krupple-like factor is erythroid-specific.
• Regulation of gene function by trans-acting chromosomes is at Xp22.2 on the X chromosome.
• Manipulation of the hemoglobin switch is a dominant theme in the treatment of sickle cell disease.

Hemoglobin and Oxygen

• Each individual globin chain envelopes and stabilizes the oxygen-binding heme moiety.
• The globin chains interact with each other under the influence of heterotophic ligands.
• Oxygen and carbon monoxide (CO) are considered homotrophic ligands.
• Hemoglobin influences the solubility of carbon dioxide in plasma by the release of protons (Bohr effect) when hemoglobin is deoxygenated.
• 2,3-bisphosphoglycerate is synthesized in the red cell and stabilizes deoxyhemoglobin.
• The interaction between hemoglobin and these heterotrophic ligands changes hemoglobin affinity for oxygen and alters the shape of the molecule through allosteric effects.
• In the presence of heterotropic ligands, the hemoglobin molecule is in the deoxygenated or “tense state" (T structure).
• In the presence of oxygen, the hemoglobin molecule is in the “relaxed state" (R structure).
• Classic allosteric enzyme exhibiting cooperativity is described by the sigmoid plot of the oxygen dissociation curve.
• Polymerization of sickle hemoglobin only occurs with hemoglobin in the T, deoxygenated, state heterotrophic ligands increase polymerization.
• CO and heme oxygenase, and nitric oxide (NO) are being appreciated to play a role in sickle cell disease.
• The polymerization of deoxygenated sickle hemoglobin is the pathognomonic event in sickle cell disease.
• A patient presented with fever, dehydration, metabolic acidosis, and relative hypoxia due to anemia and pneumonia.
• Acidosis lead to a decreased ability to take up oxygen in the pulmonary capillaries.
• Regional hypoxia leads to V/Q mismatch.
• She then developed hemoglobin polymerization in her lungs, creating acute chest syndrome.
• In dilute solutions hemoglobin A and S have identical oxygen-binding curves but hemoglobin's S is decreased due to its concentration in the red cell.
• During the journey of red cells through the hypoxic microcirculation, there is an increase in T-state hemoglobin and nucleation formation of sickle hemoglobin. Polymerization proceeds with new nuclei on the surface of the existing polymer.
• Seven double strands make up each hemoglobin fiber, which has which has a helical arrangement with a periodicity of 22 Å.

Hemoglobin Interactions

• Hemoglobin S occurs in axial and lateral planes, in that the Beta-6 sickle mutation is involved in the lateral contacts between the Beta-globin chains.
• Both hemoglobin A and hemoglobin C can copolymerize with hemoglobin S.
• Hemoglobin F nor hemoglobin A2 copolymerize with sickle hemoglobin and both inhibit sickling.
• Polymerization and sickle hemoglobin affect the red cell membrane, which interacts with the microvascular endothelium and molecular environment within the circulatory system.
• Sickle hemoglobin itself causes oxidative damage to the cell membrane by creating hemichrome that can be seen microscopically within the red cell as Heinz bodies on the inner membrane.
• Hemoglobin fibers cause red cell membrane-cytoskeleton uncoupling, and the circulation activates the coagulation cascade.
• Vascular endothelial cells become activated in patients with sickle cell disease and increases expression of adhesion molecules and activation of leukocytes, leading to a procoagulant state.
• Catastrophic events are the direct result of sepsis, hepatic sequestration of red blood cells, and acute chest syndrome.
• Sickle cell disease can be characterized as a state of abnormally activated vascular endothelium that promotes increased adhesion of the red cells and leukocytes as well as a procoagulant state.
• Vaso-occlusion leads to vessel occlusion, and tissue damage from necrosis which causes, pain, stroke pulmonary hypertension etc.

Therapy

• Therapy for sickle cell disease has changed dramatically since the mid-1990s.
• In 1986, the Penicillin Prophylaxis Study provided that intervention with penicillin prevented 80% of life-threatening infections by Streptococcus pneumoniae.
• Penicillin was subsequently established as a therapy for newborns and children with sickle cell disease.
• In 1984, hydroxyurea was shown to be effective in increasing fetal hemoglobin levels and decrease the incidence of most complications.
• Hydroxyurea is a drug that inactivates ribonucleoside reductase and blocks the synthesis of deoxynucleotides, thus inhibiting DNA synthesis.
• Other chemotherapeutic agents in trials include magnesium, clotrimazole, arginine, compounds that decrease cell adherence, and agents to increase fetal hemoglobin.
• Blood transfusion Decreases morbidity in acute chest syndrome and surgery, and decreases the recurrence of stroke stroke.
• Blood donors and patients with sickle cell disease are ethnically diverse, and the red cell antigens on the majority of donor red cells occur at different frequencies than in most recipients.
• Stem cell transplantation has been used to cure sickle cell disease.