Clinical Hematology II (HML2143) - PDF

Clinical Hematology II HML2143 1 1 LO1: Describe the hemoglobinopathies with particular regard to their prevalence and importance in the UAE. Week 1: Describe the cause, pathophysiology and clinical effects of sickle cell trait, disease and other hemoglobin vari...

Clinical Hematology II HML2143 1 1 LO1: Describe the hemoglobinopathies with particular regard to their prevalence and importance in the UAE. Week 1: Describe the cause, pathophysiology and clinical effects of sickle cell trait, disease and other hemoglobin variants, together with currently available treatment options. 2  Sickle Cell Anemia Hb disorder with inherited sickle β- globin gene The sickle β-globin abnormality is caused by: Substitution of Glutamic acid (Glu) by valine (Val) at 6th position in β-chain (β-6 glu  val) Producing Hb S & the cell is called Sickle cell “Drepanocytes”  Sickle Cell Anemia An autosomal recessive inherited disorder Homozygous: Hb SS (most common) SCD or SCA Heterozygous: Hb AS…called “Sickle cell trait” The severity of SCD is based on whether it is homozygous (Hb SS) or heterozygous (Hb AS).  Sickle Cell Anemia Hb S (α2β2s): 1) In normal O2 tension→ causes no problem & it releases oxygen to the tissues easily, compared with Hb A (shift to the right with low affinity to O2). 2) In low O2 tension: Hb S is insoluble that forms crystals. So, deoxygenated sickle Hb → polymerizes into long fibers→ forming sickled RBCs. These are rigid cells → may block different areas of microcirculation (thrombosis) or large vessels (organ failure) https://www.dnalc.org/resources/3d/17-sickle-cell.html Pathogenicity of SCD  Pathophysiology Of Sickle Cell Anemia Hemoglobin S (HbS) (β -6 glu  val) Conformational changes upon exposure to low oxygen tension Hb-S polymerization (Long fibers of double cross-linked strands) Red cell sickling (Rigid cells that are unable to pass through microcirculation)  Clinical Features Of Hb S The clinical presentation is variable, some patients have normal life, free of crises. While others develop severe crises even as infants & may die in early childhood or as young adults. Crises may be: 1. Painful vaso-occlusive crises: - Usually associated with severe pain - Caused by sickle cell in small vessels - Resulting in tissue damage & organs infarction Swelling of feet and hands happens - Example: Hand-foot syndrome due to sickle cell getting stuck in the blood vessels in the hands & feet in - It lasts from 4-6 days or maybe weeks. which causes the blood to accumulate causing pain & swelling. Clinical Features Of Hb S Hand-foot syndrome Hand-foot syndrome: 1. Shorting of right middle finger 2. Caused by small bones infarction 3. Frequently the first presentation of the disease  Clinical Features Of Hb S 2. Visceral sequestration crises: - Caused by sickling within organs. - Acute sickle chest syndrome: the most common cause of death after puberty. - Splenic sequestration: seen in infants with enlarged spleen. 3. Aplastic crises: Splenic sequestration is - Occurs as a result of infection with parvovirus or folate deficiency. a blockage of the - Sudden fall in Hb and retics due to marrow aplasia. splenic vessels by sickle cells causing a 4. Hemolytic crises: sudden, painful - Increased rate of hemolysis with fall in Hb and rise in retics. enlargement of the spleen. - Usually accompanies with a painful crisis.  Summery Of Crisis Of Hb S 1) The clinical expression of SCA is very variable, (Some patients have normal life, while others may develop severe crises & die early): a) Painful vascular crises (sickling of small vessels of brain, hip, shoulders, vertebrae) b) Visceral sequestration crises (sickling within organs) → lung, liver & spleen c) Aplastic crises→ (due to parvovirus infection)→↓ Hb & ↓ retics d) Hemolytic crises→ (↓ Hb but ↑ retics)  Other Clinical Features Severe hemolytic anemia Splenomegaly In infancy and early childhood but later is often reduced in size (splenic atrophy) due to infarction. Pigment gallstones Due to increase bilirubin Infections Abnormal growth. Eye Problems: Blindness. Stroke Skin ulcers. SCA can cause open sores, called ulcers, on your legs.  Lab Diagnosis Of Hb S 1) CBC: Hb: 6 – 9 g/dl MCV: 90 – 95 fl WBC often increased 2) Blood film: Normocytic normochromic anemia Anisocytosis and poikilocytosis Sickle/Boat cells usually < 10%, target cells, Howell- Jolly bodies Polychromasia Increased retics (5 -20%), but decreases during aplastic crises. Nucleated RBCs  Lab Diagnosis Of Hb S 3) Screening tests for sickling (Hb S): 1) Sickling test: positive When oxygen is removed from whole blood Hb S within the red cells forms crystals RBCs form sickles. Oxygen is removed from red cells by incubation in a wet preparation on a slide after the addition of a strong reducing agent, sodium metabisulphite.  Lab Diagnosis Of Hb S 2- Solubility Test: RBCs are lysed by saponin and the released Hb is reduced by Sodium dithionite in a high phosphate buffer. Hb S is insoluble when its in the reduced form. It forms crystals that produce a turbid solution. Results of Solubility Test: Positive tests: lines on card cannot be seen through the test solution. Negative test: lines on card can be seen through the test solution. After centrifugation, positive tests will show: Dark red band at the top with pink or colorless solution below.  Lab Diagnosis Of Hb S 4. Hb electrophoresis: To confirm the presence of Hb S when solubility test is positive. 1) Alkaline Electrophoresis (pH 8.9) on cellulose acetate: Some abnormal hemoglobins have the same mobility at pH 8.9, (S and D together, and A2, C and E together).  Lab Diagnosis Of Hb SS Acid Electrophoresis (pH 6.0) Electrophoresis at pH 6.0 on agarose gel helps to solve these problems. Hb S (80% predominate) Hb F (5 -15%) & Hb A2 (2-5%) …No Hb A  Electrophoresis results in SCD: Alkaline + Acid Electrophoresis in : SC trait: Hb-A: 60% Hb-S: 40% Sickle cell disease:  Hb-A: 0% Absent A B C A B C  Hb-A2: 2-5%  Hb-F: 5-15% A: Control B: Normal  Hb-S: > 80% C: Patient with Hb AS Hemoglobin Electrophoresis  Lab Diagnosis Of Hb S 5. High Performance Liquid Chromatography (HPLC) Run it to confirm the diagnosis of HbS HPLC can be also used to measure the levels of the various types of hemoglobin Principle: Hemolysed red cell samples, controls, calibrators and test samples are held in an automatic sample chamber at 12⁰ C. As Hb passes through a spectrophotometer cuvette, the absorbance of the solution is recorded. The absorbance is measured at 415 nm and at 690 nm. A chromatogram plot of A against time records the various Hb types & concentration. The area under the curve = concentration. A calibrator sample allows the apparatus to be calibrated to identify each Hb type.  Treatment of sickle cell disease Supportive Anti-infection drugs – infections may cause a sickle cell crisis Folic acid Regular blood transfusions – for severe anemia, and those who suffer frequent crises Hydration– intravenous normal saline used to treat vaso-occlusive crisis Therapeutic Drugs used to reduce the effects of Hb S, or which may help to produce more Hb F (protects against the effects of Hb S) e.g. hydroxyurea increases gamma glubin chain synthesis Curative Bone marrow transplant – selected patients Gene therapy – insertion of a normal β-globin gene into stem cells using a retrovirus  Sickle Cell Trait Benign condition with no anemia Normal RBCs mainly, sickled RBCs might be seen Sickle cell Hematuria: most common symptoms (minor renal infarction) Care must be taken during pregnancy, high altitudes, severe exercises, heavy smoking ,etc Electrophoresis in Sickle cell trait: Hb-A: 60% Hb-S: 40% (20-50)% Hb-A2: slightly ↑ Hb-F: within normal limits  Management/ Antenatal care Lab testing is important in identifying couples at risk for significant hemoglobinopathies. For difficult cases, access to molecular testing can facilitate complete diagnosis. 1. Prenatal diagnosis: may be offered, and will require molecular testing. (next slide) 2. Pre-implantation diagnosis is an alternative option. It offers the advantage of avoiding the need for pregnancy termination. Requires in vitro fertilization (IVF) to obtain embryos for evaluation.  Management/ Antenatal care Prenatal Diagnosis: (before birth) 1. Chorionic villus sampling (CVS) 10–12 weeks (1st trimester) 2. Amniocentesis 14–20 weeks (2nd trimester) Both invasive tests Risk of miscarriage of 0.5 – 1%. The most important to recognize for antenatal care: SS, SC, SD, Punjab, SE, SOArab, S/Lepore, S/β Thal, S/δβ Thal , β Thal major/Intermedia, Hb E/ β Thalassaemia & Hb H disease  The End Watch this short video cartoon about sickle cell https://www.youtube.com/watch?v=R4-c3hUhhyc Read this article from the UAE Genetics Association about Sickle Cell disease. http://www.uaegda.ae/web/guest/sickle-cell-anemia Read this article about Sickle Cell disease and the incidence in the Arab world. http://www.cags.org.ae/gme1bdensickle.pdf  References McKenzie, Shirlyn/ Landis-Piwowar; Kristin/ Williams, Lynne (2019). Clinical Laboratory Hematology. 4th Ed. Pearson Education. ISBN 9780134709239 Kaushansky/ et al, (2016) Williams Hematology, 9th Ed. McGraw-Hill. ISBN 9780071833011 Lewis, S., Bain, B., Bates, I. (2012) Dacie and Lewis, Practical Haematology, 11th Ed., Churchill Livingstone Elsevier. ISBN: 978-0-7020-3407-7 Hoffbrand, A., Moss, P. (2011) Essential Haematology, 6th Ed. Wiley Blackwell. ISBN: 978-1-4051- 9890-5 Moss, Paul, Pettit, Hoffbrand. Essential Haematology, 5th Ed. Blackwell Science. 2006. ISBN: 9781405136495 Moore, Gary/ Knight, Gavin (2013) Essential Haematology, Oxford University Press ISBN: 9780191666711 800 MyHCT (800 69428) [email protected] www.hct.ac.ae Happiness Center PO Box 25026 Abu Dhabi, UAE HCT_UAE hctuae 31