Laboratory Diagnosis of Thalassemia dr. mehdhrdad vanaki consultant of QA & QMS in medical lab 5 ABAN 1390 1 سخنی زيبا از آنتوان سنت اگزوپری من به ھيچ وجه خدا را لمس نکردم ولی خدائی که قابل لمس باشد که ديگر خدا نيست . اگر ھر دعائی را ھم اجابت کند ھمينطور . ھمان جا بود که برای نخستين بار حدس زدم که عظمت دعا بيش از ھر چيز در اين امر ن ھفته استات که پاسخاخی به آن داده نمی شود و زشتی سوداگری را به اين مبادله راھی نيستنت . اين را ھم دريافتم که آموختن دعا اموختن سکوت است و عشق فقط از جائی شروع می شود که ديگرير ھيچ انتظاری برایبری گرفترنن ھيچ چيز وجود نداشته باشد . عشق تمرين نيايش است و نيايش تمرين سکوت thal assemi a Genetics. y In the first 8 weeks of embryonic life the predominant forms of hemoglobin are: ◦ Hb Gower 1 (ζ2ε2). ◦ Hb Gower 2 (α2ε2). ◦ Hb Portland 1 (ζ2γ2). y By the 12th week embryonic hemoglobin is replaced by Hb F (α2γ2) which represents 70 – 100% of hemoglobin in fetal life. Genetics (2). y Adult hemoglobin Hb A (α2β2) detectable from 16/40, replaces Hb F as predominant hemoglobin by 6/12 after birth, up to 30% of Hb in fetal life. y Hemoglobin HbA2 (α2Δ2) is present in utero but only very minor in normal adults. y In normal adltdults 96 – 98% of hemog lo bin is HbA, Hb A2 (2 – 3%) and HbF (<1%) constitute a minor component of the total hemoglobin. Copyright ©1997 BMJ Publishing Group Ltd. α2∆2 α2γ2 What is thalassemia? y Genetic blood disorder resulting in a mutation or del e tion of the genes ththtat control globi n production. y Normal hemoggplobin is composed of 2 alpha and 2 beta globins y Mutations in a given globin gene can cause a decrease in production of that globin, resulting in deficiency y aggreg ates become oxidized Æ damage the cell membrane, leading either to hemolysis, ineffective erythropoiesis, or both. y 2 types of thalassemia: alpha and beta. Alpha Thalassemia y mutation of 1 or more of the 4 alpha globin genes on chromosome 16 y severity of disease depends on numbe r of genes affected y results in an excess of beta globins Silent Carriers (heterozygotes +/-) y 3 functional alpha globin genes y No symptoms, but thalassemia could poten ti ally appear in off spring Alpha ThalassemiaTrait y 2 functional globin genes y results in smaller blood cells that are liggtehter in colou r y no serious symptoms, except slight anemia Hemoglobin H Disease y 1 functional globin gene y results in very lightly coloured red blood cells and possib le severe ane mia y hemoglobin H is susceptible to oxidation, therefore oxidant drugs and foods are avoided y treated with folate to aid blood cell production Alpha Thalassemia Major y no functional globin genes y death before birth (embryonic lethality) Beta Thalassemia y mutations on chromosome 11 y hundreds of mutations possible in the beta ggoblobin gene, the re fo re beta thalassemia is more diverse y results in excess of alpha globins Beta ThalassemiaTrait y slight lack of beta globin y smaller red blood cells that are lighter in colou rdue to lack of hemog lob in y no major symptoms except slight anemia Beta Thalassemia Intermedia y lack of beta globin is more significant y bony deformities due to bone marrow trying to make more blood cells to replace defective ones y causes late dldevelopment, exercise intolerance, and high levels of iron in blood due to reabsorption in the GI tract y if unable to maintain hemoglobin levels between 6 gm/dl – 7 gm/dl, transfusion or splenectomy is recommended Beta Thalassemia Major y complete absence of beta globin y enlarged spleen, lightly coloured blood cells y severe anemia y chronic transfusions required, in conjunction with chelation therapy to reduce iron (desferoxamine) Variant Hemoglobin Classification. y The variant haemoglobins are disorders of globin chain synthesis. y Normal αβ ratio so most have normal MCV and MCH. y There are over 1000 mutations associated with the haemoglobinopathies most of which will produce variants. Variant Hemoglobin Classification. y Initiallyyg recognized forms were classified alphabetically (Hb C, D, E), subsequent naminggy after the location of discovery. y The most common forms in Australia include Hb S, Hb E, Hb Constant Spring and Hb C. y Usually caused by point mutations. Laboratory Diagnosis of Thalassemia dr. mehdhrdad vanaki consultant of QA & QMS in medical lab 5 ABAN 1390 24 Laboratory Diagnosis of Thalassemia y Need to start with patient's individual history and familyyy history. Ethnic background important. y Perform ppyhysical examination: ◦ Pallor indicating anemia. ◦ Jaundice indicating hemolysis. ◦ Splenomegaly due to pooling of abnormal cells. ◦ Skeletal deformity, especially in beta thalassemia major. 25 CBC with Differential y See decrease in hemoglobin, hematocrit, mean corpuscular volume (MCV), and mean corpuscular hemoglobin (MCH). See normal to slightly decreased Mean Corpuscular Hemoglobin Concentration (MCHC). y Will see microcytic, hypochromic pattern. y Have normal or elevated RBC count with a normal red cell vol ume di st rib uti on (RDW) . y Decrease in MCV very noticeable when compared to decrease in Hb and Hct. 26 CBC with Differential y Elevated RBC count with markedly decreased MCV differentiates thalassemia from iron deficiency anemia. y On differential, see microcytic, hypochromic RBCs (except in carrier states). See mild to moderate poikilocytosis. y In more severe cases, see markdked number of target cells and ellip tocy tes. Will see polhlychromas ia, basophilic stippling, and NRBCs. 27 PBS in thalassemia y Thalassaemias are typically microcytic and hypochromic anemia's. y Thalasse mia causes a uuoniform microcytosis without increase in RDW (cf iron deficiency). y Hb H and Δβ thal however can cause an increased RDW. PBS y RBC often increased in thal but decreased in iron deficiency y Hb typica lly norma l in ttahalminor but decreased in intermedia and major syndromes. y MCV is the most valuable parameter in predicting thal. Alpha Thalassaemia Major - target cells, microcytosis, hypochromia, NRBCs, poikilocytes, polychromasia, basophilic stipling, tear drops. βThal Major anisocytosis, poikilocytosis, targets, tear drops, fragments, hypochromaia, basophilic stippling. In th e setti ng of Hb H di sease, a disor der in w hic h three of four a-globin chain genes are Hb H INCLUSION BODIES IN BCB STAIN nonexpressed,30–100,RETICULOCYTE:5-10%% of red cells contain typical inclusions. y HEINZ BODIES y GOLF BALL APPEARING RBC y RETICULOCYTE y HEINZ BODY IN GIEMSA STAIN GOLF BALL APPEARING RBC In α-thalassemia minor may be associated with as few as 1 inclusion-containing cell in 1000–10000 cells . The absence of Hb H inclusions therefore does not exclude thalassemia trait, but the presence of typical inclusions may be helpful in confirming a presumptive diagnosis. Iron Studies. y Except in the urgent situation of pregnancy iron deficiency should always be excluded and treated prior to work up for thal . y MCV and MCH are influenced by iron dfiideficiency. y Hb A2 can be lowered by iron deficiency Î falsely normal results if tested when iron deficient (false negatives). Table 1: Laboratory features in different clinical states. Iron Chronic Iron Thalassemia Deficiency Disease Overload Haemoglobin N or ↓ ↓ N N or ↓ Serum Fe ↓ ↓ ↑ ↑ or N Transferrin ↑ ↓ or N ↓ N Receptor Transferrin ↓↓ ↑N Sat. Ferritin ↓↑or N ↑ ↑ or N MCV ↓ ↓ or N N ↓ Marrow Fe ↓↑ ↑↑ Hb H Inclusions. y Hb H is an insoluble tetramer consitiisting of four bbteta glbilobinchihains, due to a lack of alpha chains in alpha thal major. y Oxidation of these tetramers provokes precipitation which can be visualized microscopically as ‘golf ball’ inclusions. y Oxidation can be precipitated by oxidative dyes (although there is significant batch to batch variabilityyg making controls essential) . Hb H Inclusions (2). y In Hb H disease 30 – 100% of RBCS contain Hb H inclusions. y In aaplpha ttahalminor tteehere is oeone cell wwtith Hb H inclusions per 1000 – 10,000 RBCS. y Other nucleic acid and protein precipitates also stain (without ‘golf ball’ pattern). Routine screening tests for diff IDA & minor BB--thalassemiathalassemia 9RBC count 9Serum iron & ferritin & Iron store BM 9TIBC & TS index 9Hb A2 9Free erythrocyte protoporphihyrin 9CBC index ( simple & rapid) Differentiative index y Iron deficiencyy() anemia (IDA) and beta thalassemia minor (BTM) are the most common causes of hypochromic microcytic anemia. y Many indices have been defined for rapid differentiation of these diseases via red blood cell indices IDA T.T. MCV-(5.Hb)-RBC-3.4 = England formula >0 0< 99.2% 69.5% MCV/RBC = Mentzer formula >13 <13 94.6% 95.5 % MCH/RBC = Srivastava formula >3.8 <3.8 86.5% 85.7% Kawakami formula RBC <5000000 >5000000 86.2% 98% MCV.MCH / RBC 301-370 250-300 24.6% 9/7% = Kerman I MCVMCHMCV.MCH / RBC. MCHC 8.1-13 <10.5 62% 98.7% = Kerman I MCV-10.RBC = Kermanshahan >15 <15 90% 95.5% MINOR β--THALASSEMIATHALASSEMIA vs. IDA Discriminant β- formulas THALASSEMIA IDA Mentzer <<1313 =>=>1313 (MCV/RBC) BeningtonBenington--SrivastavaSrivastava <<44..44=>=>44..44 (MCH/RBC) Green & King <65 =>65 MCV2 x RDW/RDW/HbHb x 100 Ratio of >>00..99<<00..99 micro./hypo. MEAN DENSITY Hb PER LITER, MDHL MDHL: MCHD x RBC (Mean Cell Hb Density= MCH/MCV) SUBJECT NORMAL β--TmTm IDA MDHL M:M:11..7575+/+/--00..1212 F:F:11..55+/+/--00..1111 Differentiative index y 1- Kawakami index(RBC count index): <5 possible IDA >5 possible BTm Specifity +sensitivity =84% Differentiative index y 2- mentzer index (()MCV /RBC): >13 IDA <13 BTm Specifity +sensitivity =90% Differentiative index y 3- index Srivastava (()MCH /RBC): >3.8 IDA <3.8 BTm Specifity +sensitivity