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Hb electrophoresis

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Mohammed Bahgat Mohammed Sofyan
Mohammed Bahgat Mohammed Sofyan

ربط الشرح على ليوتيوب 1- https://youtu.be/1DcAW_WRIQc 2-https://youtu.be/RJBHBwrxPeE 3- https://youtu.be/xLtkHjE_Elk

Education
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BY Dr: Mohammed Bahgat Mohammed Sofyan Assistant lecturer Faculty of medicine, Al-Azhar university (Assiut branch) 1
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‫صورة‬ ‫كل‬ ‫على‬ ‫اضغط‬ ‫الشاش‬ ‫عرض‬ ‫وضع‬ ‫في‬ ‫ة‬ ‫قنواتنا‬ ‫على‬ ‫وادعمنا‬ ‫اإلنترنت‬ ‫على‬
‫على‬ ‫ملفي‬ ‫في‬ ‫هنا‬ ‫أرفعها‬ ‫التي‬ ‫شرائحي‬ ‫ومذاكرة‬ ‫بالتعلم‬ ‫وأسعد‬ ‫وأرحب‬ ‫أسمح‬ Slide share website ‫وال‬ ‫مانع‬ ‫أيضا‬ ‫من‬ ‫نسخ‬ ‫شريحة‬ ‫او‬ ‫اثنين‬ ‫عند‬ ‫الضرورة‬ ، ‫وال‬ ‫مانع‬ ‫من‬ ‫شرح‬ ‫البوربو‬ ‫ينت‬ ‫الخاصة‬ ‫بي‬ ‫للغير‬ ‫بشرط‬ ‫عدم‬ ‫إزالة‬ ‫اسمي‬ ‫من‬ ‫البوربوينت‬ ، ‫فال‬ ‫أسمح‬ ‫أبدا‬ ‫بإزالة‬ ‫اسمي‬ ‫من‬ ‫على‬ ‫الباوربوينت‬ ‫ووضع‬ ‫اسمك‬ ‫بدال‬ ‫منه‬ ‫لتصبح‬ ‫وكأنك‬ ‫من‬ ‫صممتها‬ ‫فهذه‬ ‫سرقة‬ ‫ال‬ ‫أسمح‬ ‫بها‬ ‫وتضييع‬ ‫لحق‬ ‫من‬ ‫تعب‬ ‫في‬ ‫عملها‬ . ‫وفقكم‬ ‫هللا‬ ‫وإياي‬ ‫للتعلم‬ ‫ونفع‬ ‫اآلخرين‬ I allow, welcome, and be happy to learn and study my slides that I upload here in my profile on Slide share website There is also no objection to copying one or two slides when necessary, and there is no objection to explaining my PowerPoint to others on the condition that my name is not removed from the PowerPoint. I never allow my name to be removed from PowerPoint and to replace it with yours, Make it look like you designed it. This is theft that I do not allow and a waste of the right of those who are tired in this work. May God bless you and me for learning and benefiting others 5
‫ال‬ Hb ‫من‬ ‫مكون‬ ‫اإلنسان‬ ‫جسم‬ ‫في‬ ‫اللي‬ heme ‫و‬ globin ‫ال‬ heme ‫مكون‬ iron ‫و‬ protoporphyrin ‫ال‬ globin ‫من‬ ‫يتكون‬ 2 polypeptide chain ( Alfa and Beta, delta or gamma,…). ‫ال‬ Alfa chain ‫من‬ ‫تتكون‬ 141 AA ‫الكروموسوم‬ ‫علي‬ ‫ممثلة‬ 16 ‫الزوجين‬ ‫علي‬ ‫جينات‬ ‫بأربع‬ . ‫ال‬ ‫بينما‬ Beta chain ‫من‬ ‫تتكون‬ 146 AA ‫الكروموسوم‬ ‫علي‬ ‫بجينين‬ ‫ممثلة‬ 11 . ‫ال‬ ‫حسب‬ ‫علي‬ Polypeptide chain in hemoglobin 6
Composition of Hemoglobin ( Hb) HB heme iron protoporphyrin globin 4 polypeptide chain 7
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11
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NORMAL HB HbA composed of Alfa 2 and beta 2 globin chain HbA2 composed of Alfa 2 and delta 2 globin chain HbF composed of Alfa 2 and gamma 2 globin chain 13
normal 14
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When is it order? • as part of newborn screening. In addition, it may be used for prenatal screening when a parent is at high risk or when parents have a child who has a hemoglobinopathy. • when results of a complete blood count (CBC) and/or blood smear suggest that you have an abnormal form of hemoglobin. • when a healthcare practitioner suspects that your signs and symptoms are the result of abnormal hemoglobin production. Abnormal forms of hemoglobin often lead to hemolytic anemia 18
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Proteins (Hb) are amphoteric compounds and are therefore either positively or negatively charged because they contain both acidic and basic residues. In an alkaline buffer (pH 8.4), Hb gain negative charges and migrate to the positive electrode (anode). On a support media, HBs are separated according to their shape, size and net surface charge , they migrate to anode. 21
• sample are put on a supporting medium between two electrodes. • When the electric current is connected, the charged molecules move from one electrode to another at different migration rates according to their size and charge, allowing their separation. 22
• The supporting medium (cellulose acetate gel) is soaked in alkaline buffer. • RBCs is washed, packed, lysed, diluted and then applied on to the gel. • The electric current is then connected (200 V for 30 minutes) to allow separation of different Hb molecules into bands which are then stained with protein/heme stain. • The stained bands are then scanned (densitometer) and the density of each band is roughly correlated with percentage of the variant in the sample. • A graph is generated representing the bands by corresponding peaks. • Control sample is run simultaneously in the same gel. 23
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STAIN 25
DETECION AND QUANTIFICATION 26
1_ Cellulose acetate electrophoresis at alkaline pH ( 8.2_8.6 ) Sample ➡ whole blood best performed on packed RBCs especially in hypergammaglobulinaemia whole blood on EDTA tube. It can be stored at 2-8 oC for 7 days Bands of cellulose acetate electrophoresis at alkaline pH : H➡ and others as Hb bart's,..... A➡ F ➡ S ➡ and others as Hb D , HbG, Hb lepor C ➡ and others as HbA2 , HbE, HbO 27
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Disadvantage Can’t be used to quantify small variants e.g., Hb A2. many variants have same migration point. 33
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Ion Exchange chromatography ‫هذه‬ ‫الطريقة‬ ‫بتفصل‬ ‫األحماض‬ ‫األمينية‬ ‫والهيموجلوبين‬ ‫وغيره‬ ... ‫نجعل‬ ‫الوسط‬ ‫حمضي‬ ‫فستصبح‬ ‫األحماض‬ ‫األمينية‬ ‫المراد‬ ‫فصلها‬ ‫موجبة‬ ، ‫ونجعل‬ ‫مثال‬ ‫الصوديوم‬ ‫قليل‬ ( ‫ما‬ ‫دة‬ ‫توجد‬ ‫في‬ ‫الجزء‬ ‫المتحرك‬ ) ‫وبوضع‬ ‫مادة‬ ‫صمغية‬ ‫عليها‬ ‫ايونات‬ ‫سالبة‬ ‫في‬ ‫الجزء‬ ‫الثابت‬ ، ‫فعند‬ ‫مرور‬ ‫األحماض‬ ‫األميني‬ ‫ة‬ ‫الموجية‬ ‫تمسك‬ ‫في‬ ‫األيونات‬ ‫السالبة‬ ‫التي‬ ‫في‬ ‫الجزء‬ ‫الثابت‬ ‫بدال‬ ‫عن‬ ‫الصوديوم‬ ‫ألنه‬ ‫قليل‬ ‫فتزيحه‬ ‫وتمسك‬ ‫باأل‬ ‫يونات‬ ‫السالبه‬ ‫الموجودة‬ ‫علي‬ ‫الجزء‬ ‫الثابت‬ ‫بدال‬ ‫من‬ ‫الصوديوم‬ Ion Exchange ‫ما‬ ‫رأيك‬ ‫لو‬ ‫جعلنا‬ ‫الوسط‬ ‫قلوي‬ ‫بدال‬ ‫من‬ ‫حمضي‬ ‫وزودنا‬ ‫ايونات‬ ‫الصوديوم؟‬ Adjust pH and ionic strength ‫في‬ ‫الوسط‬ ‫القلوي‬ ‫البروتينات‬ ‫ستصبح‬ ‫سالبة‬ ‫وستتنافر‬ ‫مع‬ ‫األيونات‬ ‫السالبه‬ ‫الموجودة‬ ‫علي‬ ‫الجزء‬ ‫ال‬ ،‫ثابت‬ ‫ولو‬ ‫زودنا‬ ‫ايونات‬ ‫الصوديوم‬ ‫ايضا‬ ‫ستحل‬ ‫محل‬ ‫األحماض‬ ‫األمينية‬ ‫وبالتالي‬ ‫يحصل‬ Elution to amino acids gradually The main application of adsorption chromatography is separation of different amino acids hemoglobin variants and isoenzymes. 36
37
High Performance Liquid Chromatography (HPLC) for separation of Hb variants ‫ايه‬ ‫فكرة‬ ‫عمل‬ ‫فصل‬ ‫أنواع‬ ‫الهيموجلوبين‬ ‫بجهاز‬ ‫ال‬ HPLC ?? ‫نضع‬ ‫عينة‬ ‫الدم‬ ‫في‬ ‫الجهاز‬ ‫مع‬ ‫ال‬ Mobile phase ‫إيه‬ ‫اللي‬ ‫هيحصل؟‬ ‫ال‬ column that contain stationary phase ‫يمتز‬ ‫الهيموجلوبين‬ ‫ألن‬ ‫الهيموجلوبين‬ positive ‫وال‬ Stationary phase contains negative charge ‫كده‬ ‫الهيموجلوبين‬ ‫ذو‬ ‫الشحنة‬ ‫الموجبة‬ ‫مسك‬ ‫في‬ ‫ال‬ column ‫ذو‬ ‫الشحنة‬ ‫السالبة‬ 38
‫بعد‬ ‫ذلك‬ ‫نزيد‬ ‫من‬ ‫مرور‬ Buffer that contain cations in mobile phase and change pH ‫تقوم‬ ‫ال‬ cations ‫تتنافس‬ ‫مع‬ ‫الشحنات‬ ‫الموجبة‬ ‫الموجودة‬ ‫على‬ ‫الهيموجلوبين‬ ‫على‬ ‫الشحنات‬ ‫السالبة‬ ‫الموجو‬ ‫دة‬ ‫على‬ ‫ال‬ Stationary phase. The cations in the mobile phase compete with the adsorbed proteins ( Hb ) for the anionic binding sites present in the stationary phase. ‫يقوم‬ ‫ال‬ cations ‫تحل‬ ‫الهيموجلوبين‬ ‫على‬ ‫الجزء‬ ‫الثابت‬ ‫ويحصل‬ gradual elution to Hb variants ‫علي‬ ‫حسب‬ Their affinity for the stationary phase ( polarity ) ‫طبعا‬ ‫كل‬ ‫ما‬ ‫زادت‬ ‫الشحنة‬ ‫الموجبة‬ ‫في‬ ‫نوع‬ ‫معين‬ ‫من‬ ‫الهيموجلوبين‬ ‫كل‬ ‫ما‬ ‫مسك‬ ‫أكثر‬ ‫بالجزء‬ ،‫الثابت‬ ‫وبالت‬ ‫الي‬ ‫يحصل‬ ‫له‬ elution ‫أبطأ‬ ‫وبالتالي‬ ‫يصل‬ ‫لل‬ detector ‫متأخرا‬ ‫وبالتالي‬ High retention time. ‫وكل‬ ‫ما‬ ‫زادت‬ ‫الشحنة‬ ‫السالبة‬ ‫في‬ ‫نوع‬ ‫معين‬ ‫من‬ ‫الهيموجلوبين‬ ‫كل‬ ‫ما‬ ‫حصل‬ ‫له‬ elution ‫أسرع‬ ‫وبالتالي‬ ‫يصل‬ ‫لل‬ detector ‫أسرع‬ ‫وبالتالي‬ Low retention time 39
‫طب‬ ‫الجهاز‬ ‫عارف‬ ‫الوقت‬ ‫اللي‬ ‫هيوصل‬ ‫عنده‬ ‫كل‬ ‫نوع‬ ‫من‬ ‫أنواع‬ ‫الهيموجلوبين‬ ‫والذي‬ ‫يس‬ ‫مي‬ ‫ال‬ retention time ‫كل‬ ‫نوع‬ ‫من‬ ‫الهيموجلوبين‬ ‫له‬ Specific retention time ‫محدد‬ ‫الجهاز‬ ‫عارفه‬ ‫وبالتالي‬ ‫الجهاز‬ ‫يعرف‬ ‫ما‬ ‫هو‬ ‫البروتين‬ ‫أو‬ ‫الهيموجلوبين‬ ‫اللي‬ ‫وص‬ ‫ل‬ ‫في‬ ‫هذا‬ ‫الوقت‬ ‫وتركيزه‬ ‫يساوي‬ ‫كم؟‬ . ‫كل‬ ‫ده‬ ‫واضح‬ ‫على‬ ‫الورقة‬ ‫بعد‬ ‫كده‬ ‫يسجل‬ ‫الكالم‬ ‫على‬ ‫هيئة‬ peaks ‫عن‬ ‫طريق‬ ‫جهاز‬ ‫ال‬ densometer ‫او‬ ‫نعملها‬ elution followed by spectrophotometer assay. ‫أعلم‬ ‫وهللا‬ . 40
is a process in which a mixture of molecules (such as normal and abnormal hemoglobin variants ) with a net positive charge is separated into its components by their adsorption onto a negatively charged stationary phase in a chromatography column, followed by their elution by a mobile phase. The mobile phase is a liquid with an increasing concentration of cations flowing through the column; the cations in the mobile phase compete with the adsorbed proteins for the anionic binding sites. 41
Thus, the adsorbed positively charged hemoglobin molecules are eluted from the column into the liquid phase at a rate related to their affinity for the stationary phase. When separated in this way, they can be detected optically in the eluate, provisionally identified by their retention time, and quantified by computing the area under the corresponding peak in the elution profile. The more positively charged hemoglobin (e.g., hemoglobin S and C) have a longer retention time. 42
43
Variant with weak positive charge will adsorb weakly to the column and is eluted rapidly from the column with the injection of low strength elution buffer. Variant with strong positive charge will adsorb strongly to the column and is eluted slowly from the column with the injection of high strength elution buffer. Procedure: HPLC is fully automated. Samples are loaded in a rack and automatically processed one by one (about 6 minutes/sample). Result: Data are graphically represented as absorbance vs time (peaks). Retention time is the time from injection to the top of the peak. 44
‫له‬ ‫الهيموجلوبين‬ ‫من‬ ‫نوع‬ ‫كل‬ Specific Retention time ‫ال‬ ‫يطلع‬ ‫ولما‬ ،‫الحالة‬ ‫للجهاز‬ ‫تدخل‬ ‫فإنت‬ ،‫عارفه‬ ‫الجهاز‬ Its Retention time ‫ال‬ ‫بمعلومية‬ ‫فورا‬ ‫الهيموجلوبين‬ ‫نوع‬ ‫يقولك‬ ‫يقوم‬ Its retention time. ‫ال‬ ‫نفس‬ ‫لها‬ ‫اللي‬ ‫األنواع‬ ‫من‬ ‫بالك‬ ‫وخلي‬ retention time ‫ال‬ ‫ومشاكل‬ Glycated Hb and HbS with HbA2..... etc. ‫العيوب‬ ‫في‬ ‫الحقا‬ ‫ستذكر‬ ‫كما‬ . 45
46
47
48
disadvantages of HPLC 1_ expensive 2_ Small part of HbS coelute with Hb A2 leads to falsely high HbA2 value in Sickle cell variant as Retention time of them are near • ‫لذلك‬ ‫ال‬ ‫يستحب‬ ‫في‬ ‫التفرقة‬ ‫بين‬ HbSS and HbSBo 3_ Retention time of glycated and non glycated hemoglobin are different ➡ leads to misinterpretation i.e. Glycosylated hemoglobin S may have a retention time the same as, or very similar to, that of hemoglobin A, so that patients with sickle cell anemia may be thought to have a small amount of hemoglobin A. 4 _ Glycated Hb may measured as HbF falsely elevate HbF in HPLC. 5_ although many variant hemoglobin can be separated from each other, they are some that overlap with each other as Hb lepor usually overlaps with HbA2 and HbE 6_ Certain artefacts need to be recognized for example, increased bilirubin in the plasma may lead to a sharp peak in the same general area as hemoglobin H. The nature of any variant hemoglobin detected by HPLC should be confirmed by an alternative technique. 49
Hb variant on HPLC • Hb C elutes in C window in HPLC (C/O). • Hb E elutes in A2 window in HPLC (A2/E/Lepore). • Hb D elutes in D window in HPLC (D/G). • Small part of HbS coelute with Hb A2 leads to falsely high HbA2 value in Sickle cell variant as Retention time of them are near so you cant differentiate between HBSS and HBSB0 • Certain artefacts need to be recognized for example, increased bilirubin in the plasma may lead to a sharp peak in the same general area as hemoglobin H. • Retention time of glycated and non glycated hemoglobin are different ➡ leads to misinterpretation i.e. Glycosylated hemoglobin S may have a retention time the same as, or very similar to, that of hemoglobin A, so that patients with sickle cell anemia may be thought to have a small amount of hemoglobin A. • Glycated Hb may measured as HbF falsely elevate HbF in HPLC. 50
51
‫زي‬ ‫ما‬ ‫في‬ ‫الصورة‬ ‫هنستخدم‬ ‫هنا‬ ‫بدال‬ ‫من‬ ‫الجل‬ Very narrow silica tube with alkaline pH buffer The interior of the tube is negatively charged attract positive charge from buffer to create diffuse layer ‫نستخدم‬ High voltage with 8 minutes ‫كده‬ ‫هينتج‬ 2 forces 52
‫كده‬ ‫هينتج‬ 2 forces 1_ electrophoretic force ‫تجعل‬ ‫الهيموجلوبين‬ ‫السالب‬ ‫يتجه‬ ‫نحو‬ ‫القطب‬ ‫الموجب‬ 2_electro-osmotic force ‫تجعل‬ ‫الهيموجلوبين‬ ‫السالب‬ ‫يتجه‬ ‫نحو‬ ‫القطب‬ ‫السالب‬ ! ‫والمفارقة‬ ‫أن‬ ‫القوة‬ ‫األكبر‬ ‫هي‬ Electro-osmotic force ‫التى‬ ‫تجعل‬ ‫الهيموجلوبين‬ ‫السالب‬ ‫يتجه‬ ‫نحو‬ ‫القطب‬ ‫السالب‬ ‫وبالتالي‬ ‫سأضع‬ ‫أنا‬ ‫عينة‬ ‫الهيموجلوبين‬ ‫ناحية‬ ‫القطب‬ ‫الموجب‬ ‫حتى‬ ‫تتجه‬ ‫نحو‬ ‫القطب‬ ،‫السالب‬ ‫و‬ ‫عند‬ ‫القطب‬ ‫السالب‬ ‫أضع‬ detector for detection of separated analytes ‫يرسم‬ ‫رسمة‬ ‫مكونة‬ ‫من‬ 15 zones . In each zone, many Hb variants are located. 53
54
Advantages of capillary electrophoresis 1_ fully automated 2_ Efficient separation with high resolution (separate Hb A2 from Hb E). 3_ Adequate quantification of Hb variants (e.g., Hb A2 and Hb Bart’s). 4_ Easy interpretation of results (minimal overlap of common Hb variants). 55
56
Hb variant on capillary electrophoresis • Hb S migrates to S zone in capillary electrophoresis • Hb C migrates to C zone in capillary electrophoresis • Hb E migrates to E zone in capillary electrophoresis • Hb lepor migrates to LEPOR zone in capillary electrophoresis • Hb D migrates to D zone in capillary electrophoresis (D/G) • Hb O-Arab migrates to A2 zone in capillary electrophoresis (A2/O) 57
58
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Hb variant on HPLC • Hb C elutes in C window in HPLC (C/O). • Hb E elutes in A2 window in HPLC (A2/E/Lepore). • Hb D elutes in D window in HPLC (D/G). • Small part of HbS coelute with Hb A2 leads to falsely high HbA2 value in Sickle cell variant as Retention time of them are near • Certain artefacts need to be recognized for example, increased bilirubin in the plasma may lead to a sharp peak in the same general area as hemoglobin H. • Retention time of glycated and non glycated hemoglobin are different ➡ leads to misinterpretation i.e. Glycosylated hemoglobin S may have a retention time the same as, or very similar to, that of hemoglobin A, so that patients with sickle cell anemia may be thought to have a small amount of hemoglobin A. • Glycated Hb may measured as HbF falsely elevate HbF in HPLC. 60
Hb variant in capillary electrophoresis • Hb S migrates to S zone in capillary electrophoresis • Hb C migrates to C zone in capillary electrophoresis • Hb E migrates to E zone in capillary electrophoresis • Hb lepor migrates to LEPOR zone in capillary electrophoresis • Hb D migrates to D zone in capillary electrophoresis (D/G) • Hb O-Arab migrates to A2 zone in capillary electrophoresis (A2/O) 61
Hb variants on different electrophoresis Hb C migrates to A2 region in gel electrophoresis (A2/C/E/O), to C zone in capillary electrophoresis and elutes in C window in HPLC (C/O). Hb E migrates to A2 region in gel electrophoresis (A2/C/E/O), to E zone in capillary electrophoresis and elutes in A2 window in HPLC (A2/E/Lepore). Hb D migrates to S region in gel electrophoresis (S/G/D/Lepore), to D zone in capillary electrophoresis (D/G) and elutes in D window in HPLC (D/G). Hb O-Arab migrates to A2 region in gel electrophoresis (A2/C/E/O), to A2 zone in capillary electrophoresis (A2/O) and elutes in C window in HPLC (C/O). Hb lepor migrates to S region in gel electrophoresis (S/G/D/Lepore), to LEPOR zone in capillary electrophoresis and elutes in A2 window in HPLC (A2/E/Lepore). 62
63
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Composition of Hemoglobin ( Hb) HB heme iron protoporphyrin globin 4 polypeptide chain 65
NOTES 1_ Hemoglobinopathy is autosomal recessive disorders so to be appeared the 2 genes should be affected . 3_ Hemoglobinopathy defined as a family of diseases caused by production of : • A _ synthesis of insufficient quantities of normal Hemoglobin ( thalassemia) • B _ structurally abnormal hemoglobin as Sickle cell anemia • C_ or both 66
67
Definition: quantitative defect in α chain production with globin chain imbalance ‫ال‬ Alfa Chain ‫من‬ ‫تتكون‬ 141 AA ‫الكروموسوم‬ ‫علي‬ ‫ممثلة‬ 16 ‫جينات‬ ‫بأربع‬ ‫لو‬ ‫حصل‬ deletion in these one or more of these genes these leads to decrease expression of Alfa genes leads to Alfa thalassemia . It may be due to another gene abnormalities as point mutation,......... Which called non deletional Alfa thalassemia 68
pathophysiology Low expression of Alfa globin gene ➡ ⬇ Alfa globin chain ➡ imbalance between Alfa and Beta globin chain ➡ excess free Beta and Gamma chain . Excess free Beta and Gamma chain ➡ ⬆ HbH and Hb Bart's 69
70
71
1-silent carrier silent carrier (_, Alfa/Alfa, Alfa) Deletion in one gene silent carrier can detected by genetic testing. It is Fully compensated. No/minimal free beta. Normal clinical lab. 72
Alfa thalassemia trait (_, Alfa/_, Alfa) with high free beta chain Deletion of two genes -There are thalassemic red cell indices as : -Mild or no anemia -Moderate or marked microcytosis and hypochromia of RBCs -Erythrocytosis above 5 X 10^6 RDW normal 73
Differentiation a _ Iron deficiency anemia by normal ferritin ( in case not acute I. e. Normal CRP) b _ beta thalassemia trait by excluded by high A2 in Hb electrophoresis. 74
HbH (Alfa_,_ _ ) type of thalassemia intermedia (moderate anemia with marked microcytosis and hypochromia no need for repeated blood transfusions) and extravascular hemolysis Hb electrophoresis Hb H + less Hb Bart's HbH is faster than HbA in electrophoresis , Hb H are usually ~ 10% with dominant HbA HbH formed of 4Beta forming HbH Reticulocyte shows golf ball appearance ‫طبعا‬ ‫فيها‬ ‫كل‬ ‫عالمات‬ ‫ال‬ hemolytic anemia 75
76
Hb Bart’s 4 Gamma chain (_ _, _ _) ‫المريض‬ ‫بيموت‬ ‫في‬ ‫بطن‬ ‫أمه‬ ‫أو‬ ‫عندالوالدة‬ Hb electrophoresis Hb Bart's + Hb H Early diagnosis: suspected by US examination or family genetic study and is confirmed by genetic testing via chorionic villous sample, amniocentesis or cordocentesis. IU transfusion might be lifesaving!!!! 77
78
α + thalassemia ‫ودي‬ ‫بيبقي‬ ‫المشكلة‬ ‫في‬ ‫جين‬ ‫واحد‬ ‫من‬ ‫األربع‬ ‫جينات‬ ‫أو‬ ‫اتنين‬ ‫بس‬ ‫علي‬ ‫كروموسين‬ ‫يعني‬ ‫كل‬ ‫كروموسوم‬ ‫عليه‬ ‫جين‬ ‫والتاني‬ ‫اتشال‬ . αo thalassemia ‫يبقي‬ ‫المشكلة‬ ‫في‬ ‫جينين‬ ‫من‬ ‫علي‬ ‫كروموسوم‬ ‫واحد‬ ‫والتاني‬ ‫سليم‬ ‫لو‬ ‫اتشال‬ 3 ‫ده‬ HbH ‫لو‬ ‫اتشال‬ 4 ‫ده‬ Hb Bart's 79
The degree of microcytosis and hypochromia is roughly correlated with number of gene deletion. 1- Silent: (-α/αα) = Heterozygous α+ 2- Mild: ▪ (--/αα) = Heterozygous α0 ▪ (-α/-α) = Homozygous α+ ▪ (αTα/αα) = Heterozygous non-deletional α thalassemia 3- Moderate: ▪ (--/-α) = Double heterozygous α0 and α+ ▪ (--/ αTα) = Double heterozygous α0 and non-deletional α thalassemia ▪ (αTα/αTα) = Homozygous or double heterozygous non-deletional α thalassemia. 4- Severe: ▪ (--/--) = Homozygous α0 ▪ (--/αTα) = Double heterozygous α0 and non-deletional α thalassemia. 80
Alfa thalassemia oNormal lab in silent carrier, detected by genetic testing. oLow MCV and MCH in silent and alfa thalassemia trait oHBH in HBH disease moderate anemia with marked microcytosis and hypochromia o Hb Bart’s in Hb Bart’s disease oGenetic testing 81
B thalassemia Definition: quantitative defect in β chain production with globin chain imbalance Beta thalassemia is found in Southeast Asia, Mediterranean area and Middle East. Occasionally seen in Africa (usually β+). Low expression of B globin gene ➡ ⬇ B globin chain ➡ imbalance between Alfa and Beta globin chain ➡ excess free Alfa chain Delta and gamma chain are increased to compensate imbalance between Alfa and Beta globin chain ➡ ⬆ HbA2 and HbF 82
Clinical features 1- Silent/almost silent beta thalassemia: no clinical ± laboratory abnormality. 2- Thalassemia minor: mild or no anemia. 3- Thalassemia intermedia: moderate chronic hemolytic anemia + complications. 4- Thalassemia major: marked chronic hemolytic anemia + complications. 83
B thalassemia major Homozygous state Symptoms and signs of : 1_ chronic hemolytic anemia ➡ anemia, jaundice, hepatosplenomegaly, failure of growth, delay puberty. 2_ iron overload ➡ cardiac failure, arrhythmia, liver affection and DM. 3_ BM expansion ➡ skeletal deformity, iron overload. 84
B thalassemia major Homozygous state Manifestations of hemolytic anemia as : 1_ severe ( less than 7 mg/dl) microcytic hypochromic Anemia 2_ Indirect hyperbilirubinemia more than 80% of total bilirubin 3_ Increased urobilinogen in urine 4_ Normal urine colure 5 _ High uric acid 85
86
87
B thalassemia major (Homozygous state) Hb electrophoresis : (Bo Bo) : HbA ➡absent HbA2➡ increased HbF ➡ about 96% . B+ Bo Or B+ B+ : HbA ➡10_30% HbA2➡ increased HbF ➡ about 70_90 % Iron profile : Increase ➡ serum iron, ferritin, iron saturation Decrease ➡ TIBC 88
89
B thalassemia minor (Heterozygous state) thalassemic red cell indices as • Mild or no anemia • Moderate or marked microcytosis and hypochromia of RBCs • Erythrocytosis above 5 X 10^6 • RDW ➡ normal Hb electrophoresis: B Bo, B B+ • HbA2➡ increased in 100% cases ( 3.5_8%) • HbF ➡ increased in 50% of cases ( 1-7%) • HbA ➡ more than 90% 90
91
B thalassemia intermedia moderate chronic hemolytic anemia + complications. Hb F from 20 – 40 % ‫االتنين‬ ‫بين‬ ‫وسط‬ ‫مرحلة‬ 92
NOTES ‫في‬ ‫حاالت‬ ‫ممكن‬ ‫ال‬ HbA2 ،‫اليزيد‬ ‫لو‬ ‫الحاالت‬ ‫دي‬ ‫أصال‬ ‫ي‬ ‫قل‬ ‫فيها‬ ‫ال‬ HbA2 ‫مثل‬ Beta thalassemia trait with normal HbA2 1- iron deficiency anemia, folate deficiency, hypothyroidism 2- Delta Beta thalassemia or Alfa beta thalassemia ‫في‬ ‫حاجة‬ ‫اسمها‬ silent B thalassemia and almost silent B thalassemia ‫ودي‬ ‫بيبقى‬ ‫مشهور‬ ‫فيها‬ -101 C mutation ‫وبيبقى‬ ‫فيها‬ HbA2 ‫طبيعي‬ ‫ومفيش‬ ‫أعراض‬ almost silent B thalassemia has normal HbA2 and abnormal red cell indices while, silent B thalassemia has normal HbA2 and normal red cell indices. 93
‫الخالصة‬ B thalassemia B thalassemia trait thalassemic red cell indices with high HBA2 more than 3.7 except reasons mentioned above, Hb F (1-7%) B thalassemia major moderate to marked MHA Blood film ‫فراخ‬ ‫نعكشة‬ Sign and symptoms of hemolytic anemia Hb separation: increased Hb F (becomes dominant) ± increase in Hb A2. Hb A is absent in β0 NB : DON’T MISS SILENT AND ALMOST SILENT B THALASSEMIA 94
Delta B thalassemia ‫زي‬ ‫ال‬ B thalassemia ‫ولكن‬ ‫ال‬ Delta ‫قلت‬ ‫فهيقل‬ HbA2 ‫ودي‬ ‫فعال‬ ‫في‬ Heterozygous delta beta thalassemia similar to B thalassemia minor clinically but low or normal HbA2 HbF from 5_ 15 % Homozygous delta beta thalassemia similar to B thalassemia intermedia clinically with absent HbA2 HbF is 100 % 95
Clinical phenotypes and genetic causes of beta thalassemia 1_ Silent: certain mutations e.g., -101 C→T mutation. 2_ Mild disease: β+βA, β0βA or β0βHPFH 3_ Moderate disease: ▪ β0β+, β0βD-Punjab, β0βO-Arab or β0βC (DOC). ▪ Autosomal dominant beta thalassemia. ▪ Homozygous Hb Lepore. 3_ Severe disease: ▪ β0β0 ▪ β0βE ▪ β0βLepore ▪ β0 with quadruple alpha (αααα). 96
TERMS BA➡ ‫ال‬ ‫تعني‬ B ‫تخرج‬ ‫التى‬ ‫العادية‬ B chain β+ ➡ ‫ال‬ ‫تعني‬ B ‫ناقصه‬ β0➡ ‫ال‬ ‫تعنى‬ B ‫شغاله‬ ‫مش‬ βO or D or E or Lepor ➡ ‫إن‬ ‫معناه‬ ‫الكروموسوم‬ ‫ال‬ ‫من‬ ‫بدل‬ ‫تخرج‬ ‫لكن‬ ‫شغال‬ B chain E or O or Lepor Hb chain 97
98
NOTES 1_ Hemoglobinopathy is autosomal recessive disorders so to be appeared the 2 genes should be affected . 2_ 90 % of structural variant hemoglobinopathy are point mutation. 3_ Hemoglobinopathy defined as a family of diseases caused by production of : • A _ synthesis of insufficient quantities of normal Hemoglobin ( thalassemia) • B _ structurally abnormal hemoglobin as Sickle cell anemia • C_ or both 99
Hemoglobin S ‫لو‬ ‫حصل‬ Point mutation ‫واتغير‬ ‫ال‬ AA ‫رقم‬ 6 ‫اللي‬ ‫هو‬ glutamate ‫في‬ ‫ال‬ Beta chain ‫في‬ ‫الكروموسوم‬ 11 ⬅ ‫اتغير‬ ‫إلي‬ Valine ‫ينتج‬ Hb ‫غريب‬ ‫سموه‬ HbS ‫ويكون‬ ‫مرض‬ ‫ال‬ Sickle diseases ‫وهو‬ ‫من‬ ‫عائلة‬ structural gene defect hemoglobinopathy α2β2 6Glu→Val ‫لو‬ ‫أصيب‬ ‫الجينين‬ ‫في‬ . Beta chain ‫يبقي‬ HbSS ‫لو‬ ‫جين‬ ‫واحد‬ ‫يبقي‬ HbAS ‫لو‬ ‫حصل‬ double heterozygous mutation ‫ممكن‬ ‫ينتج‬ HbSC _ HbSE ‫وغيره‬ . ‫وطبعا‬ ‫ده‬ ‫هيغير‬ ‫مكانهم‬ ‫في‬ electrophoresis ‫فيه‬ ‫يحصل‬ ‫الجينين‬ ‫أحد‬ ‫ممكن‬ mutation ‫الي‬ ‫ويتحول‬ S ‫ال‬ ‫ينعدم‬ ‫واآلخر‬ expression ‫ويبقي‬ ‫بتاعه‬ Bo ‫ال‬ ‫يقل‬ ‫او‬ expression ‫ويبقي‬ ‫بتاعه‬ + B ‫اما‬ ‫وينتج‬ BoS ‫او‬ B+S 100
101
Hb S is the most common hemoglobinopathy. It has a Worldwide distribution. The highest frequency is seen in West Africa, Kuwait, East of KSA and east of India. 102
103
Heterozygous sickle cell trait a. CBC: is normal (except if associated alpha thalassemia). b. Smear: is normal (occasional boat shape cells). c. Reticulocytes: are normal. d. Hb separation: shows dominant Hb A normal or slightly increased Hb A2 and or Hb F Hb S around 40% (less if with α thalassemia or IDA). e. Sickling tests are positive 104
105
Homozygous sickle cell anemia CBC Mild to moderate normocytic normochromic anemia with anisocytosis. • Anemia occasionally severe and rarely absent. • Microcytic if associated α thalassemia (higher Hb level) or IDA (lower Hb level). • Macrocytic if there are reticulocytosis, megaloblastic crisis (lower Hb level) or hydroxyurea therapy (higher Hb level). b. Smear: shows, i. Anispoikilocytosis, sickle cells, boat shape cells, target cells ± irregularly contracted cells, blister cells, RBCs fragments and/or NRBCs. ii. Howell–Jolly bodies and Pappenheimer bodies (hyposplenism) are usually seen beyond infancy. 106
C. Reticulocytes: are increased (it falls if aplastic or megaloblastic crises and hydroxyurea). d. Hb separation: dominant Hb S (>90 %) normal Hb A2, slightly increased Hb F (5-10%) with absence of Hb A i.Hb F shows higher percentage in infancy (delayed switch), with therapy (hydroxycarbamide) and in some cases (Arab-Indian). ii. Hb A2 may increase in Arab-Indian haplotype (up to 5%). ii. Hb A level depends on transfusion. e. Other: i. Sickling tests are positive. ii. Hemolysis chemistry: LDH, indirect bilirubin, haptoglobin and hemopexin. iii. For complications: e.g., cranial US, eGFR, urine albumin… Homozygous sickle cell anemia 107
108
o Asymptomatic: βS/βA and βS/HPFH. o Mild disease: βS/β+ and βS/βE. o Moderate disease: βS/βC, βS/βD and βS/βLepore o Severe disease: βS/βS, βS/β0 and βS/βO-Arab. 109
‫الخالصة‬ Homozygous sickle cell anemia : Moderate to marked NNA hemolytic anemia with symptoms of hemolytic anemia Blood smear shows sickle cell Hb electrophoresis is HbS more than 90 % . HbF slightly increased (5-10%). HbA2 is normal Heterozygous sickle cell trait : HbS around 40 % decreased in alfa thalassemia and IDA , normal or slightly increased Hb A2 and or Hb F SB+ HbS becomes more than HbA , with high HbF and HbA2 SB0 absent HbA with high HbF and HbA2 Hb A level depends on transfusion. 110
Hemoglobin C ‫حصل‬ ‫لو‬ Point mutation ‫ال‬ ‫واتغير‬ AA ‫رقم‬ 6 ‫هو‬ ‫اللي‬ glutamate ‫ال‬ ‫في‬ Beta chain ‫الكروموسوم‬ ‫في‬ 11 ⬅ ‫إلي‬ ‫اتغير‬ Lysine ‫ينتج‬ Hb ‫سموه‬ ‫غريب‬ HbC ‫ويكون‬ ‫عائلة‬ ‫من‬ ‫مرض‬ structural gene defect hemoglobinopathy ‫في‬ ‫الجينين‬ ‫أصيب‬ ‫لو‬ . Beta chain ‫يبقي‬ HbCC ‫يبقي‬ ‫واحد‬ ‫جين‬ ‫لو‬ HbAC ‫حصل‬ ‫لو‬ double heterozygous mutation ‫ينتج‬ ‫ممكن‬ HbSC ‫في‬ ‫مكانه‬ ‫هيغير‬ ‫ده‬ ‫وطبعا‬ HB electrophoresis ‫فينه‬ ‫يحصنل‬ ‫الجيننين‬ ‫أحند‬ ‫ممكنن‬ mutation ‫الني‬ ‫ويتحنول‬ C ‫ال‬ ‫ينعندم‬ ‫واآلخنر‬ expression ‫ويبقني‬ ‫بتاعنه‬ Bo ‫ال‬ ‫يقنل‬ ‫او‬ expression ‫ويبقني‬ ‫بتاعنه‬ + B ‫امنا‬ ‫ويننتج‬ BoC ‫او‬ B+C 111
Clinical features 1- Asymptomatic in heterozygous state. 2- Mild to moderate chronic hemolytic anemia ± splenomegaly in homozygous state. 3- Epidemiology. Hb C is seen frequently in West Africa 112
Heterozygous Hb C (Hb C trait) a. CBC: shows mild microcytosis, hypochromia and anisocytosis (MCV may be normal and MCHC is high normal). b. Smear: is normal or shows some target cells and irregularly contracted cells. c. Reticulocytes: are normal. d. Hb separation: Dominant Hb A normal or slightly increased Hb A2 and/or Hb F Hb C around 40% (less if associated with α thalassemia or IDA). Hb C migrates to A2 region in gel electrophoresis (A2/C/E/O), to C zone in capillary electrophoresis and elutes in C window in HPLC (C/O). 113
114
Homozygous Hb C (Hb C disease) a. CBC: shows mild to moderate anemia with marked microcytosis and hypochromia. MCHC is at high normal range. Platelet count may increase. b. Smear: shows many target cells and irregularly contracted cells with occasional NRBCs and Hb C crystals (tetragonal or hexagonal, hyperdense mass with eccentric position). c. Reticulocytes: are slightly increased. d. Hb separation: dominant Hb C (>95%) normal or slightly increased Hb A2 and/or Hb F with absence of Hb A. e. Other: i. Hemolysis chemistry: LDH, indirect bilirubin, haptoglobin and hemopexin. ii. For complications: iron level, abdominal US… iii. Decreased osmotic fragility. iv. Genetic testing 115
116
Hemoglobin E ‫لو‬ ‫حصل‬ Point mutation ‫واتغير‬ ‫ال‬ AA ‫رقم‬ 26 ‫اللي‬ ‫هو‬ glutamate ‫في‬ ‫ال‬ Beta chain ‫في‬ ‫الكروموسوم‬ 11 ⬅ ‫اتغير‬ ‫إلي‬ Lysine ‫ينتج‬ Hb ‫غريب‬ ‫سموه‬ Hb-E ‫ويكون‬ ‫مرض‬ ‫من‬ ‫عائلة‬ structural gene defect hemoglobinopathy ‫لو‬ ‫أصيب‬ ‫الجينين‬ ‫في‬ . Beta chain ‫يبقي‬ HbEE ‫لو‬ ‫جين‬ ‫واحد‬ ‫يبقي‬ HbAE ‫لو‬ ‫حصل‬ double heterozygous mutation ‫ممكن‬ ‫ينتج‬ . HbSE ‫وطبعا‬ ‫ده‬ ‫هيغير‬ ‫مكانه‬ ‫في‬ HB electrophoresis ‫ممكن‬ ‫أحد‬ ‫الجينين‬ ‫يحصل‬ ‫فيه‬ mutation ‫ويتحول‬ ‫الي‬ E ‫واآلخر‬ ‫ينعدم‬ ‫ال‬ expression ‫بتاعه‬ ‫ويبقي‬ Bo ‫او‬ ‫يقل‬ ‫ال‬ expression ‫بتاعه‬ ‫ويبقي‬ + B ‫وينتج‬ ‫اما‬ BoE ‫او‬ B+E 117
• It is the 2nd most common hemoglobinopathy. • Epidemiology: found more in Southeast Asia. • Clinical features : • 1- Asymptomatic in both heterozygous and homozygous state (homozygous state rarely shows mild hemolysis). • 2- The clinical significance of Hb E is in double heterozygous with beta thalassemia leading to moderate to severe disease. Hemoglobin E 118
Heterozygous Hb E (Hb E trait) a. CBC: shows erythrocytosis, mild microcytosis and hypochromia ± anemia. CBC is occasionally normal. b. Smear: is normal or shows some target cells and irregularly contracted cells. c. Reticulocytes: are normal. d. Hb separation: Dominant Hb A normal or slightly increased Hb A2 and/or Hb F Hb E around 30% (less if associated with α thalassemia or IDA). Higher percentage if Hb E/β thalassemia). Hb E migrates to A2 region in gel electrophoresis (A2/C/E/O), to E zone in capillary electrophoresis and elutes in A2 window in HPLC (A2/E/Lepore). 119
120
Homozygous Hb E (Hb E disease) a. CBC: shows erythrocytosis with marked microcytosis and hypochromia ± mild anemia. b. Smear: shows many target cells and irregularly contracted cells c. Reticulocytes: are normal or slightly elevated. d. Hb separation: dominant Hb E normal or slightly increased Hb A2 and/or Hb F with absence of Hb A. e. Other: i. Hemolysis chemistry: LDH, indirect bilirubin, haptoglobin and hemopexin. ii. For complications: iron level, abdominal US… iii. Decreased osmotic fragility. iv. Genetic testing 121
Hemoglobin D ‫حصل‬ ‫لو‬ Point mutation ‫ال‬ ‫واتغير‬ AA ‫رقم‬ 121 ‫هو‬ ‫اللي‬ glutamate ‫ال‬ ‫في‬ Beta chain ‫في‬ ‫الكروموسوم‬ 11 ⬅ ‫إلي‬ ‫اتغير‬ glutamine ‫ينتج‬ Hb ‫سموه‬ ‫غريب‬ Hb-D ‫عائلة‬ ‫من‬ ‫مرض‬ ‫ويكون‬ structural gene defect hemoglobenopathy ‫في‬ ‫الجينين‬ ‫أصيب‬ ‫لو‬ . Beta chain ‫يبقي‬ HbDD ‫يبقي‬ ‫واحد‬ ‫جين‬ ‫لو‬ HbAD ‫حصل‬ ‫لو‬ double heterozygous mutation ‫ينتج‬ ‫ممكن‬ . HbSD ‫في‬ ‫مكانه‬ ‫هيغير‬ ‫ده‬ ‫وطبعا‬ HB electrophoresis ‫فيه‬ ‫يحصل‬ ‫الجينين‬ ‫أحد‬ ‫ممكن‬ mutation ‫الي‬ ‫ويتحول‬ D ‫ال‬ ‫ينعدم‬ ‫واآلخر‬ expression ‫ويبقي‬ ‫بتاعه‬ Bo ‫ال‬ ‫يقل‬ ‫او‬ expression ‫ويبقي‬ ‫بتاعه‬ + B ‫اما‬ ‫وينتج‬ BoD ‫او‬ B+D 122
Epidemiology and Clinical features worldwide distribution with highest incidence in India (Punjab). 1- Asymptomatic in both heterozygous and homozygous state (homozygous state sometime shows mild hemolysis). 2- The clinical significance of Hb D is in double heterozygous with Hb S leading to severe disease. 123
Heterozygous Hb D (Hb D trait) a. CBC: is normal. b. Smear: is normal c. Reticulocytes: are normal. d. Hb separation: Dominant Hb A normal Hb A2 and/or Hb F Hb D around 40% (less if associated with α thalassemia or IDA). Hb D migrates to S region in gel electrophoresis (S/G/D/Lepore), to D zone in capillary electrophoresis (D/G) and elutes in D window in HPLC (D/G). 124
125
Homozygous Hb D (Hb D disease) a. CBC: is normal or shows mild normocytic normochromic anemia. b. Smear: shows many target cells and irregularly contracted cells c. Reticulocytes: are normal or slightly elevated. d. Hb separation: dominant Hb E (>95%) normal Hb A2 and/or Hb F with absence of Hb A. e. Other: i. Decreased osmotic fragility. ii. Genetic testing 126
Hemoglobin O-Arab ‫حصل‬ ‫لو‬ Point mutation ‫ال‬ ‫واتغير‬ AA ‫رقم‬ 121 ‫هو‬ ‫اللي‬ glutamate ‫ال‬ ‫في‬ Beta chain ‫الكروموسوم‬ ‫في‬ 11 ⬅ ‫إلي‬ ‫اتغير‬ lysin ‫ينتج‬ Hb ‫سموه‬ ‫غريب‬ Hb-O ‫عائلة‬ ‫من‬ ‫مرض‬ ‫ويكون‬ structural gene defect hemoglobenopathy ‫في‬ ‫الجينين‬ ‫أصيب‬ ‫لو‬ . Beta chain ‫يبقي‬ HbOO ‫يبقي‬ ‫واحد‬ ‫جين‬ ‫لو‬ HbAO ‫حصل‬ ‫لو‬ double heterozygous mutation ‫ينتج‬ ‫ممكن‬ . HbSO ‫في‬ ‫مكانه‬ ‫هيغير‬ ‫ده‬ ‫وطبعا‬ Hb electrophoresis ‫فيه‬ ‫يحصل‬ ‫الجينين‬ ‫أحد‬ ‫ممكن‬ mutation ‫الي‬ ‫ويتحول‬ O ‫ال‬ ‫ينعدم‬ ‫واآلخر‬ expression ‫ويبقي‬ ‫لديه‬ Bo ‫ال‬ ‫يقل‬ ‫او‬ expression ‫ويبقي‬ + B ‫اما‬ ‫وينتج‬ BoO ‫او‬ B+O 127
Epidemiology and Clinical features Epidemiology: Hb-O is seen in African, Arab and East Europe. Clinical features : 1- Asymptomatic in heterozygous state. 2- Mild anemia in some patient with homozygous state ± jaundice and splenomegaly. 3- The clinical significance of Hb O is in double heterozygous with Hb S leading to severe disease 128
Heterozygous Hb O (Hb O trait) a. CBC: is normal. b. Smear: is normal c. Reticulocytes: are normal. d. Hb separation: Dominant Hb A normal Hb A2 and/or Hb F Hb O around 40% (less if associated with α thalassemia or IDA). Hb O-Arab migrates to A2 region in gel electrophoresis (A2/C/E/O), to A2 zone in capillary electrophoresis (A2/O) and elutes in C window in HPLC (C/O). 129
130
Homozygous Hb O (Hb O disease) a. CBC: shows mild microcytosis and hypochromic ± anemia. b. Smear: shows many target cells and occasionally NRBCs. c. Reticulocytes: slightly elevated. d. Hb separation: dominant Hb O (>95%) normal Hb A2, Hb F occasionally increases, with absence of Hb A. e. Other: i. Decreased osmotic fragility. ii. Genetic testing 131
‫الخالصة‬ ‫الجميع‬ ‫في‬ HbS,G,D,C,E,O If homozygous : ‫حدود‬ ‫في‬ ‫طبيعي‬ ‫الغير‬ ‫الهيموجلوبن‬ ‫يكون‬ 90 % ‫الهيموجلوبين‬ ‫وينعدم‬ ‫أكثر‬ ‫أو‬ A . ‫الهيموجلوبين‬ ‫ويزداد‬ F ‫الهيموجلوبين‬ ‫ويكون‬ ، ‫يزداد‬ ‫ال‬ ‫وقد‬ A2 ‫يزداد‬ ‫وقد‬ ‫طبيعي‬ . If heterozygous : ‫حوالي‬ ‫الطبيعي‬ ‫غير‬ ‫الهيموجلوبين‬ ‫نسبة‬ ‫تكون‬ 40 % ‫وجد‬ ‫لو‬ ‫تقل‬ Alfa thalassemia and IDA HbA more than abnormal Hb normal or slightly increased Hb A2 and or Hb F NB : in hb E Hb E is usually ~ 30%. Hb G is usually ~ 20%. If associated B+ thalassemia (ex SB+) ‫الهيموجلوبين‬ ‫من‬ ‫أكثر‬ ‫يكون‬ ‫طبيعي‬ ‫الغير‬ ‫الهيموجلوبين‬ A If associated B0 thalassemia (ex SB0 ) ‫الهيموجلوبين‬ ‫ينعدم‬ A ‫جدا‬ ‫عالي‬ ‫طبيعي‬ ‫الغير‬ ‫الهيموجلوبين‬ ‫ويكون‬ ‫الهيموجلوبين‬ ‫من‬ ‫كال‬ ‫يزداد‬ ‫كالهما‬ ‫وفي‬ F,A2 132
133
134
‫الكروموسوم‬ ‫إن‬ ‫المشكلة‬ 11 ‫فيه‬ ‫بيحصل‬ deletion ‫يشيل‬ ‫معينة‬ ‫منطقة‬ ‫في‬ B and delta gene and silencer of Gamma gene ‫ال‬ ‫فيظل‬ gamma gene ‫الهيموجلوبين‬ ‫فيظل‬ ‫شغال‬ F ‫ال‬ ‫ويقل‬ ‫عالي‬ HbA and HbA2 May non deletional enhancement of promotor of Gamma gene. Hb F has a higher O2 affinity ➔ relative hypoxia ➔ ↑ EPO ➔ ↑ erythropoiesis. Heterozygous ➡ • Clinical pictures are normal • Normal CBC, blood smear, and reticulocyte • Hb separation : • HbF (15-30%) with normal Hb A2 (occasionally low). 135
Homozygous ➡ • Clinical pictures are normal • CBC shows polycythemia and may MHA Hb separation: increased Hb F (100%) with absent Hb A and Hb A2. HPFH /B thalassemia Hb separation: dominant Hb F (~ 70%), increased Hb A2 (>3.5%) with variable levels of Hb A (absent in β0). 136
Clinical pictures are normal Heterozygous HPFH : • HbF (15-30%) with normal Hb A2 (occasionally low). Homozygous HPHF increased Hb F (100%) with absent Hb A and Hb A2. HPFH /B thalassemia Hb separation: dominant Hb F (~ 70%), increased Hb A2 (>3.5%) with variable levels of Hb A (absent in β0). 137
A variant hemoglobin formed by an unequal crossover and fusion of the beta and delta genes. A single copy of the variant gene causes thalassemia minor. Homozygotes have thalassemia intermedia. 138
group of abnormal Hb's that N terminal portion of the δ chain joined to the C- terminal portion of the β chain, due to cross over between the genes for β and δ chains. ‫ينتج‬ ‫عن‬ ‫ذلك‬ ‫بروتين‬ ‫مختلف‬ ‫مشكلته‬ ‫إنه‬ ‫ممكن‬ ‫يظهر‬ ‫في‬ ‫مكان‬ ‫مختلف‬ Hb separation Heterozygotes form about 10% Hb Lepore, dominant HbA normal amounts of Hb A2, and moderately increased amounts of Hb F and usually have mild anemia, microcytosis, and hypochromia; Homozygous form moderate to severe anemia. Hb Lepore 10-20%, absent Hb A and Hb A2 with dominant Hb F Hb lepor migrates to S region in gel electrophoresis (S/G/D/Lepore), to LEPOR zone in capillary electrophoresis and elutes in A2 window in HPLC (A2/E/Lepore). 139
140
Hb lepor B lepor / B thalassemia : • Severe anemia. • dominant HbF (> 90%), normal HbA2 ( 2_3%), Hb Lepor around 5% absent HbA • ‫الخالصة‬ • Hb Lepor from 10_20% ➡ it may be homozygous Hb Lepor • Hb Lepor around 10 % ➡ it may be heterozygous Hb Lepor • Hb Lepor around 5 % ➡ it may be B lepor / B thalassemia. 141
• Replacement of proximal or distal histidine residue in haem pocket by tyrosine ➡ form iron phenolate complex ➡ stabilize iron in ferric state ➡methemoglobin ➡can't carry oxygen easily ➡May causes cyanosis especially if globin chain is affected. 1_ Increased free radicals and reactive oxygen species ( ROS) ➡ oxidation of ferrous iron to ferric 2_ some drugs as Sulfonamides, nitrites and nitrate 3_ reduced activities of NADH-metHb reductase that reverse normal formed met Hb to normal Hb. 142
• Normally, Hb is stable and is protected from oxidation by reducing mechanisms. • In unstable hemoglobin, however, amino acid substitution affecting the stability of Hb molecule ➔ spontaneous or oxidant induced denaturation ➔ precipitation of Heinz bodies ➔ impairment of red cells deformability and shortening of their life span. • >100 variants, the most common are: • o Hb Köln )β98Val → Met, found in German people). • o Hb Hasharon )α47Asp → His, found in Jewish and Italian people). 143
• Normally, O2 is unloaded easily at the tissue site. • In high affinity Hb➡ amino acid substitution at O2 loading/unloading site in the globin chain ➔ stabilization of Hb molecule at R sate (relaxed/oxy) configuration ± decreased affinity to 2, 3-BPG ➔ impaired O2 release ➔ functional anemic with compensatory increased erythropoietin ➔ polycythemia. • >100 variants (α and β(. • They are autosomal dominant. Homozygous state is incompatible with life. 144
‫فيه‬ ‫هل‬ ‫بشوفها‬ ‫حاجة‬ ‫أول‬ S region ‫ايه؟‬ ‫وال‬ A_ If there is S : ‫فيه‬ ‫هل‬ ‫نفسي‬ ‫أسأل‬ A ‫موجودة‬ ‫مش‬ ‫وال‬ 1- if there's A If A larger than S ➡ it's AS (Sickle cell trait) If S larger than A ➡ it's SB+ NB➡ In Sickle cell trait the expected HbS representation is 35_45%, but if represented less than 30 % it may AS/ Alfa thalassemia 145
2_ If there isn't A ➡ look at A2 : S band with high A2 ➡ it's SB0 S band and Normal A2 ➡ it's SS 146
B_ if there isn't S and there's F : • ‫ال‬ ‫األكثر‬ ‫هل‬ ‫بسأل‬ ‫سؤال‬ ‫أول‬ F ‫أم‬ A ? 1_ if HbF predominate than HbA a _ with HbA ➡ B+ thalassemia major or newborn b _ without HbA ➡ Bo thalassemia major (high A2) homozygous Delta Beta thalassemia ( absent A2 ) homozygous HPHF ( absent A2 ) 147
2_ If HbA is predominate than HbF • ‫يعني‬ HBF ‫بسيطة‬ ‫زيادة‬ ‫زايد‬ . a _ with increased HbA2 ➡ B thalassemia minor( trait ) (3_8%) • ‫زيادة‬ ‫المرض‬ ‫هذا‬ ‫في‬ ‫األصل‬ A2 ‫وليس‬ F !!!!! b_ without increased HbA2 ➡ Heterozygous Delta Beta thalassemia Heterozygous HPHF all causes of acquired increased HbF as aplastic, MDS, and megaloblastic anemia,.... 148
B thalassemia intermedia ➡ has MHA Heterozygous HPHF ➡no anemia 149
C_ if there isn't S nor F : ‫علي‬ ‫هبص‬ ‫هنا‬ A2 1_ If normal A2 ➡ normal Hb electrophoresis 2_ If increased A2 (3_8%) ➡ May be thalassemia trait ‫من‬ ‫بأكثر‬ ‫يكون‬ ‫التشخيص‬ ‫علشان‬ ‫الزم‬ ‫طبعا‬ 2 technique as HPLC and capillary electrophoresis. 150
HbF HbF 0_ 5 and high HbA2 ➡ • Beta thalassemia minor ( trait ) HbF 5_15 ➡ • Heterozygous Delta Beta thalassemia trait HbF 15_40 ➡ • Heterozygous HPFH ( normal CBC) • B thalassemia Intermedia HbF about 70 ➡ • B+ thalassemia major or intermedia (high A2) • B+/HPFH HbF about 96 and HbA2 3_5 ➡ • Bo thalassemia major (high A2) • Bo / HPFH. HbF 100 ➡ • Homozygous HPFH ( almost normal CBC) • Homozygous Delta Beta thalassemia major 151
Stress erythropoiesis (causes of reticulocytosis i.e., hemorrhage, hemolysis, nutritional anemia under therapy). Clonal diseases: erythroleukemia, MDS, PV, JMML, PNH and some non-hematological neoplasms. Drugs: hydroxyurea, erythropoietin, Na valproate. Other: ketoacidosis, thyrotoxicosis and pregnancy. 152
153
Approach to diagnosis Personal history: • Age: to justify the percentage of Hb variant. • Ethnicity: African, Asian, Mediterranean... Present history: • Asymptomatic, hemolysis, sickling, polycythemia or cyanosis. Past history: 1- Transfusion. 2- Medication e.g., hydroxycarbamide. 3- Operation e.g., splenectomy or cholecystectomy. Family history: • Similar condition in sibling or known parents with inherited Hb disorder. 154
155
156
157
Alfa thalassemia oNormal lab in silent carrier, detected by genetic testing. oLow MCV and MCH in silent and alfa thalassemia trait oHBH in HBH disease moderate anemia with marked microcytosis and hypochromia o Hb Bart’s in Hb Bart’s disease oGenetic testing 158
‫الخالصة‬ B thalassemia B thalassemia trait thalassemic red cell indices with high HBA2 more than 3.7 except reasons mentioned above, Hb F (1-7%) B thalassemia major moderate to marked MHA Blood film ‫فراخ‬ ‫نعكشة‬ Sign and symptoms of hemolytic anemia Hb separation: increased Hb F ( 70- 100 ) ± increase in Hb A2. Hb A is absent in β0 159
B thalassemia intermedia moderate chronic hemolytic anemia + complications. Hb F from 20 – 40 % ‫االتنين‬ ‫بين‬ ‫وسط‬ ‫مرحلة‬ 160
‫الخالصة‬ Homozygous sickle cell anemia : Moderate to marked NNA hemolytic anemia with symptoms of hemolytic anemia Blood smear shows sickle cell Hb electrophoresis is HbS more than 90 % . HbF slightly increased (5-10%). HbA2 is normal Heterozygous sickle cell trait : HbS around 40 % decreased in alfa thalassemia and IDA , normal or slightly increased Hb A2 and or Hb F SB+ HbS becomes more than HbA , with high HbF and HbA2 SB0 Absent HbA with high HbF and HbA2 Hb A level depends on transfusion. 161
‫الخالصة‬ ‫الجميع‬ ‫في‬ HbS,E,C,D,O,G If homozygous : ‫حدود‬ ‫في‬ ‫طبيعي‬ ‫الغير‬ ‫الهيموجلوبن‬ ‫يكون‬ 90 % ‫الهيموجلوبين‬ ‫وينعدم‬ ‫أكثر‬ ‫أو‬ A . ‫الهيموجلوبين‬ ‫ويزداد‬ F ‫الهيموجلوبين‬ ‫ويكون‬ ، ‫يزداد‬ ‫ال‬ ‫وقد‬ A2 ‫يزداد‬ ‫وقد‬ ‫طبيعي‬ . If heterozygous : ‫حوالي‬ ‫الطبيعي‬ ‫غير‬ ‫الهيموجلوبين‬ ‫نسبة‬ ‫تكون‬ 40 % ‫وجد‬ ‫لو‬ ‫تقل‬ Alfa thalassemia and IDA HbA more than abnormal Hb normal or slightly increased Hb A2 and or Hb F NB : in hb E Hb E is usually ~ 30%. Hb G is usually ~ 20%. If associated B+ thalassemia (SB+) ‫الهيموجلوبين‬ ‫من‬ ‫أكثر‬ ‫يكون‬ ‫طبيعي‬ ‫الغير‬ ‫الهيموجلوبين‬ A If associated B0 thalassemia ( SB0 ) ‫الهيموجلوبين‬ ‫ينعدم‬ A ‫جدا‬ ‫عالي‬ ‫طبيعي‬ ‫الغير‬ ‫الهيموجلوبين‬ ‫ويكون‬ ‫الهيموجلوبين‬ ‫من‬ ‫كال‬ ‫يزداد‬ ‫كالهما‬ ‫وفي‬ F,A2 162
Hb lepor • ‫الخالصة‬ • Hb Lepor from 10_20% ➡ it may be homozygous Hb Lepor • Hb Lepor around 10 % ➡ it may be heterozygous Hb Lepor • Hb Lepor around 5 % ➡ it may be B lepor / B thalassemia. 163
Clinical pictures are normal Heterozygous HPFH : • HbF (15-30%) with normal Hb A2 (occasionally low). Homozygous HPHF increased Hb F (100%) with absent Hb A and Hb A2. HPFH /B thalassemia Hb separation: dominant Hb F (~ 70%), increased Hb A2 (>3.5%) with variable levels of Hb A (absent in β0). 164
HbF HbF 0_ 5 and high HbA2 ➡ • Beta thalassemia minor ( trait ) HbF 5_15 ➡ • Heterozygous Delta Beta thalassemia trait HbF 15_30 ➡ • Heterozygous HPFH ( normal CBC) • B thalassemia Intermedia HbF about 70 ➡ • B+ thalassemia major or intermedia (high A2) • B+/HPFH HbF about 96 and HbA2 3_5 ➡ • Bo thalassemia major (high A2) • Bo / HPFH. HbF 100 ➡ • Homozygous HPFH ( almost normal CBC) • Delta Beta thalassemia major 165
166
167
168
169
170
171
172
173
174
175
176
177
178
Types of mutations 1_ small mutation that affect single gene : • A_ point mutation • B_ Deletion • C_ Insertion 2_ Large-scale mutation : It is abnormality in • A_ chromosomal number • B_ chromosomal structure 179
Point mutation 1- Silent mutation : as UGU ➡ UGC all coded for Cysteine ‫اتغيرت‬ ‫النيكليوتيد‬ ‫ولكن‬ ‫الحمض‬ ‫األميني‬ ‫كما‬ ،‫هو‬ ‫فيبقي‬ ‫كأنك‬ ‫ياأبوزيد‬ ‫ما‬ ‫غزيت‬ 😎 2- Non-Sense mutation : as UGU coded for cysteine ➡ UGA coded for stop codon result in shorter protein can't function at all ‫اتغيرت‬ ‫النيكليوتيد‬ ‫فتوقف‬ ،‫النسخ‬ ‫فينتج‬ ‫بروتين‬ ‫ناقص‬ ‫متغير‬ ‫تماما‬ ‫ال‬ ‫يعمل‬ . Example: a. CAG in exon 2 normally codes for glutamine. C→T mutation turns it to stop codon (TAG) resulting in β0 thalassemia. b. Non-sense mutation in exon 3 of β gene: leading to severe unstable beta chain (dominant thalassemia phenotype). 180
Point mutation 3- Sense mutation : as UGA coded for stop codon ➡ UGU coded for cysteine result in longer protein can't function at all ‫ت‬ ‫ومتغير‬ ‫األصلي‬ ‫من‬ ‫أكبر‬ ‫بروتين‬ ‫فنتج‬ ،‫النسخ‬ ‫يتوقف‬ ‫فلم‬ ‫النيكليوتيد‬ ‫اتغيرت‬ ‫يعمل‬ ‫وال‬ ‫ماما‬ . Examples: b. Hb Constant Spring: α chain of 172 amino acids instead of 141 (excess 31 amino acids). 4- Missense mutation : as UGU coded for cysteine ➡ UAU coded for Tyrosine, it may be : ‫علي‬ ‫سيؤثر‬ ‫التغيير‬ ‫هذا‬ ‫هل‬ ‫لكن‬ ،‫األميني‬ ‫الحمض‬ ‫معها‬ ‫وتغير‬ ‫النيكليوتيد‬ ‫اتغيرت‬ ‫هنا‬ ‫الحمض‬ ‫األميني؟‬ ⬅ 181
Missense mutations 1 - No apparent Missense mutation : ‫بال‬ ‫فقط‬ ‫ونعرفه‬ ‫تغيير‬ ‫أي‬ ‫الوظيفة‬ ‫في‬ ‫يحدث‬ ‫لم‬ ‫ولكن‬ ،‫متغير‬ ‫بروتين‬ ‫ونتج‬ ‫النيكلوتيدة‬ ‫تغيرت‬ ‫لو‬ DNA sequencing as Hb-Milwaukee and Hb-Bristol 2 - Acceptable Missense mutation : ‫يالحظ‬ ‫ال‬ ‫الوظيفة‬ ‫في‬ ‫مقبول‬ ‫طفيف‬ ‫تغير‬ ‫وحدث‬ ‫متغير‬ ‫بروتين‬ ‫ونتج‬ ‫النيكلوتيدة‬ ‫تغيرت‬ ‫لو‬ . As Hb Hikari 3- Partially Acceptable Missense mutation : ‫بقصو‬ ‫لكن‬ ‫يعمل‬ ‫البروتين‬ ‫زال‬ ‫ما‬ ‫لكن‬ ،‫معروف‬ ‫مرض‬ ‫هذا‬ ‫عن‬ ‫ونتج‬ ‫الوظيفة‬ ‫في‬ ‫شديد‬ ‫تغير‬ ‫وحدث‬ ‫متغير‬ ‫بروتين‬ ‫ونتج‬ ‫النيكلوتيدة‬ ‫تغيرت‬ ‫لو‬ ‫ف‬ ‫كما‬ ‫ر‬ ‫ي‬ HBS 4- Unacceptable Missense mutation : ‫فق‬ ‫البروتين‬ ‫لكن‬ ،‫معروف‬ ‫مرض‬ ‫هذا‬ ‫عن‬ ‫ونتج‬ ،‫الوظيفة‬ ‫في‬ ‫شديد‬ ‫تغير‬ ‫وحدث‬ ‫متغير‬ ‫بروتين‬ ‫ونتج‬ ‫النيكلوتيدة‬ ‫تغيرت‬ ‫لو‬ ‫تماما‬ ‫العمل‬ ‫علي‬ ‫القدرة‬ ‫د‬ ‫في‬ ‫كما‬ HBM 182
Insertion or deletion • ‫من‬ ‫المعلوم‬ ‫أن‬ ‫كل‬ ‫ثالثة‬ ‫نيوكليوتيدات‬ ‫علي‬ ‫ال‬ mRNA ‫تمثل‬ ‫بحمض‬ ‫أميني‬ ،‫واحد‬ • ‫لو‬ ‫أضفت‬ ‫أو‬ ‫حذفت‬ ‫ثالثة‬ ‫نيكلوتيدات‬ ‫أو‬ ‫مضاعفتها‬ ‫هنا‬ ‫كل‬ ‫اللي‬ ‫هيحصل‬ ‫إضافة‬ ‫أو‬ ‫حذف‬ ‫حمض‬ ‫أميني‬ ‫واحد‬ ‫أو‬ ‫أكثر‬ ‫وسيحدث‬ Non-Frame shift mutation. • ‫أما‬ ‫لو‬ ‫أضفت‬ ‫أو‬ ‫حذفت‬ ‫غير‬ ‫الثالثة‬ ‫نيكلوتيدات‬ ‫ومضاعفتها‬ ‫ك‬ 1 ' 2 ' 4 ' 5 ' ... ‫هيحصل‬ ‫تغيير‬ ‫لكل‬ ‫البروتين‬ ‫ما‬ ‫بعد‬ ‫الطفرة‬ ‫وسيتغير‬ ‫البروتين‬ ‫تماما‬ ‫ويحدث‬ Frame shift mutation 183
small mutation that affect single gene 184
185
186
187
188
189
2_ Large-scale mutation 190
2_ Large-scale mutation •A_ chromosomal number •B_ chromosomal structure It's abnormality in 191
A_ abnormality in chromosomal number • It's due to errors in gametes formation aneuploidy Polyploidy 192
Aneuploidy for example • 1_ Turner syndrome ➡ 45,X or 45,X0). • 2_ Klinefelter syndrome ➡ 47,XXY • 3_Edwards S➡trisomy of chromosome 18. • 4_ Down S ➡ trisomy of chromosome 21. • 5_ Patau S➡ trisomy of chromosome 13. • 6_ Trisomy 9. 193
Down Syndrome Turner syndrome 194
Polyploidy 1_ Triploidy : 3 set of chromosome (3n) 69 chromosomes 2_ Tetraploidy : 4 set of chromosomes (4n) 92 chromosomes It is non compatible with life, if occurs in cells the cell is abnormal. 195
Triploidy 196
B_ Abnormality in chromosomal structure It's due to errors in gametes formation or mutagenes ( physical, biological). •for example : 1_ Deletion ➡ as Cri du chat deletion syndrome, 1P36 2_ Duplication 3_ Inversion ➡ inv (3) ( q21;q34) 4_ translocation➡ Philadelphia Chromosome in CML 197
Abnormality in chromosomal structure 198
Philadelphia chromosome 199
200
Molecular application in hemoglobenopathy The most important application of DNA analysis molecular techniques are : 1_ The conformation of the diagnosis of Alfa thalassemia trait, particularly for genetic counseling 2_ The prenatal diagnosis of serious disorders of globin chain in the fetus as HbS 201
Techniques PCR Sequencing Southern blot analysis GAP PCR PCR with allele specific primer ( ARMS ) Reverse dot blot sequencing RFLP linkage analysis Denaturing gradient gel electrophoresis ( DGGE ) mass spectrometer 202
Lecture titles 1_ Hb separation 2_ cellulose acetate electrophoresis 3_ High Performance Liquid Chromatography (HPLC) 4_ Capillary electrophoresis 5_ interference in Hb separation 6_ Hb variants 7_ Structural abnormal hemoglobinopathy as HbS,C,E,O,… 8_ Insufficient of hemoglobin synthesis as Alfa & Beta thalassemia 9_ 0ther hemoglobinopathy as HbM, unstable Hb, High affinity Hb, HPFH, Hb lepor 10_ Hb variants interpretation & Interferance 11_ Detail & Summary of Hb variants 12_ cases 13_ Genetics & Molecular 203
REFERENCES • _ https://labtestsonline.org • _ https://www.medscape.com • _ https://www.wikipedia.org • _ Haemoglobinopathy Diagnosis Barbara J. Bain , Third Edition 2020 • _ Lecture Notes Clinical Biochemistry, 9th Edition Walker, Simon, 2103. • _ https ps://www.labcorp.com • _ https://www.uptodate.com • _ https://www.ncbi.nlm.nih.gov Home - PubMed – NCBI • _TIETZ textbook of clinical chemistry and molecular diagnostics, sixth edition 2018. • _Essential of clinical pathology book; 1st edition; Shirish M Kawthalkar; 2010. • _ Harper's illustrated biochemistry 30th edition 2015. • _ Lippincott's illustrated review of biochemistry sixth edition 2014. • _Many audios and videos from Well-known, trusted professors who study from accredited books. 204
‫اليوتيوب‬ ‫علي‬ ‫البحث‬ ‫أو‬ ،‫اليوتيوب‬ ‫علي‬ ‫قناتي‬ ‫دي‬ ‫د‬ ‫باسم‬ . ‫سفيان‬ ‫بهجت‬ ‫محمد‬ ، ‫هللا‬ ‫شاء‬ ‫إن‬ ‫نستمر‬ ‫حتى‬ ‫بالقناة‬ ‫وتشتركوا‬ ‫تتابعوني‬ ‫ياريت‬ https://www.youtube.com/channel/UCaYs1d8s0ntZvteHS3mMmGA ‫ب‬ ‫الفيس‬ ‫في‬ ‫البحث‬ ‫أو‬ ،‫انضمامكم‬ ‫يشرفنا‬ ‫الفيسبوك‬ ‫علي‬ ‫بتاعتي‬ ‫الصفحة‬ ‫دي‬ DMBMS2018 ، ‫هللا‬ ‫شاء‬ ‫إن‬ ‫نستمر‬ ‫حتى‬ ‫تتابعوني‬ ‫ياريت‬ https://www.facebook.com/DMBMS2018/ ‫باسم‬ ‫التلجرام‬ ‫علي‬ ‫قناتي‬ ‫ودي‬ ِ ‫للناس‬ ‫أنفعهم‬ ‫الناس‬ ‫خير‬ 📚 ‫هللا‬ ‫شاء‬ ‫إن‬ ‫نستمر‬ ‫حتى‬ ‫تتابعوني‬ ‫ِريت‬‫ا‬‫ي‬ https://t.me/DMBMS2020 ‫على‬ ‫بروفايلي‬ ‫لينكيدإن‬ ‫وتعملولي‬ ‫تتابعوني‬ ‫ياريت‬ endorsement ‫مهاراتي‬ ‫على‬ https://www.linkedin.com/in/mohammed-bahgat-sofyan-8ba012142/ ‫موقع‬ ‫على‬ ‫بروفايلي‬ ‫وده‬ SlideShare ‫تتابعونا‬ ‫ياريت‬ https://www.slideshare.net/MohammedBahgatMohamm1 ‫وده‬ ‫بروفايلي‬ ‫الشخصي‬ ‫الفيسبوك‬ ‫علي‬ https://www.facebook.com/mohammed.bahgat.165 ‫الفيسبوك‬ ‫على‬ ‫بنا‬ ‫الخاص‬ ‫اللوجوا‬ ‫وده‬ # ‫دمحمد‬ _ ‫بهجت‬ _ ‫سفيان‬ _ medical_biochimestry 205
My logo # ‫دمحمد‬ _ ‫بهجت‬ _ ‫سفيان‬ _ medical_biochimestry 206
THANK YOU Meet on the best later, God willing 207

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Hb electrophoresis

  • 1. BY Dr: Mohammed Bahgat Mohammed Sofyan Assistant lecturer Faculty of medicine, Al-Azhar university (Assiut branch) 1
  • 2. 2
  • 3. 3
  • 4. ‫صورة‬ ‫كل‬ ‫على‬ ‫اضغط‬ ‫الشاش‬ ‫عرض‬ ‫وضع‬ ‫في‬ ‫ة‬ ‫قنواتنا‬ ‫على‬ ‫وادعمنا‬ ‫اإلنترنت‬ ‫على‬
  • 5. ‫على‬ ‫ملفي‬ ‫في‬ ‫هنا‬ ‫أرفعها‬ ‫التي‬ ‫شرائحي‬ ‫ومذاكرة‬ ‫بالتعلم‬ ‫وأسعد‬ ‫وأرحب‬ ‫أسمح‬ Slide share website ‫وال‬ ‫مانع‬ ‫أيضا‬ ‫من‬ ‫نسخ‬ ‫شريحة‬ ‫او‬ ‫اثنين‬ ‫عند‬ ‫الضرورة‬ ، ‫وال‬ ‫مانع‬ ‫من‬ ‫شرح‬ ‫البوربو‬ ‫ينت‬ ‫الخاصة‬ ‫بي‬ ‫للغير‬ ‫بشرط‬ ‫عدم‬ ‫إزالة‬ ‫اسمي‬ ‫من‬ ‫البوربوينت‬ ، ‫فال‬ ‫أسمح‬ ‫أبدا‬ ‫بإزالة‬ ‫اسمي‬ ‫من‬ ‫على‬ ‫الباوربوينت‬ ‫ووضع‬ ‫اسمك‬ ‫بدال‬ ‫منه‬ ‫لتصبح‬ ‫وكأنك‬ ‫من‬ ‫صممتها‬ ‫فهذه‬ ‫سرقة‬ ‫ال‬ ‫أسمح‬ ‫بها‬ ‫وتضييع‬ ‫لحق‬ ‫من‬ ‫تعب‬ ‫في‬ ‫عملها‬ . ‫وفقكم‬ ‫هللا‬ ‫وإياي‬ ‫للتعلم‬ ‫ونفع‬ ‫اآلخرين‬ I allow, welcome, and be happy to learn and study my slides that I upload here in my profile on Slide share website There is also no objection to copying one or two slides when necessary, and there is no objection to explaining my PowerPoint to others on the condition that my name is not removed from the PowerPoint. I never allow my name to be removed from PowerPoint and to replace it with yours, Make it look like you designed it. This is theft that I do not allow and a waste of the right of those who are tired in this work. May God bless you and me for learning and benefiting others 5
  • 6. ‫ال‬ Hb ‫من‬ ‫مكون‬ ‫اإلنسان‬ ‫جسم‬ ‫في‬ ‫اللي‬ heme ‫و‬ globin ‫ال‬ heme ‫مكون‬ iron ‫و‬ protoporphyrin ‫ال‬ globin ‫من‬ ‫يتكون‬ 2 polypeptide chain ( Alfa and Beta, delta or gamma,…). ‫ال‬ Alfa chain ‫من‬ ‫تتكون‬ 141 AA ‫الكروموسوم‬ ‫علي‬ ‫ممثلة‬ 16 ‫الزوجين‬ ‫علي‬ ‫جينات‬ ‫بأربع‬ . ‫ال‬ ‫بينما‬ Beta chain ‫من‬ ‫تتكون‬ 146 AA ‫الكروموسوم‬ ‫علي‬ ‫بجينين‬ ‫ممثلة‬ 11 . ‫ال‬ ‫حسب‬ ‫علي‬ Polypeptide chain in hemoglobin 6
  • 7. Composition of Hemoglobin ( Hb) HB heme iron protoporphyrin globin 4 polypeptide chain 7
  • 8. 8
  • 9. 9
  • 10. 10
  • 11. 11
  • 12. 12
  • 13. NORMAL HB HbA composed of Alfa 2 and beta 2 globin chain HbA2 composed of Alfa 2 and delta 2 globin chain HbF composed of Alfa 2 and gamma 2 globin chain 13
  • 15. 15
  • 16. 16
  • 17. 17
  • 18. When is it order? • as part of newborn screening. In addition, it may be used for prenatal screening when a parent is at high risk or when parents have a child who has a hemoglobinopathy. • when results of a complete blood count (CBC) and/or blood smear suggest that you have an abnormal form of hemoglobin. • when a healthcare practitioner suspects that your signs and symptoms are the result of abnormal hemoglobin production. Abnormal forms of hemoglobin often lead to hemolytic anemia 18
  • 19. 19
  • 20. 20
  • 21. Proteins (Hb) are amphoteric compounds and are therefore either positively or negatively charged because they contain both acidic and basic residues. In an alkaline buffer (pH 8.4), Hb gain negative charges and migrate to the positive electrode (anode). On a support media, HBs are separated according to their shape, size and net surface charge , they migrate to anode. 21
  • 22. • sample are put on a supporting medium between two electrodes. • When the electric current is connected, the charged molecules move from one electrode to another at different migration rates according to their size and charge, allowing their separation. 22
  • 23. • The supporting medium (cellulose acetate gel) is soaked in alkaline buffer. • RBCs is washed, packed, lysed, diluted and then applied on to the gel. • The electric current is then connected (200 V for 30 minutes) to allow separation of different Hb molecules into bands which are then stained with protein/heme stain. • The stained bands are then scanned (densitometer) and the density of each band is roughly correlated with percentage of the variant in the sample. • A graph is generated representing the bands by corresponding peaks. • Control sample is run simultaneously in the same gel. 23
  • 24. 24
  • 27. 1_ Cellulose acetate electrophoresis at alkaline pH ( 8.2_8.6 ) Sample ➡ whole blood best performed on packed RBCs especially in hypergammaglobulinaemia whole blood on EDTA tube. It can be stored at 2-8 oC for 7 days Bands of cellulose acetate electrophoresis at alkaline pH : H➡ and others as Hb bart's,..... A➡ F ➡ S ➡ and others as Hb D , HbG, Hb lepor C ➡ and others as HbA2 , HbE, HbO 27
  • 28. 28
  • 29. 29
  • 30. 30
  • 31. 31
  • 32. 32
  • 33. Disadvantage Can’t be used to quantify small variants e.g., Hb A2. many variants have same migration point. 33
  • 34. 34
  • 35. 35
  • 36. Ion Exchange chromatography ‫هذه‬ ‫الطريقة‬ ‫بتفصل‬ ‫األحماض‬ ‫األمينية‬ ‫والهيموجلوبين‬ ‫وغيره‬ ... ‫نجعل‬ ‫الوسط‬ ‫حمضي‬ ‫فستصبح‬ ‫األحماض‬ ‫األمينية‬ ‫المراد‬ ‫فصلها‬ ‫موجبة‬ ، ‫ونجعل‬ ‫مثال‬ ‫الصوديوم‬ ‫قليل‬ ( ‫ما‬ ‫دة‬ ‫توجد‬ ‫في‬ ‫الجزء‬ ‫المتحرك‬ ) ‫وبوضع‬ ‫مادة‬ ‫صمغية‬ ‫عليها‬ ‫ايونات‬ ‫سالبة‬ ‫في‬ ‫الجزء‬ ‫الثابت‬ ، ‫فعند‬ ‫مرور‬ ‫األحماض‬ ‫األميني‬ ‫ة‬ ‫الموجية‬ ‫تمسك‬ ‫في‬ ‫األيونات‬ ‫السالبة‬ ‫التي‬ ‫في‬ ‫الجزء‬ ‫الثابت‬ ‫بدال‬ ‫عن‬ ‫الصوديوم‬ ‫ألنه‬ ‫قليل‬ ‫فتزيحه‬ ‫وتمسك‬ ‫باأل‬ ‫يونات‬ ‫السالبه‬ ‫الموجودة‬ ‫علي‬ ‫الجزء‬ ‫الثابت‬ ‫بدال‬ ‫من‬ ‫الصوديوم‬ Ion Exchange ‫ما‬ ‫رأيك‬ ‫لو‬ ‫جعلنا‬ ‫الوسط‬ ‫قلوي‬ ‫بدال‬ ‫من‬ ‫حمضي‬ ‫وزودنا‬ ‫ايونات‬ ‫الصوديوم؟‬ Adjust pH and ionic strength ‫في‬ ‫الوسط‬ ‫القلوي‬ ‫البروتينات‬ ‫ستصبح‬ ‫سالبة‬ ‫وستتنافر‬ ‫مع‬ ‫األيونات‬ ‫السالبه‬ ‫الموجودة‬ ‫علي‬ ‫الجزء‬ ‫ال‬ ،‫ثابت‬ ‫ولو‬ ‫زودنا‬ ‫ايونات‬ ‫الصوديوم‬ ‫ايضا‬ ‫ستحل‬ ‫محل‬ ‫األحماض‬ ‫األمينية‬ ‫وبالتالي‬ ‫يحصل‬ Elution to amino acids gradually The main application of adsorption chromatography is separation of different amino acids hemoglobin variants and isoenzymes. 36
  • 37. 37
  • 38. High Performance Liquid Chromatography (HPLC) for separation of Hb variants ‫ايه‬ ‫فكرة‬ ‫عمل‬ ‫فصل‬ ‫أنواع‬ ‫الهيموجلوبين‬ ‫بجهاز‬ ‫ال‬ HPLC ?? ‫نضع‬ ‫عينة‬ ‫الدم‬ ‫في‬ ‫الجهاز‬ ‫مع‬ ‫ال‬ Mobile phase ‫إيه‬ ‫اللي‬ ‫هيحصل؟‬ ‫ال‬ column that contain stationary phase ‫يمتز‬ ‫الهيموجلوبين‬ ‫ألن‬ ‫الهيموجلوبين‬ positive ‫وال‬ Stationary phase contains negative charge ‫كده‬ ‫الهيموجلوبين‬ ‫ذو‬ ‫الشحنة‬ ‫الموجبة‬ ‫مسك‬ ‫في‬ ‫ال‬ column ‫ذو‬ ‫الشحنة‬ ‫السالبة‬ 38
  • 39. ‫بعد‬ ‫ذلك‬ ‫نزيد‬ ‫من‬ ‫مرور‬ Buffer that contain cations in mobile phase and change pH ‫تقوم‬ ‫ال‬ cations ‫تتنافس‬ ‫مع‬ ‫الشحنات‬ ‫الموجبة‬ ‫الموجودة‬ ‫على‬ ‫الهيموجلوبين‬ ‫على‬ ‫الشحنات‬ ‫السالبة‬ ‫الموجو‬ ‫دة‬ ‫على‬ ‫ال‬ Stationary phase. The cations in the mobile phase compete with the adsorbed proteins ( Hb ) for the anionic binding sites present in the stationary phase. ‫يقوم‬ ‫ال‬ cations ‫تحل‬ ‫الهيموجلوبين‬ ‫على‬ ‫الجزء‬ ‫الثابت‬ ‫ويحصل‬ gradual elution to Hb variants ‫علي‬ ‫حسب‬ Their affinity for the stationary phase ( polarity ) ‫طبعا‬ ‫كل‬ ‫ما‬ ‫زادت‬ ‫الشحنة‬ ‫الموجبة‬ ‫في‬ ‫نوع‬ ‫معين‬ ‫من‬ ‫الهيموجلوبين‬ ‫كل‬ ‫ما‬ ‫مسك‬ ‫أكثر‬ ‫بالجزء‬ ،‫الثابت‬ ‫وبالت‬ ‫الي‬ ‫يحصل‬ ‫له‬ elution ‫أبطأ‬ ‫وبالتالي‬ ‫يصل‬ ‫لل‬ detector ‫متأخرا‬ ‫وبالتالي‬ High retention time. ‫وكل‬ ‫ما‬ ‫زادت‬ ‫الشحنة‬ ‫السالبة‬ ‫في‬ ‫نوع‬ ‫معين‬ ‫من‬ ‫الهيموجلوبين‬ ‫كل‬ ‫ما‬ ‫حصل‬ ‫له‬ elution ‫أسرع‬ ‫وبالتالي‬ ‫يصل‬ ‫لل‬ detector ‫أسرع‬ ‫وبالتالي‬ Low retention time 39
  • 40. ‫طب‬ ‫الجهاز‬ ‫عارف‬ ‫الوقت‬ ‫اللي‬ ‫هيوصل‬ ‫عنده‬ ‫كل‬ ‫نوع‬ ‫من‬ ‫أنواع‬ ‫الهيموجلوبين‬ ‫والذي‬ ‫يس‬ ‫مي‬ ‫ال‬ retention time ‫كل‬ ‫نوع‬ ‫من‬ ‫الهيموجلوبين‬ ‫له‬ Specific retention time ‫محدد‬ ‫الجهاز‬ ‫عارفه‬ ‫وبالتالي‬ ‫الجهاز‬ ‫يعرف‬ ‫ما‬ ‫هو‬ ‫البروتين‬ ‫أو‬ ‫الهيموجلوبين‬ ‫اللي‬ ‫وص‬ ‫ل‬ ‫في‬ ‫هذا‬ ‫الوقت‬ ‫وتركيزه‬ ‫يساوي‬ ‫كم؟‬ . ‫كل‬ ‫ده‬ ‫واضح‬ ‫على‬ ‫الورقة‬ ‫بعد‬ ‫كده‬ ‫يسجل‬ ‫الكالم‬ ‫على‬ ‫هيئة‬ peaks ‫عن‬ ‫طريق‬ ‫جهاز‬ ‫ال‬ densometer ‫او‬ ‫نعملها‬ elution followed by spectrophotometer assay. ‫أعلم‬ ‫وهللا‬ . 40
  • 41. is a process in which a mixture of molecules (such as normal and abnormal hemoglobin variants ) with a net positive charge is separated into its components by their adsorption onto a negatively charged stationary phase in a chromatography column, followed by their elution by a mobile phase. The mobile phase is a liquid with an increasing concentration of cations flowing through the column; the cations in the mobile phase compete with the adsorbed proteins for the anionic binding sites. 41
  • 42. Thus, the adsorbed positively charged hemoglobin molecules are eluted from the column into the liquid phase at a rate related to their affinity for the stationary phase. When separated in this way, they can be detected optically in the eluate, provisionally identified by their retention time, and quantified by computing the area under the corresponding peak in the elution profile. The more positively charged hemoglobin (e.g., hemoglobin S and C) have a longer retention time. 42
  • 43. 43
  • 44. Variant with weak positive charge will adsorb weakly to the column and is eluted rapidly from the column with the injection of low strength elution buffer. Variant with strong positive charge will adsorb strongly to the column and is eluted slowly from the column with the injection of high strength elution buffer. Procedure: HPLC is fully automated. Samples are loaded in a rack and automatically processed one by one (about 6 minutes/sample). Result: Data are graphically represented as absorbance vs time (peaks). Retention time is the time from injection to the top of the peak. 44
  • 45. ‫له‬ ‫الهيموجلوبين‬ ‫من‬ ‫نوع‬ ‫كل‬ Specific Retention time ‫ال‬ ‫يطلع‬ ‫ولما‬ ،‫الحالة‬ ‫للجهاز‬ ‫تدخل‬ ‫فإنت‬ ،‫عارفه‬ ‫الجهاز‬ Its Retention time ‫ال‬ ‫بمعلومية‬ ‫فورا‬ ‫الهيموجلوبين‬ ‫نوع‬ ‫يقولك‬ ‫يقوم‬ Its retention time. ‫ال‬ ‫نفس‬ ‫لها‬ ‫اللي‬ ‫األنواع‬ ‫من‬ ‫بالك‬ ‫وخلي‬ retention time ‫ال‬ ‫ومشاكل‬ Glycated Hb and HbS with HbA2..... etc. ‫العيوب‬ ‫في‬ ‫الحقا‬ ‫ستذكر‬ ‫كما‬ . 45
  • 46. 46
  • 47. 47
  • 48. 48
  • 49. disadvantages of HPLC 1_ expensive 2_ Small part of HbS coelute with Hb A2 leads to falsely high HbA2 value in Sickle cell variant as Retention time of them are near • ‫لذلك‬ ‫ال‬ ‫يستحب‬ ‫في‬ ‫التفرقة‬ ‫بين‬ HbSS and HbSBo 3_ Retention time of glycated and non glycated hemoglobin are different ➡ leads to misinterpretation i.e. Glycosylated hemoglobin S may have a retention time the same as, or very similar to, that of hemoglobin A, so that patients with sickle cell anemia may be thought to have a small amount of hemoglobin A. 4 _ Glycated Hb may measured as HbF falsely elevate HbF in HPLC. 5_ although many variant hemoglobin can be separated from each other, they are some that overlap with each other as Hb lepor usually overlaps with HbA2 and HbE 6_ Certain artefacts need to be recognized for example, increased bilirubin in the plasma may lead to a sharp peak in the same general area as hemoglobin H. The nature of any variant hemoglobin detected by HPLC should be confirmed by an alternative technique. 49
  • 50. Hb variant on HPLC • Hb C elutes in C window in HPLC (C/O). • Hb E elutes in A2 window in HPLC (A2/E/Lepore). • Hb D elutes in D window in HPLC (D/G). • Small part of HbS coelute with Hb A2 leads to falsely high HbA2 value in Sickle cell variant as Retention time of them are near so you cant differentiate between HBSS and HBSB0 • Certain artefacts need to be recognized for example, increased bilirubin in the plasma may lead to a sharp peak in the same general area as hemoglobin H. • Retention time of glycated and non glycated hemoglobin are different ➡ leads to misinterpretation i.e. Glycosylated hemoglobin S may have a retention time the same as, or very similar to, that of hemoglobin A, so that patients with sickle cell anemia may be thought to have a small amount of hemoglobin A. • Glycated Hb may measured as HbF falsely elevate HbF in HPLC. 50
  • 51. 51
  • 52. ‫زي‬ ‫ما‬ ‫في‬ ‫الصورة‬ ‫هنستخدم‬ ‫هنا‬ ‫بدال‬ ‫من‬ ‫الجل‬ Very narrow silica tube with alkaline pH buffer The interior of the tube is negatively charged attract positive charge from buffer to create diffuse layer ‫نستخدم‬ High voltage with 8 minutes ‫كده‬ ‫هينتج‬ 2 forces 52
  • 53. ‫كده‬ ‫هينتج‬ 2 forces 1_ electrophoretic force ‫تجعل‬ ‫الهيموجلوبين‬ ‫السالب‬ ‫يتجه‬ ‫نحو‬ ‫القطب‬ ‫الموجب‬ 2_electro-osmotic force ‫تجعل‬ ‫الهيموجلوبين‬ ‫السالب‬ ‫يتجه‬ ‫نحو‬ ‫القطب‬ ‫السالب‬ ! ‫والمفارقة‬ ‫أن‬ ‫القوة‬ ‫األكبر‬ ‫هي‬ Electro-osmotic force ‫التى‬ ‫تجعل‬ ‫الهيموجلوبين‬ ‫السالب‬ ‫يتجه‬ ‫نحو‬ ‫القطب‬ ‫السالب‬ ‫وبالتالي‬ ‫سأضع‬ ‫أنا‬ ‫عينة‬ ‫الهيموجلوبين‬ ‫ناحية‬ ‫القطب‬ ‫الموجب‬ ‫حتى‬ ‫تتجه‬ ‫نحو‬ ‫القطب‬ ،‫السالب‬ ‫و‬ ‫عند‬ ‫القطب‬ ‫السالب‬ ‫أضع‬ detector for detection of separated analytes ‫يرسم‬ ‫رسمة‬ ‫مكونة‬ ‫من‬ 15 zones . In each zone, many Hb variants are located. 53
  • 54. 54
  • 55. Advantages of capillary electrophoresis 1_ fully automated 2_ Efficient separation with high resolution (separate Hb A2 from Hb E). 3_ Adequate quantification of Hb variants (e.g., Hb A2 and Hb Bart’s). 4_ Easy interpretation of results (minimal overlap of common Hb variants). 55
  • 56. 56
  • 57. Hb variant on capillary electrophoresis • Hb S migrates to S zone in capillary electrophoresis • Hb C migrates to C zone in capillary electrophoresis • Hb E migrates to E zone in capillary electrophoresis • Hb lepor migrates to LEPOR zone in capillary electrophoresis • Hb D migrates to D zone in capillary electrophoresis (D/G) • Hb O-Arab migrates to A2 zone in capillary electrophoresis (A2/O) 57
  • 58. 58
  • 59. 59
  • 60. Hb variant on HPLC • Hb C elutes in C window in HPLC (C/O). • Hb E elutes in A2 window in HPLC (A2/E/Lepore). • Hb D elutes in D window in HPLC (D/G). • Small part of HbS coelute with Hb A2 leads to falsely high HbA2 value in Sickle cell variant as Retention time of them are near • Certain artefacts need to be recognized for example, increased bilirubin in the plasma may lead to a sharp peak in the same general area as hemoglobin H. • Retention time of glycated and non glycated hemoglobin are different ➡ leads to misinterpretation i.e. Glycosylated hemoglobin S may have a retention time the same as, or very similar to, that of hemoglobin A, so that patients with sickle cell anemia may be thought to have a small amount of hemoglobin A. • Glycated Hb may measured as HbF falsely elevate HbF in HPLC. 60
  • 61. Hb variant in capillary electrophoresis • Hb S migrates to S zone in capillary electrophoresis • Hb C migrates to C zone in capillary electrophoresis • Hb E migrates to E zone in capillary electrophoresis • Hb lepor migrates to LEPOR zone in capillary electrophoresis • Hb D migrates to D zone in capillary electrophoresis (D/G) • Hb O-Arab migrates to A2 zone in capillary electrophoresis (A2/O) 61
  • 62. Hb variants on different electrophoresis Hb C migrates to A2 region in gel electrophoresis (A2/C/E/O), to C zone in capillary electrophoresis and elutes in C window in HPLC (C/O). Hb E migrates to A2 region in gel electrophoresis (A2/C/E/O), to E zone in capillary electrophoresis and elutes in A2 window in HPLC (A2/E/Lepore). Hb D migrates to S region in gel electrophoresis (S/G/D/Lepore), to D zone in capillary electrophoresis (D/G) and elutes in D window in HPLC (D/G). Hb O-Arab migrates to A2 region in gel electrophoresis (A2/C/E/O), to A2 zone in capillary electrophoresis (A2/O) and elutes in C window in HPLC (C/O). Hb lepor migrates to S region in gel electrophoresis (S/G/D/Lepore), to LEPOR zone in capillary electrophoresis and elutes in A2 window in HPLC (A2/E/Lepore). 62
  • 63. 63
  • 64. 64
  • 65. Composition of Hemoglobin ( Hb) HB heme iron protoporphyrin globin 4 polypeptide chain 65
  • 66. NOTES 1_ Hemoglobinopathy is autosomal recessive disorders so to be appeared the 2 genes should be affected . 3_ Hemoglobinopathy defined as a family of diseases caused by production of : • A _ synthesis of insufficient quantities of normal Hemoglobin ( thalassemia) • B _ structurally abnormal hemoglobin as Sickle cell anemia • C_ or both 66
  • 67. 67
  • 68. Definition: quantitative defect in α chain production with globin chain imbalance ‫ال‬ Alfa Chain ‫من‬ ‫تتكون‬ 141 AA ‫الكروموسوم‬ ‫علي‬ ‫ممثلة‬ 16 ‫جينات‬ ‫بأربع‬ ‫لو‬ ‫حصل‬ deletion in these one or more of these genes these leads to decrease expression of Alfa genes leads to Alfa thalassemia . It may be due to another gene abnormalities as point mutation,......... Which called non deletional Alfa thalassemia 68
  • 69. pathophysiology Low expression of Alfa globin gene ➡ ⬇ Alfa globin chain ➡ imbalance between Alfa and Beta globin chain ➡ excess free Beta and Gamma chain . Excess free Beta and Gamma chain ➡ ⬆ HbH and Hb Bart's 69
  • 70. 70
  • 71. 71
  • 72. 1-silent carrier silent carrier (_, Alfa/Alfa, Alfa) Deletion in one gene silent carrier can detected by genetic testing. It is Fully compensated. No/minimal free beta. Normal clinical lab. 72
  • 73. Alfa thalassemia trait (_, Alfa/_, Alfa) with high free beta chain Deletion of two genes -There are thalassemic red cell indices as : -Mild or no anemia -Moderate or marked microcytosis and hypochromia of RBCs -Erythrocytosis above 5 X 10^6 RDW normal 73
  • 74. Differentiation a _ Iron deficiency anemia by normal ferritin ( in case not acute I. e. Normal CRP) b _ beta thalassemia trait by excluded by high A2 in Hb electrophoresis. 74
  • 75. HbH (Alfa_,_ _ ) type of thalassemia intermedia (moderate anemia with marked microcytosis and hypochromia no need for repeated blood transfusions) and extravascular hemolysis Hb electrophoresis Hb H + less Hb Bart's HbH is faster than HbA in electrophoresis , Hb H are usually ~ 10% with dominant HbA HbH formed of 4Beta forming HbH Reticulocyte shows golf ball appearance ‫طبعا‬ ‫فيها‬ ‫كل‬ ‫عالمات‬ ‫ال‬ hemolytic anemia 75
  • 76. 76
  • 77. Hb Bart’s 4 Gamma chain (_ _, _ _) ‫المريض‬ ‫بيموت‬ ‫في‬ ‫بطن‬ ‫أمه‬ ‫أو‬ ‫عندالوالدة‬ Hb electrophoresis Hb Bart's + Hb H Early diagnosis: suspected by US examination or family genetic study and is confirmed by genetic testing via chorionic villous sample, amniocentesis or cordocentesis. IU transfusion might be lifesaving!!!! 77
  • 78. 78
  • 79. α + thalassemia ‫ودي‬ ‫بيبقي‬ ‫المشكلة‬ ‫في‬ ‫جين‬ ‫واحد‬ ‫من‬ ‫األربع‬ ‫جينات‬ ‫أو‬ ‫اتنين‬ ‫بس‬ ‫علي‬ ‫كروموسين‬ ‫يعني‬ ‫كل‬ ‫كروموسوم‬ ‫عليه‬ ‫جين‬ ‫والتاني‬ ‫اتشال‬ . αo thalassemia ‫يبقي‬ ‫المشكلة‬ ‫في‬ ‫جينين‬ ‫من‬ ‫علي‬ ‫كروموسوم‬ ‫واحد‬ ‫والتاني‬ ‫سليم‬ ‫لو‬ ‫اتشال‬ 3 ‫ده‬ HbH ‫لو‬ ‫اتشال‬ 4 ‫ده‬ Hb Bart's 79
  • 80. The degree of microcytosis and hypochromia is roughly correlated with number of gene deletion. 1- Silent: (-α/αα) = Heterozygous α+ 2- Mild: ▪ (--/αα) = Heterozygous α0 ▪ (-α/-α) = Homozygous α+ ▪ (αTα/αα) = Heterozygous non-deletional α thalassemia 3- Moderate: ▪ (--/-α) = Double heterozygous α0 and α+ ▪ (--/ αTα) = Double heterozygous α0 and non-deletional α thalassemia ▪ (αTα/αTα) = Homozygous or double heterozygous non-deletional α thalassemia. 4- Severe: ▪ (--/--) = Homozygous α0 ▪ (--/αTα) = Double heterozygous α0 and non-deletional α thalassemia. 80
  • 81. Alfa thalassemia oNormal lab in silent carrier, detected by genetic testing. oLow MCV and MCH in silent and alfa thalassemia trait oHBH in HBH disease moderate anemia with marked microcytosis and hypochromia o Hb Bart’s in Hb Bart’s disease oGenetic testing 81
  • 82. B thalassemia Definition: quantitative defect in β chain production with globin chain imbalance Beta thalassemia is found in Southeast Asia, Mediterranean area and Middle East. Occasionally seen in Africa (usually β+). Low expression of B globin gene ➡ ⬇ B globin chain ➡ imbalance between Alfa and Beta globin chain ➡ excess free Alfa chain Delta and gamma chain are increased to compensate imbalance between Alfa and Beta globin chain ➡ ⬆ HbA2 and HbF 82
  • 83. Clinical features 1- Silent/almost silent beta thalassemia: no clinical ± laboratory abnormality. 2- Thalassemia minor: mild or no anemia. 3- Thalassemia intermedia: moderate chronic hemolytic anemia + complications. 4- Thalassemia major: marked chronic hemolytic anemia + complications. 83
  • 84. B thalassemia major Homozygous state Symptoms and signs of : 1_ chronic hemolytic anemia ➡ anemia, jaundice, hepatosplenomegaly, failure of growth, delay puberty. 2_ iron overload ➡ cardiac failure, arrhythmia, liver affection and DM. 3_ BM expansion ➡ skeletal deformity, iron overload. 84
  • 85. B thalassemia major Homozygous state Manifestations of hemolytic anemia as : 1_ severe ( less than 7 mg/dl) microcytic hypochromic Anemia 2_ Indirect hyperbilirubinemia more than 80% of total bilirubin 3_ Increased urobilinogen in urine 4_ Normal urine colure 5 _ High uric acid 85
  • 86. 86
  • 87. 87
  • 88. B thalassemia major (Homozygous state) Hb electrophoresis : (Bo Bo) : HbA ➡absent HbA2➡ increased HbF ➡ about 96% . B+ Bo Or B+ B+ : HbA ➡10_30% HbA2➡ increased HbF ➡ about 70_90 % Iron profile : Increase ➡ serum iron, ferritin, iron saturation Decrease ➡ TIBC 88
  • 89. 89
  • 90. B thalassemia minor (Heterozygous state) thalassemic red cell indices as • Mild or no anemia • Moderate or marked microcytosis and hypochromia of RBCs • Erythrocytosis above 5 X 10^6 • RDW ➡ normal Hb electrophoresis: B Bo, B B+ • HbA2➡ increased in 100% cases ( 3.5_8%) • HbF ➡ increased in 50% of cases ( 1-7%) • HbA ➡ more than 90% 90
  • 91. 91
  • 92. B thalassemia intermedia moderate chronic hemolytic anemia + complications. Hb F from 20 – 40 % ‫االتنين‬ ‫بين‬ ‫وسط‬ ‫مرحلة‬ 92
  • 93. NOTES ‫في‬ ‫حاالت‬ ‫ممكن‬ ‫ال‬ HbA2 ،‫اليزيد‬ ‫لو‬ ‫الحاالت‬ ‫دي‬ ‫أصال‬ ‫ي‬ ‫قل‬ ‫فيها‬ ‫ال‬ HbA2 ‫مثل‬ Beta thalassemia trait with normal HbA2 1- iron deficiency anemia, folate deficiency, hypothyroidism 2- Delta Beta thalassemia or Alfa beta thalassemia ‫في‬ ‫حاجة‬ ‫اسمها‬ silent B thalassemia and almost silent B thalassemia ‫ودي‬ ‫بيبقى‬ ‫مشهور‬ ‫فيها‬ -101 C mutation ‫وبيبقى‬ ‫فيها‬ HbA2 ‫طبيعي‬ ‫ومفيش‬ ‫أعراض‬ almost silent B thalassemia has normal HbA2 and abnormal red cell indices while, silent B thalassemia has normal HbA2 and normal red cell indices. 93
  • 94. ‫الخالصة‬ B thalassemia B thalassemia trait thalassemic red cell indices with high HBA2 more than 3.7 except reasons mentioned above, Hb F (1-7%) B thalassemia major moderate to marked MHA Blood film ‫فراخ‬ ‫نعكشة‬ Sign and symptoms of hemolytic anemia Hb separation: increased Hb F (becomes dominant) ± increase in Hb A2. Hb A is absent in β0 NB : DON’T MISS SILENT AND ALMOST SILENT B THALASSEMIA 94
  • 95. Delta B thalassemia ‫زي‬ ‫ال‬ B thalassemia ‫ولكن‬ ‫ال‬ Delta ‫قلت‬ ‫فهيقل‬ HbA2 ‫ودي‬ ‫فعال‬ ‫في‬ Heterozygous delta beta thalassemia similar to B thalassemia minor clinically but low or normal HbA2 HbF from 5_ 15 % Homozygous delta beta thalassemia similar to B thalassemia intermedia clinically with absent HbA2 HbF is 100 % 95
  • 96. Clinical phenotypes and genetic causes of beta thalassemia 1_ Silent: certain mutations e.g., -101 C→T mutation. 2_ Mild disease: β+βA, β0βA or β0βHPFH 3_ Moderate disease: ▪ β0β+, β0βD-Punjab, β0βO-Arab or β0βC (DOC). ▪ Autosomal dominant beta thalassemia. ▪ Homozygous Hb Lepore. 3_ Severe disease: ▪ β0β0 ▪ β0βE ▪ β0βLepore ▪ β0 with quadruple alpha (αααα). 96
  • 97. TERMS BA➡ ‫ال‬ ‫تعني‬ B ‫تخرج‬ ‫التى‬ ‫العادية‬ B chain β+ ➡ ‫ال‬ ‫تعني‬ B ‫ناقصه‬ β0➡ ‫ال‬ ‫تعنى‬ B ‫شغاله‬ ‫مش‬ βO or D or E or Lepor ➡ ‫إن‬ ‫معناه‬ ‫الكروموسوم‬ ‫ال‬ ‫من‬ ‫بدل‬ ‫تخرج‬ ‫لكن‬ ‫شغال‬ B chain E or O or Lepor Hb chain 97
  • 98. 98
  • 99. NOTES 1_ Hemoglobinopathy is autosomal recessive disorders so to be appeared the 2 genes should be affected . 2_ 90 % of structural variant hemoglobinopathy are point mutation. 3_ Hemoglobinopathy defined as a family of diseases caused by production of : • A _ synthesis of insufficient quantities of normal Hemoglobin ( thalassemia) • B _ structurally abnormal hemoglobin as Sickle cell anemia • C_ or both 99
  • 100. Hemoglobin S ‫لو‬ ‫حصل‬ Point mutation ‫واتغير‬ ‫ال‬ AA ‫رقم‬ 6 ‫اللي‬ ‫هو‬ glutamate ‫في‬ ‫ال‬ Beta chain ‫في‬ ‫الكروموسوم‬ 11 ⬅ ‫اتغير‬ ‫إلي‬ Valine ‫ينتج‬ Hb ‫غريب‬ ‫سموه‬ HbS ‫ويكون‬ ‫مرض‬ ‫ال‬ Sickle diseases ‫وهو‬ ‫من‬ ‫عائلة‬ structural gene defect hemoglobinopathy α2β2 6Glu→Val ‫لو‬ ‫أصيب‬ ‫الجينين‬ ‫في‬ . Beta chain ‫يبقي‬ HbSS ‫لو‬ ‫جين‬ ‫واحد‬ ‫يبقي‬ HbAS ‫لو‬ ‫حصل‬ double heterozygous mutation ‫ممكن‬ ‫ينتج‬ HbSC _ HbSE ‫وغيره‬ . ‫وطبعا‬ ‫ده‬ ‫هيغير‬ ‫مكانهم‬ ‫في‬ electrophoresis ‫فيه‬ ‫يحصل‬ ‫الجينين‬ ‫أحد‬ ‫ممكن‬ mutation ‫الي‬ ‫ويتحول‬ S ‫ال‬ ‫ينعدم‬ ‫واآلخر‬ expression ‫ويبقي‬ ‫بتاعه‬ Bo ‫ال‬ ‫يقل‬ ‫او‬ expression ‫ويبقي‬ ‫بتاعه‬ + B ‫اما‬ ‫وينتج‬ BoS ‫او‬ B+S 100
  • 101. 101
  • 102. Hb S is the most common hemoglobinopathy. It has a Worldwide distribution. The highest frequency is seen in West Africa, Kuwait, East of KSA and east of India. 102
  • 103. 103
  • 104. Heterozygous sickle cell trait a. CBC: is normal (except if associated alpha thalassemia). b. Smear: is normal (occasional boat shape cells). c. Reticulocytes: are normal. d. Hb separation: shows dominant Hb A normal or slightly increased Hb A2 and or Hb F Hb S around 40% (less if with α thalassemia or IDA). e. Sickling tests are positive 104
  • 105. 105
  • 106. Homozygous sickle cell anemia CBC Mild to moderate normocytic normochromic anemia with anisocytosis. • Anemia occasionally severe and rarely absent. • Microcytic if associated α thalassemia (higher Hb level) or IDA (lower Hb level). • Macrocytic if there are reticulocytosis, megaloblastic crisis (lower Hb level) or hydroxyurea therapy (higher Hb level). b. Smear: shows, i. Anispoikilocytosis, sickle cells, boat shape cells, target cells ± irregularly contracted cells, blister cells, RBCs fragments and/or NRBCs. ii. Howell–Jolly bodies and Pappenheimer bodies (hyposplenism) are usually seen beyond infancy. 106
  • 107. C. Reticulocytes: are increased (it falls if aplastic or megaloblastic crises and hydroxyurea). d. Hb separation: dominant Hb S (>90 %) normal Hb A2, slightly increased Hb F (5-10%) with absence of Hb A i.Hb F shows higher percentage in infancy (delayed switch), with therapy (hydroxycarbamide) and in some cases (Arab-Indian). ii. Hb A2 may increase in Arab-Indian haplotype (up to 5%). ii. Hb A level depends on transfusion. e. Other: i. Sickling tests are positive. ii. Hemolysis chemistry: LDH, indirect bilirubin, haptoglobin and hemopexin. iii. For complications: e.g., cranial US, eGFR, urine albumin… Homozygous sickle cell anemia 107
  • 108. 108
  • 109. o Asymptomatic: βS/βA and βS/HPFH. o Mild disease: βS/β+ and βS/βE. o Moderate disease: βS/βC, βS/βD and βS/βLepore o Severe disease: βS/βS, βS/β0 and βS/βO-Arab. 109
  • 110. ‫الخالصة‬ Homozygous sickle cell anemia : Moderate to marked NNA hemolytic anemia with symptoms of hemolytic anemia Blood smear shows sickle cell Hb electrophoresis is HbS more than 90 % . HbF slightly increased (5-10%). HbA2 is normal Heterozygous sickle cell trait : HbS around 40 % decreased in alfa thalassemia and IDA , normal or slightly increased Hb A2 and or Hb F SB+ HbS becomes more than HbA , with high HbF and HbA2 SB0 absent HbA with high HbF and HbA2 Hb A level depends on transfusion. 110
  • 111. Hemoglobin C ‫حصل‬ ‫لو‬ Point mutation ‫ال‬ ‫واتغير‬ AA ‫رقم‬ 6 ‫هو‬ ‫اللي‬ glutamate ‫ال‬ ‫في‬ Beta chain ‫الكروموسوم‬ ‫في‬ 11 ⬅ ‫إلي‬ ‫اتغير‬ Lysine ‫ينتج‬ Hb ‫سموه‬ ‫غريب‬ HbC ‫ويكون‬ ‫عائلة‬ ‫من‬ ‫مرض‬ structural gene defect hemoglobinopathy ‫في‬ ‫الجينين‬ ‫أصيب‬ ‫لو‬ . Beta chain ‫يبقي‬ HbCC ‫يبقي‬ ‫واحد‬ ‫جين‬ ‫لو‬ HbAC ‫حصل‬ ‫لو‬ double heterozygous mutation ‫ينتج‬ ‫ممكن‬ HbSC ‫في‬ ‫مكانه‬ ‫هيغير‬ ‫ده‬ ‫وطبعا‬ HB electrophoresis ‫فينه‬ ‫يحصنل‬ ‫الجيننين‬ ‫أحند‬ ‫ممكنن‬ mutation ‫الني‬ ‫ويتحنول‬ C ‫ال‬ ‫ينعندم‬ ‫واآلخنر‬ expression ‫ويبقني‬ ‫بتاعنه‬ Bo ‫ال‬ ‫يقنل‬ ‫او‬ expression ‫ويبقني‬ ‫بتاعنه‬ + B ‫امنا‬ ‫ويننتج‬ BoC ‫او‬ B+C 111
  • 112. Clinical features 1- Asymptomatic in heterozygous state. 2- Mild to moderate chronic hemolytic anemia ± splenomegaly in homozygous state. 3- Epidemiology. Hb C is seen frequently in West Africa 112
  • 113. Heterozygous Hb C (Hb C trait) a. CBC: shows mild microcytosis, hypochromia and anisocytosis (MCV may be normal and MCHC is high normal). b. Smear: is normal or shows some target cells and irregularly contracted cells. c. Reticulocytes: are normal. d. Hb separation: Dominant Hb A normal or slightly increased Hb A2 and/or Hb F Hb C around 40% (less if associated with α thalassemia or IDA). Hb C migrates to A2 region in gel electrophoresis (A2/C/E/O), to C zone in capillary electrophoresis and elutes in C window in HPLC (C/O). 113
  • 114. 114
  • 115. Homozygous Hb C (Hb C disease) a. CBC: shows mild to moderate anemia with marked microcytosis and hypochromia. MCHC is at high normal range. Platelet count may increase. b. Smear: shows many target cells and irregularly contracted cells with occasional NRBCs and Hb C crystals (tetragonal or hexagonal, hyperdense mass with eccentric position). c. Reticulocytes: are slightly increased. d. Hb separation: dominant Hb C (>95%) normal or slightly increased Hb A2 and/or Hb F with absence of Hb A. e. Other: i. Hemolysis chemistry: LDH, indirect bilirubin, haptoglobin and hemopexin. ii. For complications: iron level, abdominal US… iii. Decreased osmotic fragility. iv. Genetic testing 115
  • 116. 116
  • 117. Hemoglobin E ‫لو‬ ‫حصل‬ Point mutation ‫واتغير‬ ‫ال‬ AA ‫رقم‬ 26 ‫اللي‬ ‫هو‬ glutamate ‫في‬ ‫ال‬ Beta chain ‫في‬ ‫الكروموسوم‬ 11 ⬅ ‫اتغير‬ ‫إلي‬ Lysine ‫ينتج‬ Hb ‫غريب‬ ‫سموه‬ Hb-E ‫ويكون‬ ‫مرض‬ ‫من‬ ‫عائلة‬ structural gene defect hemoglobinopathy ‫لو‬ ‫أصيب‬ ‫الجينين‬ ‫في‬ . Beta chain ‫يبقي‬ HbEE ‫لو‬ ‫جين‬ ‫واحد‬ ‫يبقي‬ HbAE ‫لو‬ ‫حصل‬ double heterozygous mutation ‫ممكن‬ ‫ينتج‬ . HbSE ‫وطبعا‬ ‫ده‬ ‫هيغير‬ ‫مكانه‬ ‫في‬ HB electrophoresis ‫ممكن‬ ‫أحد‬ ‫الجينين‬ ‫يحصل‬ ‫فيه‬ mutation ‫ويتحول‬ ‫الي‬ E ‫واآلخر‬ ‫ينعدم‬ ‫ال‬ expression ‫بتاعه‬ ‫ويبقي‬ Bo ‫او‬ ‫يقل‬ ‫ال‬ expression ‫بتاعه‬ ‫ويبقي‬ + B ‫وينتج‬ ‫اما‬ BoE ‫او‬ B+E 117
  • 118. • It is the 2nd most common hemoglobinopathy. • Epidemiology: found more in Southeast Asia. • Clinical features : • 1- Asymptomatic in both heterozygous and homozygous state (homozygous state rarely shows mild hemolysis). • 2- The clinical significance of Hb E is in double heterozygous with beta thalassemia leading to moderate to severe disease. Hemoglobin E 118
  • 119. Heterozygous Hb E (Hb E trait) a. CBC: shows erythrocytosis, mild microcytosis and hypochromia ± anemia. CBC is occasionally normal. b. Smear: is normal or shows some target cells and irregularly contracted cells. c. Reticulocytes: are normal. d. Hb separation: Dominant Hb A normal or slightly increased Hb A2 and/or Hb F Hb E around 30% (less if associated with α thalassemia or IDA). Higher percentage if Hb E/β thalassemia). Hb E migrates to A2 region in gel electrophoresis (A2/C/E/O), to E zone in capillary electrophoresis and elutes in A2 window in HPLC (A2/E/Lepore). 119
  • 120. 120
  • 121. Homozygous Hb E (Hb E disease) a. CBC: shows erythrocytosis with marked microcytosis and hypochromia ± mild anemia. b. Smear: shows many target cells and irregularly contracted cells c. Reticulocytes: are normal or slightly elevated. d. Hb separation: dominant Hb E normal or slightly increased Hb A2 and/or Hb F with absence of Hb A. e. Other: i. Hemolysis chemistry: LDH, indirect bilirubin, haptoglobin and hemopexin. ii. For complications: iron level, abdominal US… iii. Decreased osmotic fragility. iv. Genetic testing 121
  • 122. Hemoglobin D ‫حصل‬ ‫لو‬ Point mutation ‫ال‬ ‫واتغير‬ AA ‫رقم‬ 121 ‫هو‬ ‫اللي‬ glutamate ‫ال‬ ‫في‬ Beta chain ‫في‬ ‫الكروموسوم‬ 11 ⬅ ‫إلي‬ ‫اتغير‬ glutamine ‫ينتج‬ Hb ‫سموه‬ ‫غريب‬ Hb-D ‫عائلة‬ ‫من‬ ‫مرض‬ ‫ويكون‬ structural gene defect hemoglobenopathy ‫في‬ ‫الجينين‬ ‫أصيب‬ ‫لو‬ . Beta chain ‫يبقي‬ HbDD ‫يبقي‬ ‫واحد‬ ‫جين‬ ‫لو‬ HbAD ‫حصل‬ ‫لو‬ double heterozygous mutation ‫ينتج‬ ‫ممكن‬ . HbSD ‫في‬ ‫مكانه‬ ‫هيغير‬ ‫ده‬ ‫وطبعا‬ HB electrophoresis ‫فيه‬ ‫يحصل‬ ‫الجينين‬ ‫أحد‬ ‫ممكن‬ mutation ‫الي‬ ‫ويتحول‬ D ‫ال‬ ‫ينعدم‬ ‫واآلخر‬ expression ‫ويبقي‬ ‫بتاعه‬ Bo ‫ال‬ ‫يقل‬ ‫او‬ expression ‫ويبقي‬ ‫بتاعه‬ + B ‫اما‬ ‫وينتج‬ BoD ‫او‬ B+D 122
  • 123. Epidemiology and Clinical features worldwide distribution with highest incidence in India (Punjab). 1- Asymptomatic in both heterozygous and homozygous state (homozygous state sometime shows mild hemolysis). 2- The clinical significance of Hb D is in double heterozygous with Hb S leading to severe disease. 123
  • 124. Heterozygous Hb D (Hb D trait) a. CBC: is normal. b. Smear: is normal c. Reticulocytes: are normal. d. Hb separation: Dominant Hb A normal Hb A2 and/or Hb F Hb D around 40% (less if associated with α thalassemia or IDA). Hb D migrates to S region in gel electrophoresis (S/G/D/Lepore), to D zone in capillary electrophoresis (D/G) and elutes in D window in HPLC (D/G). 124
  • 125. 125
  • 126. Homozygous Hb D (Hb D disease) a. CBC: is normal or shows mild normocytic normochromic anemia. b. Smear: shows many target cells and irregularly contracted cells c. Reticulocytes: are normal or slightly elevated. d. Hb separation: dominant Hb E (>95%) normal Hb A2 and/or Hb F with absence of Hb A. e. Other: i. Decreased osmotic fragility. ii. Genetic testing 126
  • 127. Hemoglobin O-Arab ‫حصل‬ ‫لو‬ Point mutation ‫ال‬ ‫واتغير‬ AA ‫رقم‬ 121 ‫هو‬ ‫اللي‬ glutamate ‫ال‬ ‫في‬ Beta chain ‫الكروموسوم‬ ‫في‬ 11 ⬅ ‫إلي‬ ‫اتغير‬ lysin ‫ينتج‬ Hb ‫سموه‬ ‫غريب‬ Hb-O ‫عائلة‬ ‫من‬ ‫مرض‬ ‫ويكون‬ structural gene defect hemoglobenopathy ‫في‬ ‫الجينين‬ ‫أصيب‬ ‫لو‬ . Beta chain ‫يبقي‬ HbOO ‫يبقي‬ ‫واحد‬ ‫جين‬ ‫لو‬ HbAO ‫حصل‬ ‫لو‬ double heterozygous mutation ‫ينتج‬ ‫ممكن‬ . HbSO ‫في‬ ‫مكانه‬ ‫هيغير‬ ‫ده‬ ‫وطبعا‬ Hb electrophoresis ‫فيه‬ ‫يحصل‬ ‫الجينين‬ ‫أحد‬ ‫ممكن‬ mutation ‫الي‬ ‫ويتحول‬ O ‫ال‬ ‫ينعدم‬ ‫واآلخر‬ expression ‫ويبقي‬ ‫لديه‬ Bo ‫ال‬ ‫يقل‬ ‫او‬ expression ‫ويبقي‬ + B ‫اما‬ ‫وينتج‬ BoO ‫او‬ B+O 127
  • 128. Epidemiology and Clinical features Epidemiology: Hb-O is seen in African, Arab and East Europe. Clinical features : 1- Asymptomatic in heterozygous state. 2- Mild anemia in some patient with homozygous state ± jaundice and splenomegaly. 3- The clinical significance of Hb O is in double heterozygous with Hb S leading to severe disease 128
  • 129. Heterozygous Hb O (Hb O trait) a. CBC: is normal. b. Smear: is normal c. Reticulocytes: are normal. d. Hb separation: Dominant Hb A normal Hb A2 and/or Hb F Hb O around 40% (less if associated with α thalassemia or IDA). Hb O-Arab migrates to A2 region in gel electrophoresis (A2/C/E/O), to A2 zone in capillary electrophoresis (A2/O) and elutes in C window in HPLC (C/O). 129
  • 130. 130
  • 131. Homozygous Hb O (Hb O disease) a. CBC: shows mild microcytosis and hypochromic ± anemia. b. Smear: shows many target cells and occasionally NRBCs. c. Reticulocytes: slightly elevated. d. Hb separation: dominant Hb O (>95%) normal Hb A2, Hb F occasionally increases, with absence of Hb A. e. Other: i. Decreased osmotic fragility. ii. Genetic testing 131
  • 132. ‫الخالصة‬ ‫الجميع‬ ‫في‬ HbS,G,D,C,E,O If homozygous : ‫حدود‬ ‫في‬ ‫طبيعي‬ ‫الغير‬ ‫الهيموجلوبن‬ ‫يكون‬ 90 % ‫الهيموجلوبين‬ ‫وينعدم‬ ‫أكثر‬ ‫أو‬ A . ‫الهيموجلوبين‬ ‫ويزداد‬ F ‫الهيموجلوبين‬ ‫ويكون‬ ، ‫يزداد‬ ‫ال‬ ‫وقد‬ A2 ‫يزداد‬ ‫وقد‬ ‫طبيعي‬ . If heterozygous : ‫حوالي‬ ‫الطبيعي‬ ‫غير‬ ‫الهيموجلوبين‬ ‫نسبة‬ ‫تكون‬ 40 % ‫وجد‬ ‫لو‬ ‫تقل‬ Alfa thalassemia and IDA HbA more than abnormal Hb normal or slightly increased Hb A2 and or Hb F NB : in hb E Hb E is usually ~ 30%. Hb G is usually ~ 20%. If associated B+ thalassemia (ex SB+) ‫الهيموجلوبين‬ ‫من‬ ‫أكثر‬ ‫يكون‬ ‫طبيعي‬ ‫الغير‬ ‫الهيموجلوبين‬ A If associated B0 thalassemia (ex SB0 ) ‫الهيموجلوبين‬ ‫ينعدم‬ A ‫جدا‬ ‫عالي‬ ‫طبيعي‬ ‫الغير‬ ‫الهيموجلوبين‬ ‫ويكون‬ ‫الهيموجلوبين‬ ‫من‬ ‫كال‬ ‫يزداد‬ ‫كالهما‬ ‫وفي‬ F,A2 132
  • 133. 133
  • 134. 134
  • 135. ‫الكروموسوم‬ ‫إن‬ ‫المشكلة‬ 11 ‫فيه‬ ‫بيحصل‬ deletion ‫يشيل‬ ‫معينة‬ ‫منطقة‬ ‫في‬ B and delta gene and silencer of Gamma gene ‫ال‬ ‫فيظل‬ gamma gene ‫الهيموجلوبين‬ ‫فيظل‬ ‫شغال‬ F ‫ال‬ ‫ويقل‬ ‫عالي‬ HbA and HbA2 May non deletional enhancement of promotor of Gamma gene. Hb F has a higher O2 affinity ➔ relative hypoxia ➔ ↑ EPO ➔ ↑ erythropoiesis. Heterozygous ➡ • Clinical pictures are normal • Normal CBC, blood smear, and reticulocyte • Hb separation : • HbF (15-30%) with normal Hb A2 (occasionally low). 135
  • 136. Homozygous ➡ • Clinical pictures are normal • CBC shows polycythemia and may MHA Hb separation: increased Hb F (100%) with absent Hb A and Hb A2. HPFH /B thalassemia Hb separation: dominant Hb F (~ 70%), increased Hb A2 (>3.5%) with variable levels of Hb A (absent in β0). 136
  • 137. Clinical pictures are normal Heterozygous HPFH : • HbF (15-30%) with normal Hb A2 (occasionally low). Homozygous HPHF increased Hb F (100%) with absent Hb A and Hb A2. HPFH /B thalassemia Hb separation: dominant Hb F (~ 70%), increased Hb A2 (>3.5%) with variable levels of Hb A (absent in β0). 137
  • 138. A variant hemoglobin formed by an unequal crossover and fusion of the beta and delta genes. A single copy of the variant gene causes thalassemia minor. Homozygotes have thalassemia intermedia. 138
  • 139. group of abnormal Hb's that N terminal portion of the δ chain joined to the C- terminal portion of the β chain, due to cross over between the genes for β and δ chains. ‫ينتج‬ ‫عن‬ ‫ذلك‬ ‫بروتين‬ ‫مختلف‬ ‫مشكلته‬ ‫إنه‬ ‫ممكن‬ ‫يظهر‬ ‫في‬ ‫مكان‬ ‫مختلف‬ Hb separation Heterozygotes form about 10% Hb Lepore, dominant HbA normal amounts of Hb A2, and moderately increased amounts of Hb F and usually have mild anemia, microcytosis, and hypochromia; Homozygous form moderate to severe anemia. Hb Lepore 10-20%, absent Hb A and Hb A2 with dominant Hb F Hb lepor migrates to S region in gel electrophoresis (S/G/D/Lepore), to LEPOR zone in capillary electrophoresis and elutes in A2 window in HPLC (A2/E/Lepore). 139
  • 140. 140
  • 141. Hb lepor B lepor / B thalassemia : • Severe anemia. • dominant HbF (> 90%), normal HbA2 ( 2_3%), Hb Lepor around 5% absent HbA • ‫الخالصة‬ • Hb Lepor from 10_20% ➡ it may be homozygous Hb Lepor • Hb Lepor around 10 % ➡ it may be heterozygous Hb Lepor • Hb Lepor around 5 % ➡ it may be B lepor / B thalassemia. 141
  • 142. • Replacement of proximal or distal histidine residue in haem pocket by tyrosine ➡ form iron phenolate complex ➡ stabilize iron in ferric state ➡methemoglobin ➡can't carry oxygen easily ➡May causes cyanosis especially if globin chain is affected. 1_ Increased free radicals and reactive oxygen species ( ROS) ➡ oxidation of ferrous iron to ferric 2_ some drugs as Sulfonamides, nitrites and nitrate 3_ reduced activities of NADH-metHb reductase that reverse normal formed met Hb to normal Hb. 142
  • 143. • Normally, Hb is stable and is protected from oxidation by reducing mechanisms. • In unstable hemoglobin, however, amino acid substitution affecting the stability of Hb molecule ➔ spontaneous or oxidant induced denaturation ➔ precipitation of Heinz bodies ➔ impairment of red cells deformability and shortening of their life span. • >100 variants, the most common are: • o Hb Köln )β98Val → Met, found in German people). • o Hb Hasharon )α47Asp → His, found in Jewish and Italian people). 143
  • 144. • Normally, O2 is unloaded easily at the tissue site. • In high affinity Hb➡ amino acid substitution at O2 loading/unloading site in the globin chain ➔ stabilization of Hb molecule at R sate (relaxed/oxy) configuration ± decreased affinity to 2, 3-BPG ➔ impaired O2 release ➔ functional anemic with compensatory increased erythropoietin ➔ polycythemia. • >100 variants (α and β(. • They are autosomal dominant. Homozygous state is incompatible with life. 144
  • 145. ‫فيه‬ ‫هل‬ ‫بشوفها‬ ‫حاجة‬ ‫أول‬ S region ‫ايه؟‬ ‫وال‬ A_ If there is S : ‫فيه‬ ‫هل‬ ‫نفسي‬ ‫أسأل‬ A ‫موجودة‬ ‫مش‬ ‫وال‬ 1- if there's A If A larger than S ➡ it's AS (Sickle cell trait) If S larger than A ➡ it's SB+ NB➡ In Sickle cell trait the expected HbS representation is 35_45%, but if represented less than 30 % it may AS/ Alfa thalassemia 145
  • 146. 2_ If there isn't A ➡ look at A2 : S band with high A2 ➡ it's SB0 S band and Normal A2 ➡ it's SS 146
  • 147. B_ if there isn't S and there's F : • ‫ال‬ ‫األكثر‬ ‫هل‬ ‫بسأل‬ ‫سؤال‬ ‫أول‬ F ‫أم‬ A ? 1_ if HbF predominate than HbA a _ with HbA ➡ B+ thalassemia major or newborn b _ without HbA ➡ Bo thalassemia major (high A2) homozygous Delta Beta thalassemia ( absent A2 ) homozygous HPHF ( absent A2 ) 147
  • 148. 2_ If HbA is predominate than HbF • ‫يعني‬ HBF ‫بسيطة‬ ‫زيادة‬ ‫زايد‬ . a _ with increased HbA2 ➡ B thalassemia minor( trait ) (3_8%) • ‫زيادة‬ ‫المرض‬ ‫هذا‬ ‫في‬ ‫األصل‬ A2 ‫وليس‬ F !!!!! b_ without increased HbA2 ➡ Heterozygous Delta Beta thalassemia Heterozygous HPHF all causes of acquired increased HbF as aplastic, MDS, and megaloblastic anemia,.... 148
  • 149. B thalassemia intermedia ➡ has MHA Heterozygous HPHF ➡no anemia 149
  • 150. C_ if there isn't S nor F : ‫علي‬ ‫هبص‬ ‫هنا‬ A2 1_ If normal A2 ➡ normal Hb electrophoresis 2_ If increased A2 (3_8%) ➡ May be thalassemia trait ‫من‬ ‫بأكثر‬ ‫يكون‬ ‫التشخيص‬ ‫علشان‬ ‫الزم‬ ‫طبعا‬ 2 technique as HPLC and capillary electrophoresis. 150
  • 151. HbF HbF 0_ 5 and high HbA2 ➡ • Beta thalassemia minor ( trait ) HbF 5_15 ➡ • Heterozygous Delta Beta thalassemia trait HbF 15_40 ➡ • Heterozygous HPFH ( normal CBC) • B thalassemia Intermedia HbF about 70 ➡ • B+ thalassemia major or intermedia (high A2) • B+/HPFH HbF about 96 and HbA2 3_5 ➡ • Bo thalassemia major (high A2) • Bo / HPFH. HbF 100 ➡ • Homozygous HPFH ( almost normal CBC) • Homozygous Delta Beta thalassemia major 151
  • 152. Stress erythropoiesis (causes of reticulocytosis i.e., hemorrhage, hemolysis, nutritional anemia under therapy). Clonal diseases: erythroleukemia, MDS, PV, JMML, PNH and some non-hematological neoplasms. Drugs: hydroxyurea, erythropoietin, Na valproate. Other: ketoacidosis, thyrotoxicosis and pregnancy. 152
  • 153. 153
  • 154. Approach to diagnosis Personal history: • Age: to justify the percentage of Hb variant. • Ethnicity: African, Asian, Mediterranean... Present history: • Asymptomatic, hemolysis, sickling, polycythemia or cyanosis. Past history: 1- Transfusion. 2- Medication e.g., hydroxycarbamide. 3- Operation e.g., splenectomy or cholecystectomy. Family history: • Similar condition in sibling or known parents with inherited Hb disorder. 154
  • 155. 155
  • 156. 156
  • 157. 157
  • 158. Alfa thalassemia oNormal lab in silent carrier, detected by genetic testing. oLow MCV and MCH in silent and alfa thalassemia trait oHBH in HBH disease moderate anemia with marked microcytosis and hypochromia o Hb Bart’s in Hb Bart’s disease oGenetic testing 158
  • 159. ‫الخالصة‬ B thalassemia B thalassemia trait thalassemic red cell indices with high HBA2 more than 3.7 except reasons mentioned above, Hb F (1-7%) B thalassemia major moderate to marked MHA Blood film ‫فراخ‬ ‫نعكشة‬ Sign and symptoms of hemolytic anemia Hb separation: increased Hb F ( 70- 100 ) ± increase in Hb A2. Hb A is absent in β0 159
  • 160. B thalassemia intermedia moderate chronic hemolytic anemia + complications. Hb F from 20 – 40 % ‫االتنين‬ ‫بين‬ ‫وسط‬ ‫مرحلة‬ 160
  • 161. ‫الخالصة‬ Homozygous sickle cell anemia : Moderate to marked NNA hemolytic anemia with symptoms of hemolytic anemia Blood smear shows sickle cell Hb electrophoresis is HbS more than 90 % . HbF slightly increased (5-10%). HbA2 is normal Heterozygous sickle cell trait : HbS around 40 % decreased in alfa thalassemia and IDA , normal or slightly increased Hb A2 and or Hb F SB+ HbS becomes more than HbA , with high HbF and HbA2 SB0 Absent HbA with high HbF and HbA2 Hb A level depends on transfusion. 161
  • 162. ‫الخالصة‬ ‫الجميع‬ ‫في‬ HbS,E,C,D,O,G If homozygous : ‫حدود‬ ‫في‬ ‫طبيعي‬ ‫الغير‬ ‫الهيموجلوبن‬ ‫يكون‬ 90 % ‫الهيموجلوبين‬ ‫وينعدم‬ ‫أكثر‬ ‫أو‬ A . ‫الهيموجلوبين‬ ‫ويزداد‬ F ‫الهيموجلوبين‬ ‫ويكون‬ ، ‫يزداد‬ ‫ال‬ ‫وقد‬ A2 ‫يزداد‬ ‫وقد‬ ‫طبيعي‬ . If heterozygous : ‫حوالي‬ ‫الطبيعي‬ ‫غير‬ ‫الهيموجلوبين‬ ‫نسبة‬ ‫تكون‬ 40 % ‫وجد‬ ‫لو‬ ‫تقل‬ Alfa thalassemia and IDA HbA more than abnormal Hb normal or slightly increased Hb A2 and or Hb F NB : in hb E Hb E is usually ~ 30%. Hb G is usually ~ 20%. If associated B+ thalassemia (SB+) ‫الهيموجلوبين‬ ‫من‬ ‫أكثر‬ ‫يكون‬ ‫طبيعي‬ ‫الغير‬ ‫الهيموجلوبين‬ A If associated B0 thalassemia ( SB0 ) ‫الهيموجلوبين‬ ‫ينعدم‬ A ‫جدا‬ ‫عالي‬ ‫طبيعي‬ ‫الغير‬ ‫الهيموجلوبين‬ ‫ويكون‬ ‫الهيموجلوبين‬ ‫من‬ ‫كال‬ ‫يزداد‬ ‫كالهما‬ ‫وفي‬ F,A2 162
  • 163. Hb lepor • ‫الخالصة‬ • Hb Lepor from 10_20% ➡ it may be homozygous Hb Lepor • Hb Lepor around 10 % ➡ it may be heterozygous Hb Lepor • Hb Lepor around 5 % ➡ it may be B lepor / B thalassemia. 163
  • 164. Clinical pictures are normal Heterozygous HPFH : • HbF (15-30%) with normal Hb A2 (occasionally low). Homozygous HPHF increased Hb F (100%) with absent Hb A and Hb A2. HPFH /B thalassemia Hb separation: dominant Hb F (~ 70%), increased Hb A2 (>3.5%) with variable levels of Hb A (absent in β0). 164
  • 165. HbF HbF 0_ 5 and high HbA2 ➡ • Beta thalassemia minor ( trait ) HbF 5_15 ➡ • Heterozygous Delta Beta thalassemia trait HbF 15_30 ➡ • Heterozygous HPFH ( normal CBC) • B thalassemia Intermedia HbF about 70 ➡ • B+ thalassemia major or intermedia (high A2) • B+/HPFH HbF about 96 and HbA2 3_5 ➡ • Bo thalassemia major (high A2) • Bo / HPFH. HbF 100 ➡ • Homozygous HPFH ( almost normal CBC) • Delta Beta thalassemia major 165
  • 166. 166
  • 167. 167
  • 168. 168
  • 169. 169
  • 170. 170
  • 171. 171
  • 172. 172
  • 173. 173
  • 174. 174
  • 175. 175
  • 176. 176
  • 177. 177
  • 178. 178
  • 179. Types of mutations 1_ small mutation that affect single gene : • A_ point mutation • B_ Deletion • C_ Insertion 2_ Large-scale mutation : It is abnormality in • A_ chromosomal number • B_ chromosomal structure 179
  • 180. Point mutation 1- Silent mutation : as UGU ➡ UGC all coded for Cysteine ‫اتغيرت‬ ‫النيكليوتيد‬ ‫ولكن‬ ‫الحمض‬ ‫األميني‬ ‫كما‬ ،‫هو‬ ‫فيبقي‬ ‫كأنك‬ ‫ياأبوزيد‬ ‫ما‬ ‫غزيت‬ 😎 2- Non-Sense mutation : as UGU coded for cysteine ➡ UGA coded for stop codon result in shorter protein can't function at all ‫اتغيرت‬ ‫النيكليوتيد‬ ‫فتوقف‬ ،‫النسخ‬ ‫فينتج‬ ‫بروتين‬ ‫ناقص‬ ‫متغير‬ ‫تماما‬ ‫ال‬ ‫يعمل‬ . Example: a. CAG in exon 2 normally codes for glutamine. C→T mutation turns it to stop codon (TAG) resulting in β0 thalassemia. b. Non-sense mutation in exon 3 of β gene: leading to severe unstable beta chain (dominant thalassemia phenotype). 180
  • 181. Point mutation 3- Sense mutation : as UGA coded for stop codon ➡ UGU coded for cysteine result in longer protein can't function at all ‫ت‬ ‫ومتغير‬ ‫األصلي‬ ‫من‬ ‫أكبر‬ ‫بروتين‬ ‫فنتج‬ ،‫النسخ‬ ‫يتوقف‬ ‫فلم‬ ‫النيكليوتيد‬ ‫اتغيرت‬ ‫يعمل‬ ‫وال‬ ‫ماما‬ . Examples: b. Hb Constant Spring: α chain of 172 amino acids instead of 141 (excess 31 amino acids). 4- Missense mutation : as UGU coded for cysteine ➡ UAU coded for Tyrosine, it may be : ‫علي‬ ‫سيؤثر‬ ‫التغيير‬ ‫هذا‬ ‫هل‬ ‫لكن‬ ،‫األميني‬ ‫الحمض‬ ‫معها‬ ‫وتغير‬ ‫النيكليوتيد‬ ‫اتغيرت‬ ‫هنا‬ ‫الحمض‬ ‫األميني؟‬ ⬅ 181
  • 182. Missense mutations 1 - No apparent Missense mutation : ‫بال‬ ‫فقط‬ ‫ونعرفه‬ ‫تغيير‬ ‫أي‬ ‫الوظيفة‬ ‫في‬ ‫يحدث‬ ‫لم‬ ‫ولكن‬ ،‫متغير‬ ‫بروتين‬ ‫ونتج‬ ‫النيكلوتيدة‬ ‫تغيرت‬ ‫لو‬ DNA sequencing as Hb-Milwaukee and Hb-Bristol 2 - Acceptable Missense mutation : ‫يالحظ‬ ‫ال‬ ‫الوظيفة‬ ‫في‬ ‫مقبول‬ ‫طفيف‬ ‫تغير‬ ‫وحدث‬ ‫متغير‬ ‫بروتين‬ ‫ونتج‬ ‫النيكلوتيدة‬ ‫تغيرت‬ ‫لو‬ . As Hb Hikari 3- Partially Acceptable Missense mutation : ‫بقصو‬ ‫لكن‬ ‫يعمل‬ ‫البروتين‬ ‫زال‬ ‫ما‬ ‫لكن‬ ،‫معروف‬ ‫مرض‬ ‫هذا‬ ‫عن‬ ‫ونتج‬ ‫الوظيفة‬ ‫في‬ ‫شديد‬ ‫تغير‬ ‫وحدث‬ ‫متغير‬ ‫بروتين‬ ‫ونتج‬ ‫النيكلوتيدة‬ ‫تغيرت‬ ‫لو‬ ‫ف‬ ‫كما‬ ‫ر‬ ‫ي‬ HBS 4- Unacceptable Missense mutation : ‫فق‬ ‫البروتين‬ ‫لكن‬ ،‫معروف‬ ‫مرض‬ ‫هذا‬ ‫عن‬ ‫ونتج‬ ،‫الوظيفة‬ ‫في‬ ‫شديد‬ ‫تغير‬ ‫وحدث‬ ‫متغير‬ ‫بروتين‬ ‫ونتج‬ ‫النيكلوتيدة‬ ‫تغيرت‬ ‫لو‬ ‫تماما‬ ‫العمل‬ ‫علي‬ ‫القدرة‬ ‫د‬ ‫في‬ ‫كما‬ HBM 182
  • 183. Insertion or deletion • ‫من‬ ‫المعلوم‬ ‫أن‬ ‫كل‬ ‫ثالثة‬ ‫نيوكليوتيدات‬ ‫علي‬ ‫ال‬ mRNA ‫تمثل‬ ‫بحمض‬ ‫أميني‬ ،‫واحد‬ • ‫لو‬ ‫أضفت‬ ‫أو‬ ‫حذفت‬ ‫ثالثة‬ ‫نيكلوتيدات‬ ‫أو‬ ‫مضاعفتها‬ ‫هنا‬ ‫كل‬ ‫اللي‬ ‫هيحصل‬ ‫إضافة‬ ‫أو‬ ‫حذف‬ ‫حمض‬ ‫أميني‬ ‫واحد‬ ‫أو‬ ‫أكثر‬ ‫وسيحدث‬ Non-Frame shift mutation. • ‫أما‬ ‫لو‬ ‫أضفت‬ ‫أو‬ ‫حذفت‬ ‫غير‬ ‫الثالثة‬ ‫نيكلوتيدات‬ ‫ومضاعفتها‬ ‫ك‬ 1 ' 2 ' 4 ' 5 ' ... ‫هيحصل‬ ‫تغيير‬ ‫لكل‬ ‫البروتين‬ ‫ما‬ ‫بعد‬ ‫الطفرة‬ ‫وسيتغير‬ ‫البروتين‬ ‫تماما‬ ‫ويحدث‬ Frame shift mutation 183
  • 184. small mutation that affect single gene 184
  • 185. 185
  • 186. 186
  • 187. 187
  • 188. 188
  • 189. 189
  • 191. 2_ Large-scale mutation •A_ chromosomal number •B_ chromosomal structure It's abnormality in 191
  • 192. A_ abnormality in chromosomal number • It's due to errors in gametes formation aneuploidy Polyploidy 192
  • 193. Aneuploidy for example • 1_ Turner syndrome ➡ 45,X or 45,X0). • 2_ Klinefelter syndrome ➡ 47,XXY • 3_Edwards S➡trisomy of chromosome 18. • 4_ Down S ➡ trisomy of chromosome 21. • 5_ Patau S➡ trisomy of chromosome 13. • 6_ Trisomy 9. 193
  • 194. Down Syndrome Turner syndrome 194
  • 195. Polyploidy 1_ Triploidy : 3 set of chromosome (3n) 69 chromosomes 2_ Tetraploidy : 4 set of chromosomes (4n) 92 chromosomes It is non compatible with life, if occurs in cells the cell is abnormal. 195
  • 197. B_ Abnormality in chromosomal structure It's due to errors in gametes formation or mutagenes ( physical, biological). •for example : 1_ Deletion ➡ as Cri du chat deletion syndrome, 1P36 2_ Duplication 3_ Inversion ➡ inv (3) ( q21;q34) 4_ translocation➡ Philadelphia Chromosome in CML 197
  • 198. Abnormality in chromosomal structure 198
  • 200. 200
  • 201. Molecular application in hemoglobenopathy The most important application of DNA analysis molecular techniques are : 1_ The conformation of the diagnosis of Alfa thalassemia trait, particularly for genetic counseling 2_ The prenatal diagnosis of serious disorders of globin chain in the fetus as HbS 201
  • 202. Techniques PCR Sequencing Southern blot analysis GAP PCR PCR with allele specific primer ( ARMS ) Reverse dot blot sequencing RFLP linkage analysis Denaturing gradient gel electrophoresis ( DGGE ) mass spectrometer 202
  • 203. Lecture titles 1_ Hb separation 2_ cellulose acetate electrophoresis 3_ High Performance Liquid Chromatography (HPLC) 4_ Capillary electrophoresis 5_ interference in Hb separation 6_ Hb variants 7_ Structural abnormal hemoglobinopathy as HbS,C,E,O,… 8_ Insufficient of hemoglobin synthesis as Alfa & Beta thalassemia 9_ 0ther hemoglobinopathy as HbM, unstable Hb, High affinity Hb, HPFH, Hb lepor 10_ Hb variants interpretation & Interferance 11_ Detail & Summary of Hb variants 12_ cases 13_ Genetics & Molecular 203
  • 204. REFERENCES • _ https://labtestsonline.org • _ https://www.medscape.com • _ https://www.wikipedia.org • _ Haemoglobinopathy Diagnosis Barbara J. Bain , Third Edition 2020 • _ Lecture Notes Clinical Biochemistry, 9th Edition Walker, Simon, 2103. • _ https ps://www.labcorp.com • _ https://www.uptodate.com • _ https://www.ncbi.nlm.nih.gov Home - PubMed – NCBI • _TIETZ textbook of clinical chemistry and molecular diagnostics, sixth edition 2018. • _Essential of clinical pathology book; 1st edition; Shirish M Kawthalkar; 2010. • _ Harper's illustrated biochemistry 30th edition 2015. • _ Lippincott's illustrated review of biochemistry sixth edition 2014. • _Many audios and videos from Well-known, trusted professors who study from accredited books. 204
  • 205. ‫اليوتيوب‬ ‫علي‬ ‫البحث‬ ‫أو‬ ،‫اليوتيوب‬ ‫علي‬ ‫قناتي‬ ‫دي‬ ‫د‬ ‫باسم‬ . ‫سفيان‬ ‫بهجت‬ ‫محمد‬ ، ‫هللا‬ ‫شاء‬ ‫إن‬ ‫نستمر‬ ‫حتى‬ ‫بالقناة‬ ‫وتشتركوا‬ ‫تتابعوني‬ ‫ياريت‬ https://www.youtube.com/channel/UCaYs1d8s0ntZvteHS3mMmGA ‫ب‬ ‫الفيس‬ ‫في‬ ‫البحث‬ ‫أو‬ ،‫انضمامكم‬ ‫يشرفنا‬ ‫الفيسبوك‬ ‫علي‬ ‫بتاعتي‬ ‫الصفحة‬ ‫دي‬ DMBMS2018 ، ‫هللا‬ ‫شاء‬ ‫إن‬ ‫نستمر‬ ‫حتى‬ ‫تتابعوني‬ ‫ياريت‬ https://www.facebook.com/DMBMS2018/ ‫باسم‬ ‫التلجرام‬ ‫علي‬ ‫قناتي‬ ‫ودي‬ ِ ‫للناس‬ ‫أنفعهم‬ ‫الناس‬ ‫خير‬ 📚 ‫هللا‬ ‫شاء‬ ‫إن‬ ‫نستمر‬ ‫حتى‬ ‫تتابعوني‬ ‫ِريت‬‫ا‬‫ي‬ https://t.me/DMBMS2020 ‫على‬ ‫بروفايلي‬ ‫لينكيدإن‬ ‫وتعملولي‬ ‫تتابعوني‬ ‫ياريت‬ endorsement ‫مهاراتي‬ ‫على‬ https://www.linkedin.com/in/mohammed-bahgat-sofyan-8ba012142/ ‫موقع‬ ‫على‬ ‫بروفايلي‬ ‫وده‬ SlideShare ‫تتابعونا‬ ‫ياريت‬ https://www.slideshare.net/MohammedBahgatMohamm1 ‫وده‬ ‫بروفايلي‬ ‫الشخصي‬ ‫الفيسبوك‬ ‫علي‬ https://www.facebook.com/mohammed.bahgat.165 ‫الفيسبوك‬ ‫على‬ ‫بنا‬ ‫الخاص‬ ‫اللوجوا‬ ‫وده‬ # ‫دمحمد‬ _ ‫بهجت‬ _ ‫سفيان‬ _ medical_biochimestry 205
  • 207. THANK YOU Meet on the best later, God willing 207