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  1. 1. Tinjauan Pustaka Hematologi<br />ASPEK LABORATORIS ANEMIA MEGALOBLASTIK<br />dr. Sri Kartika Sari/dr. Arifoel Hajat, SpPK<br />Rabu, 16 Desember 2009<br />
  2. 2. Pendahuluan<br />Gangguan sintesis DNA<br />Anemia megaloblastik<br />Sel megaloblastik<br />Ketidaksinkronan maturasi inti<br />2<br />
  3. 3. Penyebab anemia megaloblastik<br />3<br />
  4. 4. Vitamin B₁₂<br />Biokimia vitamin B₁₂<br />Methylcobalamin terbanyak dalam<br /> serum<br />Protein transport : transcobalamin (TC)<br />TC I, II dan III<br />4<br />
  5. 5. P<br />TISSUE<br />Protein source<br />Vitamin B₁₂<br />Oral intake<br />Celullar uptake<br />TC receptor<br />STOMACH<br />Parietal cell<br />Pancreatic protease<br />OH-Cbl<br />CN-Cbl<br />IF-binding<br />LIVER<br />IF-Cbl<br />Holo-TC II<br />Cubilin<br />TC II<br />Intestinal uptake<br />storage<br />5<br />Gbr. Absorbsi, penyimpanan dan transportasi Vitamin B₁₂ <br />
  6. 6. Folat<br />Biokimia folat<br />Terlibat dalam fungsi metabolisme penting :<br /><ul><li>Serine/glicine
  7. 7. Thymidilate
  8. 8. Histidine
  9. 9. Methionine
  10. 10. Purine</li></ul>Methyltetrahydrofolate<br /> terbanyak di serum<br />6<br />
  11. 11. Metabolisme folat<br />DNA<br />dATP<br />dGTP<br />dTTP<br />dCTP<br />dTDP<br />DHF polyglutamate<br />THF polyglutamate<br />dTMP<br />5,10 methylene THF polyglutamate<br />THF<br />dUMP<br />methionine<br />homocysteine<br />Membran sel<br />Methyl THF<br />Methyl THF<br />plasma<br />Folat dari makanan<br />Usus halus<br />7<br />
  12. 12. 8<br />Gbr. Konversi Methylmalonil-coA<br /> dan sintesis methionine<br />
  13. 13. Defisiensi vitamin B₁₂<br />9<br />
  14. 14. Intake kurang<br />Kebutuhan <br />malabsorbsi<br />Defisiensi folat<br />Gangguan metabolik<br />kehilangan<br />10<br />
  15. 15. Gambaran klinis<br />Anemia berjalan lambat, memburuk perlahan<br />Inefektif eritropoiesis<br />Kelainan epitel<br />Gejala defisiensi vitamin B₁₂ :<br />Neuropati progresif gangguan metilasi mielin<br />Gejala pada ibu hamil defek tabung saraf pada janin<br />Peningkatan homocysteine miokard infark, vascular disease, trombosis vena.<br />Kemandulan<br />11<br />
  16. 16. Pemeriksaan laboratorium<br />MCV<br />Hapusan Darah Tepi<br />Vitamin B₁₂ serum<br />Folat serum dan eritrosit<br />Methylmalonic acid (MMA) dan homocysteine<br />Antibodi antiparietal cell dan anti IF<br />Tes Schilling<br />12<br />
  17. 17. Mean Corpuscular Volume (MCV)<br />MCV > 100 fL<br />Penyebab makrositosis :<br />Megaloblastik atau non megaloblastik<br />MCV meningkat sebelum terjadi penurunan kadar Hb.<br />Bila defisiensi vitamin B₁₂ bersamaan dengan defisiensi besi MCV normal<br />MCV kurang spesifik<br />13<br />
  18. 18. Oval makrosit kesan penyakit megaloblastik<br />Stomatosit dengan makrosit alkoholism<br />Hipersegmentasi sensitif dan spesifik untuk anemia megaloblastik<br />Hapusan darah tepi<br />14<br />
  19. 19. Vitamin B₁₂ serum<br />Digunakan untuk diagnosis dan monitoring terapi defisiensi vitamin B₁₂<br />Batas bawah normal biasanya sekitar 148 pmol/L (200 pg/mL)<br />Belum ada baku emas  sensitivitas dan spesifisitasnya bervariasi lebar.<br />Metode pemeriksaan : automated non-isotopic procedure menggunakan chemiluminescence<br />15<br />
  20. 20. Lanjutan vitamin B₁₂ serum....<br />Pada gagal ginjal vitamin B₁₂ serum  tinggi<br />Defisiensi vitamin B₁₂ pada penderita penyakit mieloproliferatif, gangguan ginjal/liver  kadar vitamin B₁₂ bisa normal/tinggi<br />Kolonisasi bakteri usus  tinggi palsu<br />16<br />
  21. 21. Folat serum dan folat eritrosit<br />Batas bawah normal folat serum : 6,8 nmol/L<br />Kadar folat serum  dipengaruhi diet.<br />Kadar folat eritrosit  lebih mencerminkan simpanan folat jaringan.<br />Belum ada metode baku emas<br />17<br />
  22. 22. Methylmalonic acid (MMA) dan homocysteine<br />Defisiensi vitamin B₁₂  MMA dan homocysteine <br />Defisiensi folat  homocysteine<br />MMA dan homocysteine  sensitif untuk diagnosis defisiensi vitamin B₁₂<br />Hati-hati interpretasi peningkatan metabolit bila tanda-tanda defisiensi (-)<br />Setelah 7-14 hari pemberian terapi  kadar metabolit menjadi normal<br />18<br />
  23. 23. 19<br />
  24. 24. Antibodi anti sel parietal dan anti Intrinsic Factor (IF)<br />Antibodi anti sel parietal :<br />Non spesifik<br />Terdapat pada penderita autoimun, orang sehat<br />Antibodi anti IF :<br />Tidak sensitif, namun cukup spesifik, walaupun masih mungkin ada pada penderita Graves disease.<br />20<br />
  25. 25. Tes Schilling<br />Untuk konfirmasi gangguan absorbsi vitamin<br />Terdiri dari 2 tahap :<br />21<br />
  26. 26. Interpretasi hasil tes Schilling pada defisiensi vitamin B₁₂<br />Tahap I : N (≥8%)<br />Tahap I : Abn (<8%);<br />Tahap II : N (≥8%)<br />Tahap I dan II : Abn (<8%)<br />Kemungkinan :<br /><ul><li> Defisiensi diet
  27. 27. Malabsorbsi </li></ul> kompleks protein-<br /> Cbl :<br /><ul><li>Hipokhlorhidria
  28. 28. Partial </li></ul> gastrectomy<br /><ul><li> Kongenital </li></ul> defisiensi TC II<br />Kemungkinanan :<br /><ul><li> Ileal disease
  29. 29. Anemia pernisiosa dgn</li></ul> disfungsi ileal sekunder<br /><ul><li> Anemia pernisiosa dgn </li></ul> pengumpulan urine tidak <br /> adequat<br /><ul><li> Renal insufisiensi
  30. 30. Pengumpulan urine tidak</li></ul> adequat<br /><ul><li> Bacterial overgrowth </li></ul> syndromes<br /><ul><li> Fish tapeworm infestation
  31. 31. Insufisiensi pankreas</li></ul>Kemungkinan :<br /><ul><li> Anemia pernisiosa
  32. 32. Gastrectomy
  33. 33. Disfungsi/ IF tidak</li></ul> ada (kongenital)<br /><ul><li> Pengumpulan </li></ul> urine tahap I <br /> tidak adequat<br />22<br />
  34. 34. Pendekatan diagnosis penderita dengan kelainan hematologi<br />Vitamin B₁₂<br />< 74 pmol/L<br />>221 pmol/L<br />74-221 pmol/L<br />Folat <br />Folat N<br />Folat N<br />Folat <br />Folat <br />Folat N<br />Defisiensi vitamin B₁₂<br />Defisiensi vitamin B₁₂ dan folat<br />Bukan Defisiensi vitamin B₁₂ dan folat<br />Def. folat<br />Defisiensi vitamin B₁₂<br />Tx. folat<br />Indeterminate<br />Gbr.7<br />Tes Schilling<br />Penyebab lain<br />Gbr.7<br />Tes Schilling<br />23<br />
  35. 35. 24<br />Gbr. 7. Pendekatan penderita dengan vitamin B₁₂ < 74 pmol/L<br />
  36. 36. Tabel 6. Evaluasi penderita dengan kelainan hematologi dan vitamin B₁₂ <74 pmol/L<br />25<br />
  37. 37. Gbr. 8. Evaluasi penderita yang diduga defisiensi vitamin B₁₂ dengan CBC normal<br />26<br />
  38. 38. Diagnosis defisiensi vitamin B₁₂ dan folat<br />hal yang kompleks.<br />Belum ada tes yang mudah sebagai baku emasnya<br />Perlu integrasi hasil laboratorium, klinis, respon terapi<br />27<br />
  39. 39. TERIMA KASIH<br />28<br />
  40. 40. 29<br />Gambar 6. Prinsip Beckman ACCESS Immunoassay System<br /> untuk pengukuran Vitamin B12. Serum.5<br />
  41. 41. 30<br />Treatment<br />B12 can be supplemented in healthy subjects by oral pill; sublingual pill, liquid, or strip; intranasal spray; or by injection. B12 is available singly or in combination with other supplements. B12 supplements are available in forms including cyanocobalamin, hydroxocobalamin, methylcobalamin, and adenosylcobalamin (sometimes called "cobamamide" or "dibencozide"). Oral treatments involve giving 250 ug to 1 mg of B12 daily.[28]<br />Vitamin B12 can be given as intramuscular injections of hydroxycobalamin, methylcobalamin, or cyanocobalamin. Body stores (in the liver) are refilled with half a dozen injections in the first couple of weeks and then maintenance with monthly to quarterly injections throughout the life of the patient.<br />B12 has traditionally been given parenterally to ensure absorption. However, oral replacement is now an accepted route, as it has become increasingly appreciated that sufficient quantities of B12 are absorbed when large doses are given. This absorption does not rely on the presence of intrinsic factor or an intact ileum. Generally 1 to 2 mg daily is required as a large dose [3]. By contrast, the typical Western diet contains 5–7 µg of B12 (Food and Drug Administration (FDA) Daily Value [29]).<br />
  42. 42. 31<br />
  43. 43. 32<br />
  44. 44. 33<br />Hematological findings<br />The blood film can point towards vitamin deficiency:<br />Decreased red blood cell (RBC) count and hemoglobin levels[citation needed]<br />Increased mean corpuscular volume (MCV, >95 fl) and mean corpuscular hemoglobin (MCH)<br />Normal mean corpuscular hemoglobin concentration (MCHC, 32-36 g/dL)<br />The reticulocyte count is decreased due to destruction of fragile and abnormal megaloblastic erythroid precursor.<br />The platelet count may be reduced.[citation needed]<br />Neutrophil granulocytes may show multisegmented nuclei ("senile neutrophil"). This is thought to be due to decreased production and a compensatory prolonged lifespan for circulating neutrophils, which increase numbers of nuclear segments with age.[citation needed]<br />Anisocytosis (increased variation in RBC size) and poikilocytosis (abnormally shaped RBCs).<br />Macrocytes (larger than normal RBCs) are present.<br />Ovalocytes (oval-shaped RBCs) are present.<br />Howell-Jolly bodies (chromosomal remnant) also present.<br />Blood chemistries will also show:<br />In increased lactic acid dehydrogenase (LDH) level. The isozyme is LDH-2 which is typical of the serum and hematopoetic cells.<br />Increased homocysteine and methylmalonic acid in B12 deficiency<br />Increased homocysteine in folat<br />
  45. 45.
  46. 46. 35<br />
  47. 47. 36<br />
  48. 48. 37<br />
  49. 49. Consequences of impaired folate status or metabolism<br />Metabolic Disruption<br />Biochemical Markers<br />Clinical Associations<br />CancerCVDdemyelinationNTDs<br />S-adenosylmethionine (MTHFR, MS, B12 deficiency)<br />Thymidylate<br />Purines (A,G)<br />Hypomethylated DNA<br />Elevated homocysteine<br />Reduced methylation<br />CancerNTDsAnemia<br />Increased uracil in DNADecreased DNA synthesis & reduced cell division<br />Decreased DNA synthesis & reduced cell division<br />Anemia<br />
  50. 50. 39<br />
  51. 51. 40<br />5 jenis sel kelenjar lambung:<br /><ul><li> sel chief : enzim </li></ul> pepsin dan lipase<br /><ul><li> sel parietal (oxyntic) :</li></ul> asam lambung dan IF<br /><ul><li> sel leher mukosa :</li></ul> mukus<br /><ul><li> sel stem
  52. 52. sel neuroendokrin :</li></ul> gastrin <br />
  53. 53. 41<br />Vitamin B12 Analogues<br />Vitamin B12 is a coenzyme: it is needed for enzymes to do their job of changing one molecule into another. As vitamins go, B12 is large. One part of its structure is known as the corrin nucleus, which holds an atom of cobalt. The corrin resembles the heme of hemoglobin which holds an atom of iron. Any molecule that contains a corrin nucleus is considered a corrinoid. <br />The corrin plus other atoms make up the cobalamin part of B12. There are many different cobalamins and they are named after their attachments. For example, methylcobalamin is cobalamin with a methyl group (one carbon and three hydrogens) attached. <br />All corrinoids (including all cobalamins) are considered B12 analogues. Many corrinoids, and possibly even some cobalamins, are not useable by human B12 enzymes. These are considered inactive B12 analogues.<br />
  54. 54. Vitamin B12 Deficiency<br />homocysteine and methylmalonyl CoA<br />Increase in methylmalonyl CoA<br />Increased enzyme activity in fatty acid synthesis<br />Build up of odd fatty acids around peripheral nerves<br />Increase in homocysteine<br />Vascular/nervous problems<br />
  55. 55. Transcobalamin I<br /><ul><li>R-type binding protein
  56. 56. 33% is carbohydrate
  57. 57. Molecular weight = 125,000-150,000
  58. 58. Beta globulin
  59. 59. Contains more sialic acid than transcobalamin III
  60. 60. Carries ~80% of Vitamin B12 in blood
  61. 61. Vitamin B12 has half-life of 10-12 days when bound to it</li></li></ul><li>Transcobalamin II<br /><ul><li>Molecular weight = 38,000
  62. 62. Alpha globulin
  63. 63. NOT a glycoprotein
  64. 64. Carries less than 25% of Vitamin B12 in blood
  65. 65. Vitamin B12 has half-life of under 1 ½ hours when bound to it
  66. 66. Encourages absorption in a number of tissues
  67. 67. Degenerates once B12 is released
  68. 68. B12 then recirculates
  69. 69. Transcobalamin II deficiency results in pernicious anemia</li></li></ul><li>Antibacterial Roles of Transcobalamin I & III<br /><ul><li>Binds to large amounts of vitamin B12 and carries it to liver
  70. 70. Excreted in bile
  71. 71. Prevents bacteria from using the vitamin for growth</li></li></ul><li>
  72. 72. 47<br />
  73. 73. 48<br />
  74. 74. 49<br />Reference Values Folat serum : > or = 3.5 ug/L <br />
  75. 75. 50<br />
  76. 76. 51<br />
  77. 77. 52<br />
  78. 78. 53<br />
  79. 79. 54<br />
  80. 80. 55<br />
  81. 81. 56<br />
  82. 82. 57<br />Methylmalonic acid (MMA) is a dicarboxylic acid that is a C-methylated derivative of malonate.<br />Pathology<br />Increased methylmalonic acid levels may indicate a vitamin B12 deficiency. However, it is sensitive without being specific. MMA is elevated in 90-98% of patients with B12 deficiency. This test may be overly sensitive, as 25-20% of patients over the age of 70 have elevated levels of MMA, but 25-33% of them do not have B12 deficiency. For this reason, MMA is not routinely recommended in the elderly. [1]<br />An excess is associated with methylmalonicacidemia.<br />MMA concentrations in blood are measured by Gas chromatographicMass spectrometry<br />
  83. 83. 58<br />Folic acid and vitamin B12Large amounts of folic acid can mask the damaging effects of vitamin B12 deficiency by correcting the megaloblastic anemia caused by vitamin B12 deficiency [3,5] without correcting the neurological damage that also occurs [1,31]. Moreover, preliminary evidence suggests that high serum folate levels might not only mask vitamin B12 deficiency, but could also exacerbate the anemia and worsen the cognitive symptoms associated with vitamin B12 deficiency [6,11]. Permanent nerve damage can occur if vitamin B12 deficiency is not treated. For these reasons, folic acid intake from fortified food and supplements should not exceed 1,000 mcg daily in healthy individuals [5].<br />
  84. 84. Diet<br />Methyltetrahydrofolate<br />Vitamin B12<br />Methionine<br />Methyl B12<br />Homocysteine<br />CH3<br />tetrahydrofolate<br />Serine <br />Dihydrofolate<br />Purine and pyrimidine synthesis<br />+ B6<br />DNA<br />Glycine<br />Thymidylate<br />Deoxyuridylate<br />5,10 methylenetetrahydrofolate<br />Gambar 4. Jalur metabolik asam folat dan vitamin B12.2.<br />59<br />
  85. 85. Symptoms/Effects of Vitamin B12 Deficiency<br /><ul><li>Pernicious Anemia (Vitamin B12 is necessary for RBC production)
  86. 86. Lethargy
  87. 87. Weight loss
  88. 88. Weakness
  89. 89. Dementia
  90. 90. Leucopenia
  91. 91. Thrombocytopenia
  92. 92. Axonal degeneration
  93. 93. Demyelination
  94. 94. Urethral Sphincter problems
  95. 95. Depression
  96. 96. Alzheimer’s Disease
  97. 97. Increased liver weight
  98. 98. Fat accumulation around heart, liver, peripheral nerves</li></li></ul><li>Symptoms/Effects of Vitamin B12 Deficiency<br /><ul><li>Increase
  99. 99. Homocysteine
  100. 100. MMA
  101. 101. Bilirubin excretion
  102. 102. LDH
  103. 103. Liver glycogen
  104. 104. Mitochondrial cristae in liver
  105. 105. Hepatic citrate synthase
  106. 106. Propionic acid
  107. 107. Succinate dehydrogenase
  108. 108. Cytochrome c activity
  109. 109. Propionyl CoA
  110. 110. Amino Acids
  111. 111. Cell metabolism
  112. 112. Protein synthesis
  113. 113. Fatty acid synthesis enzymes
  114. 114. ATP citrate lyase
  115. 115. Causes rise in Kreb’s cycle
  116. 116. Decrease
  117. 117. Transcobalamin II
  118. 118. Intrinsic factor</li></li></ul><li>62<br />
  119. 119. 63<br />The present invention relates to a new method named the COBASORB test, which can be used for testing the cause of cobalamin malabsorption in humans. The COBASORB test contains three separate tests (first, second and third test) than can be performed separately, sequentially or in random order and number. The first test use non-radioactive cobalamin for ingestion, the second test uses non-radioactive cobalamin and recombinant intrinsic factor for ingestion and the third test uses recombinant haptocorrin saturated with cobalamin for ingestion. All three tests involve analysis of changes in the concentration of cobalamin saturated transcobalamin (holo-TC) and cobalamin saturated haptocorrin (holo-HC) in the blood. Also disclosed are fits suitable for use in these methods.<br />
  120. 120. 64<br />Salivary haptocorrin, also known as the R-protein, binds strongly to Vitamin B12 (after it is released from food by gastric pepsin), stabilizing it and preventing its breakdown in the low-pH environment of the stomach. The complex is absorbed by ilealvilli into the blood. HC accounts for 10-40% of B12 serum level.<br />