Megaloblastic anemias are disorders caused by impaired DNA synthesis due to the substances of DNA synthesis deficiency, such as folic acid and Vit B12,etc. It is characterized by the presence of megaloblastic cells in the bone marrow and macrocytic anemia.

Definition

Classification of MA

1.Vitamin B12 deficiency

2.Folate deficiency

3.Penicious anemia

The important role of folate and Vitamin B12 pl

aying in the pathways of deoxynucleotide and DNA synthesis:

reductase

purine synthesis

pyrimidine synthesis

thymidylate synthetase(N5,N10-methylene FH4)

UTP(UR PPP ) → dUTP(UdRPPP)

CTP(CR PPP) → dCTP(CdR PPP)

ATP(AR PPP ) → dATP(AdR PPP)

GTP(GR PPP) → dGTP(GdR PPP)

Ribonucleotide

dUMP

D NA

dTMP dTTP

1. De novo synthesis of purine:

C

N

N C

C

C C

N

N

1

2

7

8

N10-formyl FH4

FH4

2 . Thymidylate synthesis

FH2 reductase

Glycine

N5,N10-methylene FH4

thymidylate synthetase

FH2

FH4

d UMP d TMP

Serine

ATP

FH4 methionine PPi+Pi

N5-methyl FH4 S-adenosyl methionine

homocysteine RH

adenosyl

R-CH3

H2O S-adenosyl homocysteine

N5-methyl FH4 methyltransferase (Vit B12)

3. Methionion synthesis

Trap

dUMP dTMP dTTP DNA 正常合成

亚甲基四氢叶酸 二氢叶酸

四氢叶酸二氢叶酸还原酶

叶酸缺乏

5- 甲基四氢叶酸高半胱氨酸

甲硫蛋氨酸Vit B12 缺乏

叶酸和 Vit B12 缺乏对 DNA 合成的影响

dUTP 合成异常DNA

巨幼变细胞

Diet

Methyltetrahydrofolate

Tetrahydrofolate

Dihydrofolate

N5,N10-methylene FH4

VitaminB12

methylB12 CH3

Methionine

Homocysteine

Thymidylate DNA

Deoxyuridylate

purine and pyrimidine synthesis

Serine

B6

glycine

Megaloblastic Anemias

Morphologically and functionally change

Megaloblastic cells

Defective DNA synthesis

Different disorders

Deficiency of folate and

Vit B12,etc. Non-deficiency

Ineffective erythropoiesis

Pathogenesis of MgA Etiology of MgA

单谷氨酸叶酸

Enterohepatic cycle

叶酸的吸收、利用、贮存和排泄

γ- 谷氨酰胺羧基肽酶

甲基 // 四氢叶酸

DNA 合成蛋氨酸循环

+Vit B12

tissue

FBP 叶酸结合蛋白

blood

Excretion

urine

Food folate (daily needs 200μg)

多聚谷氨酸叶酸 Folate stores (5-20mg)

TFH- 多谷氨酸盐liver

绿叶蔬菜、柠檬、香蕉、瓜类、香菇、酵母及动物内脏（尤其是肝脏）等都含大量叶酸。

1.The causes of folic acid deficiency

I inadequate intake(diet)

II intestinal malabsorption

III increased demand

IV inability to utilize folate due to action of folate antagonists

Food Vit B12 (daily needs 2 ～ 5μg)Vit B12 stores

(4 ～ 5mg) liver

Vit B12 的吸收、利用、贮存和排泄

TcII-B12

tissue

+ 甲基叶酸

蛋氨酸循环

Vit B12

Enterohepatic cycle

Vit B12 –IF

IF Vit B12

–R

蛋白

blood

释放内因子Excretionurinefaecestearsalivamilk

动物的肝、肾、肉类、禽蛋、乳类和海洋生物等 B12 含量较多。

2. The causes of Vitamin B12 deficiency

I. inadequate intake

II. intestinal malabsorption

III. vitamin B12 destruction

IV. congenital defects

LaboratoryFeatures Clinical Features

1. Common manifestations

• Anemia develops slowly, severe anemia with

weakness, palpitation, fatigue, light-headedness,

and shortness of breath.

• Slight jaundice

• Glossitis

Clinical Features

2. Different features caused by different disorders

Vit B12 deficiency:

•peripheral neuropathy and subacute combined degeneration of the spinal cord with nervous system symptoms such as numbness and tingling of the extremities, loss of position sense, muscle weakness and decreased tendon reflexes.

Folate deficiency :

not produce nervous system manifestations.

Pernicious anemia

• abdominal pain, diarrhea, nausea, and vomiting

• nervous system symptoms.

甲基丙二酰辅酶A

琥珀酰辅酶 A

腺苷钴胺ATP+VitB12 adenosylcobalamin

Methylmalonyl CoA Succinyl CoA

合成神经鞘磷脂代谢产物堆积，血、尿可测

Blood

•macrocytic anemia, with MCV↑(100~150fl or more).

•But coexisting IDA, Thalassemia trait, or inflammation may prevent macrocytosis.

•Erythrocytes show marked anisocytosis and poikilocytosis, with many oval macrocytes and in severe cases, basophilic stippling, Howell-Jolly bodies, and Cabot rings

•Megaloblastic normoblasts may be seen.

•Ret count is low.

Laboratory Features

•Leukopenia and thrombocytopenia are frequently present.

•“Hypersegmented neutrophils” (more than five lobes) are an early sign of megaloblastosis.

--Platelets:smaller and vary more widely in size.(PDW increase)

Bone Marrow:

--Moderate or marked hypercellularity.

-- M/E ratio ↓

--Erythroid hyperplasia with striking megaloblasts (more than 10%). Promegaloblasts and early megalonormoblasts increase with mitotic figures abundant in severe cases.

•basophilic stippling, Howell-Jolly bodies, and Cabot rings may be seen.

The characteristics of megaloblasts:

1. Cells and nuclei are larger in size and the chromatin is more looser.

2. Marked nuclear-cytoplasmic asynchrony.

The development of the nucleus is behind that of cytoplasm, called “young nucleus and old cytoplasm”

Impaired synthesis of one or more deoxyribonucleotides

DNA replication and cell division are blocked,while synthesis of cytoplasm(RNA and protein) proceeds normally.

RNA/DNA ratio rises and cytoplasm is basophilic

Chramatin is looser

Cells are larger in size

Pathogenesis of MgA

pronormoblast

megalopronormoblast

Early normablast

Polychromatic megalonormoblast

Normoblasts and megalonormoblast

Orthochromatic normoblast

Megaloblastic changes 核畸形、多核、核碎裂的巨幼红细胞

Howell-Jolley bodies

Basophilic stippling normoblast

The megaloblastic myeloid cells may be larger in size, deforming nucleus with looser chromatin; specific granules reduce and vacuoles appear in cytoplasm. Hpersegmented neutrophils (also be seen in the peripheral blood) and giant metamyelocytes and bands present.

Myeloid megaloblastic changes

Megakaryocytes are usually present either in normal or slightly increased numbers, but occasionally they are decreased in number, some are atypical and have a deeply basophilic agranular cytoplasm or hypersegmented nucleus.

Megakaryocytic megaloblastic changes

--Serum vitB12 (RIA <74~103 pmol/L)

--Urinary excretion of methylmalonic acid

--Serum folate assay( <6.91nmol/L or <3 ng /ml)

--The red cell folate assay (RIA)< 227 nmol/L

--Folic acid therapy test

--Intrinsic factor and its antibody

Tests for determining the causes of MA

【 Diagnosis 】 Two steps:

1.To establish the anemia is of MA

2.To determine the cause of the anemia.

Diagnosis of typical MA:

--Macrocytic—normochromic anemia

--Megaloblastic haemopoiesis of erythroid.

--Myeloid and megakaryocytic series are involved also.

When MA is atypical, how to diagnose it?

Clinical features: history, signs, and specific tests

Myeloid megaloblastic changes

--Minimal anemia or no anemia, erythroid changes have not appeared when the myeloid megaloblastic changes present.

--Dimorphic anemia(MA +IDA), the blood will show a small number of oval macrocytes and almost always neutrophil hypersegmentation ; the marrow usually show intermmidiate megaloblasts and giant stab forms.

Why myeloid megaloblastic changes take an important role

--After treatment, the myeloid changes still present when erythroid megaloblasts disappear.

--If MgA with hypocellularity when pregnancy, megaloblastic normoblasts and megakaryocytes are seldom seen, megaloblastic changes in myeloid are distinctive.

Questions:

1. What are the characteristics of megaloblastic cells?

2. Why myeloid megaloblastic changes sometimes take an important role in the diagnosis of MA?

3. What are the common cause of MA?

4. How to diagnose MA caused by folic acid deficiency, Vit B12 deficiency,or pernicious anemia?

ANEMIA

贫血的病因及发病机制分类

贫 血红细胞生成减少 红细胞破坏过多 红细胞丢

失增加 骨髓造血功能障碍

造血物质缺乏或利用障碍

铁缺乏和铁利用障碍

维生素 B12

或叶酸缺乏

红细胞 内在缺陷 外在异常

干细胞增殖分化障碍

骨髓被异常组织侵害

骨髓造血功能低下

免疫因素

非免疫因素

急性失血性贫血

慢性失血性贫血

微血管病性溶血性贫血

化学、物理、生物因素致溶血

脾功能亢进

各种原因致免疫性 溶血性贫血

巨幼细胞贫血等

缺铁性贫血

铁粒幼细胞性贫血等

再障，纯红再障等

骨髓增生异常综合征等

膜异常 酶异常 Hb 异常

珠蛋白生成障碍性贫血

异常血红蛋白病

不稳定血红蛋白病

葡萄糖6磷酸脱氢酶缺乏症

丙酮酸激酶缺乏症等

遗传性球形红细胞增多症

遗传性椭圆红细胞增多症等

阵发性睡眠性血红蛋白尿症

肾病、肝病、感染性疾病、

内分泌疾病等

白血病、骨髓瘤、癌转移、

骨髓纤维化等

Anemia: weakness,fatigue, listlessness,palpitation, pallor jaundice, splenomegaly

MCV increase normal decrease

MCH MA IDA,SA,Thala

MCHC infection

decrease ret increase acute loss blood hypopoiesis HA (Coomb’s)

decrease PL normal IHA

infection extracellular defect intra-

WBC chronic renal disease osmotic fragility

decrease increase increase normal decrease

AA,MF AM HS,HE, G-6PD, PK Thala

PNH (ham’s) abnormalHb

Diagnostic steps of HA:

history,infection, underlying diseases,drugs,pallor,weakness

dark urine, jaundice,hepatosplenomegaly

blood film

spherocytes, autoagglutination, red cell fragments

ret increased immune assay

cold agglutinin test Coomb’s test

positive

CAS AIHA negative cold warm

hemolysis test

transfusion reaction infection, congenital syphilis

Normocytic normochromic anemia

history, underlying diseases, pallor,weakness

blood film

decrease/ increase ret increase

morphologic features acute blood loss HA

normal abnormal

secondary hypoplasia abnormal proliferation

anemia infiltration in marrow

Infection AA MDS leukemia

renal disease MF

liver disease metastatic cancer

endocrinic disease

Microcytic hypochromic anemia:

history, underlying diseases, anemia,MCV,MCH

Blood film

SI

increased normal/ increased decreased

Iron stain Hb eletrophoresis SF

HbA2,F

increased normal/ increased decreased

SA Thalasemia anemia of chronic IDA

HbC,S, D,E disorders

etc.

Differentiation of macrocytic anemias:MCV,MCH

history, anemia ,underlying diseases,

drugs,nutrition,neurologic signs, hepatosplenomegly

blood film

increased Ret normal / decreased

marrow morpholoy

acute blood loss HA non-MA MA

erythroblastic anemia abnormal proliferation

Alchohol poisoning MDS Folate deficiency

Liver disease VitB12 deficiency

Pernicious anemia