1. Mechanism of hemolysis Characteristics in clinical and laboratory
Immune Hemolytic Anemias (IHA)
Definition
Classification
Mechanism of hemolysis
Characteristics in clinical and laboratory
Diagnostic process

2. Definition of IHA
The shortened red cells life span is caused by the antigens existing on the surface of erythrocytes reacting with relative antibodies. This acquired hemolytic anemia is termed immune hemolytic anemia.

3. 2. According to the nature of Ab
Classification
IHA
1. According to the cause of Ab production
2. According to the nature of Ab
Autoimmunehemolytic anemia (AIHA)
Drug-induced immune hemolytic anemia (DIHA)
Allo-immune hemolytic anemia (ALIHA)
temperature
Warm Ab type
(37 ℃，IgG)
Cold Ab type
(less than 37 ℃, IgM)
Mixed Ab type

4. I. Autoimmunehemolytic anemias (AIHAs)
Definition
AIHAs are a group of acquired hemolytic disorders that are the result of antibodies or complement binding to specific antigens on the RBC membrane, which leads to a shortened RBC life span and an increased RBC destruction.

5. Antierythrocyte antibodies can be divided into three general categories:
1. IgG Warm Abs bind to RBCs at 37 ℃ but fail to agglutinate the RBCs.
2. Cold agglutinins almost always are of IgM subtype and clump RBCs at cold tempratures. (CAS) .
3. Donath-Landsteiner(IgG) Abs bind to RBCs in the cold and activate the hemolytic complement cascade when the RBCs are warmed to 37 ℃. (PCH)

6. AIHA : Warm Antibody Type
overview:
a group of diseases resulting from the binding of IgG to the RBC membrane.
The IgG-bound RBCs either are trapped by macrophages as they pass through the spleen or may first have part of their membrane removed, then destroyed in the circulation.
70% of AIHAs.

7. Pathogenesis of hemolysis:
37°C
IgG
macrophages
FcR
Spleen sinus
C3bR
spleen
extravascular and/or intravascular

8. Etiology: Idiopathic: females more than males? secondary causes:
Lymphoprolitive diseases (e.g., CLL , non-Hodgkin lymphoma )
Connective tissue diseases(e.g., SLE)
Immune deficiency disorders( e.g., AIDS)
Drugs(e.g., penicillin, quinidine, methyldopa, cephalosporins)

9. Clinical Features
1. jaundice, anemia(e.g., fatigue, shortness of breath, light-headedness) or asymptomatic
2 . PE: jaundice, splenomegaly, and some signs relate to underlying illness , such as fever, lymphadenopathy, skin rash,, hypertension, renal failure, petechiae or ecchymoses ( PC decrease)
Evans syndrome is the combination of warm AIHA and idiopathic thrombocytopenia purpura.
Highly variable symptoms that are unrelated to temperature.

10. Blood： Hb, PCV , RBC( vary from normal to low)
Lab Findings
Blood： Hb, PCV , RBC( vary from normal to low)
Ret , “3M” (MCV ?) and RDW fall
Morphology：polychromatic RBCs, macrocytosis,
nucleated RBCs, or microspherocytes.
WBC? PC: N or low
Bone marrow: hyperplasia figure
Serum bilirubin and urobilinogen(urine):elevated
DAT usually positive. ( confirm test)

11. Blood smear may present macrocytes, spherocytes.

12. Bone marrow reveals erythroid hyperplasia
Bone marrow reveals erythroid hyperplasia. Polychromatic and orthochromatic normoblasts increaes, M/E decreased, erythroid mitosis increased; mature RBC?

13. Coombs Test 1. Direct Anti-immunoglobin Test ( DAT ) serum
Poly-
Anti-IgG
Anti-complement

14. overview Cold AIHA Cold Agglutinin Syndrome (CAS)
a group of disorders caused by IgM auto-antibodies (usually against the I/i antigen)
cold agglutinins bind to RBCs at cold temperatures (4 ℃ - 18 ℃).

15. cold agglutinins-IgM binding at low temperature
Hemolytic mechanism of CAS :
0 to 5 (4~18) ℃
agglutinate
cold agglutinins-IgM binding at low temperature
anti-C3+
activate complement, and C3b fixation( at higher tempratures.)
Extravascular
or Intravascular

16. Specific clinical features of CAS
1. Cold-induced acrocyanosis (Raynaud’s phenomenon)：
blue color of the skin at fingertips, toes, nose, and ear lobes) due to vascular sludding arising from agglutination.
2. cold-associated hemoglobinuria
3. Secondary to upper respiratory tract infection
4. Symptoms worse in cold weather and
higher IgM titer and activity)

17. Lab Findings: --anemia: Hb --Hemolysis: ret, bilirubin, LDH,
--blood smear: RBC agglutination
--bone marrow
--DAT +: for C3 only. (screening)
--The Cold agglutinin test (confirm)

18. Coombs Test Direct Anti-immunoglobin Test ( DAT ) serum
Anti –C3 +
Anti-immunoglobin
Poly-
serum

19. Cold agglutinin test autoerythrocytes or O type RBC Patient serum
IgM
autoerythrocytes
or O type RBC
or the same type RBC
37℃

20. 【Result】 Normal: titer<1:40, CAS: titer>1:64

21. III. Paroxysmal cold hemoglobinuria (PCH)
IgG autoantibodies are specific for the P antigen.
D-L Ab: mono- or polyclonal; IgG;
syphilis
paroxysms of fever, back pain, leg pain,
abdominal cramps; rigors (after exposure to cold temp.) hemoglobinuria
hemolysis: intravascular , less than 20 ℃
children often , secondary to viral disorder

22. Lab findings: 1. Hb, PCV (severity of anemia)
2. Ret low in episode; elevated in recovery phase.
3. Blood smear: anisocytosis, poikilocytosis, polychromatophilia, spherocytes, nucleated RBCs.
4. FHb , Hp ; hemoglobinuria, methemoglobin (urin)
5 DAT negative usually, or C3 + only, IAT+
6 Donath-Landstainer Test : D-L Ab +

23. Indirect Anti-immunoglobin Test ( IAT )
Sample of patient
T<20 ℃
Anti-IgG，IgM or / and C

24. Cold Warm Hemolysis Test
(Donath-Landstainer Test)
【Principle】
At lower temperature(<20℃), D-L antibody may attach to the surface of the patient’s red cell. When temperature up to 37℃, the red cells with D-L antibodies lysis rapidly with the help of activated complements.
【Result】 Negative: no lysis in normal people.

25. 37℃
<20℃

26. Diagnostic process of IHA

27. Anemia and type of anemia
Hb, RBC, PCV;
3M, RDW
Fatigue, weakness
Anemia and type of anemia
jaundice, hemoglobinuria
Splenomegaly, fever, chilly
Ret
morphology
serum and urine
Hemolytic anemia and its type
underlying diseases and specific featrues?
IHA
Coombs test
Anti-C3
Anti-C3, ITA+
DAT+
cold agglutinin test
cold warm hemolysis test
titer>1:64
Warm type of AIHA
PCH
PLC
CAS
Evan syndrome

28. Questions :
Summarize the features of blood and bone marrow smear in IHA?
How do you know the patient may have IHA?

29. Case
A female, 45 years old
Complaintof “having been fatigue, weakness for about 6 months. Sometimes had black urine.”
PE: moderate splenomegaly and slightly enlarged liver.
Lab: Hg 80g/l, Hct 0.30 , RBC 2.5X1012 , RDW 16.7
What do you know from these information?

30. Blood smear

31. Marrow cellularity (×100)

32. Marrow smear (×1000)

33. No.5 is the result of Coombs test of the patient1 2 3 4 5
No.5 is the result of Coombs test of the patient
DAT(anti IgG+anti C3)

34. Discussing questions:
1. What is your primitive impression of the patient?
Describe your evidence?
2. What clinical information do you need to support your diagnosis?
3. Which further tests do you need to complete the diagnosis?
4. Why the patient had black urine.

35. Secondary anemia
MYELOPATHIC ANEMIAS
These anemias are due to the bone marrow infiltration or replacement by abnormal tissues such as malignant metastases, myelosclerosis, leukemia and myeloma.

36. Pathogenesis of myelopathic anemia:
--bone marrow damage
--hemorrhage
--hemolysis

37. Clinical Features
--severe anemia
--bleeding problems may occur
--splenomegaly and hepatomegaly is common
--symptoms referable to underlying disease

38. Lab Findings
Blood
--varying degrees of anemia (most normocytic; slightly macrocytic)
--distinct anisocytosis and poikilocytosis
--leukoerythroblastic anemia:
--reticulocytosis and polychromatophilia
--The WBC count may be variable
--The platelet count is often low, giant, bizarre shaped with abnormal function.

39. What is leukoerythroblastic anemia?
The patients suffering from anemias with nucleated RBCs and immature granulocytes in the peripheral blood.
disruption of marrow sinusoids
hematopoiesis in extramedullary sites
Why?

40. .Bone marrow
--The marrow aspiration may fail or show metastatic cells and so on.
--The marrow biopsy is necessary to establish the diagnosis.
Other tests:
--x-ray
--NAP
--CD

43. ANEMIA
REVIEW

44. 贫 血 贫血的病因及发病机制分类 红细胞生成减少 红细胞破坏过多 红细胞丢失增加 内在缺陷 外在异常 骨髓造血功能障碍
贫 血
红细胞生成减少
红细胞破坏过多
红细胞丢失增加
红细胞
内在缺陷 外在异常
骨髓造血功能障碍
造血物质缺乏或利用障碍
急性失血性贫血
慢性失血性贫血
膜异常
酶异常
Hb异常
免疫因素
非免疫因素
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
铁缺乏和铁利用障碍
维生素B12或叶酸缺乏
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
葡萄糖6磷酸脱氢酶缺乏症
丙酮酸激酶缺乏症等
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
各种原因致免疫性 溶血性贫血
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
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
再障，纯红再障等
骨髓增生异常综合征等
缺铁性贫血
铁粒幼细胞性贫血等
巨幼细胞贫血等
贫血的病因及发病机制分类

45. 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

46. transfusion reaction infection, congenital syphilis
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

47. 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

48. increased normal/ increased decreased Iron stain Hb eletrophoresis SF
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.

49. history, anemia ,underlying diseases,
Differentiation of macrocytic anemias:MCV, MCH
history, anemia ,underlying diseases,
drugs,nutrition,neurologic signs, hepatosplenomegly
blood film
increased Ret normal / decreased
marrow morphology
acute blood loss HA non-MA MA
erythroblastic anemia abnormal proliferation
Alchohol poisoning MDS Folate deficiency
Liver disease VitB12 deficiency
Pernicious anemia