HEMATO-IMMUNOLOGY BLOCK

Autoimmune haemolytic anemia (AIHA)

First Session

Case Title : Miss. Hely

Miss Hely presented to the Emergency Department with main complaint of fatigue.

Page 2 session 1

Miss Hely is a 15-year-old girl and she is a student on the Junior High School.

Page 3 session 1

Recent additional complaint

There is another complain such as nausea, headache and yellowish discoloration of eyes of 5
days duration. Sometimes Miss Hely feel fever.

Page 4 session 1

History of disease :

Miss Hely had no bleeding per rectum, dark stool, bleeding from the gums, no cough of blood or
hematemesis.

Page 5 session 1

Physical examination :

Vital sign : Blood Pressure = 120/80 mmHg, Pulse = 92b/min, regular

Temperature = 37,3 ‘C (rectal) RR = 22 x/min

Page 6 session 1
  Head : Eye : conjunctiva palpebral marked pallor, and sclera icterus.
 Neck : There was no lymphadenopathy
 Thorax : Cor : within normal limit

Lung : Lung fields were clear

 Abdomen ; A non tender soft hepatomegaly with a span of 14 cm and a soft

splenomegaly 3 cm below the left costal margin was noted.
 Extremity : There was no edema, rash, petechiae or bruises.
Neurological examination was normal.

OBJECTIVES

At the end of the first session the students should be able to :

1. Make identification of problem

2. Describe the definitions of the difficult words
3. Describe several hypothesis this problems
4. Explain approach diagnosis of anemia
5. Mention the lymphatic system
6. Explain the anatomy of Spleen
7. Explain about the hematopoiesis in histology
8. Describe the histological structure of erythrocytes
9. Explain the life span of erythrocyte

Guiding Questions:
 1. Identify the patient problems :
- A 15-year-old girl
- Main complaint : fatigue
- Additional complaint : nausea, headache, fever and yellowish

2. Describe the definition of pallor!

Pallor : paleness especially of the face that is caused by illness

3. Describe the definition of icterus!

Icterus : jaundice, yellowish

4. Describe the definition of hepatomegaly!

Hepatomegaly : enlargement of the liver (at physical examination = liver spam more 12
cm)

5. Describe the definition of splenomegaly!

Splenomegaly : abnormal enlargement of the spleen (clinical description = Hacket score
or Schuffnerr Score)

6. Hypothesis this problems :

- Hematologic disturbance : Anemia
- Icterus = Infections : Hepatitis, Weill disease
Haemolytic : Malarial parasite, autoimmune haemolytic
- Chronic diseases
- Vitamin deficiency

What further information do you need?

About laboratory finding

Page 7 session 1

Laboratory findings :

- Hb = 5 g/dl, HCt (PCV) = 16%, Leucocyte = 5000 / ml, thrombocyte = 295.000 / ml

- Liver function tests showed a total serum bilirubin (TSB) of 4.5 mg/dl with an
indirect fraction of 3.2 mg/dl and SGOT = 30 IU/L; SGPT = 30 IU/L
- Malarial parasite was absent
- Urine : pH = 6,8; color = like tea; reduction (-), protein (-); Sediment = erythrocyte (0
– 1); leucocyte (2 – 3), bilirubin (+)
- Stool/feces : erythrocyte = 0-1; leucocyte = 0 – 1, parasite = (-), bacteria (-)

7. What is patient’s problem now?

 Anemia : conjunctiva palpebral marked pallor, Hb = 5 g/dl
 Total serum bilirubin (TSB) of 4.5 mg/dl with an indirect fraction of 3.2 mg/dl,
Bilirubin urine (+) -> Icterus pre hepatic (elevated indirect bilirubin more dominant),
malarial parasite (-) -> Hemolytic without malaria
 Probable diagnosis : Haemolytic Anemia

8. How is approach diagnosis of anemia?

9. Mention the anatomy of lymphatic system! (Anatomy)
 The lymphatic system include lymph, lymphatic vessels, lymphatic tissue, lymphatic
nodules, lymph nodes, tonsils, the spleen, the thymus
Seeley’s Principles Anatomy & Physiology
P:566

10. Explain the anatomy of Spleen!

 The Spleen is a soft, vascular (sinusoidal) mass with a relatively delicate
fibroelastic capsule, entirely surrounded by peritoneum except at the splenic
hilum, where the splenic branches of the splenic and vein enter and leave.
 Consequently, it is capable of marked expansion and some relatively rapid
contraction. It is a mobile organ although it normally does not descend inferior to
the costal (rib) region; it rests on the left colic flexure.
 It is associated posteriorly with the left 9th-11th ribs and separated from them by
the diaphragm and the costodiaphragmatic recess the cleft-like extension of the
pleural cavity between the diaphragm and the lower part of the thoracic cage.
 The relations of the spleen are
 Anteriorly, the stomach.
 Posteriorly, the left part of the diaphragm, which separates it from the
pleura, lung, and ribs 9-11.
 Inferiorly, the colic flexure.
 Medially, the left kidney.
 The spleen varies considerably in size, weight, and shape; however, it is usually
approximately 12 cm long and 7 cm wide. (A nonmetric memory device exploits
odd numbers; the spleen is 1 inch thick, 3 inches wide, 5 inches long, and weighs
7 ounces.). The diaphragmatic surface of the spleen is convexly curved to fit the
concavity of the diaphragm. The anterior and superior borders of the spleen are
sharp and often notched, whereas its posterior (medial) end inferior border are
rounded.
Figure 2.46. Pancreas, duodenum, biliary ducts, and spleen. A. The pancreas, extrahepatic bile
passages, pancreatic ducts, and duodenum are shown. B. The entry of the bile duct and
pancreatic duct into the duodenum through the hepatopancreatic ampulla the dilation within the
major duodenal papilla that normally receives the bile duct and the main pancreatic duct is
shown. Smooth muscle sphincters encircle the bile and pancreatic ducts and the hepatopancreatic
ampulla. C. The interior of the descending part of the duodenum reveals the major and minor
duodenal papillae. The accessory pancreatic duct opens on the minor duodenal papilla. D.
11. Explain etiology of icteric (jaundice)?
- Pre hepatic / haemolytic jaundice
ETHIOLOGY: HEMOLYSIS
- Hepatic/parenchymal jaundice
ETHIOLOGY: Hepatitis virus, Hepatitis drug induced
- Post Hepatic/(obstructive) jaundice
ETHIOLOGY: Cholestasis, Tumor abdomen

12. Describe the definition of hemolysis!

Hemolysis : lysis of red blood cells with liberation of hemoglobin

13. What is the definition of hematopoiesis in histology? (histo)

 Hematopoiesis is the result of simultaneous, continuous proliferation and
differentiation of cells derived from stem cells whose potentially is reduced as
differentiation progress. This process can be observed in both in vivo studies, in
which colonies of cells derived from stem cells with various potentialities appear.
Colonies derived from a myeloid stem cell can produce erythrocytes,
granulocytes, monocytes, and megakaryocytes, all in the same colony. The origin
and maturation of these cells are termed, respectively, erythropoiesis (Gr.
erythros, red, + poiesis), granulopoises, monocytopoisis, and
megakaryocytopoiesis.
 Haemoglobin synthesis begins during the early normoblast (basophilic
erythroblast) stage and is complete by the end of the reticulocyte stage.
 Cell division ceases with the early normoblast stage, after which the nucleus
progressively condenses and is finally extruded ate the late normoblast
(orthochromatic erythroblast) stage.

14. What are series of rubricytic/erythroid in every developmental stage in bone

marrow? (histo)
 Proerythroblast is the first recognizable erythrocyte precursor; the cell is a large
cell, with loose, lacy chromatin and clearly visible nucleoli; its cytoplasm is
basophilic
 Basophilic Erythroblast with a strongly basophilic cytoplasm and a condensed
nucleus that has no visible nucleolus. The basophilia of these two cell types is
caused by the large number of polyribosomes involved in the synthesis of
hemoglobin.
  Polychromatophilic Erythroblast. Polyribosomes decrease, and are of the
cytoplasm begin to be filled with hemoglobin. At this stage, staining causes
several colors to appear in the cell.
 Orthochromatophilic Erythroblast.
The Nucleus continues to condense and no cytoplasmic basophilia is evident,
resulting in a uniformly acidophilic cytoplasm (Janquiera, 11th ed)

15. How is the mechanism of erythropoiesis (red cell formation) in histology?

 RBC production controlled by hormones, especially erythropoietin (EPO)
from the kidney.
 The process of erythropoiesis is directed towards producing a cell devoid of
organelles but packed with hemoglobin. The first recognizable erythrocyte
precursor is known as the proerythroblast, a large cell with numerous cytoplasmic
organelles and no hemoglobin.
Further stages of differentiation are characterized by three main features:
1) Decreasing cell size and nuclear extrusion
2) Progressive loss of organelle; the presence of numerous ribosomes at early
stages accounts for the marked cytoplasmic basophilia (blue staining) which
steadily decreases as the number of ribosomes falls
3) Progressive increase in the cytoplasmic haemoglobin content; this
accounts for the increasing eosinophilia (pink staining) of the cytoplasm
towards maturity (histo, Wethers 5th ed).

16. What is the histology structure of erythrocytes? (histo)

 Erythrocytes (red blood cells), which are anucleate, are packed with the O2-
carrying protein hemoglobin. Under normal conditions, these corpuscles never
leave the circulatory system.
 When suspended in an isotonic medium, most mammalian erythrocytes are
biconcave disks without nuclei. Human erythrocytes are 7.5 μm in diameter, 2.6
 μm thick at the rim, and 0.8 μm thick in the center. The biconcave shape
provides erythrocytes with a large surface to-volume ratio.
 Erythrocytes with diameters greater than 9 μm are called macrocytes, and
those with diameters less than 6 μm are called microcytes. The presence of a
high percentage of erythrocytes with great variations in size is called
anisocytosis (Gr. aniso, uneven, + kytos).
 The normal concentration of erythrocytes in blood is approximately 3.9-5.5
million per microliter in women and 4.1-6 million per microliter in men
(Junquiera, 11th ed).

17. Explain the life span of erythrocytes!

 When RBCs are delivered from the bone marrow into the circulatory system,
they normally circulate an average of 120 days before being destroyed. Even
though mature RBCs do not have a nucleus, mitochondria, or endoplasmic
reticulum, they do have cytoplasmic enzymes that are capable of metabolizing
glucose and forming small amounts of adenosine triphosphate. These
enzymes also (1) maintain pliability of the cell membrane, (2) maintain
membrane transport of ions, (3) keep the iron of the cells’ hemoglobin in the
ferrous form rather than ferric form, and (4) prevent oxidation of the
proteins in the RBCs. Even so, the metabolic system of old RBCs become
progressively less active and the cells become more and more fragile,
presumably because their life processes wear out.
 Once the RBCs membrane becomes fragile, the cell ruptures during passage
through some tight spot of the circulation. Many of the RBCs self-destruct in
the spleen, where they squeeze through the red pulp of the spleen. There, the
spaces between the structural trabeculae of the red pulp, though which most of the
cells must pass, are the only 3 micrometers wide, in comparison with the 8-
micrometer diameter of the RBC. When the spleen is removed, the number of old
abnormal RBCs circulating in the blood increases considerably.
 When RBCs burst and release their hemoglobin, the hemoglobin is
phagocytized almost immediately by macrophages in many parts of the body,
but especially by the Kupffer cells of the liver and macrophages of the spleen
and bone marrow. During the next few hours to days, the macrophages
release iron from the hemoglobin and pass it back into the blood, to be
carried by transfferin either to the bone marrow for the production of new
RBCs or to the liver and other tissues for storage in the form of ferritin. The
porphyrin portion of the hemoglobin molecule is converted by the
macrophages, through a series of stages, into the bile pigment bilirubin, which
 is released into the blood and later removed from the body by secretion
through the liver into the bile.

a. Red cell formation and destruction. RBC, red blood cells.

b. Iron transport and metabolism.

 18. What further information do you need to confirm this diagnosis haemolytic anemia?
- Blood Smear to watch morphology of red cells
- Reticulocyte count
- Direct anti globulin test (DAT)

HEMATO-IMMUNOLOGY BLOCK

Autoimmune haemolytic anemia (AIHA)

Second session

CASE TITLE : Miss Hely

Update Laboratory finding :

Blood smear : anisopoikilocytosis with predominant macrocytes, hypochromia and nucleated
red blood cells
Reticulocyte count = 10%
Direct anti globulin test (DAT) was strongly positives.

OBJECTIVES
At the end of the second session the students should be able to:

1. Explain about the function of erythrocyte and hemoglobin

2. Explain about the erythrogenesis (erythropoiesis) and its regulations
3. Explain clinical features (symptom and sign) Haemolytic anemia
4. Explain how to confirm diagnosis AIHA
5. Describe of laboratory finding haemolytic anemia

Guiding Question:

1. Explain about the function of erythrocyte and hemoglobin.

 The major function of red blood cells, also known as erythrocytes, is to transport
hemoglobin, which in turn carries oxygen from the lungs to the tissues. Therefore, for
hemoglobin to remain in the human blood stream, it must exist inside red blood cells.
 The red blood cells have other functions besides transport of hemoglobin. For instance,
they contain a large quantity of carbonic anhydrase, an enzyme that catalyzes the
reversible reaction between carbon dioxide (CO2) and water to form carbonic acid
(H2CO3), increasing the rate of this reaction several thousandfold.
 The rapidity of this reaction makes it possible for the water of the blood to transport
enormous quantities of CO2 in the form of bicarbonate ion (HCO3) from the tissues to the
lungs, where it is reconverted to CO2 and expelled into the atmosphere as a body waste
product.
 The hemoglobin in the cells is an excellent acid-base buffer (as is true of most proteins),
so that red blood cells responsible for most of the acid-base buffering power of whole
blood.

2. Explain about the production of the red blood cells.

 In the early weeks of embryonic life, primitive, nucleated red blood cells are
produced in the yolk sac.
 During the middle trimester of gestation, the liver is the main organ for production
of red blood cells, but reasonable numbers are also produced in the spleen and
lymph nodes.
 Then, during the last month or so of gestation and after birth, red blood cells are
produced exclusively in the bone marrow.
 The bone marrow of essentially all bones produces red blood cells until a person is 5
years old.
 The marrow of the long bones, except for the proximal portions of the humeri and
tibiae, becomes quite fatty and produces no more red blood cells after about age 20
years. Beyond this age, most red cells continue to be produced in the marrow of the
membranous bones, such as the vertebrae, sternum, ribs, and ilia. Even in these
bones, the marrow becomes less productive as age increases.
3. Explain about the regulation of the red blood cells production.
 In a normal person, the total volume of circulating erythrocytes remains remarkably
constant because of the reflexes that regulate the bone marrow’s production of these
cells. We stated that iron, folic acid, and vitamin B12 must be present for normal
erythrocyte production. However, none of these substances constitutes the signal that
regulates the production rate.
 The direct control of erythrocyte production (erythropoiesis) is exerted primarily by a
hormone called erythropoietin, which is secreted into the blood mainly by a
particular group of hormone-secreting connective tissue cells in the kidneys (the
liver also secretes this hormone, but to a much lesser extent). Erythropoietin acts on the
bone marrow to stimulate the proliferation of erythrocyte progenitor cells and their
differentiation into mature erythrocytes.
 Erythropoietin is normally secreted in relatively small amounts, which stimulate the
bone marrow to produce erythrocytes at a rate adequate to replace the usual loss. The
erythropoietin secretion rate is increased markedly above basal values when there is a
decreased oxygen delivery to the kidneys. Situation in which this occurs include
insufficient pumping of blood by the heart, lung disease, anemia (a decrease in
number of erythrocytes or in hemoglobin concentration), and exposure to high
altitude. As a result of the increase in the erythropoietin secretion, plasma
erythropoietin concentration, erythrocyte production, and the oxygen carrying
capacity of the blood all increase. Therefore, oxygen delivery to the tissues returns
toward normal.
 Renal tissue hypoxia leads to increased tissue levels of hypoxia-inducible factor-1
(HIF-1), which serves as a transcription factor for a large number of hypoxia-
inducible genes, including the erythropoietin gene. HIF-1 binds to a hypoxia response
element residing in the erythropoietin gene, inducing transcription of messenger RNA
and, ultimately, increased erythropoietin synthesis.
 At times, hypoxia in other parts of the body, but not in the kidneys, stimulates
kidney erythropoietin secretion, which suggests that there might be some non-renal
sensor that sends an additional signal to the kidneys to produce this hormone. In
particular, both norepinephrine and epinephrine and several of the prostaglandins
stimulate erythropoietin production.
 When both kidneys are removed from a person or when the kidneys are destroyed
by renal disease, the person invariably becomes very anemic because the 10 percent of
the normal erythropoietin formed in other tissues (mainly in the liver) is sufficient
to cause only one third to one half the RBC formation needed by the body.
 When an animal or a person is placed in an atmosphere of low oxygen, erythropoietin
begins to be formed within minutes to hours, and it reaches maximum production
within 24 hours. Yet almost no new red blood cells appear in the circulating blood
until about 5 days later. From this fact, as well as other studies, it has been determined
that the important effect of erythropoietin is to stimulate the production of
proerythroblasts from hematopoietic stem cells in the bone marrow.
 In the absence of erythropoietin, few red blood cells are formed by the bone marrow.
At the other extreme, when large quantities of erythropoietin are formed available,
and if there is plenty of iron and other required nutrients available, the rate of red
blood cell production can rise to perhaps 10 or more times normal. Therefore, the
erythropoietin mechanism for controlling red blood cell production is a powerful one.
 Testosterone, the male sex hormone, also stimulates the release of erythropoietin. This
accounts in part for the higher hematocrit in men than in women.
4. What is confirm diagnosis now?
Haemolytic Anemia

5. Explain Classification of Hemolytic Anemia?

6. Explain Causes (etiology) of Haemolytic Anemia?

Causes of Acquired hemolytic anemia

I. Entrapment
II. Immune
- Warm reactive (IgG) antibody
- Cold reactive IgM antibody
- Cold reactive IgG antibody
- Drug dependent antibody : * autoimmune
* haptene

III. Traumatic hemolytic anemia

- Impact hemolysis
- Macrovascular defect
- Microvascular : * hemolytic uremic syndrome
* other causes of microvascular abnormalities
* Disseminated intravascular hemolysis

IV. Hemolytic anemia due to toxic effects on the membrane

 - Spur cell anemia
- External toxins: * animal bites
* metals
* organics compounds

V. Paroxysmal Nocturnal hemoglobinuria

7. Explain pathophysiology of hemolytic anemia in this case!

Pathophysiology

Hemolysis can be due to hereditary and acquired disorders. The etiology of premature
erythrocyte destruction is diverse and can be due to conditions such as intrinsic
membrane defects, abnormal hemoglobin, erythrocyte enzymatic defects, immune
destruction of erythrocytes, mechanical injury, and hypersplenism.

Hemolysis may be an extravascular or an intravascular phenomenon. Autoimmune

hemolytic anemia and heredity spherocytosis are examples of extravascular hemolysis
because the red blood cells are destroyed in the spleen and other reticuloendothelial
tissues. Intravascular hemolysis occurs in hemolytic anemia due to the following:

 Prosthetic cardiac valves

 Glucose-6-phosphate dehydrogenase (G6PD) deficiency
 Thrombotic thrombocytopenic purpura
 Disseminated intravascular coagulation
 Transfusion of ABO incompatible blood
 Paroxysmal nocturnal hemoglobinuria (PNH)

Hemolysis may also be intramedullary, when fragile red blood cell (RBC) precursors are
destroyed in the bone marrow prior to release into the circulation. Intramedullary
hemolysis occurs in pernicious anemia and thalassemia major.

Hemolysis is associated with a release of RBC lactate dehydrogenase (LDH).

Hemoglobin released from damaged RBCs leads to an increase in indirect bilirubin and
urobilinogen levels.
 A patient with mild hemolysis may have normal hemoglobin levels if increased RBC
production matches the rate of RBC destruction. However, patients with mild hemolysis
may develop marked anemia if their bone marrow erythrocyte production is transiently
shut off by viral (parvovirus B-19) or other infections. This scenario would be an aplastic
crisis since the bone marrow can no longer compensate for ongoing hemolysis.

Skull and skeletal deformities can occur in childhood due to a marked increase in
hematopoiesis and resultant bone marrow expansion in disorders such as thalassemia.

8. Explain Clinical presentation (symptom and sign) of iron deficiency anemia?

The patient may complain of fatigue and other symptom of anemia
Symptoms
- Symptoms anemia : weakness, loss of stamina, breathlessness
- Hemolytic anemia e.c Malaria : fever, night sweats, jaundice, dark urine

Physical examination

- Lymphadenopathy
- Splenomegaly
- Jaundice

9. How is confirm diagnosis of AIHA?

Diagnosis of AIHA is based on based on evidence of haemolytic anemia consisting of
 Anemia,
 Jaundice,
 Splenomegaly,
 Reticulocytosis,
 Raised serum bilirubin
 Positive DAT (Direct anti globulin test) -> autoimun
10. Describe of laboratory finding of Haemolytic anemia?
 Laboratory evaluation/finding in Hemolytic anemia:
o Request for CBC : anemia is constant finding, hemoglobin decrease can reach
less than 5 g/dL.
o Reticulocyte (young RBC) count; enumeration of reticulocyte will be a
measure of increased erythropoiesis stimulated by erythropoietin.
 Corrected reticulocyte count :
Reticulocyte count (Normal value 0.5-1.5%) times patient hematocrit (%) /normal
hematocrit (%).
 Reticulocyte index :
Laboratory assessment for the accelerated release of erythrocyte from the marrow;
an effect of erythropoietin increase in anemia. The normal reticulocyte maturation
time in the blood is one day, this varies inversely with hematocrit (Hct).

Hct (%) Reticulocyte maturation time (days)

45 1.0

35 1.5

25 2.0

15 2.5

Correction for anemia :

Patient retic. Count (%)/Normal retic. Count (%) x Patient Hct (%)/Normal Hct
(%), for this patient = 10/1x16/45 = 3.55.
Correction for shift :
Corrected retic. Index x 1/Maturation time = 3.55/2.5 = 1.42
These correction are necessary to assess the degree of RBC production in respons
to anemia. In our patient the increase in RBC production only 1.42 times normal.
 Evaluation of blood smear, usually blood smear from hemolytic anemia show
some RBC degradation : spherocyte (round form of erythrocyte before cell lysis),
helmet cell/schistocyte, polychromatophilic erythrocyte (reticulocyte appearance
in Wright staining), nucleated RBC (normoblast).
 DAT (Direct Antiglobulin Test):
 This laboratory test detect auto antibody against self-antigen (non-blood group
antigen) or complement on the surface of RBC. Positive DAT means there’s IgG
or complement attaching on RBC surface.

11. What kind of the diagnosis hemolytic anemia?

Auto immune Hemolytic Anemia (AIHA)

HEMATO-IMMUNOLOGY BLOCK

Autoimmune haemolytic anemia (AIHA)

Third session
CASE TITLE : Miss Hely

Miss Hely hospitality for fifth days. He was gived four pack PCR (Packed Red Cell) transfusion.
Laboratory finding = Hb : 9,6 mg/dl, HCt (PCV) = 16%, Leucocyte = 5000/ml, Thrombocyte =
125.000/ml

OBJECTIVES

At the end of the second session the students should be able to:

1. Explain the management of haemolytic anemia

2. Explain the prognosis of haemolytic anemia
3. Explain the indication transfusion of haemolytic anemia

Guiding Questions:

1. How is management Haemolytic anemia?

Treatment hemolytic anemia depend of CAUSES
 Anti Malaria : if caused by malaria infestation
 Glucocorticoid : if caused by immunologic
 Splenectomy : if caused by hereditary hemolytic anemia
 Blood Transfusion is Limited benefit

2. How is prognosis of Haemolytic anemia?

 The prognosis for patients with hemolytic anemia depends on the underlying cause.
Overall, mortality rates are low in hemolytic anemias. However, the risk is greater in
older patients and patients with cardiovascular impairment. Morbidity depends on the
etiology of the hemolysis and the underlying disorder, such as sickle cell anemia or
malaria.

3. Explain the indication transfusion of hemolytic anemia?

Indications of blood transfusion can be summed up as:
(http://www.mahasbtc.com/indications-blood-transfusion)
 Anaemia
 Major Surgical Operation
 Accidents resulting in considerable blood loss
 Cancer patients requiring therapy
 Women in childbirth and newborn babies in certain cases
 Patients of hereditary disorders like Haemophilia and Thalassaemia
 Severe burn victims.