Northern Caribbean University

College of Allied Health and Nursing

Department of Medical Technology

MTCH 311: Fundamentals of Haematology and Haemostasis

Sectional # 4

Name Deneisha Mckella ID 18085298 Date 13/4/11

1. Complete the following table (12 pts)

Anemia cause Test Treatment

Iron deficiency CBC, Reticulocyte count, serum Iron absorption enhancers
iron, serum ferritin, transferrin
(TIBC).

Beta Thalassaemia CBC, Hb electrophoresis Transfusion chelation

Sickle cell anaemia Hb electrophoresis, CBC, Prophylactic penicillin

Hereditary Elliptocytosis
G6PD-Deficiency
Reticulocyte count.
CBC, Reticulocyte count.
CBC, methemoglobin reduction test, The most important
ascorbate-cyanide test.
therapy – Twice daily
administration of oral
penicillin
Administration of age-
appropriate immunizations
should be given, including
pneumococcal, conjugated
H. influenza and hepatitis B
vaccines.
Splenectomy
Patients with compensated
mild HE and no
splenomegaly possess a
benign disorder and require
no therapeutic intervention.
measure is prevention –
avoidance of the drugs and
foods that cause hemolysis.
Vaccination against some
common pathogens (for e.g.
hepatitis A and hepatitis B)
 Hereditary Spherocytosis CBC, osmotic fragility test, Splenectomy, prophylactic
autohemolysis test. antibiotic treatment,
pneumococcal vaccine.

Pyruvate Kinase Deficiency Reticulocyte count, fluorescence Blood transfusion, removal

screening test, osmotic fragility test, of the spleen
autohemolysis test.

2. Classify the haemolytic anaemias as follows NB. Answers require more than three

responses (4 pts each)

a. Haemolysis due to abnormal haemoglobin

-
Sickle Cell Anemia
-
Thalassemia
-
Hemoglobin C Disease
-
Hemoglobin D Disease
-
Hemoglobin E Disease 1

b. Haemolysis due to abnormal membrane

-
Hereditary Spherocytosis
-
Hereditary Elliptocytosis
-
Hereditary Stomatocytosis
-
Hereditary Xerocytosis 1

c. Haemolysis due to abnormal metabolism

-
Glucose-6-Phosphate Dehydrogenase Deficiency
-
Pyruvate Kinase Deficiency
-
Methemoglobin Reductase Deficiency
-
Pernicious anemia
 -
Phosphoglycerate Kinase Deficency 1

d. Haemolysis due to Stem cell defect

-
Aplastic anemia
-
Thalassemia
-
PHN

e. Immunohaemolytic
-
Alloimmune Hemolytic Anemia
-
Autoimmune Hemolytic Anemia
-
Hemolytic Disease of the Newborn
-
Warm Autoimmune Hemolytic Anemia
-
Drug Induced Hemolytic Anemia 1

f. Micoangiopathic Haemolysis
-
Thrombotic Thrombocytopenic Purpura
-
Hemolytic Uremic Syndrome
-
Eclampsia
-
Preeclampsia
-
HELLP syndrome 1
-
Autoimmune Hemolytic Anemia
-
Alloimmune Hemolytic Anemia

g. Hemolysis due to erythrocyte damage

-
Sickle cell anemia
-
Thalassemia
-
G6PD deficiency, hereditary spherocytosis
 3. Discuss the following (7 pts each):

a. Paroxysmal Nocturnal Hemoglobinuria

Paroxysmal Nocturnal Hemoglobinuria is a rare acquired stem cell disorder that

results from abnormalities of the red cell membrane1. This occurs as there is increased

susceptibility of the red cells to complement lysis which is directly related to a clonal

membrane defect2. In this condition RBC’s are destroyed while patients sleep because of

their increased sensitivity to complement lysis, and upon arising the patient notices bloody

urine or hemoglobinuria2. PNH occurs because of a somatic mutation in the hematopoietic

stem cells designated as phosphatidylinositol glycan class A (PIGA). PIGA is an X-linked

mutation that is essential for the synthesis of the glycosylphosphatidylinositol (GPI)-

anchored proteins which is present in all cell lines2. Defects in the GPI anchoring protein

seen in PNH, causes variable deficiencies in at least 17 cell surface proteins. This may affect

the function of the hematopoietic cells1. CD55 decay accelerating factor and CD59

membrane inhibitor are essential for the protection to red cells against lysis by

complement, therefore when these are missing intravascular lysis occurs thus causing

hemoglobinuria. Patients having this condition may show clinical manifestations such as

marrow failure, severe anemia, pancytopenia with elevated reticulocyte levels, neutropenia,

and absence of stainable iron due to continued lysis2.

Screening tests usually done in the diagnosis of PNH are the sugar water test, the Ham’s

test, and flow cytometry. The treatment options for patients with PNH include transfusion

support and, in selected younger patients, bone marrow transplant. Iron therapy may also
be used once the patient’s iron status has been assessed. A new drug, eculixumab, which

blocks complement activity by binding to C5 and prevents hemolysis is also used.

b. Warm antibody Autoimmune hemolysis

Warm antibody Autoimmune haemolytic anemia (WAIHA) is one of the most common

causes of hemolyic anemia in adults and in more prevalent in adults over 40 years. This

condition is usually secondary to another disease process such as lymphoid neoplasm,

autoimmune o chronic disorder, and viral infection1. Signs and symptoms are not usually

declares until severe anemia is developed. These include pallor, weakness, dizziness,

dyspenia, jaundice, and unexplained fever1. Hemolysis in this condition is usually

extravascular and occurs in the spleen. The onset of WAIHA is usually gradual and may be

precipitated by infections, especially viral infection, or after transfusion or organ

transplantation.

Patients with this condition usually show a positive DAT with polyspecific AHG reagent

and when monospecific AHG is used there is usually the presence of both complement C3d

and IgG1. WAIHA can cause several problems in serologic testing which include patient’s

RBCs being strongly coated with autoantibodies, which causes interferences with

phenotyping. Also, autoantibodies present in serum may mask underlying alloantibodies 1.

In the laboratory diagnosis of patients with WAIHA there is usually the presence of

microcytic anemia with increased reticulocyte count. Polychromasia reflecting

reticulocytosis and spherocytosis may be present on the blood smear. Patients may also

show increased hyperbilirubinemia and increased LD1. The treatment options are usually
aimed at treating the underlying conditions if one is present. Corticosteroids are usually the

first line of treatment and if this is ineffective then splenectomy is done.

c. Cold agglutinin autoimmune hemolysis

Cold autoantibodies (autoagglutinins) are present in normal human sera. Pathologic

cold agglutinins can be divided in to primary cold agglutinin disease (primary CAD), cold

agglutinin syndrome (CAD), and paroxysmal cold hemoglobinuria (PCH)1. CAD is a

hemolytic anemia produced by an autoantibody that reacts optimally at 40C but has wide

thermal amplitude, reacting at temperatures greater than 300C as well. The antibody is

usually an IgM immunoglobulin which efficiently activates complement.

The clinical ciiteria that are required for the diagnosis of CAD include clinical signs of

an hemolytic anemia, a positive DAT result using polyspecific AHG, a positive result using

monospecific C3 AHG and a negative result using monospecific IgG AHG1. The presence of

reactivity in the patient’s serum owing to a cold autoantibody and a cold agglutinin titer of

1:1000 or greater in saline at 40C with visible autoagglutination of anticoagulated blood at

room temperature is also required for diagnosis.

4. Differentiate between Acute and Chronic haemolysis (be sure to include laboratory

findings) (5 pts)

5. Explain the sequence of events that follows intravascular and extra vascular haemolysis.

(10 pts)

Extravascular hemolysis is where there is red blood cell (RBC) destruction in the spleen,

liver, lymph nodes and bone marrow. RBC contents are release and the internal products of

hemoglobin, heme and globin are released. When globin chains are released amino acids from

this chain are recycled into the amino acid pool 2. Products of heme, such as iron and the
protoporhyrin ring, are taken through different pathways. The iron is transported via the plasma

protein carrier, transferrin, to the bone marrow to be used in the synthesis of new hemoglobin 2.

The protoporhyrin ring reacts with hemoxygenase to yield the by product biliverdin. This

biliverdin is converted to Bilirubin and carried by albumin to the liver. Once it reaches the liver it

is conjugated to Bilirubin glucuronide and excreted with bile into the intestines 1. In the

intestines it is further conjugated to Urobilinogen by bacterial action and excreted into the stool

while small amounts are reabsorbed into the enterohepatic circulation, filtered by kidneys and

excreted in urine 1.

Intravascular hemolysis occurs as a result of RBC breakdown in the lumen of the vessels.

The RBC ruptures and hemoglobin is released which dissociates into α and β dimers and picked

up by haptoglobin 1, 2. This haptoglobin-hemoglobin complex is not able to be absorbed by the

kidney so prevents renal excretion thus the dimers are transported to the liver cell for catabolism

so that it can be destroyed and broken down into Bilirubin. Once converted it is excreted with

bile into the intestines. In the intestines it is further conjugated to Urobilinogen by bacterial

action and excreted into the stool while small amounts are reabsorbed into the enterohepatic

circulation, filtered by kidneys and excreted in urine 1.

6. Discuss the laboratory evidence of intravascular and extravascular Haemolysis.(10 pts)

In intravascular and extravascular hemolysis similar laboratory findings will be evident

except only in intravascular hemolysis there is hemoglobinemia and hemoglobinuria and only in

extravascular hemolysis there is hepatosplenomegaly. These laboratory findings are as follows 2:

-
Decreased hemoglobin, hematocrit and RBC count.
-
Decreased serum haptoglobin.
 -
Elevated reticulocyte count.
-
Elevated serum Bilirubin.
-
Elevated Lactate dehydrogenase (LDH) 2.

For both extravascular and intravascular hemolysis the following will cause these

laboratory results. The CBC will show decreased hemoglobin, hematocrit and RBC count as

there is excessive red cell destruction so lesser red blood cells, more hemoglobin-haptoglobin

complexes formed and destroyed thus lowering hemoglobin levels. Also decreased hemoglobin

occurs as a result of hemoglobin released being disassembled and the alpha and beta chains

released. Once there is a decrease in the hemoglobin levels there will be a decrease in hematocrit

as the hemoglobin is a third of the hematocrit 1. There is a decrease in serum haptoglobin as the

more hemoglobin that is released from the destroyed RBCs, the more hemoglobin-haptoglobin

complexes are formed thus they are removed via catabolism in the liver. Reticulocytes are

elevated as there is an increase in the premature release of reticulocytes from the bone marrow in

response to the decreased RBC count due to the anemic condition 2. When hemoglobin is

released and its contents disassembled it goes through as sequence of events as explained in the

previous question. The release of increased biliverdin due to the reaction with hemoxygenase

causes an increase in serum Bilirubin as more biliverdin is released and conjugated to Bilirubin 1,
2
. LDH is increased as this enzyme is released once red blood cells lyse prematurely. Thus the

more severe the hemolysis, the more LDH released.

In intravascular hemolysis hemoglobinemia and hemoglobinuria occurs. Hemoglobinemia

occurs as the cells lyse directly in the vessels so plasma will appear pink to red based on severity

of hemolysis. This occurs as a result of hemoglobin dimers being present in the plasma 2.

Hemoglobinuria occurs as a result of the free hemoglobin being present in the urine via filtration
by the kidney. Hepatosplenomegaly occurs due to damaged red cells being sequestered in the

liver and spleen.

7. Discuss Sickle cell anaemia with respect to epidemiology, etiology, clinical manifestation

(Explain pathophysiology associated with each manifestation) and laboratory findings.

(10 pts).

Sickle cell anemia is a homozygous disease in which the individual inherits twice the

abnormal gene that codes for Hemoglobin S (HbS) 1. With this hemoglobin there is an amino

acid substitution of valine for glutamic acid in the β chain 1. Sickle cell anemia is most

commonly found in individuals who are of African ancestry and also those who live in the

Caribbean, South and Central America and the Middle East 1. The clinical features of sickle cell

anemia include chronic hemolytic anemia, vaso-occlusion (recurrent painful attacks), bacterial

infections and deterioration of tissue and organ function 1, 2. Chronic hemolytic anemia is

characterized by a hypercellular bone marrow as red cells only live 10 to 20 days, with elevated

production of reticulocytes, increased Bilirubin due to severe hemolysis. The anemia is usually

characterised with hematocrits lower than 37 % 1. Complications arising from this hemolytic

anemia occur in the form of aplastic anemia or splenic sequestration crisis. Acute aplastic anemia

may develop as a result of infection, when the already overworked bone marrow fails to produce

cells 2.

Tissue infarctions and sickling in small vessels produce several painful target points. There

are several features that may predispose to a crisis event which includes fever, dehydration, cold,

and stress 2. When this occurs, if centered in the bones, individuals experience tenderness,

warmth, and swelling and some bone necrosis. Infarctions at the joint lead to swelling, pain, and

loss of mobility. Bacterial infections occur as these sickling individuals are more prone. These
infections, septicemia and meningitis, can be caused by encapsulated Streptococcus pneumoniae
1
. This occurs as the sickling has splenic dysfunction which predisposed them to these infections.

The spleen bears the burden of the sickle process as it loses its ability to clear abnormalities

from red cells. Repeated infarctions and congestion of the spleen will lead to autosplenectomy,

producing a fibrosed and shriveled organ 2. This scarred organ is dysfunctional, lacking the basic

and most important splenic functions. Two consequences may develop: overwhelming sepsis and

splenic sequestration. The lungs are particularly susceptible to occlusions in the

microenvironment of the pulmonary space. During the course of disease, patients may

experience clinical lung conditions that are chronic or acute. Children with sickle cell anemia are

more susceptible to pneumonia than are other members of the pediatric population. As was stated

before, there is the occurrence of fever, chest pain, hypoxia, and pulmonary infiltrates.

The following laboratory tests are performed on a patient who is sickle: CBC, reticulocyte

count, peripheral smear evaluation, hemoglobin electrophoresis and the measurement of

Hemoglobin A2 and F by HPLC 1. The following results will be seen normocytic normochromic

cells with a decreased hemoglobin level, hematocrit, and red cell count. The reticulocyte count is

elevated which may lead to a slightly increased MCV. Bilirubin and LDH are increased, while

haptoglobin is decreased, indicating extravascular hemolysis. The peripheral smear will show

marked polychromasia, many nucleated RBCs, target cells, and the presence of irreversible and

reversible sickle cells. Hemoglobin S precipitates in high molarity buffered phosphate solutions

and the amount of hemoglobin S is insignificant in this screening. After electrophoresis two

bands, hemoglobin F and hemoglobin S, will be seen in patients with sickle cell anemia because

hemoglobin F is predominant in neonates 1.

8. Discuss the Sickle Variants. (15 pts)

 Sickle cell variants include SS, Sβ0 thal, Sβ+ thal, SC, and S HPFH. Hemoglobin SC

disease is a combination of two abnormal hemoglobins, hemoglobin S and haemoglobin C.

Individuals that are affected have a moderate anemia, with average hemoglobin of 8 to

10g/dL, with a slight reticulocytosis and the life span of Red cell is reduced to

approximately 29 days2. Although the disease is less severe than sickle cell anemia,

individuals with this condition may experience a painful crisis. In the clinical

manifestations of this condition the peripheral smear shows high numbers of target cells;

few reversible sickled cells, and folded cells, with a peculiar crystal shaped like the

Washington Monument or a gloved hand showing in some cells2.

Hemoglobin S-Beta Thalassemia includes Sβ0 thal and Sβ+ thal. The severity of HBS

combined with β thalassemia depends on the degree of suppression of β- globin chain

synthesis1.

This combination hemoglobin may produce clinical manifestations as severe as sickle

cell anemia, with virtually no hemoglobin A present. The clinical manifestations of RBCs

include microcytic hypochromic cells, showing the influence of the thalassemia gene, with

nRBCs, target cells, polychromasia, and sickle cells2. There is also the presence of

anisocytosis, poikillocytosis and sickle cells for Sβ0 thal. However, for Sβ+ thal there are no

sickle cells present but the clinical manifestations are the same. There is an increase in the

RDW as well as the reticulocyte count. As opposed to the usual presentation of sickle cell

anemia, splenomegaly is usually present.

References:

1. Harmening DM. Clinical hematology and fundamentals of hemostasis. 5th ed.

Philadelphia (PA): F.A Davis; 2009. p. 77-78, 177-298.

2. Ciesla B. Hematology in practice. Philadelphia (PA): F.A Davis; 2007. p. 55-58, 114-

119.