SAN PEDRO COLLEGE

Bachelor in Medical Laboratory Science

THIRD YEAR

UTILIZATION OF LASER-BASED FLOW CYTOMETRY:

LABORATORY EVALUATION OF ANISOCYTOSIS
IN ECULIZUMAB-DEPENDENT PATIENT

A case study in
HEMATOLOGY LABORATORY II

AGTING, Kazee Ulaiza K.

AGUSTIN, John Kenneth C.
ARINZOL, Christian T.
ARTOS, Trisha May D.
FLAUTA, Re-anne Liegh E.
MAMON, Abigail Thea C.
SORIANO, Maria Trisha A.
SUNIO, Ray Brandon B.

May 24, 2022

 CHAPTER I
(Prepared by: John Kenenth C. Agustin)
INTRODUCTION

Paroxysmal nocturnal hemoglobinuria (PNH) is a bone marrow clonal disorder

marked by the absence (total or partial) of all proteins normally connected to the cell
membrane by the glycosylphosphatidylinositol (GPI) anchor. A somatic mutation of the
PIG-A gene, which encodes a protein required for the production of the anchor GPI, causes
this cellular abnormality in hematopoietic stem cells 1.

Intravascular hemolytic anemia, venous thrombosis (especially in the belly), and

cytopenia (reduced blood cell production) are some of the clinical symptoms. Using
monoclonal antibodies and flow cytometry, the diagnosis is confirmed by confirming the lack
of GPI-anchored proteins on blood cells. The mutation's cause is usually unclear; the
condition frequently occurs in the context of aplastic anemia. PNH is an uncommon disorder
with a prevalence of 1-1.5 cases per million at the most 1.

Treatment is symptomatic (transfusions, erythropoietin, glucocorticoids,

anticoagulants) or includes bone marrow transplants in severe cases. Monoclonal
antibody-based techniques for suppressing complement activation are currently being
developed and promise to be very promising 1.

Contrary to the aforementioned paragraph, the majority of individuals have

nonspecific and diverse symptoms that do not fit into any one diagnosis. Patients with
smooth muscle dystonia commonly experience constitutional symptoms such as fatigue,
malaise, dyspnea, dark urine due to marked hemoglobinuria, renal insufficiency from
hemosiderin deposition leading to tubulointerstitial inflammation, dysphagia or esophageal
spasms, abdominal pain, back pain, and erectile dysfunction. Because of the wide range of
indications and symptoms, health experts frequently struggle to diagnose the disorder,
causing diagnosis to be delayed 2.

Flow cytometry is the gold standard test for diagnosing PNH. It uses monoclonal
antibodies and a reagent called fluorescent aerolysin reagent (FLAER) that binds directly to
GPI-anchored proteins, specifically the glycan region. This test has a high sensitivity and
specificity for screening a range of GPI-anchored proteins, most notably CD55 and CD59 2.

Flow cytometry tests come in two types: low sensitivity and high sensitivity. Although
low-sensitivity flow cytometry tests can diagnose PNH, a high-sensitivity test is superior at
detecting PNH in the context of another bone marrow illness. PNH can be classified into
three kinds based on the clinical picture and laboratory test results. 1. PNH classic. 2. PNH
in combination with another BM condition 3. There is evidence of hemolysis as well as a
primary bone marrow problem in PNH with BM abnormalities. To track the progression of the
condition, patients should be reassessed every 6 to 12 months for the size of the PNH clone.
Patients should also be reviewed if their clinical condition or laboratory tests change
significantly 2.

The most common causes of PNH are complement-mediated hemolysis and

persistent dysregulation of the alternative complement pathway. Anchoring proteins such as
CD55 and CD59 are commonly lost, causing cells to hemolyze and thrombosis, which causes
morbidity and mortality. Eculizumab, ravulizumab, and allogeneic hematopoietic stem cell
transplantation are the mainstays of contemporary PNH therapy 2.
 Generally, a healthcare professional team must take a comprehensive and integrated
approach to care to help patients obtain the best results possible. Different specialists'
involvement can improve the patient's result depending on the type of patient group
involved in PNH. A hematologist and a pediatrician should be involved if children are
affected. Similarly, if a pregnant patient is involved, excellent internal communication
between a primary care physician, a gynecologist, and a hematologist is required to offer
best care. Surgery can also trigger PNH, thus if a patient needs elective or emergent
surgery, the patient's primary hematologist should be consulted and involved in the patient's
care while they are in the hospital 3.

If a patient is taking eculizumab, frequent dosing and breakthrough hemolysis can

result in a poor quality of life. Patient and family education can help patients live a better
life. The patient's mental stress can be reduced if the family is more supportive and
understanding of what the patient is going through, and if the patient can reason about the
challenges they confront during therapy. A social worker should be involved in the treatment
if the patient cannot afford the treatment 3.

In addition, if the patient has several comorbidities and indicates a wish to focus on
comfort care, palliative care should be addressed. The palliative care team, the patient, and
the family should have a thorough discussion about the future alternatives for care, taking
into account the patient's mortality risk on a case-by-case basis 3.
 CHAPTER II
(Prepared by: John Kenenth C. Agustin)

PATIENT’S DATA

Patient information:
❖ 31 years old
❖ Caucasian woman

Patient manifestations:
❖ Tea-colored urine for seven days
❖ Menstrual period was over two weeks ago
❖ Experienced jaundice for three days
❖ Experienced intense abdominal pain for three days

Patient’s history:
❖ There was no medical history for kidney stones, weight loss, night sweats,
hemoptysis, or melena. The patient tested negative for pregnancy, and informed that
there was no drug abuse. Moreover, she also started on Eculizumab, and from that
there is a significant improvement.

Laboratory tests:

Complete blood count results:

● Hemoglobin - 102 g/L, decreased (Normal: 110-165 g/L)
● Red blood cell - 3.3 x 1012/L, decreased (Normal: 3.8-5.8 x 1012/L)
● Hematocrit - 0.36 L/L , slightly decreased (Normal: 0.37-0.47 L/L )
● Mean corpuscular volume - 108 fL, increased (Normal: 80-100 fL)
● Mean corpuscular hematocrit - 30.9 pg, normal (Normal: 26.0-32.0 pg)
● Mean corpuscular hemoglobin concentration - 283 g/L, decreased (Normal:
330-360 g/L)
● White blood cell - 4.7 x 109/L, normal (Normal: 4.0-10.0 x 109/L)
● Platelet - 67 x 109/L, decreased (Normal: 150-450 x 109/L)
● Red cell distribution width - 28%, increase (Normal: 11.5-14.5%)
● Reticulocyte - 10.8%, increased(Normal: 0.5-2.5)
● White blood cell differential
➢ Neutrophils - 63% (Absolute count: 2.961 x 109/L)
➢ Band - 5 (Absolute count: 0.235 x 109/L)
➢ Lymphocytes - 27 (Absolute count: 1.296 x 109/L)
➢ Monocytes - 4 (Absolute count: 0.188 x 109/L)
➢ Eosinophils - 1 (Absolute count: 0.047 x 109/L)
➢ Basophils - 0 (Absolute count: 0 x 109/L)
➢ Metamyelocytes, Myelocytes, Promyelocytes, Band, Promonocytes, and
nRBC/100 WBC were not reflected on the blood smear.
● Red blood cell Morphology - Moderate anisocytosis, macrocytes, mild
polychromasia; presence of target cells and elongated cells.
● White blood cell Morphology - Normal in number and morphology
● Platelet Morphology - Moderate thrombocytopenia; normal in morphology

Serum chemistry results:

● Total bilirubin - 7.5 mg/dL, increased (Normal: 0.1-1.2 mg/dL)
 ● Indirect bilirubin - 5.7 mg/dL, increased (Normal: 0.1-1.0 mg/dL)
● Aspartate Aminotransferase - 214 IU/L, increased (Normal: 4-36 IU/L)
● Lactate Dehydrogenase - 1550 IU/L (Normal: 259-613 IU/L)
● Serum haptoglobin - 10 mg/dL, decreased (Normal: 60-270 mg/dL)

Urinalysis results:
● Color - Red
● Clarity - Clear
● Specific gravity - 1.025
● pH - 6.0
● Glucose - Negative
● Albumin - +1
● Hemoglobin - +2
● Perl’s Stain on sediments - +2

Further tests:
❖ Direct Coomb’s Test - Negative
❖ Liver Ultrasound - Mild hepatomegaly; no signs of stones; no hepatic mass
❖ Immunophenotyping - CD55 and CD59 absent on 78% of RBCs
❖ Sugar Lysis Test - Positive
❖ Cytogenetics - Mutation in phosphatidylinositol-glycan complementation class A
❖ Bone Marrow Examination - Hypoplastic
❖ Osmotic Fragility Test - Negative

Ham’s tests:

1 2 3 4 5 6 7

Fresh normal serum 0.05 mL 0.05 mL 0.05 mL 0.05 mL

Patient serum 0.05 mL

Heat-inactivated 0.05 mL 0.05 mL

normal serum

0.2 N HCl 0.05 mL 0.05 mL 0.05 mL 0.05 mL 0.05 mL

50% Px red cells 0.05 mL 0.05 mL 0.05 mL 0.05 mL

50% normal red cells 0.05 mL 0.05 mL 0.05 mL

Px Result Trace ++ + 0 0 0 0
 CHAPTER III

A. ANATOMY AND PHYSIOLOGY (Prepared by: Abigail Thea C. Mamon)

I. Bone Marrow

The bone marrow, a soft and spongy tissue, also one of the largest organs in the
body, is considered as the primary lymphoid organ and a major hematopoietic organ that is
responsible for the production of bone marrow stem cells which are then converted into
erythrocytes, granulocytes, monocytes, lymphocytes and platelets. This organ is found
within the central cavities of axial and long bones and has many blood vessels in it.
Approximately this accounts for 4-5% of the body weight in humans 4.

There are actually two types of bone marrow which are red and yellow. These types
have different functions. Each type of bone marrow serves a vital purpose in our body:
1. Red Marrow. This type of bone marrow is involved in blood cell production. This is
where hematopoietic stem cells are found and can develop into a variety of different
blood cells, including: red blood cells which carry the oxygen to the tissues in the
body, platelets that prevent bleeding by creating blood clots, and white blood cells
that help fight the infection. 5
2. Yellow Marrow. Your red bone marrow is eventually replaced by yellow bone
marrow as you become older. Red bone marrow can only be present in a few bones
by adulthood, including the: skull, vertebrae, sternum, ribs, the ends of humerus,
pelvis, the ends of the femur, and the ends of the tibia. This type of bone marrow is
involved in the storage of fats which are called adipocytes where it is used as an
energy source when needed. It is yellow due to high fat content 5.

The bone marrow produces erythrocytes, granulocytes, monocytes, thrombocytes,

and lymphocytes. Lymphocytic stem cells move to the thymus and differentiate under the
influence of thymic hormones thymopoietin and thymosin. The blood cell production
depends on how much the body needs. In fact, all blood cells have limited lifespan inside
our body. White blood cells last for a few hours to a few days, platelets last for about 10
days, and 120 days for red blood cells. Normal blood cells last for a limited time since they
must be replaced constantly. Though, there are times that the production of blood cells will
be triggered due to some certain conditions unexpectedly occurring such as:

● Low level of oxygen in our body tissues

● Loss of blood or anemia
● Decreased number of red blood cells
● When kidneys produce and release erythropoietin
● Infections

In the event of severe blood loss, yellow bone marrow can be stimulated and
converted into red bone marrow. As people become older, more red bone marrow changes
into yellow bone marrow, making it more difficult to produce new blood cells. Anatomically
speaking, bone marrow consists of blood vessels in which it prevents immature blood cells
from exiting the said organ. Therefore, blood vessels act as a barrier. Only mature blood
cells have the membrane proteins needed to adhere to and pass through the endothelium of
blood vessels. Hematopoietic stem cells can also get through the bone marrow barrier and
be extracted for circulation.In the bone marrow, there is biologic compartmentalization,
which means that various cell types tend to congregate in certain locations. Erythrocytes,
macrophages, and their progenitors, for example, prefer to congregate around blood
vessels, whereas granulocytes congregate around the bone marrow's boundaries 6.

As for kidneys producing and releasing a hormone called erythropoietin, it stimulates

the action of bone marrow to produce more red blood cells since the body has to maintain a
sufficient amount of erythropoietin (EPO) hormone needed by the body. Erythropoietin also
helps in making red blood cells. The more red blood cells you have, the more it raises your
hemoglobin levels and hemoglobin is a protein in RBCs that helps blood carry oxygen
throughout the body 7.

Mesenchymal cells can also be seen in bone marrow stroma. These cells have the
capability to self-renew by dividing and grow into different variety of cell types which
includes: osteoblasts, osteoclasts, chondrocytes, myocytes, fibroblasts, macrophages,
adipocytes, and endothelial cells. Although they are not directly involved in the main
function of hematopoiesis, it does offer the microenvironment and colony-stimulating factors
required by parenchymal cells to assist the process of hematopoiesis 6.
 B. PATHOPHYSIOLOGY (Prepared by: Ray Brandon B. Sunio)

Etiology

The development of a genetic mutation in hematopoietic stem cells causes

paroxysmal nocturnal hemoglobinuria. The glycosylphosphatidylinositol (GPI) protein, which
is essential for anchoring other protein moieties to the surface of erythrocytes, is deficient
due to a mutation in the X-linked gene phosphatidylinositol glycan class A (PIGA). CD55 and
CD59, which regulate complement activity, are thereby inhibited from adhering to
PNH-affected cells. Chronic complement-mediated hemolysis of PNH cells results from the
lack of complement inhibition. If the complement system is activated by stress from
surgery, trauma, or other inflammatory causes, this persistent state of hemolysis might be
aggravated. Protein anchoring with glycosylphosphatidylinositol (GPI) is a conserved
post-translational modification in eukaryotes. GPI-anchored proteins (GPI-APs) are
expressed on the cell surface by more than 150 proteins in mammalian cells and more than
60 proteins in yeast. Because of the glycolipid anchor, GPI-APs have distinct characteristics
from typical membrane proteins. GPI is a functional molecule that modulates protein
trafficking and localization as well as an anchor that tethers changed proteins to the
membrane 8.

Development (progression)

A phosphatidylinositol (PI) molecule and a glycan core make up the GPI anchor. A
mutation in the PIGA gene, which codes for phosphatidylinositol glycan anchor biosynthesis
class A (PIG-A), also known as phosphatidylinositol N-acetylglucosaminyltransferase subunit
A, causes PNH in hematopoietic stem cells. The hematopoietic stem cell is unable to
properly generate the glycan core on phosphatidylinositol in the membrane without a fully
functional glycosyl transferase enzyme, and thus lacks membrane GPI anchors. All of the
progeny of the mutated stem cell are unable to express any of the approximately 20
GPI-anchored proteins seen on normal blood cells without GPI anchors. GPI-anchored
proteins are Complement regulators, enzymes, adhesion molecules, blood type antigens,
and receptors 9.
Two GPI-anchored proteins on the RBC membrane, decay accelerating factor (DAF, or
CD55), and membrane inhibitor of reactive lysis, are absent or defective in PNH, which is
relevant to the occurrence of hemolysis (MIRL, or CD59) 9. CD55 inhibits the C3 and C5
convertases of the complement alternative pathway, whereas CD59 hinders the
development of the membrane attack complex. When CD55 and CD59 are missing from the
surface of RBCs, the cell is unable to avoid complement activation, resulting in spontaneous
and persistent intravascular hemolysis. PIGA is the sole gene required for GPI anchor
production that is found on the X chromosome. As a result, the PNH phenotype in a stem
cell requires only one acquired mutation in the PIGA gene (males have only one X
chromosome, and in females one of the X chromosomes is inactivated) 10.
These various mutations cause varying expression of CD55 and CD59 on RBCs within
a single patient, resulting in three RBC phenotypes: type I, type II, and type III 11. Type I
RBCs are phenotypically normal, have normal levels of CD55 and CD59 expression, and
experience little or no complement-mediated hemolysis. Type II RBCs are caused by a PIGA
mutation that results in only a partial lack of CD55 and CD59, and these cells are
reasonably resistant to complement-mediated hemolysis. Type III RBCs are caused by a
PIGA mutation that results in a complete lack of the GPI anchor, therefore no CD55 or CD59
proteins are attached to the RBC surface. Type III RBCs are extremely vulnerable to
complement-induced lysis. In PNH, the most common RBC phenotype is a mix of type I and
type III cells, whereas the second most common has all three kinds. When determining the
severity of hemolysis in PNH, both the relative volume and type of circulating RBCs are
taken into account 10.
Patients with PNH may have bone marrow dysfunction in addition to hemolysis,
which contributes to the severity of the anemia. Many patients have a history of bone
marrow failure caused by acquired aplastic anemia or myelodysplastic syndrome that occurs
before or at the same time as the development of PNH, resulting in a hypoplastic PNH
manifestation 12.

Pertinent changes in the laboratory result

Decreased levels of serum haptoglobin, increased levels of lactate dehydrogenase,

hemoglobinuria, and hemosiderinuria. Low levels of hemoglobin, red blood cell, and
decreased platelet (thrombocytopenia). Reticulocytes are high, another indication of
anemia. Total bilirubin is elevated, as well as the indirect bilirubin and the AST. In
Immunophenotyping, the CD55 and CD59 are absent on 78% of RBCs . Perl's stain (2+)
result. Direct Coomb’s Test negative. In terms of cytogenetics, results showed mutation in
phosphatidylinositol-glycan complementation class A. In bone marrow examination, it is
hypoplastic.

Clinical Manifestation (Prepared by: Trisha May D. Artos)

The most common clinical manifestations and implications of PNH are those
associated with hemolytic anemia, thrombosis, and bone marrow failure. The severity of
anemia varies according to the main type of RBC, the degree of hemolysis, and the
existence of bone marrow failure. The intravascular hemolysis causes dark/tea-colored urine
(hemoglobinuria) and jaundice. Furthermore, during intravascular hemolytic events, free
hemoglobin rapidly scavenges and eliminates nitric oxide (NO). Smooth muscle dystonia
(esophageal spasms, dysphagia, erectile dysfunction, abdomen and back pain) or platelet
activation and thrombosis can result from low NO levels. Hepatic vein thrombosis is the
most prevalent thrombotic manifestation, which obstructs venous outflow from the liver,
resulting in a significant, often deadly consequence. Patients may potentially develop
chronic kidney disease as a result of renal tubule damage caused by microvascular
thrombosis and iron buildup during episodes of hemoglobinuria 9.

CLINICAL FINDING CAUSE

Symptoms of Anemia: Intravascular hemolysis

● Fatigue Bone marrow failure
● Shortness of breath

Thrombosis: Intravascular hemolysis

● Budd-Chiari syndrome (hepatic vein Platelet activation caused by depletion of
thrombosis) nitric oxide by free hemoglobin
● Deep vein thrombosis
● Portal hypertension
● Pulmonary embolism
● Stroke

Smooth muscle dystonia: Intravascular hemolysis

● Abdominal and/or back pain Depletion of nitric oxide by free hemoglobin
● Dysphagia
● Erectile dysfunction
● Esophageal spasms
● Fatigue

Dark Urine Intravascular hemolysis

Hemoglobinuria

Jaundice Increased serum indirect bilirubin

Chronic kidney disease/renal tubule Intravascular hemolysis

damage Microvascular thrombosis due to platelet
Activation of iron caused by repeated
hemoglobinuria
 CHAPTER IV

LABORATORY TEST RESULTS

(Prepared by: Re-anne Liegh E. Flauta)

Requested Test Unit Reference Interpretati Rationale

Tests Value Range on

COMPLETE BLOOD COUNT (CBC)

Hemoglobin 102 g/L 110-165 LOW This suggests that the

patient is suffering from
anemia, specifically
acquired aplastic anemia.
PNH develops in the
setting of autoimmune
bone marrow failure, as in
the majority of cases with
acquired aplastic
anemia.13

RBC 3.3 X 1012/L 3.8-5.8 LOW PNH, or Paroxysmal

nocturnal hemoglobinuria,
is a rare blood disorder
that causes red blood cells
to rupture. It occurs when
the surface of a patient's
blood cells lacks a protein
that protects them from
the immune system. It
also indicates anemia.

Hematocrit 0.36 L/L 0.37-0.47 LOW Another sign of anemia is

a low hematocrit. Iron
deficiency in PNH patients
is most commonly caused
by urinary iron losses due
to prolonged intravascular
hemolysis.14

MCV 108 fL 80-100 HIGH High MCV levels indicate

that red blood cells are
excessively large,
indicating macrocytic
anemia. Several causes,
including a lack of folate
or vitamin B12, can
contribute to this
condition.15
 MCH 30.9 pg 26.0-32.0 NORMAL MCH measures the
amount of hemoglobin per
each red blood cell.
Women's MCH may be
lower than men's since
they lose blood during
their periods.

MCHC 283 g/L 330-360 LOW Low MCHC levels might

result in hypochromia, or
paler red blood cells.
Hypochromia is a
symptom of anemia. Since
hemoglobin is low, MCHC
is also low.

WBC 4.7 X 109/L 4.0-10.0 NORMAL A normal white blood cell

count can indicate that
your immune system is
functioning properly.

Platelet 67 X 109/L 150-450 LOW Thrombocytopenia can

occur as a result of
reduced platelet
production. Patients with
PNH have bone marrow
failure such as aplastic
anemia.16

RDW 28 % 11.5-14.5 HIGH A high RDW count results

in macrocytic anemia,
which causes red blood
cells to grow larger than
normal. A high RDW and
MCV indicate a B12 or
folate deficiency,
macrocytic anemia, or
chronic liver disease. A
high RDW has been linked
to conditions such as
autoimmune disorders.17

Reticulocyte 10.8 % 0.5-2.5 HIGH If the percentage is

significantly higher,
reticulocytes are said to
be large enough to induce
an increase in mean cell
volume (MCV). In
hemolytic diseases,
whether intrinsic or
extrinsic, reticulocyte
percentages are higher.18
 WBC Differential % Absolute Count Interpretati Rationale
on

Neutrophils 63 2.961 x 109/L NORMAL A normal white blood cell

count indicates that your
Band 5 0.235 x 109/L NORMAL immune system is in good
condition. The amount of
white blood cells in the
Lymphocytes 27 1.296 x 109/L NORMAL
body at any given time
can contribute to the
Monocytes 4 0.188 x 109/L NORMAL detection of an infection.
Any errors from the
Eosinophils 1 0.047 x 109/L NORMAL normal range of WBC
counts could indicate the
presence of an underlying
Basophils 0 0 x 109/L NORMAL
disease.

Metamyelocytes 0

Myelocytes 0

Promyelocytes 0

Blasts 0

Promonocytes 0

nRBC/100 WBC 0

Test Values Rationale

RBC Morphology Moderate anisocytosis, The RBC Morphology suggests that

macrocytes, mild polychromasia; hemolytic anemia is a possibility.
presence of target Hemolytic anemia can be caused by
cells and elongated cells moderate anisocytosis, macrocytes, and
mild polychromasia. This happens when
your body is unable to produce RBCs as
quickly as they are destroyed. In
addition, the presence of target cells and
elongated cells can indicate iron
deficiency anemia.

WBC Morphology Normal in number and The number of white blood cells in the
morphology body at any given time can help in the
detection of any underlying infection.
Any variation from the typical range of
WBC counts could be due to an
underlying disease.
 Platelet Moderate thrombocytopenia; Thrombocytopenia with small platelets
Morphology normal in morphology indicates a platelet production
abnormality.
Platelet size is diagnostically important,
especially when it is set in relation to
platelet count. Thrombocytopenia with
small platelets indicates decreased
platelet production in the bone marrow,
as seen in aplastic anemia.19

(Prepared by: Kazee Ulaiza K. Agting)

Requested Normal Value Test Value Interpretation Rationale

Tests

SERUM CHEMISTRY

Total 0.1-1.2 mg/dL 7.5 HIGH A high result of total Bilirubin

Bilirubin may be a problem with the
liver if bilirubin levels are
increased. It is possible that
elevated bilirubin levels
might produce jaundice, a
yellow coloring of the eyes or
skin. This is most typically
caused by bilirubin retention,
although it can also be
caused by retention of other
chemicals. In PNH, severe
hemolysis can lead to a loss
of blood cells. Bilirubin must
be processed by the liver in
order to be eliminated from
the body. As previously
noted, conjugation of
bilirubin is necessary for this.
A low quantity of the
colorless substance, called
urobilinogen, is also expelled
in the urine as a byproduct
of bilirubin production. The
liver's function may be
normal in certain cases, or it
may be operating at its
highest capacity to make up
for other problems. This is
not the situation with hepatic
jaundice, which is caused by
an intrinsic liver abnormality
or illness. A high level of
bilirubin in the bloodstream,
 such as that observed in
acute and chronic hemolytic
anemia, is the most
prevalent cause of this
condition. Due to increased
red blood cell destruction
caused by hemolytic anemia,
the liver receives more
bilirubin from the
bloodstream.20

Indirect 0.1-1.0 mg/dL 5.7 HIGH A disease in liver is the

Bilirubin outcome of increasing
indirect bilirubin levels (may
be elevated in cases of PNH)
Albumin binds bilirubin,
which is then delivered to
the liver. The primary
pigment in bile is bilirubin,
which is formed when red
blood cells are broken down.
In this process, hemoglobin
is liberated from red blood
cells. Heme, globin, and iron
are all products of
hemoglobin degradation.
Transferrin binds the iron,
which is then recycled to the
body's iron reserves in the
liver and bone marrow. All of
the amino acids in the globin
are recycled by the body.
Bilirubin is formed from the
heme component of
hemoglobin. Unconjugated
or indirect bilirubin is the
term given to this kind of
bilirubin. The liver must first
conjugate unconjugated
bilirubin before it can be
excreted from the body.
Bilirubin total is divided by
total conjugated bilirubin to
arrive at unconjugated
bilirubin (indirect bilirubin).20

AST 4-36 IU/L 214 HIGH Damage to the liver can be

detected by an elevated
Aspartate Aminotransferase
(AST) (increased liver
enzymes). The human body
has a high concentration of
 AST. The heart, liver, and
skeletal muscle have the
largest concentrations,
whereas the kidney,
pancreas, and erythrocytes
have the lowest. Pulmonary
embolism is a common cause
of elevated AST levels. In
patients with congestive
heart failure, AST levels may
rise as a result of a lack of
blood flow to the liver. AST
levels can rise as a result of
skeletal muscle illnesses
such as muscular
dystrophies or inflammatory
conditions. A high serum AST
content is possible if
hemolysis is performed,
hence it should be avoided.20

LD 259-613 IU/L 1550 HIGH LD is a widely distributed

enzyme in the human body.
When cells in the body are
injured or killed, it is
released into the
bloodstream and acts as a
broad, nonspecific indicator
of cellular injury. Anemia,
renal disease, and liver
disease can all cause
excessive levels of lactate
dehydrogenase (Laboratory
findings in PNH include signs
of hemolysis such as
negative direct antiglobulin
test, elevated levels of
serum lactate
dehydrogenase, elevated
reticulocyte counts, low or
absent serum haptoglobin,
and hemoglobinuria).
Metastatic liver cancer may
result in elevated serum
levels. Blood hemoglobinuria
may be caused by illnesses,
alcohol or a variety of other
factors such as stress and
exercise. Thrombosis in veins
is the leading cause of
mortality and serious
complications in individuals
 with PNH, accounting for up
to 50% of cases.21

Serum 60-270 mg/dL 10 LOW Many inflammatory disorders

Haptoglobin are accompanied by a rise in
haptoglobin, an acute-phase
positive protein. It is not
common practice to utilize
haptoglobin levels to
diagnose or monitor these
disorders. When haptoglobin
is present, it binds to free
hemoglobin and prevents the
loss of iron in the urine.
Haptoglobin concentrations
are primarily used to
evaluate the possibility of
hemolytic anemia and
distinguish it from anemias
caused by other reasons.
Low Red blood cells from
serum Haptoglobins are
depleting faster than they
can be produced.
Haptogoblin's Serum. The
reticuloendothelial system
destroys haptoglobin in
patients with hemolytic
anemia, resulting in a lower
haptoglobin content. In
cases when the haptoglobin
concentration is normal and
the reticulocyte count is
high, the anemia is likely
caused by the spleen and
liver destroying red blood
cells. To ensure that the
haptoglobin concentrations
are within the reference
range, the hemoglobin is not
released into the circulation.
It is probable that the
anemia is not related to red
blood cell breakdown if both
the haptoglobin
concentration and the
reticulocyte count are within
the reference range. The
liver may not be making
enough haptoglobin if
haptoglobin concentrations
 fall without any evidence of
hemolytic anemia.20

Requested Normal Test Value Interpretation Rationale

Tests Value

URINALYSIS

Color Yellow Red ABNORMAL

(light/pale to The presence of blood is a
dark/deep common factor in cases in
amber) which urine seems to be an
abnormal color. When blood
is present in the urine, it will
often become red. The
breakdown of skeletal
muscle will eventually lead
to the formation of
myoglobin. In addition to
anemia and
thrombocytopenia, a
urinalysis revealed that there
was an excessive number of
red blood cells. If a sample
is both red and clear,
respectively, this indicates
the presence of hemoglobin
and myoglobin 22. An
enlarged prostate, malignant
and non-cancerous tumors
of the bladder and urinary
system as well as kidney or
bladder stones can all lead
to hematuria (blood in the
urine) 23.

Clarity Clear Clear NORMAL

It is quite likely that urine
that has been normally
released and has not been
affected in any way will be
clear. The presence of urine
that is perfectly transparent
and free of any particles that
can be seen is indicative of a
healthy urinary tract. It's not
always normal to have
crystal-clear urine. Most
abnormalities in clear urine
 will be found in microscopic
analysis 22.

Specific 1.010 - 1.030 1.025 NORMAL Urine samples are

Gravity considered to be
hypersthenuric if they have a
specific gravity that is
greater than 1.010. It is not
always the case that urine
that has been concentrated
by the kidneys will have a
hypersthenuric quality to it.
Normal random specimens
have a range that can go
anywhere from 1.002 to
1.035, and this range is
determined by the patient's
degree of hydration 22.

pH 4.5 - 8.0 6.0 NORMAL A healthy first morning

specimen typically has a pH
of 5.0 to 6.0, with a higher
alkaline pH detected after
eating (alkaline tide). In a
typical random sample, the
acidity level can range from
about 4.5 to about 8.0. No
typical values are assigned
to the urine pH, and it must
be assessed in conjunction
with other patient
information, such as blood
acid–base content, renal
function, presence or
absence of a urinary tract
infection, the patient's diet
and age of specimen
collection 22.

Glucose Negative / Negative NORMAL

0 - 15 mg/dL Negative results may
indicate either a normal or
critically low blood glucose
level which is called
hypoglycemia. Dumping
syndrome frequently results
in hypoglycemia. There is
usually no glucose within
urine. As a result, further
examination is needed 22.
 Albumin Negative +1 ABNORMAL Kidney disease can cause
albuminuria, which implies
that the patient is excreting
too much albumin in their
urine. Among the several
serum proteins that may be
identified in human urine,
albumin is the most
prevalent. Because the
glomerulus does not filter
most albumin that enters it,
and because the tubules
reabsorb much of the filtered
albumin, the typical urine
albumin concentration is low,
despite its high plasma
concentration. The selective
filtration of the glomerulus
may be overridden by higher
blood pressure entering the
glomerulus, resulting in
more albumin entering the
filtrate. Disorders of tubular
reabsorption can also induce
an increase in albumin since
the usually filtered albumin
is unable to be reabsorbed
22
.

Hemoglobin Negative +2 ABNORMAL Hemoglobin, the result of the

breakdown of red blood
cells, may be seen in the
urine. Hemoglobinuria - or
hemoglobin in the urine - is
the medical term for this
condition. If more than five
cells per microliter of urine
are found to contain blood,
visual inspection cannot be
relied upon to identify the
presence of hemoglobin, the
red portion of the
hemoglobin red blood cells.
Dilute, alkaline urine has
been shown to cause
hemoglobinuria by causing
red blood cells to lyse.
Normally, massive
hemoglobin-haptoglobin
complexes in the circulation
prevents the glomerular
filtration of hemoglobin. In
 hemolytic anemias, the
quantity of free hemoglobin
surpasses the haptoglobin
level 22.

Perl’s Stain Negative +2 ABNORMAL

on Staining with Perl's Prussian
sediments Blue identifies granules of
hemosiderin in cells and
casts that contain iron. The
presence of yellow-brown
granules in renal tubular
epithelial cells, casts, or the
urine sediment is a sign of
kidney disease. Hemosiderin
should be shown. An
acquired hemolytic condition
called paroxysmal nocturnal
hemoglobinuria (PNH) is
characterized by
hemosiderinuria, which is
produced by intravascular
hemolysis (PNH) 22.

(Prepared by: Christian T. Arinzol)

Requested Normal Test Value Interpretatio Rationale

Tests Value n

OTHER / FURTHER TESTS

Direct Negative Negative NORMAL A positive direct Coomb’s test

Coomb’s Test indicates that immunoglobulin
bound in vivo to RBCs has
been detected 24. In this case,
having a negative direct
coomb’s test result makes
sense as the hemolysis in PNH
is not caused by antibodies,
but caused by a mutation in
the PIGA gene, leading to the
absence of GPI-anchored
proteins which ultimately
causes cellular abnormalities,
such as the deficiency of CD55
and CD59 13.

Liver No Mild ABNORMAL This could possibly align with

Ultrasound hepatomegaly hepatomegal the elevated AST results as
; no stones; y; no signs there is mild hepatomegaly.
 no hepatic of stones; Hematologic disorders are
mass no hepatic likely to be accompanied with
mass liver involvement 25.

A journal mentioned that in

PNH is a common serious
cause of portal vein thrombosis
25
. In this case, it is still at its
mild stage. The patient’s
reported jaundice, however, is
indicative that there is slight
impairment in hepatocellular
function 26 and may require
further investigation.

Immuno- Presence of CD55 and ABNORMAL CD55 and CD59 are two
phenotyping CD55 and CD59 absent GPI-anchored proteins that
CD59 on 78% of play a role in controlling
RBCs complement activation 13. Due
to a mutation in the PIGA
gene, deficiency of these
GPI-anchored proteins
happens– complement action
becomes uninhibited and this
leads to the
complement-mediated
hemolysis characteristic of PNH
27
.

Sugar Lysis Negative Positive ABNORMAL The sugar lysis test or sucrose
Test lysis test is one of the
traditional complement-based
tests used to screen for PNH.
[28] With the patient testing
positive for this test, it is a
possible diagnosis for PNH.

However, as these are

traditional tests, they are now
obsolete as this test is
considered tedious and may
give false positive results 28.
Therefore, when this test is
performed, a suggested further
testing with flow cytometry is
done to confirm diagnosis.

Cytogenetics No mutation Mutation in PNH, most of the time, is

phosphatidyl known to be caused by a
inositol-glyc ABNORMAL mutation in a gene called
an phosphatidylinositol glycan
anchor biosynthesis, class A or
 complement PIG-A 28. This PIG-A gene is
ation class A required in GPI-anchor
biosynthesis 13, which helps
control complement action.

Therefore, by having a
mutation in this gene as
detected in this test performed
on the patient, PNH
manifestations occur and it
now leads us to a possible
diagnosis of PNH.

Bone Marrow Normal Hypoplastic ABNORMAL PNH may manifest with bone
Examination marrow failure 28.PNH in itself
is a case of bone marrow
failure disorder as a mutation
in the PIG-A gene in a clone of
bone marrow stem cells leads
to a defective production of
GPI-anchored proteins, hence
the hemolysis manifestations
29
.

Bone marrow transplantation is

the most preferred cure for
PNH, but is only reserved for
patients that are not that
responsive with Eculizumab 13.

Osmotic Negative Negative NORMAL PNH has normal OFT as there

Fragility Test is no problem with the cell
structure when not triggered
by complement activation.
Therefore, it tests as negative
here as the components of the
cell required to keep it stable
in a hypotonic condition are
not affected.

Comparing this to the sugar

lysis test, the added solution of
sucrose activates the
complement pathway,
therefore,
complement-sensitive cells are
lysed.
Ham’s Test:

1 2 3 4 5 6 7 Rationale

Fresh normal 0.05 0.05 0.05 0.05 Ham’s test is

serum mL mL mL mL another test for
PNH, which
Patient serum 0.05 checks whether
mL red blood cells
become more
fragile when
Heat-inactivate 0.05 0.05
placed in mild
d normal serum mL mL
acid, such as the
0.2 N HCl used in
0.2 N HCl 0.05 0.05 0.05 0.05 0.05
the test.
mL mL mL mL mL
Complement
activation can be
50% Px red 0.05 0.05 0.05 0.05 triggered by low
cells mL mL mL mL pH 30, and
therefore the
50% normal 0.05 0.05 0.05 complement-medi
red cells mL mL mL ated lysis
mechanism of
PNH also
Px Result Trace ++ + 0 0 0 0
happens.

The patient’s
result tested
positive for
hemolysis as seen
in tubes 2 and 3,
containing the
patient’s red cell
suspension and
the mild acid.

The patient was started on Eculizumab and there has been a significant improvement
since then. This drug is the first drug treatment of choice for PNH patients 13, as when this is
taken around the early stage, it may prevent further complications 28. Eculizumab has also
been proven to be highly effective in the control of hemolysis associated with PNH, resulting
in the improvement of the history of PNH patients. [31] It was mentioned earlier in the
tables above that bone marrow transplant is the most effective choice for treatment of
classical PNH 14, but it should only be the option when there is minimal reaction to
Eculizumab.
 CHAPTER V
(Prepared by: Maria Trisha A. Soriano)
SUMMARY

The case of a 31 year old Caucasian woman after visiting her family doctor as she
noticed that her urine was tea-colored for the previous seven days. Knowing that her
menstrual cycle is regular but this time her monthly period had ended two weeks earlier.
She had never suffered kidney stones, weight loss, night sweats, hemoptysis, or melena
before. There was no drug misuse either. Her vital statistics were unexceptional. A complete
blood count (CBC), urinalysis and renal ultrasonography were all normal at the initial exam.
Her urine pregnancy test was negative too but then she developed jaundice and severe
abdominal pain after three days. Since the patient was receiving Eculizumab, the patient's
condition has significantly improved. The attending physician requested a complete blood
count, serum chemistry, and urinalysis, and there are other tests that are involved such as
Direct Coomb’s Test, Liver Ultrasound, Immunophenotyping, Sugar Lysis Test, Cytogenetics,
Bone Marrow Examination, Osmotic Fragility Test and Ham’s test. After evaluating her
results, it is suspected that she has PNH (Paroxysmal Nocturnal Hemoglobinuria). PNH is a
disease in which the absence of glycosylphosphatidylinositol (GPI) anchor causes red blood
cells to lyse.

The patient’s findings demonstrate a decrease in hemoglobin, RBC, and hematocrit

with high MCV indicating that she is experiencing hemolytic anemia. She also has
thrombocytopenia with small platelets in which she’s experiencing platelet production
abnormality and her reticulocyte counts are elevated indicating regeneration of new red
blood cells. She has a normal result in WBC differential. As the patient is having tea-colored
urine this may indicate that the patient is having hemolysis in her urine. The total bilirubin
and indirect bilirubin shows high results, this may result in liver disease and jaundice to the
patient. A low serum haptoglobin is one of the parameters that may also indicate that the
patient has PNH. Moreover, the patient’s immunophenotyping was abnormal as her CD55
and CD59 were absent on her RBCs, this may happen due to the mutation of PIGA gene
causing it to not produce these proteins resulting in immune system problems for her. In
addition, Cytogenetics, Sugar lysis test, and bone marrow examination revealed
abnormalities in examination.

SUGGESTED FURTHER TESTING/WORK-UP

(Prepared by: Kazee Ulaiza K. Agting,
Re-anne Liegh E. Flauta, & Christian T. Arinzol)

● FLOW CYTOMETRY
Ham’s test and sucrose lysis test are the traditional tests for diagnosing PNH
24
, however, they have become obsolete as it can be taxing, certain conditions should
be met, and requires great care in order not to produce false results 28.Flow
cytometry, on the other hand, has become the gold standard in diagnosing PNH as it
allows evaluation of population of blood cells 32. This test has a higher sensitivity in
detecting even tiny PNH populations 32. Being the gold standard test for this disease,
this will help in confirming the diagnosis of PNH.

● FLUORESCENT AEROLYSIN (FLAER)-BASED PNH TESTING

 Studies have suggested that this test is at par with flow cytometry in
detecting PNH 33. It is known to be cost-effective and is reported to have better
sensitivity in cases such as PNH 33. The FLAER test uses the Aerolysin toxin from the
bacteria Aeromonas hydrophila which was found out to be a highly specific ligand for
GPI molecule on WBCs, therefore, making it a good indicator for the absence of
GPI-deficient cells in PNH 34. It needs further study and is still far from being another
gold standard, but I believe it is worth a shot with the odds looking good.

GROUP INSIGHTS

AGTING, Kazee Ulaiza K.

Accuracy and precision is a crucial part of the job in the medical field. Even
the slightest mistake in the results could lead to a whole different diagnosis. There
are new tests that were unfamiliar to me and I had to learn it just to help diagnose
the patient in this case. PNH is quite an interesting disease to learn about, for as I
was studying it, I could follow the flow– from the mutation, to the GPI-deficiency, all
the way to its hemolysis manifestation. There are still a lot that we don’t know, and
that is a given as part of human limitation, however, new diagnostic tests for it are
being developed and it was nice to learn a little about it. From the discovery of
Eculizumab, to introducing FLAER as a possible another gold standard test for this
disease. Having PNH is potentially life-threatening, therefore, the studies advancing
the knowledge we have regarding this disease is such a big help. As far as I know,
the only cure is bone marrow transplantation and even that may have complications,
while for drug treatment, Eculizumab is an option though it only controls the disease,
not entirely cures it. I don’t know but I hope I’ll at least be alive when a cure is
discovered so I can go back to this case study and reminisce about doing it during
late nights.

AGUSTIN, John Kenneth C.

"The more you know, the more you realize you don't know," Aristotle, a
Greek philosopher, famously said of discovering the unknown. This is especially true
in the case of Paroxysmal Nocturnal Hemoglobinuria, which is a fascinating case
study of the complicated interplay between cell-intrinsic alterations caused by PIGA
loss and extrinsic variables that result in PNH clonal growth in patients. The absence
of GPI-anchored NKG2D ligands, a missing GPI antigen, a loss of costimulatory
molecules, or altered cytokine sensitivity are all plausible pathways that could
increase PNH clonal proliferation, according to landmark studies.
Due to a lack of suspicion, PNH is commonly misdiagnosed and treated as
anemia. The key diagnostic problems in resource-constrained settings are a low level
of suspicion and a lack of investigations. The cost of definitive treatment once a
diagnosis has been determined remains an even greater barrier.

ARINZOL, Christian T.
The importance of laboratory testing cannot be overstated. In situations
where any single mistake or error is not an excuse or an alternative, precise and
accurate analysis is a must. Medical professionals must confirm the accuracy of
healthcare data or outcomes with every specimen that enters the laboratory. If we
want to know which medications will be most effective for particular patients, we
need to collect more data for us to know what are the specific reasons why certain
diseases occur and can now better understand the case for Improved methods about
disease prevention, detection, and treatment. Errors in laboratories and medical
facilities can have a severe impact on a healthcare facility's image and result in
significant expenses for the institution and the government.

ARTOS, Trisha May D.

It is critical to be accurate and precise when obtaining scientific
measurements. This is significant because inaccuracies in results might be caused by
faulty equipment, inadequate data processing, or human error. It is critical to reduce
bias and error, as well as be precise and accurate in data collecting, in order to
obtain the most dependable results in a scientific investigation. Because of lack of
accuracy there has been a problem in diagnosing the patient and this can be
prevented if there is a proper accuracy and precision in order to prevent such
problems in the near future.

FLAUTA, Re-anne Liegh E.

Patients, healthcare professionals, healthcare systems, and society all benefit
from the industry's breakthrough equipment and tests. We saw how difficult it is to
diagnose a patient as we reviewed the case that was assigned to us because all of
your interpretations must be correct at all times. It is critical to thoroughly examine
the information provided when conducting a case study. There would be several
problems if you misdiagnose the patient, not only for the patient but also for the
medical technologist who was assigned to run the samples. Interpret the patient's
results and make sure they correspond to the patient's signs and symptoms to
ensure that everything is in order. Medical technologies can benefit healthcare
systems and society by helping citizens in remaining socially and economically
engaged and minimizing serious complications from chronic disease.

MAMON, Abigail Thea C.

As we studied the case that was assigned to us, it made us realize how hard it
is to diagnose a patient because you have to be accurate about all your
interpretations at all times. There are many kinds of anemia, almost all the same but
with slight differences from each other. In diagnosing a patient, you have to be sure
that the results are accurate and reliable. Once you misdiagnose the patient, there
will be a lot of consequences, not just for the patient but also for the medical
technologist assigned to run the samples. To make sure that everything’s right,
interpret the patient’s results as well as make sure that they correlate with the signs
and symptoms of the patient. Also, I realized that laboratory tests are important in
diagnosing a patient. Sometimes just by looking at one parameter, you can actually
diagnose a patient already.

SORIANO, Maria Trisha A.

 All cases are somewhat complex in terms of determining its disease but it can
be accomplished with the aid of the patient's results. In this case, examining the
patient and explaining the results as to how it was acquired in that manner is very
critical. However, this case could be quite useful in the near future if we encounter
this similar situation. In addition, this case also gives me knowledge in PNH disease,
in which it occurs when a person's body produces abnormal clone red blood cells that
lack the complement regulatory proteins CD55 and CD59. Thus, the absence of these
protective proteins causes the complement system to destroy the cells, resulting in
general hemolysis.

SUNIO, Ray Brandon B.

In conducting/doing a case study/presentation, it is really important to
carefully assess the given information. When a patient seeks medical attention, a
systematic process of obtaining information, synthesizing and interpreting that
information, and determining a working diagnosis begins. Conducting patient
interviews and assessing clinical history, physical examination, and diagnostic tests
are all ways of gathering information that may be useful in identifying a patient's
health problem. For this case, the group identified the diagnosis as Paroxysmal
Nocturnal Hemoglobinuria (PNH).
Throughout the procedure, there were assessments and opinions about
whether we had gathered enough information to make a diagnosis. Our diagnosis
was still riddled with uncertainty and doubt. However, we arrived at the diagnosis
after extensive reading and information gathering. PNH is a disease that causes
significant morbidity and mortality, and this case study allowed us to understand
why/how it arises and how it affects individuals.
 CHAPTER VI
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(Prepared by: Ray Brandon B. Sunio,
Abigail Thea C. Mamon, & Trisha May D. Artos)

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 CHAPTER VII
DOCUMENTATION

CASE STUDY GROUP MEETING LAST MAY 19, 2022

GRAMMARLY SIMILARITY INDEX REPORT