Unexpected Glycosylated Hemoglobin Results

A Case Study Presented to the Faculty of

Department of Medical Laboratory Science

In Partial Fulfillment of the Requirements in

Medical Laboratory Science Clinical Internship

Bernardino, Jayson
Gabuya, Sabrina
Geguinto, Jessa Marie
Guiloreza, Ralph
Jayuma, Meah Lou
Maloloy-on, Micaela Nina
Olayan, Christine Dennise
Pepito, Rexielyn Mae

Arvin Mikel Wee, RMT

CEP Instructor

March 2018
 OBJECTIVES

This case study aims to:

1. To identify the different types of method used for HBA1c measurement.

2. To determine the necessary actions to be done when a spurious HBA1c result

occurs in a patient.
3.
4. To recognize how the different variants of hemoglobin cause interference
with the HBA1c methods.

5. To identify the different abnormal hemoglobin responsible for the patient’s

HBA1c result and discuss the following clinical significance.
 CHAPTER I

INTRODUCTION

The HbA1c test, also known as the haemoglobin A1c or glycated

haemoglobin test is an important blood test that gives a good indication of how well
your diabetes is being controlled. Together with the fasting plasma glucose test, the
HbA1c test is one of the main ways in which type 2 diabetes is diagnosed. HbA1c
tests are not the primary diagnostic test for type 1 diabetes but may sometimes be
used together with other tests. HbA1c (glycated haemoglobin, haemoglobin A1c)
occurs when haemoglobin, the oxygen-carrying protein in red blood cells, becomes
bonded with glucose in the bloodstream. The bonding with glucose is called
glycation. The higher a person’s blood glucose levels have been, the higher the
number of red blood cells that will have become glycated, and therefore the higher
HbA1c level they will have. Note that red blood cells exist in the body for around 3
months, therefore an HbA1c levels generally reflects a person’s blood glucose levels
over the previous 8-12 weeks.

The test for hemoglobin A1c is dependent on the chemical (electrical)

charge on the molecule of HbA1c, which is different from the charges on the other
components of hemoglobin. The molecule of HbA1c also is different in size from the
other components. HbA1c may be separated by charge and size from the other
hemoglobin A components in blood by a procedure called high pressure (or
performance) liquid chromatography (HPLC). Whilst HbA1c tests are usually
reliable, there are some limitations to the accuracy of the test. For example, people
with forms of anaemia may not have sufficient haemoglobin for the test to be
accurate and may need to have a fructosamine test instead. Being pregnant or
having an uncommon form of haemoglobin (known as a haemoglobin variant) can
also return an inaccurate HbA1c, while readings can also be affected by short term
issues such as illness as they can cause a temporary rise in blood glucose. Because
of the way the HbA1c test measures blood sugar, if you have higher blood sugar
levels in the weeks leading up to your HbA1c test, this will have a greater impact on
your test result than your glucose levels 2 to 3 months before the test.

The case study presented a 55-year-old woman who went to the

internal medicine clinic for a routine visit with a history of hepatitis B,
hyperlipidemia, hypertension, anemia, and depression. Laboratory tests 3 months
previously had revealed an impaired fasting glucose concentration of 5.9 mmol/L
(106 mg/dL) [reference interval, 3.9–5.6 mmol/L (70–100 mg/dL)]. Therefore, a
hemoglobin (Hb)A1c analysis was performed. The initial Hb A1c evaluation by
cation-exchange HPLC (CE-HPLC) (Hb A1c Program on the VARIANT II TURBO Link
System; Bio-Rad Laboratories) showed an Hb A1cvalue of 115.8% (reference
interval, 4.0%–6.0%).
 CHAPTER II

PATIENT’S DATA

 Age: 55 years old

 Sex: Female

 Medical History:
o Hepatitis B
o Hyperlipidemia
o Hypertension
o Anemia
o Depression

Laboratory Test Results:

Clinical Chemistry

TESTS NORMAL VALUES PATIENT’S RESULT

Fasting Blood 3.9-5.6 mmol/L 5.9 mmol/L
Glucose
HBA1c (CE- 4.0-6.0% 115.8%
HPLC)
Citrate Agar Hb S
Electrophoresis Another band with a mobility
(QuickGel similar to Hb F
Acid; Helena
Laboratories)

Hb Variant Hb A: 95%-98% 2.7% Hb A

Analysis (Bio- Hb A2: 1.5%-3.5% 3.2% Wild-type Hb A2
Rad VARIANT Hb F: < 2% (age-dependent) <1.0% Hb F
CE-HPLC β- Hb S: Absent 37.4% Sickle cell Hb (Hb S)
Thalassemia 53.0 % P2
Short
Program)
 CHAPTER III

DEFINITION OF THE CASE, ANATOMY AND PHYSIOLOGY, PATHOPHYSIOLOGY

A. DEFINITION OF THE CASE

Hemoglobin A1c (HbA1c) is a result of the nonenzymatic attachment of a

hexose molecule to the N-terminal amino acid of the hemoglobin molecule. The
attachment of the hexose molecule occurs continually over the entire life span of
the erythrocyte and is dependent on blood glucose concentration and the duration
of exposure of the erythrocyte to blood glucose. Therefore, the HbA1c level reflects
the mean glucose concentration over the previous period (approximately 8-12
weeks, depending on the individual) and provides a much better indication of long-
term glycemic control than blood and urinary glucose determinations. Diabetic
patients with very high blood concentrations of glucose have from 2 to 3 times
more HbA1c than normal individuals.

Four basic types of methods are used most commonly to measure HbA1c:
immunoassay, ion-exchange high-performance liquid chromatography (HPLC),
boronate affinity HPLC, and enzymatic assays. Most immunoassays measure HbA1c
specifically; antibodies recognize the structure of the N-terminal glycated amino
acids (usually the first 4–10 amino acids) of the Hb β chain. Ion-exchange HPLC
separates Hb species based on charge differences between HbA1c and other
hemoglobins. With boronate affinity methods, m-aminophenylboronic acid reacts
specifically with the cis-diol groups of glucose bound to Hb. This method measures
total glycated GHB, including HbA1c and Hb glycated at other sites, and tends to
demonstrate the least interference from the presence of Hb variants and
derivatives. The enzymatic method currently available measures HbA1c by using an
enzyme that specifically cleaves the N-terminal valine.

The method initially used to analyze the patient’s Hb A1cwas a CE-HPLC

assay. If DNA sequencing of ß-globin genes is performed, there’s a possible
demonstration of Hb Raleigh which led to a falsely increased HbA1c value. Hb
Raleigh is unique in that a mutation (T to C) at the second base of the codon
encoding the first amino acid of the chain changes the N-terminal valine to an
alanine residue. This substitution would not necessarily induce any change in Hb A
charge except that N-terminal alanines are immediately acetylated to acetylalanines
soon after translation. This acetylation decreases the positive charge to one similar
to Hb A1c. Therefore, the retention times of Hb A1c and Hb Raleigh are virtually
identical; the elution peaks of these 2 hemoglobins fall in the same window in the
chromatogram.
 In their case, the patient was heterozygous for Hb A and Hb Raleigh, and the
falsely increased Hb A1c value of 115.8% included a small fraction of real Hb A1c.
In our case, the patient was heterozygous for Hb S, in which the N-terminal valine
of the ß-chain was glycated as Hb S1c, and Hb Raleigh, in which the N-terminal
acetylalanine of the ß-chain could not be glycated. Therefore, Hb A1c did not exist
in this patient. The spurious Hb A1c value (115.8%) primarily represented Hb
Raleigh.

B. ANATOMY AND PHYSIOLOGY

The liver is the largest organ in the body, normally weighing about 1.5kg
(although this can increase to over 10kg in chronic cirrhosis).
The liver is the main organ of metabolism and
energy production; its other main functions include:
 Bile production
 Storage of iron, vitamins and trace elements
 detoxification
 conversion of waste products for excretion by
the kidneys

The liver is a roughly triangular organ that

extends across the entire abdominal cavity just
inferior to the diaphragm. Most of the liver’s mass is
located on the right side of the body where it
descends inferiorly toward the right kidney. The liver
is made of very soft, pinkish-brown tissues encapsulated by a connective tissue
capsule. This capsule is further covered and reinforced by the peritoneum of the
abdominal cavity, which protects the liver and holds it in place within the abdomen.

The tubes that carry bile through the liver and gallbladder are known as bile
ducts and form a branched structure known as the biliary tree. Bile produced by
liver cells drains into microscopic canals known as bile canaliculi. The countless bile
canaliculi join together into many larger bile ducts found throughout the liver.

These bile ducts next join to form the larger left and right hepatic ducts, which
carry bile from the left and right lobes of the liver. Those two hepatic ducts join to
form the common hepatic duct that drains all bile away from the liver. The common
hepatic duct finally joins with the cystic duct from the gallbladder to form the
common bile duct, carrying bile to the duodenum of the small intestine. Most of the
bile produced by the liver is pushed back up the cystic duct by peristalsis to arrive
in the gallbladder for storage, until it is needed for digestion. The blood supply of
the liver is unique among all organs of the body due to the hepatic portal vein
system. Blood traveling to the spleen, stomach, pancreas, gallbladder, and
intestines passes through capillaries in these organs and is collected into the
hepatic portal vein. The hepatic portal vein then delivers this blood to the tissues of
the liver where the contents of the blood are divided up into smaller vessels and
processed before being passed on to the rest of the body. Blood leaving the tissues
of the liver collects into the hepatic veins that lead to the vena cava and return to
the heart. The liver also has its own system of arteries and arterioles that provide
oxygenated blood to its tissues just like any other organ. The internal structure of
the liver is made of around 100,000 small hexagonal functional units known as
lobules. Each lobule consists of a central vein surrounded by 6 hepatic portal veins
and 6 hepatic arteries. These blood vessels are connected by many capillary-like
tubes called sinusoids, which extend from the portal veins and arteries to meet the
central vein like spokes on a wheel.

Each sinusoid passes through liver tissue containing 2 main cell types: Kupffer cells
and hepatocytes.
 Kupffer cells are a type of macrophage that capture and break down old,
worn out red blood cells passing through the sinusoids.
 Hepatocytes are cuboidal epithelial cells that line the sinusoids and make up
the majority of cells in the liver. Hepatocytes perform most of the liver’s
functions – metabolism, storage, digestion, and bile production. Tiny bile
collection vessels known as bile canaliculi run parallel to the sinusoids on the
other side of the hepatocytes and drain into the bile ducts of the liver.

The liver plays an active role in the process of digestion through the production
of bile. Bile is a mixture of water, bile salts, cholesterol, and the pigment bilirubin.
Hepatocytes in the liver produce bile, which then passes through the bile ducts to
be stored in the gallbladder. When food containing fats reaches the duodenum, the
cells of the duodenum release the hormone cholecystokinin to stimulate the
gallbladder to release bile. Bile travels through the bile ducts and is released into
the duodenum where it emulsifies large masses of fat. The emulsification of fats by
bile turns the large clumps of fat into smaller pieces that have more surface area
and are therefore easier for the body to digest.

Bilirubin present in bile is a product of the liver’s digestion of worn out red blood
cells. Kupffer cells in the liver catch and destroy old, worn out red blood cells and
pass their components on to hepatocytes. Hepatocytes metabolize hemoglobin, the
red oxygen-carrying pigment of red blood cells, into the components heme and
globin. Globin protein is further broken down and used as an energy source for the
body. The iron-containing heme group cannot be recycled by the body and is
converted into the pigment bilirubin and added to bile to be excreted from the
body. Bilirubin gives bile its distinctive greenish color. Intestinal bacteria further
convert bilirubin into the brown pigment stercobilin, which gives feces their brown
color.

The hepatocytes of the liver are tasked with many of the important metabolic
jobs that support the cells of the body. Because all of the blood leaving the
digestive system passes through the hepatic portal vein, the liver is responsible for
metabolizing carbohydrate, lipids, and proteins into biologically useful materials.
 Our digestive system breaks down carbohydrates into the monosaccharide
glucose, which cells use as a primary energy source. Blood entering the liver
through the hepatic portal vein is extremely rich in glucose from digested food.
Hepatocytes absorb much of this glucose and store it as the macromolecule
glycogen, a branched polysaccharide that allows the hepatocytes to pack away
large amounts of glucose and quickly release glucose between meals. The
absorption and release of glucose by the hepatocytes helps to maintain
homeostasis and protects the rest of the body from dangerous spikes and drops in
the blood glucose level.

Fatty acids in the blood passing through the liver are absorbed by hepatocytes
and metabolized to produce energy in the form of ATP. Glycerol, another lipid
component, is converted into glucose by hepatocytes through the process of
gluconeogenesis. Hepatocytes can also produce lipids like cholesterol,
phospholipids, and lipoproteins that are used by other cells throughout the body.
Much of the cholesterol produced by hepatocytes gets excreted from the body as a
component of bile.

Dietary proteins are broken down into their component amino acids by the
digestive system before being passed on to the hepatic portal vein. Amino acids
entering the liver require metabolic processing before they can be used as an
energy source. Hepatocytes first remove the amine groups of the amino acids and
convert them into ammonia and eventually urea. Urea is less toxic than ammonia
and can be excreted in urine as a waste product of digestion. The remaining parts
of the amino acids can be broken down into ATP or converted into new glucose
molecules through the process of

As blood from the digestive organs passes through the hepatic portal circulation,
the hepatocytes of the liver monitor the contents of the blood and remove many
potentially toxic substances before they can reach the rest of the body. Enzymes in
hepatocytes metabolize many of these toxins such as alcohol and drugs into their
inactive metabolites. And in order to keep hormone levels within homeostatic limits,
the liver also metabolizes and removes from circulation hormones produced by the
body’s own glands.

The kidneys are a pair

of organs found along the
posterior muscular wall of
the abdominal cavity. The
left kidney is located slightly
more superior than the right
kidney due to the larger size
of the liver on the right side
of the body. Unlike the other
abdominal organs, the
kidneys lie behind the
peritoneum that lines the
abdominal cavity and are
thus considered to be retroperitoneal organs. The ribs and muscles of the back
protect the kidneys from external damage. Adipose tissue known as perirenal fat
surrounds the kidneys and acts as protective padding.

They are bean-shaped with the convex side of each organ located laterally
and the concave side medial. The indentation on the concave side of the kidney,
known as the renal hilus, provides a space for the renal artery, renal vein, and
ureter to enter the kidney. A thin layer of fibrous connective tissue forms the renal
capsule surrounding each kidney. The renal capsule provides a stiff outer shell to
maintain the shape of the soft inner tissues. Deep to the renal capsule is the soft,
dense, vascular renal cortex. Seven cone-shaped renal pyramids form the renal
medulla deep to the renal cortex. The renal pyramids are aligned with their bases
facing outward toward the renal cortex and their apexes point inward toward the
center of the kidney.

Each apex connects to a minor calyx, a small hollow tube that collects urine.
The minor calyces merge to form 3 larger major calyces, which further merge to
form the hollow renal pelvis at the center of the kidney. The renal pelvis exits the
kidney at the renal hilus, where urine drains into the ureter.

Each kidney contains around 1 million individual nephrons, the kidneys’

microscopic functional units that filter blood to produce urine. The nephron is made
of 2 main parts, the renal corpuscle and the renal tubule.

Responsible for filtering the blood, our renal corpuscle is formed by the
capillaries of the glomerulus and the glomerular capsule (also known as Bowman’s
capsule). The glomerulus is a bundled network of capillaries that increases the
surface area of blood in contact the blood vessel walls. Surrounding the glomerulus
is the glomerular capsule, a cup-shaped double layer of simple squamous
epithelium with a hollow space between the layers. Special epithelial cells known as
podocytes form the layer of the glomerular capsule surrounding the capillaries of
the glomerulus. Podocytes work with the endothelium of the capillaries to form a
thin filter to separate urine from blood passing through the glomerulus. The outer
layer of the glomerular capsule holds the urine separated from the blood within the
capsule. At the far end of the glomerular capsule, opposite the glomerulus, is the
mouth of the renal tubule.

The primary function of the kidneys is the excretion of waste products

resulting from protein metabolism and muscle contraction. The liver metabolizes
dietary proteins to produce energy and produces toxic ammonia as a waste
product. The liver is able to convert most of this ammonia into uric acid and urea,
which are less toxic to the body. Meanwhile, the muscles of our body use creatine
as an energy source and, in the process, produce the waste product creatinine.
Ammonia, uric acid, urea, and creatinine all accumulate in the body over time and
need to be removed from circulation to maintain homeostasis.

The glomerulus in the kidneys filter all four of these waste products out of
the bloodstream, allowing us to excrete them out of our bodies in urine. Around
50% of the urea found in the blood is reabsorbed by the tubule cells of the nephron
and returned to the blood supply. Urea in the blood helps to concentrate other more
toxic waste products in urine by maintaining the osmotic balance between urine and
blood in the renal medulla.

The kidneys are able to control the volume of water in the body by changing
the reabsorption of water by the tubules of the nephron. Under normal conditions,
the tubule cells of the nephron tubules reabsorb (via osmosis) nearly all of the
water that is filtered into urine by the glomerulus.

Water reabsorption leads to very concentrated urine and the conservation of

water in the body. The hormones antidiuretic hormone (ADH) and aldosterone both
increase the reabsorption of water until almost 100% of the water filtered by the
nephron is returned to the blood. ADH stimulates the formation of water channel
proteins in the collecting ducts of the nephrons that permit water to pass from urine
into the tubule cells and on to the blood. Aldosterone functions by increasing the
reabsorption of Na+ and Cl- ions, causing more water to move into the blood via
osmosis.

In situations where there is too much water present in the blood, our heart
secretes the hormone atrial natriuretic peptide (ANP) in order to increase the
excretion of Na+ and Cl- ions. Increased concentration of Na+ and Cl- in urine
draws water into the urine via osmosis, increasing the volume of urine produced.

The kidneys regulate the pH level of the blood by controlling the excretion of
hydrogen ions (H+) and bicarbonate ions (HCO3-). Hydrogen ions accumulate when
proteins are metabolized in the liver and when carbon dioxide in the blood reacts
with water to form carbonic acid (H2CO3). Carbonic acid is a weak acid that
partially dissociates in water to form hydrogen ions and bicarbonate ions. Both ions
are filtered out of the blood in the glomerulus of the kidney, but the tubule cells
lining the nephron selectively reabsorb bicarbonate ions while leaving hydrogen ions
as a waste product in urine. The tubule cells may also actively secrete additional
hydrogen ions into the urine when the blood becomes extremely acidic.

The reabsorbed bicarbonate ions enter the bloodstream where they can
neutralize hydrogen ions by forming new molecules of carbonic acid. Carbonic acid
passing through the capillaries of the lungs dissociates into carbon dioxide and
water, allowing us to exhale the carbon dioxide.

The kidneys maintain the homeostasis of important electrolytes by controlling their

excretion into urine.
 Sodium (Na+): Sodium is a vital electrolyte for muscle function, neuron
function, blood pressure regulation, and blood volume regulation. Over 99%
of the sodium ions passing through the kidneys are reabsorbed into the blood
from tubular filtrate. Most of the reabsorption of sodium takes place in the
proximal convoluted tubule and ascending loop of Henle.
 Potassium (K+): Just like sodium, potassium is a vital electrolyte for muscle
function, neuron function, and blood volume regulation. Unlike sodium,
 however, only about 60 to 80% of the potassium ions passing through the
kidneys are reabsorbed. Most of the reabsorption of potassium occurs in the
proximal convoluted tubule and ascending loop of Henle.
 Chloride (Cl-): Chloride is the most important anion (negatively charged ion)
in the body. Chloride is vital to the regulation of factors such as pH and
cellular fluid balance and helps to establish the electrical potential of neurons
and muscle cells. The proximal convoluted tubule and ascending loop of
Henle reabsorb about 90% of the chloride ions filtered by the kidneys.
 Calcium (Ca2+): Calcium is not only one of the most important minerals in
the body that composes the bones and teeth, but is also a vital electrolyte.
Functioning as an electrolyte, calcium is essential for the contraction of
muscle tissue, the release of neurotransmitters by neurons, and the
stimulation of cardiac muscle tissue in the heart. The proximal convoluted
tubule and the ascending loop of Henle reabsorb most of the calcium in
tubular filtrate into the blood. Parathyroid hormone increases the
reabsorption of calcium in the kidneys when blood calcium levels become too
low.
 Magnesium (Mg2+): Magnesium ion is an essential electrolyte for the proper
function of enzymes that work with phosphate compounds like ATP, DNA,
and RNA. The proximal convoluted tubule and loop of Henle reabsorb most of
the magnesium that passes through the kidney.

The kidneys are able to control blood pressure by either reabsorbing water to
maintain blood pressure or by allowing more water than usual to be excreted into
urine and thus reduce blood volume and pressure. Sodium ions in the body help to
manage the body’s osmotic pressure by drawing water towards areas of high
sodium concentration. To lower blood pressure, the kidneys can excrete extra
sodium ions that draw water out of the body with them. Conversely, the kidneys
may reabsorb additional sodium ions to help retain water in the body.

Finally, the kidneys produce the enzyme renin to prevent the body’s blood
pressure from becoming too low. The kidneys rely on a certain amount of blood
pressure to force blood plasma through the capillaries in the glomerulus. If blood
pressure becomes too low, cells of the kidneys release renin into the blood. Renin
starts a complex process that results in the release of the hormone aldosterone by
the adrenal glands. Aldosterone stimulates the cells of the kidney to increase their
reabsorption of sodium and water to maintain blood volume and pressure.

The kidneys maintain a small but important endocrine function by producing the
hormones calcitriol and erythropoietin.
 Calcitriol is the active form of vitamin D in the body. Tubule cells of the
proximal convoluted tubule produce calcitriol from inactive vitamin D
molecules. At that point, calcitriol travels from the kidneys through the
bloodstream to the intestines, where it increases the absorption of calcium
from food in the intestinal lumen.
 Erythropoietin (EPO) is a hormone produced by cells of the peritubular
capillaries in response to hypoxia (a low level of oxygen in the blood). EPO
stimulates the cells of red bone marrow to increase their output of red blood
 cells. Oxygen levels in the blood increase as more red blood cells mature and
enter the bloodstream. Once oxygen levels return to normal, the cells of the
peritubular capillaries stop producing EPO.

Several hormones produced elsewhere in the body help to control the function of
the kidneys. These are antidiuretic hormone, angiotensin II, aldosterone, atrial
natriuretic peptide.

Red blood cells play an important role in your health by carrying fresh oxygen
throughout the body. Non-nucleated formed
elements in the blood. It lacks cytoplasmic
organelles such as nucleolus, mitochondria &
ribosomes. It is biconcave and has a size of
7.2um in diameter, a of thickness 2um at the
periphery and 1um at the center . The red
color of RBC is due to the presence of
hemoglobin. Spectrin a contractile protein
maintains shape and flexibility of RBC and
antigen on cell membrane helps in blood group
classification.

Hemoglobin is the protein inside red blood

cells that carries oxygen. Red blood cells also
remove carbon dioxide from your body, transporting it to the lungs for you to
exhale. Red blood cells are made inside your bones, in the bone marrow. They
typically live for about 120 days, and then they die.

Diseases of the red blood cells include many types of anemia, a condition in
which there is too few red
blood cells to carry sufficient
oxygen throughout the body.
People with anemia may have
red blood cells that have an
unusual shape or that look
normal, larger than
normal, or smaller than
normal.

The heart is the pump

responsible for maintaining
adequate circulation of
oxygenated blood around the
vascular network of the body.
It is a four-chamber pump,
with the right side receiving
deoxygenated blood from the
body at low presure and pumping it to the lungs (the pulmonary circulation) and
the left side receiving oxygenated blood from the lungs and pumping it at high
pressure around the body (the systemic circulation).

The myocardium (cardiac muscle) is a specialised form of muscle, consisting of

individual cells joined by electrical connections. The contraction of each cell is
produced by a rise in intracellular calcium concentration leading to spontaneous
depolarisation, and as each cell is electrically connected to its neighbour,
contraction of one cell leads to a wave of depolarisation and contraction across the
myocardium.

C. PATHOPHYSIOLOGY

The liver plays a central role in lipid metabolism, serving as the center for
lipoprotein uptake, formation and export to the circulation. Alterations in hepatic
lipid metabolism can contribute to the development of chronic liver disease such as
hepatitis B. Chronic liver disease can also impact hepatic lipid metabolism leading
to alterations in circulating lipid levels contributing to hyperlipidemia- which is the
abnormally elevated levels of any or all lipids or lipoproteins in the blood. It is the
most common form of dyslipidemia. Hypertension or high blood pressure, also
occasionally labeled as arterial hypertension, is a disorder wherein a person doesn’t
demonstrate normal blood pressure. This happens when the cholesterol level in the
body surges from its normal reading. Saturated foods, processed fats, trans-fats, or
any unhealthy fats are among the fat forms connected to hypertension. Cholesterol
accumulation in kidney cells increases sodium retention. Sodium retention can
increase blood volume, which increases blood pressure resulting to hypertension.
Sodium retention also lowers endothelial cell nitric oxide production. High salt
intake in people with salt-sensitive hypertension causes a decrease in artery
elasticity and raises blood pressure.

Moreover, hepatitis characterized as a condition wherein there’s an inflammation

in the liver due to the presence of inflammatory cells in liver tissue can lead to
fibrosis or scarring and cirrhosis and can lead to another cause of hypertension
which is “decompensated cirrhosis”. This type of hypertension occurs when the
blood can no longer flow properly through the liver and there’s more pressure in the
portal vein directed to the organ.

Diabetes can be classified into two types: Type 1 and Type 2.

Type 1 diabetes mellitus is characterized by absolute insulin deficiency. In type 1A,
a cellular-mediated autoimmune destruction of beta cells of the pancreas occurs.
The disease process is initiated by an environmental factor—that is, an infectious or
noninfectious agent in genetically susceptible individuals.

Some genes in the histocompatibility leukocyte antigen (HLA) system are

thought to be crucial. A stress-induced epinephrine release, which inhibits insulin
release (with resultant hyperglycemia), sometimes occurs and may be followed by
a transient asymptomatic period known as "the honeymoon." Lasting weeks to
months, the honeymoon precedes the onset of overt, permanent diabetes.

Amylin, a beta-cell hormone that is normally cosecreted with insulin in

response to meals, is also completely deficient in persons with type 1 diabetes
mellitus. Amylin exhibits several glucoregulatory effects that complement those of
insulin in postprandial glucose regulation. Idiopathic forms of type 1 diabetes also
occur, without evidence of autoimmunity or HLA association; this subset is termed
type 1B diabetes. While Type 2 diabetes mellitus results from a defect in insulin
secretion and an impairment of insulin action in hepatic and peripheral tissues,
especially muscle tissue and adipocytes. A postreceptor defect is also present,
causing resistance to the stimulatory effect of insulin on glucose use. As a result, a
relative insulin deficiency develops, unlike the absolute deficiency found in patients
with type 1 diabetes. The specific etiologic factors are not known, but genetic input
is much stronger in type 2 diabetes than in the type 1 form.

Diabetes increases the risk of developing high blood pressure and other
cardiovascular problems, because diabetes adversely affects the arteries,
predisposing them to atherosclerosis - narrowing of the arteries.

Though the relationship between diabetes and chronic disease and

depression isn't fully understood, the rigors of managing diabetes and other chronic
diseases can be stressful and lead to symptoms of depression. Depression can lead
to poor lifestyle decisions, such as unhealthy eating, less exercise, smoking and
weight gain — all of which are risk factors for diabetes.

The loss of red blood cell elasticity is central to the pathophysiology of sickle-
cell disease. Normal red blood cells are quite elastic, which allows the cells to
deform to pass through capillaries. In sickle-cell disease, low oxygen
tension promotes red blood cell sickling and repeated episodes of sickling damage
the cell membrane and decrease the cell's elasticity. These cells fail to return to
normal shape when normal oxygen tension is restored. As a consequence, these
rigid blood cells are unable to deform as they pass through narrow capillaries,
leading to vessel occlusion and ischemia.

The actual anemia of the illness is caused by hemolysis, the destruction of

the red cells, because of their shape. Although the bone marrow attempts to
compensate by creating new red cells, it does not match the rate of
destruction. Healthy red blood cells typically function for 90–120 days, but sickled
cells only last 10–20 days. This trait is inherited from one of his or her parent and
often is asymptomatic.
 CHAPTER IV

PRESENTATION OF DATA

a. Laboratory Results

TESTS NORMAL PATIENT’S RESULT INTERP RATIONALE

VALUES RETATI
ON
1. Fasting 3.9-5.6 5.9 mmol/L High A level of 5.6 to 6.9
Blood mmol/L mmol/L means that
Glucose there is an impaired
fasting glucose, a type
of prediabetes. This
increases the risk of
developing type 2
diabetes.
2. HBA1c 4.0-6.0% 115.8% High HbA1C is widely
(CE-HPLC) accepted as the most
reliable marker for
monitoring long-term
glucose control in
diabetes.

The patient’s marked

high HBA1c result is
caused by an
interference of a
hemoglobin variant.

3. Hb Hb A: 2.7% Hb A Low The marked low result of

Variant 95%-98% Hb A is due to the
Analysis presence of
(Bio-Rad Hb A2: 3.2% Wild-type Hb A2 Normal heterozygous Hb S and
VARIANT 1.5%- Hb Raleigh.
CE-HPLC 3.5%
β-
Thalassem Hb F: < <1.0% Hb F Normal
ia Short 2%
Program)
Hb S: 37.4% Sickle cell Hb (Hb S) High
Absent An increase in Hb S of
 (0%) 35-40% with Hb A
concentration of 60-
53.0 % P2 Abnormal 65% is indicative of
Sickle Cell Trait. The
patient’s sickle cell trait
is due to heterozygous
Hb S and Hb Raleigh.
Since Hb Raleigh is just
another form of Hb A
but with a mutation of
valine alanine in the
beta chain’s 1st position,
it could still carry
oxygen and act as Hb A.

4. Citrate C > S > Hb S Abnormal

Agar A, D, G, E
Electropho > F Another band with a Abnormal Indicates the presence
resis mobility similar to Hb F of another hemoglobin
(QuickGel variant.
Acid;
Helena
Laboratori
es)

b. Suggested Medications and Their Actions

 Hyperlipidemia, hypertension and progression of impaired glucose tolerance

to diabetes can be effectively delayed or prevented by intensive lifestyle
modification, pharmacologic and non pharmacologic therapy including proper
diet, exercise, counseling related to smoking cessation and alcohol intake,
and start of anti diabetic medication such as metformin(if advised).
 Many substances have been tested as anti-sickling agents but none has
proven to be totally significant. So the prevention of sickle cell crises by
avoiding situation that triggers the precipitation of hemoglobin s is
recommended. However, if crisis does occur, patients is hydrated, pain can
be alleviated with analgesic such as aspirin, infection should be treated with
right antibiotics, and excessive sweating must be avoided since dehydration
causes sickling.
 CHAPTER V

SUMMARY, CONCLUSION AND RECOMMENDATION

SUMMARY

A 55-year-old woman with a medical history of hepatitis B,

hyperlipidemia, hypertension, anemia, and depression has an impaired fasting
glucose concentration of 5.9 mmol/L. The patient was tested for HbA1c by cation-
exchange HPLC (CE-HPLC) with markedly increased result of 115.8%. Hb variant
analysis were performed to check if the cause of the increased HbA1c is not due to
hemoglobin variants that could cause interferences and behave like the normal
HbA1c. The analysis revealed the absence of Hb A and the presence of Hb S
(37.4%), along with normal Hb A2 (3.2%) and Hb F (<1.0%). A large peak was
observed that eluted earlier than Hb A with a result of 53.0% that was called as P2.
Further tests should be conducted, and the hemoglobin present should be specified
through confirmatory tests.

CONCLUSION

After thoroughly studying the laboratory results. We have demonstrated in

this study that the commonly used cation-exchange HPLC HbA1c assay the D-10TM,
Bio-Rad Laboratories can report a falsely high value of HbA1c in the presence of
hemoglobinopathy. In our case, the patient was heterozygous for Hb S, in which
the N-terminal valine of the ß-chain was glycated as Hb S1c, and Hb Raleigh, in
which the N-terminal acetylalanine of the ß-chain could not be glycated. Therefore,
Hb A1c did not exist in this patient. The spurious Hb A1c value (115.8%) primarily
represented Hb Raleigh. Hb Raleigh is relatively rare and could be considered as a
clinically silent variant without pathological manifestations. Nevertheless,
differentiation from other clinically overt hemoglobinopathies is still essential.

RECOMMENDATION

When a spurious HbA1c result is obtained, there is a possibility of

interference such us the presence of hemoglobin variants, and the interpretation of
HbA1c results should be based on patient’s medical history and other laboratory
results.

•Identify the Hemoglobin Variant and alternative HbA1c methods that are free of
interference should be used. If there is no appropriate method for a particular Hb
variant, fructosamine, daily multiple testing of continuous glucose monitoring may
be used to monitor glycemic control. To distinguish Hemoglobin Variants it is
recommended to use:
•Turbidimetric Inhibition Immunoassay to avoid inconsistency with previous blood
test and for evaluation between the measurements of Hb Raleigh and HbA1c

•DNA Sequence of β globin gene which is more preferable

CHAPTER VI

BIBLIOGRAPHY

Chapter 18. The Cardiovascular System: Blood Retrieved March 23, 2018
from:https://opentextbc.ca/anatomyandphysiology/chapter/18-3-erythrocytes/

Human Anatomy & Physiology of the Liver By: Pharma Tips Date: 07-Oct-2013